Post-annealing microgel particle systems and methods

Abstract

Provided herein are systems and methods for delivering a dermal filler formulation to a tissue site, wherein the dermal filler formulation comprises a hydrogel that anneals in vivo to form a covalently stabilized porous scaffold under conditions sufficient to form a cellular matrix within the covalently stabilized porous scaffold, the covalently stabilized porous scaffold being effective to durably fill at least a portion of a tissue site in a subject with the cellular matrix while minimizing a foreign body response in the subject.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 481,968, filed January 27, 2023, and U.S. Provisional Patent Application No. 63 / 484,439, filed February 10, 2023, each of which is incorporated by reference in its entirety for all purposes.

[0002] Incorporation by reference of sequence listing This application is filed with an electronic Sequence Listing, which is provided as file entitled 48469-707.601.xml, created on January 26, 2024, and is 11,694 bytes in size. The information in the electronic Sequence Listing is incorporated by reference in its entirety. Summary of the Invention

[0003] In some aspects, the present disclosure provides methods for delivering a dermal filler formulation to a tissue site in a subject, the method comprising delivering to the tissue site a dermal filler formulation comprising a hydrogel that anneals in vivo to form a covalently stabilized porous scaffold under conditions sufficient to form a cellular matrix within the covalently stabilized porosity, the cellular matrix forming new tissue at the tissue site while minimizing a foreign body reaction in the subject. In some embodiments, the delivering comprises subdermal administration. In some embodiments, the delivering comprises dermal administration. In some embodiments, the delivering comprises intradermal administration. In some embodiments, the delivering comprises subcutaneous administration. In some embodiments, the delivering comprises expelling the dermal filler formulation from a syringe or needle. In some embodiments, the needle has a gauge including about 25 gauge to about 35 gauge. In some embodiments, the needle has a gauge including about 27 gauge. In some embodiments, the needle has a gauge including about 30 gauge. In some embodiments, the delivering comprises exerting an extrusion force of up to 40 Newtons (N) on the dermal filler formulation. In some embodiments, the cellular matrix comprises cells endogenous to the subject. In some embodiments, at least a portion of the tissue site is permanently filled with the cellular matrix after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, the cellular matrix comprises at least 10% of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, the cellular matrix comprises at least 25% of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, the cellular matrix forms within about 30 days after delivery. In some embodiments, the cellular matrix begins to form within the scaffold within 7 days after administration. In some embodiments, the cellular matrix forms new tissue at the tissue site of the subject prior to complete degradation of the covalently stabilized porous scaffold.In some embodiments, the new tissue is characterized by having (i) mature vascularization, (ii) characteristics of the surrounding tissue at the tissue site, (iii), or a combination thereof. In some embodiments, the characteristics of the surrounding tissue at the tissue site include cell types that are functionally differentiated from the surrounding tissue. In some embodiments, the new tissue has an in vivo survival time at the tissue site similar to the endogenous tissue surrounding the tissue site. In some embodiments, the tissue site is soft tissue. In some embodiments, the foreign body reaction is characterized by causing harm to the subject. In some embodiments, the harm is characterized by causing chronic inflammation, granuloma formation, scar tissue formation, nodule formation, swelling, pain, or any combination thereof. In some embodiments, the harm occurs at the tissue site. In some embodiments, the cellular matrix is ​​formed with minimal foreign body reaction in the subject, as measured by using histological analysis to detect the amount of granuloma at the tissue site and comparing the amount of granuloma at the tissue site to a reference tissue that does not contain the dermal filler formulation. In some embodiments, the cellular matrix is ​​formed while minimizing a foreign body response in a subject when a foreign body response is measured by using histological analysis to detect the amount of scar tissue at the tissue site and comparing the amount of scar tissue at the tissue site with reference tissue not containing the dermal filler formulation. In some embodiments, the cellular matrix is ​​formed while minimizing a foreign body response in a subject when a foreign body response is measured by using histological analysis to detect the amount of nodules at the tissue site and comparing the amount of nodules at the tissue site with reference tissue not containing the dermal filler formulation. In some embodiments, the cellular matrix is ​​formed while minimizing a foreign body response in a subject when a foreign body response is measured by using histological analysis to detect chronic inflammation at the tissue site. In some embodiments, the cellular matrix comprises an amount or type of collagen that mimics endogenous tissue at the tissue site. In some embodiments, the type of collagen comprises type I collagen, type III collagen, or a combination thereof.In some embodiments, type I collagen is present in a ratio of about 10:1 or less to type III collagen. In some embodiments, type I collagen is present in a ratio of about 6:1 or less to type III collagen. In some embodiments, type I collagen is present in a ratio of about 5:1 or less to type III collagen. In some embodiments, at least a portion of the tissue site comprises elastin after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, the dermal filler formulation is biocompatible with tissue at the tissue site as determined by one or more techniques described in ISO Standard 10993. In some embodiments, the covalently stabilized porous scaffold remains at the tissue site in an amount sufficient to fill at least a portion of the tissue site for an amount of time of 9 months or more after delivery. In some embodiments, the method further comprises delivering lidocaine to the tissue site. In some embodiments, the lidocaine is delivered at a concentration comprising about 1.0 milligrams per microliter (mg / mL) to about 5.0 mg / mL. In some embodiments, lidocaine is delivered at a concentration comprising about 3.0 mg / mL. In some embodiments, the hydrogel comprises a polymer comprising hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), collagen, polymethyl methacrylate, or any combination thereof. In some embodiments, the polymer is a copolymer comprising HA and PEG. In some embodiments, the polymer is HA. In some embodiments, the polymer is PEG. In some embodiments, the dermal filler formulation further comprises a vinyl or a derivative thereof. In some embodiments, the vinyl comprises vinyl sulfone (VS), acrylate, methacrylate, acrylamide, maleimide, norbornene, or any combination thereof. In some embodiments, the dermal filler formulation further comprises a thiol or a derivative thereof. In some embodiments, the thiol or derivative thereof comprises thiolated HA. In some embodiments, the thiol or derivative thereof comprises two or more thiols. In some embodiments, the thiol or derivative thereof comprises polyethylene glycol (PEG)-dithiol or a derivative thereof.In some embodiments, the hydrogel and PEG-dithiol or derivative thereof are delivered to a subject separately. In some embodiments, the hydrogel and PEG-dithiol or derivative thereof are delivered to a subject together. In some embodiments, the hydrogel and PEG-dithiol or derivative thereof have a shelf life of at least about 18 months when stored as a mixture in a single container. In some embodiments, the hydrogel and PEG-dithiol or derivative thereof have a shelf life of at least about 36 months when stored as a mixture in a single container at room temperature. In some embodiments, the dermal filler formulation is lyophilized. In some embodiments, the method further comprises reconstituting the dermal filler formulation before delivering the dermal filler formulation to the tissue site. In some embodiments, either the thiol or derivative thereof and the vinyl sulfone or derivative thereof are present in the dermal filler formulation in excess of the other. In some embodiments, the thiol or derivative thereof and the vinyl sulfone or derivative thereof are present in the dermal filler formulation in a 1:1 molar ratio. In some embodiments, the tissue site comprises (1) the mid-face or cheek region of a subject, (2) the cheek of a subject, (3) the chin of a subject, or (4) the lips of a subject, or (5) any combination thereof. In some embodiments, the method further comprises treating the tissue site of the subject by delivering a dermal filler formulation to the tissue site. In some embodiments, treating the tissue site comprises tissue filling, skin filling, wrinkle removal, cosmetic improvement of the skin surrounding the tissue site, tissue repair, correction of skin irregularities, treatment of one or more skin lesions, or any combination thereof. In some embodiments, tissue filling comprises building new tissue formation, generating new tissue formation, stimulating new tissue formation, or any combination thereof. In some embodiments, the one or more skin lesions comprise acne scars, basal cell carcinoma, cellulitis, epidermolysis bullosa, melanoma, Merkel cell carcinoma, scars, skin biopsies, skin cancer, squamous cell carcinoma, stretch marks, or any combination thereof.In some embodiments, treating a tissue site is achieved by delivering the dermal filler formulation to the tissue site of a subject once. In some embodiments, treating a tissue site is achieved by delivering the dermal filler formulation to the tissue site of a subject twice. In some embodiments, treating a tissue site is achieved by delivering the dermal filler formulation to the tissue site of a subject three times. In some embodiments, the covalently stabilized porous scaffold comprises a compressive modulus of about 1,000 Pascals (Pa) to about 100,000 Pa when the modulus is measured using a compression test (e.g., by Instron). In some embodiments, the covalently stabilized porous scaffold comprises a storage modulus of about 50 Pascals (Pa) to about 10,000 Pa when the storage modulus is measured using a rheometer. In some embodiments, the covalently stabilized porous scaffold comprises a plurality of pores having a median diameter comprising about 5 micrometers (μm) to about 1000 μm. In some embodiments, the dermal filler formulation further comprises a buffer. In some embodiments, the buffer comprises a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

[0004] In some aspects, the present disclosure provides a dermal filler system comprising: (a) microgel particles comprising a hydrogel polymer and a thiol or derivative thereof, wherein the hydrogel polymer comprises hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), or a combination thereof; and (b) vinyl sulfone (VS) or a derivative thereof, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold, and wherein either the thiol or derivative thereof and the vinyl sulfone or derivative thereof are present in the dermal filler formulation in excess of the other. In some aspects, the present disclosure provides a dermal filler system comprising: (a) a dermal filler formulation comprising microgel particles, wherein the microgel particles comprise a hydrogel polymer and a thiol or a derivative thereof, wherein the hydrogel polymer comprises hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), or a combination thereof; and (b) vinyl sulfone (VS) or a derivative thereof, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold comprising an elastic modulus of about 1,000 Pascals (Pa) to about 100,000 Pa. In some embodiments, the microgel particles are spherical. In some embodiments, the microgel particles comprise microspheres. In some embodiments, the microgel particles comprise a diameter of 5 μm to 1,000 μm. In some embodiments, the diameter is 50 μm to 1,000 μm. In some embodiments, the diameter is 80 μm to 140 μm. In some embodiments, the covalently stabilized porous scaffold comprises pores with a median pore size of about 5 μm or greater. In some embodiments, the pores comprise a median pore size of about 10 μm to about 35 μm. In some embodiments, the microgel particles further comprise one or more cell adhesion peptides. In some embodiments, the one or more cell adhesion peptides comprise an RGD peptide. In some embodiments, the RGD peptide comprises the amino acid sequence provided in any one of SEQ ID NOs: 1-2 or 6-9.In some embodiments, the RGD peptide comprises an amino acid sequence about 75% identical to the amino acid sequence provided in any one of SEQ ID NOs: 1-3. In some embodiments, the microgel particles further comprise one or more K peptides. In some embodiments, the one or more K peptides comprise the amino acid sequence provided in any one of SEQ ID NOs: 3, Ac-FKGGERCG-NH2. In some embodiments, the microgel particles further comprise one or more Q peptides. In some embodiments, the one or more Q peptides comprise the amino acid sequence provided in any one of SEQ ID NOs: 4. In some embodiments, the hydrogel polymer comprises a polydispersity of 0.1 or less. In some embodiments, the polydispersity is calculated based on the standard deviation and average size of the particles (e.g., PDI = (SD / mean)^2). In some embodiments, the dermal filler further comprises a buffer, and the buffer comprises a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, an acetate buffer, or any combination thereof. In some embodiments, the dermal filler system further comprises lidocaine. In some embodiments, lidocaine is present in the dermal filler system at a concentration of about 1.0 mg / mL to about 5.0 mg / mL. In some embodiments, lidocaine is present in the dermal filler system at a concentration of about 3.0 mg / mL. In some embodiments, the hydrogel polymer comprises HA and PEG. In some embodiments, the hydrogel is a copolymer of HA and PEG having approximately the same molecular weight as each of HA and PEG. In some embodiments, the HA comprises a molecular weight of 1 kilodalton (kDa) to 1 megadalton (1 MDa). In some embodiments, the HA comprises a molecular weight of 10 kDa to 250 kDa (e.g., 10, 40, 50, 150, and 250 kDa). In some embodiments, the PEG comprises a molecular weight of 1 kilodalton (kDa) to 5 kDa. In some embodiments, the hydrogel polymer comprises a thiol or a derivative thereof, or a VS or a derivative thereof, or a combination thereof. In some embodiments, the HA is modified to include a thiol or a derivative thereof to form thiolated HA.In some embodiments, PEG is modified to include VS or a derivative thereof to form PEG-VS. In some embodiments, the PEG-VS includes a multi-arm PEG-VS. In some embodiments, the multi-arm PEG-VS includes a 4-arm or 8-arm PEG-VS. In some embodiments, the VS includes divinyl sulfone. In some embodiments, the thiol or a derivative thereof and the VS or a derivative thereof are configured to interact with each other in a reaction to synthesize microgel particles. In some embodiments, the reaction includes a covalent synthesis reaction. In some embodiments, the covalent synthesis reaction includes a Michael addition (e.g., a thiol-ene Michael addition) reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof and the VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 1:1. In some embodiments, an excess of thiol or its derivative and VS or its derivative are present in the dermal filler system such that the excess of either thiol or its derivative or VS or its derivative participates in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the dermal filler system further comprises two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof. In some embodiments, the dermal filler system further comprises a molecule comprising two or more thiols or their derivatives. In some embodiments, the molecule comprises PEG. In some embodiments, the molecule comprises PEG-dithiol. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.4 kDa.In some embodiments, the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof. In some embodiments, the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol. In some embodiments, the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof of the PEG-dithiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the excess VS is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the dermal filler system further comprises PEG-divinylsulfone or a derivative thereof. In some embodiments, the PEG-divinylsulfone or a derivative thereof is configured to interact with excess thiol or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the divinyl sulfone or a derivative thereof of PEG-divinyl sulfone is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, an excess thiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the hydrogel polymer comprises HA. In some embodiments, the HA comprises a molecular weight of 1 kilodalton (kDa) to 1000 kDa. In some embodiments, the HA comprises a molecular weight of about 10 kDa to about 250 kDa. In some embodiments, the molecular weight comprises about 10, 40, 50, 150, or 250 kDa.In some embodiments, the dermal filler system further comprises glutaraldehyde or a derivative thereof, divinyl sulfone or a derivative thereof, 1,4-butanediol diglycidyl ether (BDDE) or a derivative thereof, or any combination thereof, configured to interact in a crosslinking reaction to synthesize the microgel particles. In some embodiments, HA is modified to include a thiol or a derivative thereof to form thiolated HA. In some embodiments, HA is modified to include VS or a derivative thereof to form HA-VS. In some embodiments, the thiol or a derivative thereof and VS or a derivative thereof are configured to interact in a crosslinking reaction to synthesize the microgel particles. In some embodiments, the crosslinking reaction comprises a covalent synthesis reaction. In some embodiments, the covalent synthesis reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof and the VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 1:1. In some embodiments, the thiol or derivative thereof and VS or derivative thereof are present in excess of each other in the dermal filler system, such that the excess of thiol or derivative thereof or VS or derivative thereof participates in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the dermal filler system further comprises two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof. In some embodiments, the dermal filler system further comprises a molecule comprising two or more thiols or derivatives thereof. In some embodiments, the molecule comprises PEG. In some embodiments, the molecule comprises PEG-dithiol. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.4 kDa.In some embodiments, the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof. In some embodiments, the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol. In some embodiments, the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or derivative thereof of the PEG-dithiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the excess VS is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the dermal filler system further comprises PEG-divinyl sulfone or a derivative thereof. In some embodiments, the PEG-divinyl sulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the divinyl sulfone or derivative thereof of the PEG-divinyl sulfone is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the excess thiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the hydrogel polymer comprises PEG. In some embodiments, the PEG comprises a molecular weight of 1 kilodalton (kDa) to 1000 kDa. In some embodiments, the hydrogel polymer further comprises a thiol or derivative thereof, a VS or derivative thereof, or a combination thereof. In some embodiments, the PEG comprises a thiol or derivative thereof to form a PEG-dithiol. In some embodiments, the PEG comprises a VS or derivative thereof to form a PEG-VS. In some embodiments, the PEG-VS group comprises a multi-arm PEG-VS. In some embodiments, the multi-arm PEG-VS comprises a 4-arm or 8-arm PEG-VS. In some embodiments, the VS comprises divinyl sulfone. In some embodiments, the thiol or derivative thereof and the VS or derivative thereof are configured to interact with each other in a reaction to synthesize microgel particles. In some embodiments, the reaction comprises a covalent synthesis reaction.In some embodiments, the covalent synthesis reaction comprises a Michael addition (e.g., thiol-ene Michael addition) reaction or a pseudo-Michael addition reaction. In some embodiments, a thiol or a derivative thereof is a Michael donor in the Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof and VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 1:1. In some embodiments, an excess of either the thiol or a derivative thereof and either the VS or a derivative thereof is present in the dermal filler system such that the excess of either the thiol or a derivative thereof or the VS or a derivative thereof participates in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the dermal filler system further comprises two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof. In some embodiments, the dermal filler system further comprises a molecule comprising two or more thiols or derivatives thereof. In some embodiments, the molecule comprises PEG. In some embodiments, the molecule comprises PEG-dithiol. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.4 kDa. In some embodiments, the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof. In some embodiments, the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol. In some embodiments, the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.In some embodiments, the thiol or derivative thereof of the PEG-dithiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the excess VS is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the dermal filler system further comprises PEG-divinylsulfone or a derivative thereof. In some embodiments, the PEG-divinylsulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the divinylsulfone or derivative thereof of the PEG-divinylsulfone is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the excess thiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the covalently stabilized porous scaffold is degradable in vivo by one or more degradation pathways. In some embodiments, the one or more degradation pathways include oxidative degradation, enzymatic degradation, photolysis, or hydrolysis. In some embodiments, the covalently stabilized porous scaffold remains at the tissue site for at least 18 months before complete degradation. In some embodiments, the covalently stabilized porous scaffold remains at the tissue site for at least 24 months before complete degradation. In some embodiments, the microgel particles are present in a suspension comprising microgel particles and water. In some embodiments, the suspension comprises 50% to 100% volume fraction of microgel particles. In some embodiments, when the dermal filler is formulated for needle administration, the volume fraction of microgel particles is about 50% or greater. In some embodiments, the covalently stabilized porous scaffold comprises a compressive modulus of 1,000 Pascals (Pa) to 50,000 Pa. In some embodiments, the covalently stabilized porous scaffold comprises a compressive modulus of 5,000 Pascals (Pa) to 100,000 Pa in an unswollen state.In some embodiments, the covalently stabilized porous scaffold comprises a compressive modulus in a swollen state of 1,000 Pascals (Pa) to 50,000 Pa. In some embodiments, the covalently stabilized porous scaffold comprises a storage modulus of 50 Pascals (Pa) to 10,000 Pa. In some embodiments, the covalently stabilized porous scaffold comprises a storage modulus of 60 Pa to 1,000 Pa. In some embodiments, the covalently stabilized porous scaffold comprises a loss modulus of about 10 Pascals (Pa) to 10,000 Pa. In some embodiments, when the dermal filler is formulated for needle administration, the covalently stabilized porous scaffold comprises a compressive modulus of about 50 Pa or greater. In some embodiments, the thiol or derivative thereof and the vinyl sulfone or derivative thereof are present in the dermal filler system in a molar ratio of about 0.3 to about 0.8 to achieve the compressive modulus. In some embodiments, the microgel particles are present in a suspension comprising microgel particles and water, and the volume fraction of the suspension comprises microgel particles at 50% to 100% to achieve a compressive modulus. In some embodiments, the covalently stabilized scaffold comprises an apparent viscosity of 1,000 to about 1,000,000 mPa·s. In some embodiments, the covalently stabilized porous scaffold comprises a pH of 5.0 to 9.0. In some embodiments, the covalently stabilized porous scaffold comprises a pH of 6.5 to 7.5. In some embodiments, the covalently stabilized porous scaffold comprises an osmolality of about 100 milliosmoles per kilogram (mOsmol / kg) to about 400 mOsmol / kg. In some embodiments, the hydrogel polymer comprises a degree of substitution per monomer of about 5% to about 20%. In some embodiments, the hydrogel polymer comprises modified HA. In some embodiments, the system is lyophilized. In some embodiments, the microgel particles comprise a modulus of elasticity of about 10 kPa to about 100 kPa. In some embodiments, the microgel particles comprise a modulus of elasticity of about 15 kPa to about 50 kPa.

[0005] In some aspects, the present disclosure provides a cosmetic formulation comprising a dermal filler system described herein in a suspension, the suspension comprising a buffer solution and a molecule comprising two or more thiols or derivatives thereof, two or more vinyls or derivatives thereof, or a combination thereof. In some embodiments, the buffer solution comprises a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, an acetate buffer, or any combination thereof. In some embodiments, the molecule comprises a PEG-dithiol. In some embodiments, the cosmetic formulation is formulated for administration to a subject. In some embodiments, the administration is subdermal, dermal, intradermal, or subcutaneous. In some embodiments, the administration minimizes a foreign body reaction in the subject. In some embodiments, the cosmetic formulation comprises a dosage volume of about 0.75 mL to about 1.0 mL. In some embodiments, the cosmetic formulation is sterile. In some embodiments, the cosmetic formulation further comprises lidocaine.

[0006] In some aspects, the present disclosure provides a delivery device comprising: (a) a body containing a dermal filler system described herein or a cosmetic formulation described herein; and (b) an applicator in fluid communication with the body, wherein the delivery device is sterile. In some embodiments, the delivery device is a syringe or a needle. In some embodiments, the delivery device is a microneedle patch.

[0007] In some aspects, the present disclosure provides a method of lyophilizing a dermal filler system described herein or a cosmetic formulation described herein, the method comprising lyophilizing the dermal filler system or cosmetic formulation into a powder. In some embodiments, the method further comprises reconstituting the freeze-dried dermal filler system or cosmetic formulation for delivery to a subject.

[0008] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief explanation of the drawings]

[0009] For a better understanding of the features and advantages of the present subject matter, please refer to the following detailed description and accompanying drawings that set forth illustrative embodiments. [Figure 1] FIG. 1 illustrates a workflow for initiating and performing rheology measurements, including flow curves for apparent viscosity measurements and frequency or amplitude sweeps for storage and loss modulus measurements, according to some embodiments herein. [Figure 2A] 2A-2B show viscosity curves illustrating the power law region, according to some embodiments herein. Figure 2A shows the complete viscosity curve, according to some embodiments herein. As shown in Figures 2A-2B, higher particle stiffness (15 kPa, 30 kPa, and 46 kPa) generally correlates with higher viscosity, according to some embodiments herein. [Figure 2B] 2A-2B show viscosity curves exhibiting the power law region, according to some embodiments herein. FIG. 2B shows viscosity curves relating to only the power law region, according to some embodiments herein. As shown in FIG. 2A-2B, higher particle stiffness (15 kPa, 30 kPa, and 46 kPa) generally correlates with higher viscosity, according to some embodiments herein. [Figure 3A]3A-3C show viscosity curves at constant volume fraction (VF) and different microgel particle stiffnesses, according to some embodiments herein. FIG. 3A shows viscosity curves for varying unswollen microgel particle stiffnesses at a microgel particle volume fraction (VF) of 0.75, according to some embodiments herein. As shown in FIG. 3A-3C, higher particle stiffnesses (15 kPa, 20 kPa, 30 kPa, and 46 kPa) are associated with higher viscosities, according to some embodiments herein. [Figure 3B] 3A-3C show viscosity curves at constant volume fraction (VF) and different microgel particle stiffnesses, according to some embodiments herein. FIG. 3B shows viscosity curves for varying microgel particle stiffnesses at a microgel particle volume fraction (VF) of 0.85, according to some embodiments herein. As shown in FIGS. 3A-3C, higher particle stiffnesses (15 kPa, 20 kPa, 30 kPa, and 46 kPa) are associated with higher viscosities, according to some embodiments herein. [Figure 3C] 3A-3C show viscosity curves at a constant volume fraction (VF) and different microgel particle stiffnesses, according to some embodiments herein. FIG. 3C shows viscosity curves for varying microgel particle stiffnesses at a microgel particle volume fraction (VF) of 0.95, according to some embodiments herein. As shown in FIGS. 3A-3C, higher particle stiffnesses (15 kPa, 20 kPa, 30 kPa, and 46 kPa) are associated with higher viscosities, according to some embodiments herein. [Figure 4A] 4A-4D show viscosity curves at constant microgel particle stiffness and different volume fractions, according to some embodiments herein. FIG. 4A shows viscosity curves for varying microgel particle volume fractions at an unswollen microgel particle stiffness of 15 kilopascals (kPa), according to some embodiments herein. As shown in FIGS. 4A-4D, higher volume fractions (0.75 mL / mL, 0.85 mL / mL, and 0.95 mL / mL) are associated with higher viscosities, according to some embodiments herein. [Figure 4B] Figures 4A-4D show viscosity curves at constant microgel particle stiffness and different volume fractions, according to some embodiments herein. Figure 4B shows viscosity curves for varying microgel particle volume fractions at an unswollen microgel particle stiffness of 20 kPa, according to some embodiments herein. As shown in Figures 4A-4D, higher volume fractions (0.75 mL / mL, 0.85 mL / mL, and 0.95 mL / mL) are associated with higher viscosities, according to some embodiments herein. [Figure 4C] Figures 4A-4D show viscosity curves at constant microgel particle stiffness and different volume fractions, according to some embodiments herein. Figure 4C shows viscosity curves for varying microgel particle volume fractions at an unswollen microgel particle stiffness of 30 kPa, according to some embodiments herein. As shown in Figures 4A-4D, higher volume fractions (0.75 mL / mL, 0.85 mL / mL, and 0.95 mL / mL) are associated with higher viscosities, according to some embodiments herein. [Figure 4D] 4A-4D show viscosity curves at constant microgel particle stiffness and different volume fractions, according to some embodiments herein. FIG. 4D shows viscosity curves for varying microgel particle volume fractions at an unswollen microgel particle stiffness of 45 kPa, according to some embodiments herein. As shown in FIGS. 4A-4D, higher volume fractions (0.75 mL / mL, 0.85 mL / mL, and 0.95 mL / mL) are associated with higher viscosities, according to some embodiments herein. [Figure 5] Figure 5 shows the effect of volume fraction (VF) and microgel particle stiffness on the elastic modulus (EM) of scaffolds after annealing, according to some embodiments herein. As shown in Figure 5, higher particle stiffness (15 kPa, 20 kPa, 30 kPa, and 46 kPa) is associated with higher elastic modulus of scaffolds after annealing, according to some embodiments herein. [Figure 6]Figures 6A-6F show the effect of microgel particle volume fraction (VF) and microgel particle stiffness on apparent viscosity across the shear rate range assessed (0.1 s-1 to 10 s-1), according to some embodiments. Figure 6A shows the dependence of viscosity on microgel particle volume fraction (VF) at different unswollen microgel particle stiffnesses measured at a shear rate of 0.1 inverse seconds (s-1), according to some embodiments herein. Figure 6B shows the dependence of viscosity on unswollen microgel particle stiffness at different volume fractions (VF) measured at a shear rate of 0.1 s-1, according to some embodiments herein. Figure 6C shows the dependence of viscosity on microgel particle volume fraction (VF) at different unswollen microgel particle stiffnesses measured at a shear rate of 1.0 s-1, according to some embodiments herein. Figure 6D shows the dependence of viscosity on non-swollen microgel particle stiffness at different volume fractions (VF) measured at a shear rate of 0.1 s, according to some embodiments herein. Figure 6E shows the dependence of viscosity on microgel particle volume fractions (VF) at different non-swollen microgel particle stiffnesses measured at a shear rate of 10.0 s, according to some embodiments herein. Figure 6F shows the dependence of viscosity on non-swollen microgel particle stiffness at different volume fractions (VF) measured at a shear rate of 10.0 s, according to some embodiments herein. As shown in Figures 6A, 6C, and 6E, higher particle stiffness (15 kPa, 20 kPa, 30 kPa, and 46 kPa) is associated with higher viscosity, according to some embodiments herein. As shown in Figures 6B, 6D, and 6F, according to some embodiments herein, higher volume fractions (0.75 mL / mL, 0.85 mL / mL, and 0.95 mL / mL) are associated with higher viscosities. [Figure 7] FIG. 7 shows the rheological working range of a microgel particle suspension obtained from a viscosity curve, according to some embodiments herein. [Figure 8]Figures 8A-D show exemplary anatomical injection schematics and timing of measurements used in in vivo testing according to some embodiments herein. Figure 8A shows the location of the anatomical injection site according to some embodiments herein. Figure 8B shows test design parameters according to some embodiments herein. Figure 8C shows a schematic of the injection site anatomy according to some embodiments herein. Figure 8D shows an image of the injection site in excised tissue according to some embodiments herein. [Figure 9] FIG. 9 illustrates the synthesis of microgel particles by Michael addition, according to some embodiments herein. [Figure 10] Figure 10 shows in vitro cell viability when cells in culture are exposed to the microgel particle system described herein. As shown, each grouping of bars represents, from left to right, days 1, 3, and 6, respectively. [Figure 11] 11 shows cell viability in the presence of different concentrations of PETMA, according to some embodiments herein. As shown, each grouping of bars, from left to right, represents day 1, day 3, and day 6, respectively. [Figure 12] 12 shows cell viability in the presence of various annealing reactions, according to some embodiments herein. As shown, each grouping of bars, from left to right, represents day 1, day 3, and day 6, respectively. [Figure 13] FIG. 13 shows an exemplary injection site of the dermal filler system disclosed herein in a rat, according to some embodiments herein. [Figure 14A] 14A-14B show histology of the injection site in a rat 7 days after injection, according to some embodiments herein. Figure 14A shows the histology of Formulation 4 in a rat 7 days after injection, in four insets, according to some embodiments herein. [Figure 14B]14A-14B show the histology of the injection site in a rat 7 days after injection, according to some embodiments herein. FIG. 14B shows the histology of Formulation 4 in a rat 7 days after injection, in four insets, according to some embodiments herein. [Figure 15A] 15A-15B show histology of injection sites in rats 30 days post-injection, according to some embodiments herein. Figure 15A shows the histology of Formulation 4 in rats 30 days post-injection, in four insets, according to some embodiments herein. [Figure 15B] 15A-15B show the histology of the injection site in a rat 30 days after injection, according to some embodiments herein. FIG. 15B shows the histology of Formulation 4 in a rat 30 days after injection, in four insets, according to some embodiments herein. [Figure 16A] 16A-16D show a comparison of injection sites in a rat model 30 days after injection, according to some embodiments herein. Figure 16A shows the degradation of Formulation 2, immune response, new protein deposition, and cellular infiltration, according to some embodiments herein. [Figure 16B] 16A-16D show a comparison of injection sites in a rat model 30 days after injection, according to some embodiments herein. FIG. 16B shows the degradation of Formulation 3, immune response, new protein deposition, and cellular infiltration, according to some embodiments herein. [Figure 16C] 16A-16D show a comparison of injection sites in a rat model 30 days after injection, according to some embodiments herein. FIG. 16C shows the degradation of Formulation 4, immune response, new protein deposition, and cellular infiltration, according to some embodiments herein. [Figure 16D] 16A-16D show a comparison of injection sites in a rat model 30 days after injection, according to some embodiments herein. Figure 16D shows the degradation of Juvederm, immune response, new protein deposition, and cellular infiltration, according to some embodiments herein. [Figure 17]FIG. 17 shows histology of an injection site following administration of a dermal filling system described herein, showing vascular ingrowth (red arrow), protein deposition within pores (blue arrow), and collagen bundles (green arrow), according to some embodiments herein. [Figure 18] FIG. 18 shows a scheme for thiolation of hyaluronic acid (HA) prior to synthesis of microgel particles, according to some embodiments herein. [Figure 19A] 19A-19B show the elastic modulus (EM) of swollen and non-swollen gels with various ratios of PEG-VS to SH-HA, according to some embodiments herein. Figure 19A shows the dependence of EM of swollen and non-swollen gels on SH-HA concentration (in the presence of a fixed concentration of PEG-VS) when the hyaluronic acid (HA) contains a molecular weight of 10 kDa, according to some embodiments herein. [Figure 19B] 19A-19B show the elastic modulus (EM) of swollen and non-swollen gels with various ratios of PEG-VS to SH-HA, according to some embodiments herein. Figure 19B shows the dependence of EM of swollen and non-swollen gels on SH-HA concentration (in the presence of a fixed concentration of PEG-VS) when the hyaluronic acid (HA) contains a molecular weight of 50 kDa, according to some embodiments herein. [Figure 20] FIG. 20 shows the linear dependence of thiol concentration on SH-HA concentration when the hyaluronic acid (HA) comprises either 10 kDa or 50 kDa molecular weight, according to some embodiments herein. [Figure 21] FIG. 21 shows the effect of different annealing agents on the elastic modulus (EM) of scaffolds after annealing, according to some embodiments herein. [Figure 22] FIG. 22 shows the effect of formulation pH on annealing reaction kinetics when the annealing agent comprises linear PEG-(SH)2 having a molecular weight of 3.4 kDa and a volume fraction of 80%, according to some embodiments herein. [Figure 23]FIG. 23 shows the annealing kinetics, including the elastic modulus of microgel particles after annealing, for a dermal filler system using PEG-dithiol, according to some embodiments herein. [Figure 24A] 24A-24C show the effect of lidocaine on the elastic modulus of microgel particles after annealing, according to some embodiments herein. Figure 24A shows the effect of lidocaine on the elastic modulus of a formulation, according to some embodiments herein. [Figure 24B] 24A-24C show the effect of lidocaine on the elastic modulus of microgel particles after annealing, according to some embodiments herein. FIG. 24B shows the effect of lidocaine on the elastic modulus of a formulation, according to some embodiments herein. [Figure 24C] 24A-24C show the effect of lidocaine on the elastic modulus of microgel particles after annealing, according to some embodiments herein. FIG. 24C shows the effect of lidocaine on the elastic modulus of a formulation, according to some embodiments herein. [Figure 25A] 25A-25B show the proliferation of mouse fibroblast cell line 3T3 cells after treatment with various concentrations of an annealing agent, according to some embodiments herein. Figure 25A shows the proliferation of 3T3 cells after treatment with various concentrations of an annealing agent containing 20 kDa molecular weight 4-arm PEG-SH, according to some embodiments herein. As shown, each grouping of bars, from left to right, represents day 1, day 3, and day 6, respectively. [Figure 25B] 25A-25B show the proliferation of mouse fibroblast cell line 3T3 cells after treatment with various concentrations of an annealing agent, according to some embodiments herein. FIG. 25B shows the proliferation of 3T3 cells after treatment with various concentrations of an annealing agent comprising linear PEG-(SH)2 with a molecular weight of 3.4 kDa, according to some embodiments herein. As shown, each grouping of bars, from left to right, represents day 1, day 3, and day 6, respectively. [Figure 26]FIG. 26 shows the viability of 3T3 cells 6 days after exposure to either 4-arm PEG-SH of 20 kDa molecular weight or linear PEG-(SH)2 of 3.4 kDa molecular weight, according to some embodiments herein. [Figure 27] 27 shows the growth of 3T3 cells after exposure to either 4-arm PEG-SH with free thiols or 4-arm PEG-SH fully capped with maleimide to remove free thiols of 20 kDa molecular weight, according to some embodiments herein. As a control, growth of 3T3 cells after exposure to maleimide alone is shown. As shown, each grouping of bars, from left to right, represents day 1 and day 3, respectively. [Figure 28] Figure 28 shows the growth of 3T3 cells after treatment with various dermal filler systems and annealing reactions according to some embodiments herein. Note that the data shown in Figures 29 and 30 are from separate experiments. As shown, each grouping of bars represents days 1, 3, and 6, from left to right. [Figure 29] Figure 29 shows the growth of 3T3 cells after treatment with various dermal filler systems and annealing reactions according to some embodiments herein. Note that the data shown in Figures 29 and 30 are from separate experiments. As shown, each grouping of bars represents, from left to right, days 1, 3, and 6, respectively. [Figure 30] FIG. 30 shows the proliferation of 3T3 cells after treatment with various dermal filler systems and annealing reactions in multiple experiments according to some embodiments herein, normalized to a control experiment of proliferation of cells not exposed to any dermal filler system. [Figure 31] FIG. 31 shows the degradation of HA particles over time using hyaluronidase, according to some embodiments herein. [Figure 32A]32A-32B show the break force and extrusion force before and after annealing according to some embodiments herein. Figure 32A shows the break force and extrusion force of a dermal filler system after annealing according to some embodiments herein. In this experiment, a 30G needle was used with a plunger speed of 10 mm / min. [Figure 32B] 32A-32B show the break force and extrusion before and after annealing according to some embodiments herein. FIG. 32B shows the break force and extrusion force of a dermal filler system before annealing according to some embodiments herein. In this experiment, a 30G needle was used with a plunger speed of 10 mm / min. [Figure 33] FIG. 33 shows a plot of extrusion force over time for a dermal filler system, according to some embodiments herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some embodiments, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as indicating that they are substantially different from those commonly understood in the art.

[0011] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. References herein to "or" are intended to include "and / or" unless expressly stated otherwise.

[0012] References throughout this specification to "some embodiments," "further embodiments," or "particular embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in some embodiments," "further embodiments," or "in particular embodiments" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013] The term "about," when used herein in reference to a number, refers to a number that is plus or minus 10% of that number. Ranges followed by the term "about" refer to a range of minus 10% of the minimum value and plus 10% of the maximum value.

[0014] The term "annealing agent," as used herein, refers to a substance that can initiate an annealing reaction between particles (e.g., microparticles) of the present disclosure to form an annealed scaffold (e.g., a covalently stabilized scaffold). Non-limiting examples of annealing agents include molecules having two or more reactive functional groups, including eosin Y, PETMA (pentaerythritol tetrakis(2-mercaptoacetate)), Factor XIII / Factor XIIIa, thiol (e.g., PEG-dithiol), divinyl sulfone, or a combination thereof. The annealing agent may not be covalently involved in the linking of the particles described herein when the annealing reaction is initiated. The annealing agent may be covalently linked to the particles of the annealed scaffold when the annealing reaction is initiated.

[0015] The term "annealing moiety," as used herein, refers to a substrate that is attached to the microgel particles (e.g., microparticles) of the present disclosure in an annealing reaction between the microgel particles themselves. Non-limiting examples of annealing moieties include two or more reactive functional groups including K or Q peptides, thiols or thiol derivatives, vinyl or vinyl derivatives (e.g., vinyl sulfone), methacrylate, acrylate, amine, or combinations thereof.

[0016] The term "biocompatibility," as used herein, refers to biocompatibility as determined under international standard ISO 10993-1, which is incorporated herein by reference in its entirety.

[0017] The terms "cell adhesive peptide" or "cell adhesion peptide," as used interchangeably herein, refer to peptides capable of initiating cell adhesion to synthetic materials, such as microgel particles. A non-limiting example of a cell adhesion peptide is the RGD peptide. The cell adhesion peptides disclosed herein can be found in Moral MEG, Siahaan TJ. Conjugates of Cell Adhesion Peptides for Therapeutics and Diagnostics Against Cancer and Autoimmune Diseases. Curr Top Med Chem. 2017;17(32):3425-3443, the entire contents of which are incorporated herein by reference.

[0018] The term "cellular matrix," as used herein, refers to the network of proteins or other molecules that surround, support, and / or provide structure to cells and tissues in the body.

[0019] The term "compressive modulus," as used herein, refers to the stiffness of either individual microgel particles, macroscopic hydrogels, or annealed scaffolds of microgel particles. Compressive modulus may be measured by a compression test (fail or pass) in which a hydrogel, non-annealed scaffold (microgel particles), or annealed scaffold is crushed by a compression platen with a known cross-sectional area over a known distance and speed, while the force applied to the platen is recorded by a force transducer attached to the compression platen. Compressive modulus can be mathematically calculated from the stress / strain curve recorded during the compression test.

[0020] The term "crosslinker," as used herein, refers to a reagent that participates in the crosslinking reaction of raw materials to form microgel particles (e.g., microparticles) of the present disclosure. Crosslinkers are linkers with two or more reactive functional groups (e.g., thiol, vinyl sulfone, maleimide, acrylate, methacrylate, acrylamide, methacrylamide, norbornene, amine, hydroxyl). When the crosslinker is in excess in the crosslinking reaction, the crosslinker may be an annealing component or participate in the annealing reaction between particles of the present disclosure. Non-limiting examples of crosslinkers include vinyl derivatives with two or more vinyl groups (e.g., PEG-VS), thiol derivatives with two or more thiol groups (e.g., PEG-dithiol or thiolated HA), peptides with two or more cysteines (e.g., matrix metalloproteinase (MMP)-degradable crosslinkers), or combinations thereof.

[0021] The term "crosslinking," as used herein, refers to a reaction to form the microgel particles (e.g., microparticles) of the present disclosure.

[0022] The term "derivative" with respect to "vinyl" or "thiol" refers to a vinyl- or thiol-containing chemical, respectively. Non-limiting examples of vinyl derivatives include PEG-VS, PEG-acrylate, PEG-methacrylate, and PEG-maleimide. Non-limiting examples of vinyl groups include vinyl sulfone, acrylate, methacrylate, acrylamide, maleimide, and norbornene. Non-limiting thiol derivatives include PEG-dithiol, thiolated HA, cysteine-containing peptides (e.g., matrix metalloproteinase (MMP)-degradable crosslinkers), any organosulfur compound of the form R-SH (where R represents alkyl or other organic substituents), methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, allyl mercaptan, butanethiol, tert-butyl mercaptan, pentanethiol, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, metallothionein, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 1-mercaptoindole, grapefruit mercaptan, furan-2-ylmethanethiol, 3-mercaptopropane-1,2-diol, 3-mercapto-1-propanesulfonic acid, 1-hexadecanethiol, pentachlorobenzenethiol, or combinations thereof.

[0023] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present (e.g., detecting). Such terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present, in addition to determining whether something is present or absent, depending on the context.

[0024] The term "elastic modulus," as used herein, refers to the mechanical property of a material relating to its resistance to elastic deformation when stress is applied, and can be calculated by the following equation:

[0025]

number

[0026]

number

[0027] The term "ex vivo" is used to describe events that occur outside of a subject's body. Ex vivo assays are not performed on a subject. Rather, the assay is performed on a sample that is separate from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay, or on a tissue section excised (harvested) from a subject.

[0028] The term "foreign body reaction," as used herein, refers to a fibrotic reaction resulting from a graft or dermal filler, typically characterized by chronic inflammation, granuloma formation, and / or scar tissue formation at or around the implantation site. A foreign body reaction in a subject can be detected by histological analysis of tissue at or around the implantation site by comparing the results of the histological analysis with the histology of a reference tissue that does not contain the graft or dermal filler.

[0029] The term "gel," as used herein, refers to a three-dimensional network of cross-linked polymers swollen in a solvent.

[0030] The term "HA," as used herein, refers to hyaluronic acid or hyaluronan.

[0031] The term "HEPES," as used herein, refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0032] The term "in situ," as used herein, refers to the original delivery or administration site and remains within the site of origin without invading adjacent tissue.

[0033] The term "in vitro" is used to describe events that occur within a container holding a laboratory reagent such that the reagent is isolated from the biological source from which the substance is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0034] The term "in vivo" is used to describe events that take place in a subject's body.

[0035] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence, may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula has been incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J Mol Biol. 1990 Oct 5;215(3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application. Percent sequence identity can be determined using the most recent version of BLAST as of the filing date of this application.

[0036] The term "hydrogel," as used herein, refers to a gel that is water-insoluble and capable of retaining water.

[0037] The term "K peptide," as used herein, refers to a peptide comprising an amino acid sequence that includes one or more lysine residues that serve as a substrate for an annealing agent in an annealing reaction.

[0038] The terms "microparticle" or "microsphere" as used herein refer interchangeably to particles having a size of about 0.1 to about 1000 μm.

[0039] The term "microgel particles," as used herein, refers to particles composed of gel having a size of about 0.1 to about 1000 μm.

[0040] The term "particle," as used herein, refers to a single unit of a larger system, such as, for example, a dermal filler system or composition disclosed herein.

[0041] The term "percent identity" as used herein generally refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for purposes of determining percent identity can be achieved in a variety of ways known in the relevant art. The percent identity of two sequences can be calculated by aligning a test sequence to a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0042] The term "PEG," as used herein, refers to poly(ethylene glycol).

[0043] The term "PLA" as used herein refers to polylactic acid or polylactide.

[0044] The term "polydispersity," as used herein, is a measure of the heterogeneity of particles (e.g., microgel particles) of the present disclosure based on size. Polydispersity may be measured by either laser diffraction, dynamic light scattering, small angle X-ray scattering (SAXS), small angle neutron scattering (SANS), or microscopy using a particle size analyzer.

[0045] The term "polymer," as used herein, refers to a class of materials composed of macromolecules made up of repeating monomers. Non-limiting polymers include poly(ethylene glycol) (PEG), hyaluronic acid (HA), polylactic acid (PLA), collagen, poly(methyl methacrylate) (PMMA), or any combination thereof. The polymer may be synthetic, such as PEG, PLA, or PMMA. The polymer may be natural, such as HA or chitosan, or a protein, such as collagen, gelatin, or lysozyme. The polymer may also be a modified form of the polymer, whether natural or synthetic, to contain, for example, one or more thiol or vinyl derivatives disclosed herein (e.g., PEG-dithiol, 4-arm PEG-thiol, PEG-VS, thiolated HA).

[0046] The term "pore size," as used herein, refers to the size of each individual pore in a covalently stabilized scaffold, which is defined as the interstitial void space between particles. Pore size may be measured by approximating the void area to a circle, and the diameter of each circle may be considered the size of the pore.

[0047] The terms "porosity" or "void fraction," as used herein, refer interchangeably to a measure of void (i.e., "empty") space in a material, and may be expressed as a ratio of void volume to total volume between 0 and 1, or as a percentage between 0% and 100%. For example, porosity P= 空間 volume / 総Porosity may be measured using the method disclosed in "Void volume fraction of granular scaffolds; Lindsay Riley, Grace Wei, Yijun Bao, Peter Cheng, Katrina L. Wilson, Yining Liu, Yiyang Gong, Tatiana Segura; bioRxiv 2022.06.14.496197; doi: https: / / doi.org / 10.1101 / 2022.06.14.496197," which is incorporated by reference in its entirety for all purposes.

[0048] The term "precursor solution" refers to a solution of raw materials (eg, polymers and / or peptides) used to form the microgel particles of the present disclosure.

[0049] The term "Q peptide," as used herein, refers to a peptide comprising an amino acid sequence that includes one or more glutamine residues that serve as a substrate for an annealing agent in an annealing reaction.

[0050] The term "RGD peptide," as used herein, refers to a peptide derived from an extracellular matrix protein having an RGD motif characterized by an amino acid sequence containing "Arg-Gly-Asp." Non-limiting examples of extracellular matrix proteins include fibronectin, vitronectin, fibrinogen, von Willebrand factor, laminin, and collagen. RGD peptides may be those provided in Moral MEG, Siahaan TJ, et al. RGD peptides may be modified to contain cysteine ​​for conjugation. In some embodiments, the RGD peptide comprises an amino acid sequence containing RGDSPGERCG (SEQ ID NO: 1).

[0051] The term "storage modulus," as used herein, refers to a mechanical property of a viscoelastic material related to the energy stored therein, representing the elastic portion. Storage modulus is the ratio of elastic stress to strain. The storage modulus of microgel particles may also be measured in a surrogate non-porous gel formed with the same precursor solution used to create the microgel particles but not emulsified in an oil phase to produce microspheres. Storage modulus may also be measured by performing shear modulus measurements as described above and performing shear stress amplitude and frequency sweeps in a parallel plate system. This allows for the calculation of both the storage modulus and loss modulus of the viscoelastic material (the storage modulus and loss modulus together comprise the shear modulus).

[0052] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" may be a biological entity containing expressed genetic material. The biological entity may be, for example, a plant, animal, or microorganism, including bacteria, viruses, fungi, and protozoa. A subject may be tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected of being at high risk for a disease. In some cases, a subject is not necessarily diagnosed or suspected of being at high risk for a disease.

[0053] The term "surrogate gel" refers to a macroscopic surrogate bulk gel made from the same precursor solution used to make the microgel particles disclosed herein.

[0054] The term "tissue site," as used herein, refers to a discrete location of tissue to which the dermal filler systems disclosed herein can be delivered.

[0055] As used herein, the terms "treatment" or "treating" refer to a pharmaceutical or other interventional regimen to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit, prophylactic benefit, or cosmetic benefit. Therapeutic benefit can also refer to the eradication or amelioration of the condition or underlying disease being treated. Similarly, therapeutic benefit can be achieved by the eradication or amelioration of one or more physiological symptoms associated with an underlying disease, such that an improvement is observed in a subject, even though the subject may still be affected by the underlying disease. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. In a prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease can receive treatment even if a diagnosis of the disease has not been made. Cosmetic benefits can improve the quality of a subject's appearance, including, but not limited to, the appearance of wrinkles or fine lines, moisture, volume, skin grooves, sagging skin, or other signs of aging.

[0056] The term "VS" as used herein refers to vinyl sulfone.

[0057] system Dermal fillers are gel-like substances useful for certain therapeutic and cosmetic applications. In cosmetic applications, dermal fillers are typically injected into the skin (e.g., the dermis and subdermis) to improve the skin's cosmetic qualities, such as the reduction of wrinkles or fine lines, moisture, volume, skin grooves, sagging skin, or other signs of aging. Existing dermal fillers, such as Juvederm® injectable gel filler, are made from natural polymers, such as hyaluronic acid, which expand in volume after injection into tissue. This expansion occurs due to electrostatic interactions between the polymers present in the dermal filler and interstitial fluid present in the tissue surrounding the injection site, potentially causing fluid to flow into the injection site and resulting in swelling. This volume expansion can lead to unintended changes in cosmetic results (e.g., injection of a larger volume than intended) and can actually cause unwanted tissue inflammation due to the application of gentle pressure to tissues in contact with the injection site. These products typically degrade relatively rapidly in situ. Existing dermal fillers in this class completely degrade in situ within approximately nine months, necessitating periodic reapplication, which can be both painful and inconvenient for the subject. In an attempt to address this issue, alternative dermal fillers, known as biostimulators, such as Sculptra®, have been developed. These biostimulators may contain stiff mechanical properties that intentionally induce a foreign body response, resulting in collagen deposition to fill the surrounding tissue at the injection site. While these products provide volume for a longer period of time compared to Juvederm, they are characterized by prolonged granuloma formation and chronic inflammation. This prolonged granuloma formation and chronic inflammation can cause significant discomfort, pain, and even tissue degradation in some subjects. Furthermore, the tissue produced by this foreign body response mechanism is primarily scar-like and lacks the appearance and feel of normal tissue (e.g., the tissue is stiffer and more brittle than normal tissue due to its composition, primarily consisting of collagen I). Therefore, there is a need for next generation dermal filler systems for treating tissue that provide lasting cosmetic or therapeutic benefits while minimizing volume expansion and foreign body reaction in the subject.

[0058] In some embodiments, a dermal filler system is disclosed herein that provides sustained cosmetic or therapeutic benefits while minimizing a subject's foreign body reaction. The dermal filler system disclosed herein includes microgel particles made of a hydrogel with an optimized degradation profile for cosmetic applications, containing functional groups that promote annealing of the microgel particles to form a covalently stabilized porous scaffold in situ at the site of administration (e.g., injection). The covalently stabilized porous scaffold disclosed herein allows cells endogenous to the subject to infiltrate the pores of the scaffold and develop into a cellular matrix (and ultimately new tissue) that mimics the tissue surrounding the scaffold. This new tissue is non-scar-like and has the appearance and feel of normal tissue. After complete degradation of the scaffold by endogenous degradation pathways, the new tissue formed within the scaffold fills at least a portion of the space occupied by the scaffold, providing sustained cosmetic qualities to the subject's tissue. The dermal filler systems disclosed herein optionally contain additional medications (e.g., therapeutic agents), such as local anesthetics (e.g., lidocaine), analgesics, anti-inflammatory drugs, antibiotics (e.g., penicillin, dicloxacillin, cephalexin), and others that can provide therapeutic or cosmetic benefits at the site of administration.In some embodiments, antibiotics include macrolides (e.g., erythromycin, clarithromycin, dirithromycin, roxithromycin, and azithromycin), aminoglycosides (e.g., amikacin, gentamicin, neomycin, streptomycin, tobramycin), carbapenems (e.g., doripenem, meropenem), cephalosporins, tetracyclines (e.g., doxycycline, minocycline), rifamycins (e.g., rifabutin, rifampin), fluoroquinolones ( For example, these may include ciprofloxacin, levofloxacin, delafloxacin, gemifloxacin), penicillins (e.g., amoxicillin, ampicillin, penicillin, oxacillin), oxazolidinones (e.g., linezolid, tedizolid), glycopeptides (e.g., vancomycin), polypeptides (e.g., polymyxin B, bacitracin), sulfonamides (e.g., sulfacetamide, sulfadiazine, sulfadoxine), and streptogramins (e.g., quinupristin, dalfopristin). The dermal fillers disclosed herein may be formulated with one or more agents or solvents that can improve the sterility, stability, pH, viscosity, stiffness, porosity, degradation rate, etc. of the dermal filler system, which can be fine-tuned depending on the given application. Tissue sites for administration of the dermal fillers disclosed herein include, but are not limited to, the mid-face or cheek region, cheeks, chin, lips, or any combination thereof.

[0059] Microgel particles In some embodiments, a dermal filler system is disclosed herein that includes a plurality of microgel particles. In some embodiments, the plurality of microgel particles is formed by crosslinking one or more reagents and raw materials together according to various embodiments herein. The plurality of microgel particles may be in a slurry suitable for delivery to a subject via injection. Upon delivery of the slurry to a subject at a tissue site, the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold in situ. The covalently stabilized porous scaffold includes annealed microgel particles and may be interchangeably referred to as "annealed particles," "annealed microgel particles," or "annealed scaffold." In some embodiments, the covalently stabilized porous scaffold is formed such that pores are formed between the microgel particles of the covalently stabilized scaffold. In some embodiments, when the dermal filler system is administered to a subject, the covalently stabilized scaffold allows for the growth of a cellular matrix in situ at or around the tissue site of the subject, forming new tissue, even after the covalently stabilized scaffold has completely degraded. In some embodiments, the new tissue formed is endogenous tissue of the subject. In some embodiments, the new tissue is characterized as having mature vascularization, characteristics of the surrounding tissue at the tissue site, or a combination thereof. In some embodiments, characteristics of the surrounding tissue at the tissue site include cell types that are functionally differentiated from the surrounding tissue.

[0060] In some embodiments, the microgel particles are spherical. In some embodiments, the microgel particles are spheroidal. In some embodiments, the microgel particles are substantially spherical or substantially spherical. In some embodiments, the microgel particles comprise microspheres. The microgel particles may have a substantially uniform shape such that adjacent microgel particles generate pores when they contact one another. Other shapes of microgel particles are contemplated, including, but not limited to, flat, prolate, round, granular, flake, or 3D geometric shapes.

[0061] In some embodiments, the microparticles may have a diameter or dimension (e.g., length, width, height, axis). In some embodiments, the microgel particles include a diameter or dimension between 0.1 micrometers (μm) and 1000 μm. In some embodiments, the microgel particles include a diameter or dimension between 5 micrometers (μm) and 1000 μm. In some embodiments, the diameter or dimension is between 50 μm and 1000 μm. In some embodiments, the diameter or dimension is between 80 μm and 140 μm. In some embodiments, the diameter is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, Including 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 μm or greater.In some embodiments, the diameter is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, including 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 μm or less. In some embodiments, the diameter or dimension includes a range of about 5 μm to about 1100 μm. In some embodiments, the diameter or dimension includes a range of about 10 μm to about 1090 μm. In some embodiments, the diameter or dimension includes a range of about 15 μm to about 1080 μm. In some embodiments, the diameter or dimension includes a range of about 20 μm to about 1070 μm. In some embodiments, the diameter or dimension includes a range of about 25 μm to about 1060 μm. In some embodiments, the diameter or dimension includes a range of about 30 μm to about 1050 μm. In some embodiments, the diameter or dimension includes a range of about 35 μm to about 1040 μm. In some embodiments, the diameter or dimension includes a range of about 40 μm to about 1030 μm. In some embodiments, the diameter or dimension includes a range of about 45 μm to about 1020 μm. In some embodiments, the diameter or dimension includes a range of about 50 μm to about 1010 μm. In some embodiments, the diameter or dimension includes a range of about 55 μm to about 1000 μm, hi some embodiments, the diameter or dimension includes a range of about 60 μm to about 990 μm.In some embodiments, the diameter or dimension includes a range of about 65 μm to about 980 μm. In some embodiments, the diameter or dimension includes a range of about 70 μm to about 970 μm. In some embodiments, the diameter or dimension includes a range of about 75 μm to about 960 μm. In some embodiments, the diameter or dimension includes a range of about 80 μm to about 950 μm. In some embodiments, the diameter or dimension includes a range of about 85 μm to about 940 μm. In some embodiments, the diameter or dimension includes a range of about 90 μm to about 930 μm. In some embodiments, the diameter or dimension includes a range of about 95 μm to about 920 μm. In some embodiments, the diameter or dimension includes a range of about 100 μm to about 910 μm. In some embodiments, the diameter or dimension includes a range of about 110 μm to about 900 μm. In some embodiments, the diameter or dimension includes a range of about 120 μm to about 890 μm. In some embodiments, the diameter or dimension includes a range of about 130 μm to about 880 μm. In some embodiments, the diameter or dimension includes a range of about 140 μm to about 870 μm. In some embodiments, the diameter or dimension includes a range of about 150 μm to about 860 μm. In some embodiments, the diameter or dimension includes a range of about 160 μm to about 850 μm. In some embodiments, the diameter or dimension includes a range of about 170 μm to about 840 μm. In some embodiments, the diameter or dimension includes a range of about 180 μm to about 830 μm. In some embodiments, the diameter or dimension includes a range of about 190 μm to about 820 μm. In some embodiments, the diameter or dimension includes a range of about 200 μm to about 810 μm. In some embodiments, the diameter or dimension includes a range of about 210 μm to about 800 μm. In some embodiments, the diameter or dimension includes a range of about 220 μm to about 790 μm. In some embodiments, the diameter or dimension includes a range of about 230 μm to about 780 μm. In some embodiments, the diameter or dimension includes a range of about 240 μm to about 770 μm. In some embodiments, the diameter or dimension includes a range of about 250 μm to about 760 μm. In some embodiments, the diameter or dimension includes a range of about 260 μm to about 750 μm.In some embodiments, the diameter or dimension includes a range of about 270 μm to about 740 μm. In some embodiments, the diameter or dimension includes a range of about 280 μm to about 730 μm. In some embodiments, the diameter or dimension includes a range of about 290 μm to about 720 μm. In some embodiments, the diameter or dimension includes a range of about 300 μm to about 710 μm. In some embodiments, the diameter or dimension includes a range of about 310 μm to about 700 μm. In some embodiments, the diameter or dimension includes a range of about 320 μm to about 690 μm. In some embodiments, the diameter or dimension includes a range of about 330 μm to about 680 μm. In some embodiments, the diameter or dimension includes a range of about 340 μm to about 670 μm. In some embodiments, the diameter or dimension includes a range of about 350 μm to about 660 μm. In some embodiments, the diameter or dimension includes a range of about 360 μm to about 650 μm. In some embodiments, the diameter or dimension includes a range of about 370 μm to about 640 μm. In some embodiments, the diameter or dimension includes a range of about 380 μm to about 630 μm. In some embodiments, the diameter or dimension includes a range of about 390 μm to about 620 μm. In some embodiments, the diameter or dimension includes a range of about 400 μm to about 610 μm. In some embodiments, the diameter or dimension includes a range of about 410 μm to about 600 μm. In some embodiments, the diameter or dimension includes a range of about 420 μm to about 590 μm. In some embodiments, the diameter or dimension includes a range of about 430 μm to about 580 μm. In some embodiments, the diameter or dimension includes a range of about 440 μm to about 570 μm. In some embodiments, the diameter or dimension includes a range of about 450 μm to about 560 μm. In some embodiments, the diameter or dimension includes a range of about 460 μm to about 550 μm. In some embodiments, the diameter or dimension includes a range of about 470 μm to about 540 μm. In some embodiments, the diameter or dimension includes a range of about 480 μm to about 530 μm. In some embodiments, the diameter or dimension includes a range of about 490 μm to about 520 μm. In some embodiments, the diameter or dimension includes a range of about 500 μm to about 510 μm.

[0062] The microgel particles may have an average diameter or size of about 10 μm. The microgel particles may have an average diameter or size of about 15 μm. The microgel particles may have an average diameter or size of about 25 μm. The microgel particles may have a diameter or size of about 50 μm. The microgel particles may have an average diameter or size of about 100 μm. The microgel particles may have an average diameter or size of about 150 μm. The microgel particles may have an average diameter or size of about 200 μm. The microgel particles may have a diameter or size in the range of about 10 μm to about 500 μm. The microgel particles may have a diameter or size in the range of about 10 μm to about 200 μm. The microgel particles may have a diameter or size in the range of about 15 μm to about 200 μm. The microgel particles may have a diameter or size in the range of about 15 μm to about 150 μm. The microgel particles may have a diameter or size in the range of about 30 μm to about 100 μm. The microgel particles may have an average diameter or size of 10 μm. The microgel particles may have an average diameter or size of 15 μm. The microgel particles may have an average diameter or size of 25 μm. The microgel particles may have a diameter or size of 50 μm. The microgel particles may have an average diameter or size of 100 μm. The microgel particles may have an average diameter or size of 150 μm.

[0063] The microgel particles may have an average diameter or size of 200 μm. The microgel particles may have a diameter or size in the range of 10 μm to 500 μm. The microgel particles may have a diameter or size in the range of 10 μm to 200 μm. The microgel particles may have a diameter or size in the range of 15 μm to 200 μm. The microgel particles may have a diameter or size in the range of 15 μm to 150 μm. The microgel particles may have a diameter or size in the range of 30 μm to 100 μm. In some embodiments, the diameter of the microgel particles may be measured by (1) measuring the area of ​​the microgel particles, (2) solving for the radius of the microgel particles using the equation for the area of ​​a circle (i.e., A=pi*r^2), and (3) solving for the diameter by multiplying the radius by 2 (i.e., D=2*r).

[0064] In some embodiments, the microgel particles comprise one or more cell adhesion peptides. In some embodiments, the cell adhesion peptide comprises at least a portion of an extracellular matrix protein. In some embodiments, the cell adhesion peptide comprises at least a portion of collagen. In some embodiments, the cell adhesion peptide comprises at least a portion of fibronectin. In some embodiments, the cell adhesion peptide comprises an integrin. In some embodiments, the adhesive peptide comprises a ligand for a receptor expressed on a cell. In some embodiments, the adhesive peptide comprises a cluster of differentiation (CD) protein. In some embodiments, the adhesive peptide comprises a naturally occurring peptide. In some embodiments, the adhesive peptide comprises a synthetic peptide. In some embodiments, the cell adhesion peptide may be homologous to a naturally occurring peptide. In some embodiments, the cell adhesion peptide comprises at least about 70% homology to a naturally occurring peptide. In some embodiments, the cell adhesion peptide is at least about 80% homology to a naturally occurring peptide. In some embodiments, the cell adhesion peptide comprises at least about 90% homology to a naturally occurring peptide. In some embodiments, the cell adhesive peptide comprises at least 70% homology to a naturally occurring peptide. In some embodiments, the cell adhesive peptide comprises at least 80% homology to a naturally occurring peptide. In some embodiments, the cell adhesive peptide comprises at least 90% homology to a naturally occurring peptide. In some embodiments, the cell adhesive peptide may be bound to the surface of a microgel particle. In some embodiments, the cell adhesive peptide is grafted to the surface of a microgel particle. In some embodiments, the coupling may comprise one or more chemical bonds. In some embodiments, the one or more chemical bonds are one or more covalent bonds.

[0065] As a non-limiting example, the cell adhesive peptide may comprise an RGD peptide. In some embodiments, the RGD peptide comprises RGDSPGERCG (SEQ ID NO: 1). In some embodiments, the RGD peptide comprises ACDCRGDCFCG (SEQ ID NO: 2). In some embodiments, the RGD peptide comprises GRGDSP (SEQ ID NO: 6). In some embodiments, the RGD peptide comprises cyclo(Arg-Gly-Asp-DPhe-Val) (SEQ ID NO: 7). In some embodiments, the RGD peptide comprises cyclo(Arg-Gly-Asp-DPhe-Lys)cyclo(Arg-Gly-Asp-DPhe-Cys) (SEQ ID NO: 8). In some embodiments, the RGD peptide comprises KACDCRGDCFCG (SEQ ID NO: 9). In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 7. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 7.In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 7. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in any one of SEQ ID NOS: 1-2 or 6-9. In some embodiments, the RGD peptide is provided in Moral MEG, Siahaan TJ, et al., the entire contents of which are incorporated herein by reference. In some embodiments, the RGD peptide is modified to improve binding of the RGD peptide to a substrate, such as the microgel particles disclosed herein. Non-limiting examples of modifications include the addition of a cysteine ​​residue, the addition of a linker having a thiol group at one end and an amine group at the other end, or the addition of a linker having a thiol group at one end and a carboxylic acid group at the other end. In some embodiments, the RGD peptide comprises a modification at either end (e.g., C-terminus, N-terminus). In some embodiments, the modifications are in sequences flanking the RGD motif within the RGD sequence.

[0066] In some embodiments, the microgel particles comprise a polymer. In some embodiments, the polymer is or comprises a polymer backbone. In some embodiments, the polymer backbone of the polymer is comprised of the main chain of the polymer (e.g., the polymer occupies a larger proportion of the material compared to other polymers in the material). In some embodiments, the main chain can be considered a linear chain in the polymer with any other chains pendant from this linear chain. In some embodiments, the polymer is or comprises a copolymer. In some embodiments, a copolymer comprises polymer chains containing two or more different monomers in substantially equal proportions. In some embodiments, the polymer can be crosslinked to retain large amounts of water to form a water-insoluble hydrogel. In some embodiments, the polymer is a natural polymer. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the polymer is made from both natural and synthetic polymers. Non-limiting examples of polymers include poly(ethylene glycol), hyaluronic acid, polyacrylamide, and polymethacrylate. In some embodiments, the polymer may comprise a hydrophilic polymer, an amphiphilic polymer, a synthetic or natural polymer, or a copolymer of hydrophobic and hydrophilic polymers (e.g., poly(ethylene glycol) (PEG), poly(propylene glycol), poly(hydroxyethyl methacrylate), hyaluronic acid (HA), gelatin, fibrin, chitosan, heparin, heparan, and synthetic versions of HA, gelatin, fibrin, chitosan, heparin, or heparan). In some embodiments, the polymer may be made from any natural (e.g., modified HA) or synthetic polymer (e.g., PEG) capable of forming a hydrogel. In some embodiments, the polymer may comprise nitrogen-containing natural polymers, such as proteins and derivatives, including crosslinked or modified gelatin and keratin. In some embodiments, the polymer may comprise a functional group (e.g., vinyl) at at least one end of the polymer (e.g., poly(ethylene glycol) methacrylate).In some embodiments, the polymer may include functional groups (e.g., vinyl) incorporated into the polymer backbone (e.g., poly(methacrylate)). In some embodiments, the polymer may include vinyl polymers such as, for example, poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) vinyl sulfone, poly(ethylene glycol) maleimide, poly(ethylene glycol) norbornene, and poly(ethylene glycol) allyl. In some embodiments, the polymer may include polyacrylamide or polymethacrylate. In some embodiments, the polymer may include polyester, polyamide, polyurethane, or a mixture or copolymer thereof. In some embodiments, the polymer may include a graft copolymer obtained by initiating the polymerization of a synthetic polymer onto an existing natural polymer. In some embodiments, the polymer is or includes hyaluronic acid (HA). In some embodiments, the polymer consists of HA. In some embodiments, the polymer is or includes poly(ethylene) glycol (PEG). In some embodiments, the polymer consists of PEG. In some embodiments, the microgel particles comprise two or more polymers (e.g., polymers made of different materials). In some embodiments, the two or more polymers comprise HA and PEG. In some embodiments, the two or more polymers comprise poly(lactic acid) (PLA) and HA. In some embodiments, the two or more polymers comprise PLA and PEG. In some embodiments, the two or more polymers comprise poly(methyl methacrylate) (PMMA) and HA. In some embodiments, the two or more polymers comprise PMMA and PEG. In some embodiments, the two or more polymers comprise a polymer that includes functional groups and a polymer that does not include functional groups. In some embodiments, the microgel particles comprise three or more polymers, each independently made of a material selected from the group consisting of a hydrophilic polymer, an amphiphilic polymer, a synthetic polymer, or a natural polymer.In some embodiments, the microgel particles comprise three or more polymers, each independently made from a material selected from poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(propylene glycol), poly(hydroxyethyl methacrylate), hyaluronic acid (HA), gelatin, fibrin, chitosan, heparin, heparan, and synthetic versions of HA, gelatin, fibrin, chitosan, heparin, or heparan, or modified versions of any of these. In some embodiments, when the microgel particles comprise two or more polymers, the ratio of each polymer in the microgel particles may vary. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles have a concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.8%, 0.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 It may contain more than 7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, or 20.0% by weight (wt) of HA.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles have a concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.8%, 0.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 The microgel particles may comprise less than 7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, or 20.0% by weight (wt) HA. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise between about 0.01 wt% and about 20.0 wt% HA. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 19.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 18.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 17.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 16.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 15.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise HA in a range of about 0.01 wt% to about 14.0 wt% in a swollen state.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 13.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 12.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 11.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 10.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 9.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 8.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 7.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 6.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 5.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 4.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.01 wt% to about 3.0 wt% HA. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.01 wt% to about 2.0 wt% HA.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, HA in a range of about 0.01 wt% to about 1.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, HA in a range of about 0.01 wt% to about 0.95 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, HA in a range of about 0.01 wt% to about 0.90 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, HA in a range of about 0.01 wt% to about 0.85 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, HA in a range of about 0.01 wt% to about 0.80 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise HA in a range of about 0.01 wt% to about 0.75 wt% in a swollen state. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles comprise about 0.01 wt% to about 0.75 wt% in a swollen state. % to about 0.70 wt% HA. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 0.65 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 0.60 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 0.55 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 0.50 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in a range of about 0.01 wt% to about 0.45 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.40 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.35 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.30 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.25 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.20 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.01 wt% to about 0.15 wt% HA. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.01 wt% to about 0.10 wt% HA.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.09 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.08 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.07 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.06 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise HA in the range of about 0.01 wt% to about 0.05 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles have a concentration of about 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.85 wt%, 0.9 wt%, 0. It may contain greater than 95%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, or 20.0% by weight (wt) PEG.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles have a concentration of about 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.85 wt%, 0.9 wt%, 0.0 wt%, ...0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, 0.0 wt%, The microgel particles may comprise less than 0.95%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, or 20.0% by weight (wt) of PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 20.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 19.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 18.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 17.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 16.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 15.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 14.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise PEG in a range of about 0.1 wt% to about 13.0 wt% in a swollen state.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 12.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 11.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 10.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 9.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 8.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 7.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 6.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 5.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 4.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 3.0 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 2.0 wt% PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 1.0 wt% PEG.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 0.95 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 0.90 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 0.85 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 0.80 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in a range of about 0.1 wt% to about 0.75 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in the range of about 0.1 wt% to about 0.70 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in the range of about 0.1 wt% to about 0.65 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in the range of about 0.1 wt% to about 0.60 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in the range of about 0.1 wt% to about 0.55 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles, in a swollen state, may comprise PEG in the range of about 0.1 wt% to about 0.50 wt%. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.45 wt% PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.40 wt% PEG.In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.35 wt% of PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.30 wt% of PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.25 wt% of PEG. In some embodiments, the microgel particles comprise HA and PEG, and the microgel particles may comprise, in a swollen state, about 0.1 wt% to about 0.20 wt% of PEG. In some embodiments, the microgel particles comprise a hydrogel. In some embodiments, the hydrogel comprises one or more polymers disclosed herein. For example, a PEG hydrogel is a water-insoluble hydrogel made with PEG that can retain water. In another example, HA hydrogels are water-insoluble hydrogels made of HA that can retain water. Similarly, hydrogel microparticles made of copolymers of HA and PEG can retain water. The present invention relates to a water-insoluble hydrogel made of HA and PEG, which can retain a polymeric structure. In some embodiments, the first polymer and the second polymer are copolymers having approximately the same molecular weight as each other. In some embodiments, the copolymer comprises HA and PEG. In some embodiments, the copolymer consists of HA and PEG.

[0067] In some embodiments, the microgel particles include a support material instead of or in addition to a polymer. In some embodiments, the support material is suitable for tissue engineering or regenerative medicine applications. In some embodiments, the support material is biocompatible. In some embodiments, the support material is biodegradable. Examples of support materials include, but are not limited to, natural polymeric carbohydrates, as well as nitrogen-containing natural polymers such as gelatin, agar, agarose, cross-linked alginate, chitin, substituted and cross-linked guar gum, cellulose esters, especially with nitrites and carboxylic acids, mixed cellulose esters, and cellulose ethers, cross-linked or modified gelatin, proteins and derivatives including heparin, chondroitin sulfate, glycosaminoglycans, and keratin, vinyl polymers such as poly(ethylene glycol) acrylate / methacrylate / vinylsulfone / maleimide / norbornene / allyl, polyacrylamide, polymethacrylate, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers such as polyurethanes, as well as mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating the polymerization of a synthetic polymer onto an existing natural polymer. A variety of biocompatible and biodegradable polymers are available for therapeutic use, examples include polycaprolactone, polyglycolide, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), and poly-3-hydroxybutyrate.

[0068] In some embodiments, the polymer (e.g., a copolymer (e.g., HA and PEG)) may be present in the microgel particles, the dermal filler system containing the microgel particles, the resulting covalently stabilized scaffold, or any combination thereof at about 1 weight percent (wt%) to about 50 wt%. In some embodiments, the polymer may be present in the microgel particles, the dermal filler system containing the microgel particles, the resulting covalently stabilized scaffold, or any combination thereof at about 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% or more. In some embodiments, the polymer may be present at about 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% or less. In some embodiments, the polymer may be present at about 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.5 wt%, 3 wt%, 4 wt%, or 5 wt% or more. In some embodiments, the polymer may be present at about 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.5 wt%, 3 wt%, 4 wt%, or 5 wt% or less. In some embodiments, the polymer may be present at about 5 wt% to about 45 wt%. In some embodiments, the polymer may be present at about 10 wt% to about 40 wt%. In some embodiments, the polymer may be present at about 15 weight percent (wt%) to about 35 wt%. In some embodiments, the polymer may be present at about 20 weight percent (wt%) to about 30 wt%. In some embodiments, the polymer may be present at about 0.1 wt% to about 1.5 wt%.In some embodiments, the polymer may be present at about 0.5 wt%.

[0069] In some embodiments, the polymer is modified relative to an otherwise identical polymer, but without the modification. In some embodiments, the modified polymer is modified HA, for example, HA modified to contain a thiol or its derivative. In some embodiments, the modified polymer comprises modified PEG, for example, PEG modified to contain a thiol or its derivative. Non-limiting modifications include thiolation of carboxylic acid groups on HA polymers, thiolation of primary alcohol groups on HA polymers, and thiolation of terminal alcohol groups on PEG polymers.

[0070] In some embodiments, the molecular weight of the polymer can have an effect on the properties of the microgel particles, the dermal filler system containing the microgel particles, the resulting covalently stabilized scaffold, or any combination thereof. For example, as discussed below in Example 3, the molecular weight of HA can have an effect on the concentration of one or more functional groups (e.g., thiols) disclosed elsewhere herein in the microgel particles. As a non-limiting example, as shown in Figure 20, when HA itself is present at a fixed weight percent concentration, a higher molecular weight of HA can lead to a higher thiol concentration in the dermal filler system.

[0071] In some embodiments, when the modified PEG is present at a fixed concentration, the molecular weight and concentration of the modified HA can be adjusted to achieve the desired mechanical properties of the hydrogel, including the compressive modulus and / or storage modulus. As shown in Figures 19A-19B, a higher molecular weight HA can be used to achieve a higher compressive modulus for the swollen particles. In some embodiments, the higher compressive modulus can be the result of reduced swelling due to increased crosslink density of the particles. In some embodiments, when the modified HA is present at a fixed concentration, the molecular weight and concentration of the modified PEG can be adjusted to achieve the desired mechanical properties of the hydrogel, including the compressive modulus and / or storage modulus.

[0072] In some embodiments, the HA comprises a molecular weight of about 1 kilodalton (kDa) to about 1 megadalton (1 MDa), hi some embodiments, the HA comprises a molecular weight of about 10 kDa to 250 kDa (e.g., 10, 40, 50, 150, and 250 kDa). In some embodiments, the HA is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 9 and molecular weights of 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa or greater.In some embodiments, the HA is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 9 including molecular weights of 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa or less. In some embodiments, HA comprises a molecular weight range of about 5 kDa to about 1100 kDa. In some embodiments, HA comprises a molecular weight range of about 10 kDa to about 1090 kDa. In some embodiments, HA comprises a molecular weight range of about 15 kDa to about 1080 kDa. In some embodiments, HA comprises a molecular weight range of about 20 kDa to about 1070 kDa. In some embodiments, HA comprises a molecular weight range of about 25 kDa to about 1060 kDa. In some embodiments, HA comprises a molecular weight range of about 30 kDa to about 1050 kDa. In some embodiments, HA comprises a molecular weight range of about 35 kDa to about 1040 kDa. In some embodiments, HA comprises a molecular weight range of about 40 kDa to about 1030 kDa. In some embodiments, HA comprises a molecular weight range of about 45 kDa to about 1020 kDa. In some embodiments, HA comprises a molecular weight range of about 50 kDa to about 1010 kDa. In some embodiments, HA comprises a molecular weight range of about 55 kDa to about 1000 kDa. In some embodiments, HA comprises a molecular weight range of about 60 kDa to about 990 kDa.In some embodiments, HA comprises a molecular weight range of about 65 kDa to about 980 kDa. In some embodiments, HA comprises a molecular weight range of about 70 kDa to about 970 kDa. In some embodiments, HA comprises a molecular weight range of about 75 kDa to about 960 kDa. In some embodiments, HA comprises a molecular weight range of about 80 kDa to about 950 kDa. In some embodiments, HA comprises a molecular weight range of about 85 kDa to about 940 kDa. In some embodiments, HA comprises a molecular weight range of about 90 kDa to about 930 kDa. In some embodiments, HA comprises a molecular weight range of about 95 kDa to about 920 kDa. In some embodiments, HA comprises a molecular weight range of about 100 kDa to about 910 kDa. In some embodiments, HA comprises a molecular weight range of about 110 kDa to about 900 kDa. In some embodiments, HA comprises a molecular weight range of about 120 kDa to about 890 kDa. In some embodiments, HA comprises a molecular weight range of about 130 kDa to about 880 kDa. In some embodiments, HA comprises a molecular weight range of about 140 kDa to about 870 kDa. In some embodiments, HA comprises a molecular weight range of about 150 kDa to about 860 kDa. In some embodiments, HA comprises a molecular weight range of about 160 kDa to about 850 kDa. In some embodiments, HA comprises a molecular weight range of about 170 kDa to about 840 kDa. In some embodiments, HA comprises a molecular weight range of about 180 kDa to about 830 kDa. In some embodiments, HA comprises a molecular weight range of about 190 kDa to about 820 kDa. In some embodiments, HA comprises a molecular weight range of about 200 kDa to about 810 kDa. In some embodiments, HA comprises a molecular weight range of about 210 kDa to about 800 kDa. In some embodiments, HA comprises a molecular weight range of about 220 kDa to about 790 kDa. In some embodiments, HA comprises a molecular weight range of about 230 kDa to about 780 kDa. In some embodiments, HA comprises a molecular weight range of about 240 kDa to about 770 kDa. In some embodiments, HA comprises a molecular weight range of about 250 kDa to about 760 kDa. In some embodiments, HA comprises a molecular weight range of about 260 kDa to about 750 kDa.In some embodiments, HA comprises a molecular weight range of about 270 kDa to about 740 kDa. In some embodiments, HA comprises a molecular weight range of about 280 kDa to about 730 kDa. In some embodiments, HA comprises a molecular weight range of about 290 kDa to about 720 kDa. In some embodiments, HA comprises a molecular weight range of about 300 kDa to about 710 kDa. In some embodiments, HA comprises a molecular weight range of about 310 kDa to about 700 kDa. In some embodiments, HA comprises a molecular weight range of about 320 kDa to about 690 kDa. In some embodiments, HA comprises a molecular weight range of about 330 kDa to about 680 kDa. In some embodiments, HA comprises a molecular weight range of about 340 kDa to about 670 kDa. In some embodiments, HA comprises a molecular weight range of about 350 kDa to about 660 kDa. In some embodiments, HA comprises a molecular weight range of about 360 kDa to about 650 kDa. In some embodiments, HA comprises a molecular weight range of about 370 kDa to about 640 kDa. In some embodiments, HA comprises a molecular weight range of about 380 kDa to about 630 kDa. In some embodiments, HA comprises a molecular weight range of about 390 kDa to about 620 kDa. In some embodiments, HA comprises a molecular weight range of about 400 kDa to about 610 kDa. In some embodiments, HA comprises a molecular weight range of about 410 kDa to about 600 kDa. In some embodiments, HA comprises a molecular weight range of about 420 kDa to about 590 kDa. In some embodiments, HA comprises a molecular weight range of about 430 kDa to about 580 kDa. In some embodiments, HA comprises a molecular weight range of about 440 kDa to about 570 kDa. In some embodiments, HA comprises a molecular weight range of about 450 kDa to about 560 kDa. In some embodiments, HA comprises a molecular weight range of about 460 kDa to about 550 kDa. In some embodiments, HA comprises a molecular weight range of about 470 kDa to about 540 kDa. In some embodiments, HA comprises a molecular weight range of about 480 kDa to about 530 kDa. In some embodiments, HA comprises a molecular weight range of about 490 kDa to about 520 kDa. In some embodiments, HA comprises a molecular weight range of about 500 kDa to about 510 kDa.

[0073] In some embodiments, the PEG comprises a molecular weight of about 1 kilodalton (kDa) to about 5 kDa. In some embodiments, the PEG comprises a molecular weight of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the PEG comprises a molecular weight of about 1 kilodalton (kDa) to 1000 kDa. In some embodiments, the PEG is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 9 and molecular weights of 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa or greater.In some embodiments, the PEG is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 9 including molecular weights of 0, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa or less. In some embodiments, PEG comprises a molecular weight range of about 5 kDa to about 1100 kDa. In some embodiments, PEG comprises a molecular weight range of about 10 kDa to about 1090 kDa. In some embodiments, PEG comprises a molecular weight range of about 15 kDa to about 1080 kDa. In some embodiments, PEG comprises a molecular weight range of about 20 kDa to about 1070 kDa. In some embodiments, PEG comprises a molecular weight range of about 25 kDa to about 1060 kDa. In some embodiments, PEG comprises a molecular weight range of about 30 kDa to about 1050 kDa. In some embodiments, PEG comprises a molecular weight range of about 35 kDa to about 1040 kDa. In some embodiments, PEG comprises a molecular weight range of about 40 kDa to about 1030 kDa. In some embodiments, PEG comprises a molecular weight range of about 45 kDa to about 1020 kDa. In some embodiments, PEG comprises a molecular weight range of about 50 kDa to about 1010 kDa, hi some embodiments, PEG comprises a molecular weight range of about 55 kDa to about 1000 kDa.In some embodiments, PEG comprises a molecular weight range of about 60 kDa to about 990 kDa. In some embodiments, PEG comprises a molecular weight range of about 65 kDa to about 980 kDa. In some embodiments, PEG comprises a molecular weight range of about 70 kDa to about 970 kDa. In some embodiments, PEG comprises a molecular weight range of about 75 kDa to about 960 kDa. In some embodiments, PEG comprises a molecular weight range of about 80 kDa to about 950 kDa. In some embodiments, PEG comprises a molecular weight range of about 85 kDa to about 940 kDa. In some embodiments, PEG comprises a molecular weight range of about 90 kDa to about 930 kDa. In some embodiments, PEG comprises a molecular weight range of about 95 kDa to about 920 kDa. In some embodiments, PEG comprises a molecular weight range of about 100 kDa to about 910 kDa. In some embodiments, PEG comprises a molecular weight range of about 110 kDa to about 900 kDa. In some embodiments, PEG comprises a molecular weight range of about 120 kDa to about 890 kDa. In some embodiments, PEG comprises a molecular weight range of about 130 kDa to about 880 kDa. In some embodiments, PEG comprises a molecular weight range of about 140 kDa to about 870 kDa. In some embodiments, PEG comprises a molecular weight range of about 150 kDa to about 860 kDa. In some embodiments, PEG comprises a molecular weight range of about 160 kDa to about 850 kDa. In some embodiments, PEG comprises a molecular weight range of about 170 kDa to about 840 kDa. In some embodiments, PEG comprises a molecular weight range of about 180 kDa to about 830 kDa. In some embodiments, PEG comprises a molecular weight range of about 190 kDa to about 820 kDa. In some embodiments, PEG comprises a molecular weight range of about 200 kDa to about 810 kDa. In some embodiments, PEG comprises a molecular weight range of about 210 kDa to about 800 kDa. In some embodiments, PEG comprises a molecular weight range of about 220 kDa to about 790 kDa. In some embodiments, PEG comprises a molecular weight range of about 230 kDa to about 780 kDa. In some embodiments, PEG comprises a molecular weight range of about 240 kDa to about 770 kDa. In some embodiments, PEG comprises a molecular weight range of about 250 kDa to about 760 kDa.In some embodiments, PEG comprises a molecular weight range of about 260 kDa to about 750 kDa. In some embodiments, PEG comprises a molecular weight range of about 270 kDa to about 740 kDa. In some embodiments, PEG comprises a molecular weight range of about 280 kDa to about 730 kDa. In some embodiments, PEG comprises a molecular weight range of about 290 kDa to about 720 kDa. In some embodiments, PEG comprises a molecular weight range of about 300 kDa to about 710 kDa. In some embodiments, PEG comprises a molecular weight range of about 310 kDa to about 700 kDa. In some embodiments, PEG comprises a molecular weight range of about 320 kDa to about 690 kDa. In some embodiments, PEG comprises a molecular weight range of about 330 kDa to about 680 kDa. In some embodiments, PEG comprises a molecular weight range of about 340 kDa to about 670 kDa. In some embodiments, PEG comprises a molecular weight range of about 350 kDa to about 660 kDa. In some embodiments, PEG comprises a molecular weight range of about 360 kDa to about 650 kDa. In some embodiments, PEG comprises a molecular weight range of about 370 kDa to about 640 kDa. In some embodiments, PEG comprises a molecular weight range of about 380 kDa to about 630 kDa. In some embodiments, PEG comprises a molecular weight range of about 390 kDa to about 620 kDa. In some embodiments, PEG comprises a molecular weight range of about 400 kDa to about 610 kDa. In some embodiments, PEG comprises a molecular weight range of about 410 kDa to about 600 kDa. In some embodiments, PEG comprises a molecular weight range of about 420 kDa to about 590 kDa. In some embodiments, PEG comprises a molecular weight range of about 430 kDa to about 580 kDa. In some embodiments, PEG comprises a molecular weight range of about 440 kDa to about 570 kDa. In some embodiments, PEG comprises a molecular weight range of about 450 kDa to about 560 kDa. In some embodiments, PEG comprises a molecular weight range of about 460 kDa to about 550 kDa. In some embodiments, PEG comprises a molecular weight range of about 470 kDa to about 540 kDa. In some embodiments, PEG comprises a molecular weight range of about 480 kDa to about 530 kDa.In some embodiments, PEG comprises a molecular weight range of about 490 kDa to about 520 kDa, hi some embodiments, PEG comprises a molecular weight range of about 500 kDa to about 510 kDa.

[0074] In some embodiments, microgel particles may be functionalized to include one or more functional groups. For example, microgel particles made of hydrogel may be functionalized to include functional groups attached thereto. In some embodiments, the functional groups include hydroxyl functional groups, methyl functional groups, carbonyl functional groups, carboxyl functional groups, amino functional groups, phosphate functional groups, sulfhydryl functional groups, or combinations thereof. In some embodiments, the functional groups include alkanes, alkenes, alkynes, ethers, sulfides, amines, aldehydes, ketones, imines, nitriles, or combinations thereof. In some embodiments, the functional groups may be attached to the microgel particles using bonds, linkages, interactions, or other coupling mechanisms. In some embodiments, the bonds are covalent bonds. In some embodiments, the bonds are non-covalent bonds. In some embodiments, the bonds are selected from carbon-carbon bonds, amide bonds, imine bonds, ester bonds, thioether bonds, disulfide bonds, hydrazone bonds, hydrogen bonds, and metal ligand bonds. In some embodiments, the ester bonds include cyclic boronic esters. In some embodiments, the linkage is selected from a carbamate linkage, an ester linkage, and a thioether linkage. In some embodiments, the coupling is selected from an oxime coupling and a thiourea coupling. In some embodiments, the interaction is selected from an electrostatic interaction and a van der Waals interaction. In some embodiments, the functional group comprises a thiol or a derivative thereof. In some embodiments, the functional group comprises a matrix metalloproteinase (MMP)-sensitive peptide. Non-limiting examples of thiol derivatives include any organosulfur compound of the form R-SH, where R represents an alkyl or other organic substituent.In some embodiments, the thiol derivative comprises methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, allyl mercaptan, butanethiol, tert-butyl mercaptan, pentanethiol, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, metallothionein, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 1-mercaptoindole, grapefruit mercaptan, furan-2-ylmethanethiol, 3-mercaptopropane-1,2-diol, 3-mercapto-1-propanesulfonic acid, 1-hexadecanethiol, pentachlorobenzenethiol, or a combination thereof. In some embodiments, the functional group comprises vinyl or a derivative thereof. In some embodiments, the functional group comprises vinyl sulfone (VS) or a derivative thereof. Non-limiting examples of vinyl derivatives include alkenes, including ethene, propene, butene, pentene, hexene, heptene, and octene, acrylates, methacrylates, acrylamides, methacrylamides, maleimides, norbornenes, or combinations thereof. Non-limiting examples of VS derivatives include phenyl vinyl sulfone, methyl vinyl sulfone, ethyl vinyl sulfone, or any combination thereof. In some embodiments, the functional group comprises a thiol and a VS, or a derivative of either a thiol or a VS. In some embodiments, HA is modified to contain a thiol or a derivative thereof to form thiolated HA (e.g., SH-HA). In some embodiments, PEG is modified to contain a VS or a derivative thereof to form PEG-VS. In some embodiments, HA is modified to contain a VS or a derivative thereof to form HA-VS. In some embodiments, PEG is modified to contain a thiol or a derivative thereof to form thiolated PEG (e.g., PEG-SH, PEG-dithiol). In some embodiments, the PEG-VS comprises a multi-arm PEG-VS. In some embodiments, the multi-arm PEG-VS comprises a 4-arm, or a 6-arm, or an 8-arm PEG-VS.In some embodiments, the multi-arm PEG-VS comprises a star polymer, a brush polymer, a hyperbranched polymer, a comb polymer, or a dendritic polymer PEG-VS. In some embodiments, the VS comprises vinyl sulfone.

[0075] In some embodiments, the microgel particles include functional groups that can be pH-responsive (e.g., pH-responsive microgel particles). In some embodiments, pH-responsive microgel particles may be characterized as microgel particles that must be in the presence of a desired pH range to initiate annealing of the covalently stabilized scaffold.

[0076] In some embodiments, the dermal filler system or microgel particles comprise glutaraldehyde, divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE), or any derivative thereof, or any combination thereof, one or more of which are configured to interact in a cross-linking reaction to synthesize the microgel particles.

[0077] The functional groups disclosed herein may comprise peptides. The functional groups disclosed herein may comprise amino acids. In some embodiments, the functional group comprises a K peptide. In some embodiments, the K peptide comprises an amino acid sequence comprising FKGGERCG (SEQ ID NO: 4). In some embodiments, the K peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide comprises an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the functional group comprises a Q peptide. In some embodiments, the Q peptide comprises the amino acid sequence provided in NQEQVSPLGGERCG (SEQ ID NO: 5). In some embodiments, the Q peptides comprise an amino acid sequence that is about 75% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptides comprise an amino acid sequence that is about 85% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptides comprise an amino acid sequence that is about 95% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptides comprise an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 5.

[0078] The functional group may include a non-peptide polymer. The degradable functional group may also be a random sequence, an Omi target sequence, or a heat shock protein target sequence. The functional group may include an amino acid with D chirality. The functional group may include an amino acid with L chirality. The functional group may include hydrolytically degradable natural and synthetic polymers such as heparin, alginate, poly(ethylene glycol), polyacrylamide, polymethacrylate, copolymers and terpolymers of polycondensates (e.g., polyesters, polyamides, and other polymers such as polyurethanes). The functional group may be synthetically produced or naturally isolated. The functional group may include a restriction enzyme recognition sequence, a CpG motif, a Zn finger motif, a CRISPR or Cas-9 sequence, a Talon recognition sequence, or a DNA oligonucleotide having a sequence corresponding to a transcription factor binding domain. The functional groups may be activated at at least two ends with reactive groups, defined as chemical groups that either engage crosslinkers in crosslinking reactions to form microgel particles (intramolecular crosslinking) or anneal molecules together to form covalently stabilized scaffolds (intermolecular crosslinking). These functional groups may include cysteine ​​amino acids, synthetic and naturally occurring thiol-containing molecules, carbene-containing groups, vinyl-containing groups, activated esters, acrylates, norborenes, primary amines, hydrazides, phosphenes, azides, epoxy-containing groups, SANPAH-containing groups, and diazirine-containing groups. In some embodiments, the microgel particles themselves may function as crosslinkers. In some embodiments, the functional groups may be degradable.

[0079] In some embodiments, the microgel particles may be functionalized with acrylates, methacrylates, methacrylamides, maleimides, norbornenes, or any other vinyl derivatives. For example, the dermal filler system may further include two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof.

[0080] In some embodiments, the microgel particles are drug-eluting, such that a therapeutic agent disclosed herein is released in situ by the microgel particles. In some embodiments, the therapeutic agent comprises an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-fibrotic agent, an antibiotic, or an anti-cancer therapeutic. In some embodiments, the local anesthetic is an ester. In some embodiments, the ester-type local anesthetic comprises benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is an amide. In some embodiments, the amide-type local anesthetic comprises articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, or cinchocaine, or any combination thereof. In some embodiments, the local anesthetic is or comprises lidocaine. In some embodiments, the local anesthetic comprises lidocaine. In some embodiments, the analgesic comprises codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID) or a steroid. In some embodiments, the NSAID comprises ibuprofen or naproxen. In some embodiments, the steroid comprises a corticosteroid. In some embodiments, the antibiotic comprises dicloxacillin, erythromycin, or tetracycline. In some embodiments, the anti-fibrotic agent comprises pentoxifylline. In some embodiments, the therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the anti-cancer therapeutic agent comprises an alkylating agent, a nitrosourea, an antimetabolite, a plant alkaloid (e.g., a drug made from a natural product), an antitumor antibiotic, a hormonal agent, a biological response modifier, or a combination thereof. In some embodiments, the anti-cancer therapeutic agent is an agent designed to treat skin cancer. In some embodiments, skin cancers include basal cell carcinoma, cutaneous squamous cell carcinoma, melanoma, and Merkel cell carcinoma.In some embodiments, the anticancer therapeutic agent is cisplatin, 5-fluorouracil (5-FU), Aldara (imiquimod), cemiplimab-rwlc, Efudex (fluorouracil-topical), Erivedge (vismodegib), 5-FU (fluorouracil-topical), fluorouracil-topical, imiquimod, Libtayo (cemiplimab-rwlc), Odomzo (sonidegib), sonidegib, vismodegib, cemiplimab-rwlc, Keytruda (pembrolizumab), Libtayo, pembrolizumab, aldesleukin, binimetinib, Braftovi (encorafenib), cobimetinib, Cotellic (cobimetinib), dabrafenib, dacarbazine, DTIC-Dome (dacarbazine), encorafenib, IL-2 (aldesleukin), Imlygic (talimogene laherparepvec), interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), ipilimumab, Keytruda (pembrolizumab), Kimmtrak (Tebentafusp-tebn), Mekinist (trametinib dimethyl sulfoxide), Mektovi (binimetinib), nivolumab, nivolumab and leralimab-rmbw, Opdivo (nivolumab), Opdualag (nivolumab and leralimab-rmbw), peginterferon alfa-2b, pembrolizumab These include izumab, Proleukin (aldesleukin), recombinant interferon alfa-2b, Sylatron (peginterferon alfa-2b), Tafinlar (dabrafenib), talimogen, laherparepvec, laventafusp-tebn, trametinib dimethyl sulfoxide, vemurafenib, Yervoy (ipilimumab), Zelboraf (vemurafenib), avelumab, Bavencio (avelumab), or a combination thereof.

[0081] In some embodiments, the skin cancer is basal cell carcinoma. In some embodiments, the anti-cancer therapeutic agent comprises Aldara (imiquimod), cemiplimab-rwlc, Efudex (fluorouracil-topical), Erivedge (vismodegib), 5-FU (fluorouracil-topical), fluorouracil-topical, imiquimod, Libtayo (cemiplimab-rwlc), Odomzo (sonidegib), sonidegib, vismodegib, or a combination thereof. In some embodiments, the skin cancer is cutaneous squamous cell carcinoma, and the anti-cancer therapeutic agent comprises cemiplimab-rwlc, Keytruda (pembrolizumab), Libtayo, pembrolizumab, or a combination thereof. In some embodiments, the skin cancer is melanoma and the anti-cancer therapeutic agent is aldesleukin, binimetinib, Braftovi (encorafenib), cobimetinib, Cotellic (cobimetinib), dabrafenib, dacarbazine, DTIC-Dome (dacarbazine), encorafenib, IL-2 (aldesleukin), Imlygic (talimogene laherparepvec), interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), ipilimumab, Keytruda (pembrolizumab), Kimmtrak (tebentafusp-tebn), Mekinist (trametinib dimethyl sulfoxide), Mektovi (binimetinib), nivolumab, nivolumab and leratolimab-rmbw, Opdivo (nivolumab), Opdualag (nivolumab and leratolimab-rmbw), peginterferon alfa -2b, pembrolizumab, Proleukin (aldesleukin), recombinant interferon alfa-2b, Sylatron (peginterferon alfa-2b), Tafinlar (dabrafenib), talimogene laherparepvec, tebentafusp-tebn, trametinib dimethyl sulfoxide, vemurafenib, Yervoy (ipilimumab), Zelboraf (vemurafenib), or combinations thereof.In some embodiments, the skin cancer is Merkel cell carcinoma and the anti-cancer therapy comprises avelumab, Bavencio (avelumab), Keytruda (pembrolizumab), pembrolizumab, or a combination thereof.

[0082] In some embodiments herein, the microgel particles are characterized as having a degree of polydispersity. In some embodiments, the polydispersity of the microgel particles is a measure of the heterogeneity of the microgel particles based on molecular weight. In some embodiments, the polydispersity of the microgel particles is a measure of the heterogeneity of the microgel particles based on size. In some embodiments, the microgel particles comprise a polydispersity (PDI) of 0.1 or less, where the polydispersity is calculated based on the standard deviation (SD) and average size of the microgel particles (e.g., PDI = (SD / mean)^2). In some embodiments, the microgel particles comprise a polydispersity (PDI) of 0.1 or less, where the polydispersity is calculated based on the weight average (MW) and number average molecular weight (Mn) of the microgel particles (e.g., PDI = MW / Mn). In some embodiments, the polydispersity is measured using the coefficient of variation (CV), where the coefficient of variation is calculated based on the standard deviation (SD) and average size of the microgel particles (e.g., CV = SD / mean). In some embodiments, lower polydispersity based on size or weight of the microgel particles aids in the formation of covalently stabilized scaffolds. In some embodiments, lower polydispersity based on size or weight of the microgel particles aids in achieving desired mechanical properties of the covalently stabilized scaffolds. In some embodiments, lower polydispersity increases the porosity of the composition (e.g., more polydisperse particle sizes can lead to smaller particles fitting into the pores of the covalently stabilized scaffolds).

[0083] In some embodiments, the microgel particles comprise a degree of substitution per monomer of about 5% to about 20%. In some embodiments, the microgel particles comprise a degree of substitution per monomer of about 5%, 10%, 15%, or 20% or more. In some embodiments, the microgel particles comprise a degree of substitution per monomer of about 5%, 10%, 15%, or 20% or less. In some embodiments, the microgel particles comprise modified HA with a degree of substitution per HA monomer of about 5% to about 20%. In some embodiments, the microgel particles comprise modified HA with a degree of substitution per HA monomer of about 10% to about 20%. In some embodiments, the microgel particles comprise modified HA with a degree of substitution per HA monomer of about 12%. In some embodiments, the microgel particles comprise modified PEG with a degree of substitution per HA monomer of about 80% to about 100%.

[0084] In some embodiments, the components of the dermal filler system discussed above can aid in the synthesis of the microgel particles. In some embodiments, the thiol or derivative thereof and the VS or derivative thereof are configured to interact with each other in a reaction to synthesize the microgel particles.

[0085] In some embodiments, the reaction comprises a covalent synthesis reaction. Non-limiting examples of covalent bonds are those found in carbon-carbon, amide, ester, thioether, carbamate, disulfide, oxime, thiourea, hydrazone, and imine bonds. In some embodiments, the reaction comprises a non-covalent synthesis reaction. Non-limiting examples of non-covalent bonds are those found in interactions such as electrostatic interactions, hydrogen bonding, cation-π, π-π stacking, metal-ligand bonding, and van der Waals interactions. In some embodiments, the method comprises linking two or more microgel particles together. Non-limiting examples of linking reactions include Michael addition, amide bond coupling, "click" chemistry (e.g., Diels-Alder cycloaddition, Huisgen 1,3-dipolar cycloaddition), reductive amination, carbamate linkage, ester linkage, thioether linkage, disulfide linkage, hydrazone linkage, oxime coupling, and thiourea coupling.

[0086] In some embodiments, the reaction comprises a covalent synthesis reaction. In some embodiments, the covalent synthesis reaction comprises a Michael addition (e.g., thiol-ene Michael addition, aza-Michael addition, oxa-Michael addition) reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or derivative thereof is a Michael donor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the thiol or derivative thereof and VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 1:1. In some embodiments, the thiol or derivative thereof and VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 0.3:1 thiol:VS to 1:1 thiol:VS. In some embodiments, the thiol or derivative thereof and VS or a derivative thereof are present in the dermal filler system in a molar ratio of about 0.6:1 thiol:VS to 0.8:1 thiol:VS. In some embodiments, the thiol or derivative thereof and VS or derivative thereof are present in the dermal filler system in a molar ratio of about 1:1 to about 1:2 thiol:VS. In some embodiments, the thiol or derivative thereof and VS or derivative thereof are present in the dermal filler system in a molar ratio of about 1:1 to about 1:1.4 thiol:VS. In some embodiments, the molar ratio may be defined as the molar ratio of thiol (SH) to vinyl sulfone (VS) groups. R = [SH] / [VS] = nSH / nVS, where [SH] is defined as the molar concentration of thiol, [VS] is defined as the molar concentration of VS, nSH is defined as the number of moles of thiol, and nVS is defined as the number of moles of VS. In some embodiments, an excess of either the thiol or derivative thereof and VS or derivative thereof is present in the dermal filler system such that the excess of either the thiol or derivative thereof or the VS or derivative thereof participates in an annealing reaction to form a covalently stabilized porous scaffold.

[0087] In some embodiments, synthesis of the microgel particles may be achieved through one or more physical linkage points (e.g., looping) of the polymer(s) (e.g., PEG and / or HA) comprising the microgel particles. In some embodiments, the physical linkage may include weak physical interactions. In some embodiments, the weak physical interactions may include coordinate bonds and ionic interactions. In some embodiments, the physical linkage points aid in the synthesis of the microgel particles. In some embodiments, synthesis of the microgel particles is achieved solely by reaction. In some embodiments, synthesis of the microgel particles is achieved solely by physical linkage.

[0088] Covalently stabilized scaffolds In some embodiments, disclosed herein are microgel particles that anneal together to form a covalently stabilized scaffold. In some embodiments, the formation of the covalently stabilized scaffold occurs in situ after delivery of the microgel particles to a tissue site. In some embodiments, the dermal filler system includes one or more components configured to assist the annealing reaction to form the covalently stabilized porous scaffold.

[0089] In some embodiments, the one or more components that promote or initiate annealing of the microgel particles in the dermal filler system to form a covalently stabilized scaffold include an annealing component, an annealing agent, or a combination thereof. In some embodiments, the annealing agent includes a molecule. In some embodiments, the annealing agent includes a photoinitiator. A non-limiting example of the photoinitiator may be eosin Y. In some embodiments, the annealing agent includes triethanolamine. In some embodiments, the annealing agent includes an enzyme. In some embodiments, the enzyme includes thrombin. In some embodiments, the annealing agent includes a transglutaminase enzyme. A non-limiting example of a transglutaminase enzyme is factor XIII (the active form of factor XIII). In some embodiments, the annealing agent includes a radical initiator. In some embodiments, the annealing agent includes an electron transfer agent. Examples of additional and alternative annealing agents include, but are not limited to, active esters and nucleophiles, catechols that undergo crosslinking upon oxidation, and other redox-sensitive molecules. In some embodiments, the annealing agent comprises a homo- or heterofunctional polymer containing a thiol, maleimide, vinyl sulfone, methacrylate, methacrylamide, or other vinyl functional group. In some embodiments, the annealing agent comprises a cyclodextrin, a cucurbituril, or a calixarene. In some embodiments, the annealing component comprises a K peptide, a Q peptide, or a combination thereof. In some embodiments, the annealing component comprises a vinyl group (e.g., vinyl sulfone, methyl acrylate, acrylamide), a thiol, a maleimide, or an amine.

[0090] In some embodiments, the microgel particles do not require an annealing agent for annealing. For example, the microgel particles may contain other components (e.g., functional groups) that participate in a chemical crosslinking reaction to form a covalently stabilized scaffold. In some embodiments, one or more components that promote annealing of the microgel particles in the dermal filler system to form a covalently stabilized scaffold include a thiol derivative, a vinyl derivative, or a combination thereof. For example, the microgel particles may be composed of a polymer or copolymer modified to contain one or more vinyl derivatives and one or more thiol derivatives, with either the vinyl derivative or the thiol derivative being in excess. Such vinyl derivatives and thiol derivatives may undergo a chemical crosslinking reaction to anneal the microgel particles together to form a covalently stabilized scaffold.

[0091] In some embodiments, the annealing reaction that anneals the microgel particles together to form a covalently stabilized scaffold comprises a covalent synthesis reaction. Non-limiting examples of covalent bonds are those found in carbon-carbon, amide, ester, thioether, carbamate, disulfide, oxime, thiourea, hydrazone, and imine bonds. In some embodiments, the covalent synthesis reaction comprises a Michael addition (e.g., thiol-ene Michael addition) reaction or a pseudo-Michael addition reaction. In some embodiments, a thiol derivative is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, a vinyl derivative (e.g., vinyl sulfone) is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the covalent synthesis reaction comprises an ether (oxo-Michael addition) reaction or an amine (aza-Michael addition) reaction.

[0092] In some embodiments, the functional group and one or more components supporting the annealing reaction are configured to interact to perform the annealing reaction. In some embodiments, the PEG-dithiol is configured to interact with excess VS or a derivative thereof in the annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction comprises a covalent annealing reaction. In some embodiments, the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol or a derivative thereof of the PEG-dithiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction. In some embodiments, the excess VS is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

[0093] In some embodiments, the dermal filler system further comprises PEG-divinyl sulfone or a derivative thereof. In some embodiments, the PEG-divinyl sulfone may be a component that aids in an annealing reaction. In some embodiments, the PEG-divinyl sulfone or a derivative thereof is configured to interact with an excess thiol or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the divinyl sulfone or a derivative thereof in the PEG-divinyl sulfone is a Michael acceptor in a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the excess thiol is a Michael donor in a Michael addition reaction or a pseudo-Michael addition reaction.

[0094] In some embodiments, the annealing reaction that anneals the microgel particles together to form a stabilized scaffold comprises a non-covalent synthesis reaction. Non-limiting examples of non-covalent bonds are those found in interactions such as electrostatic interactions, hydrogen bonding, cation-π, π-π stacking, metal-ligand bonding, and van der Waals interactions. Non-limiting examples of annealing reactions include Michael addition, amide bond coupling, "click" chemistry (e.g., Diels-Alder cycloaddition, Huisgen 1,3-dipolar cycloaddition), reductive amination, carbamate linkage, ester linkage, thioether linkage, disulfide linkage, hydrazone linkage, oxime coupling, and thiourea coupling.

[0095] In some embodiments, the molecule comprises PEG. In some embodiments, the molecule comprises PEG-dithiol. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 10.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 15.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 20.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.0 kDa to about 10.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.0 kDa to about 5.0 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of about 3.4 kDa. In some embodiments, the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof. In some embodiments, the PEG-thiol comprises a multi-arm PEG-thiol. In some embodiments, the multi-arm PEG-thiol comprises a 4-arm, 6-arm, or 8-arm PEG-thiol. In some embodiments, the multi-arm PEG-thiol comprises a star polymer, a brush polymer, a multi-branched polymer, a comb polymer, or a dendritic polymer PEG-thiol.

[0096] In some embodiments, the microgel particles are annealed in the presence of one or more buffers to form a covalently stabilized scaffold. Non-limiting examples of buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4), calcium chloride (CaCl), phosphate-buffered saline (PBS) (pH 6.0-8.0), TRIS, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid buffer (MES), or combinations thereof. In some embodiments, an additional stimulus, such as light or pH, is added to catalyze the reaction.

[0097] In some embodiments, the covalently stabilized scaffold is porous. In some embodiments, the covalently stabilized scaffold comprises pores having a median pore size of about 5 μm or more. In some embodiments, the pores comprise a median pore size of about 10 μm to about 35 μm. In some embodiments, the pores comprise a median pore size of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 μm or more. In some embodiments, the pores comprise a median pore size of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 μm or less. In some embodiments, the pores comprise a median pore size of about 5 μm to about 70 μm. In some embodiments, the pores comprise a median pore size of about 10 μm to about 65 μm. In some embodiments, the pores comprise a median pore diameter of about 15 μm to about 60 μm. In some embodiments, the pores comprise a median pore diameter of about 20 μm to about 55 μm. In some embodiments, the pores comprise a median pore diameter of about 25 μm to about 50 μm. In some embodiments, the pores comprise a median pore diameter of about 30 μm to about 45 μm. In some embodiments, the pores comprise a median pore diameter of about 35 μm to about 40 μm. The median pore diameter may be measured by a process that includes, for a sampling of pores, (1) measuring the area of ​​the pore, (2) solving for the radius of the pore using the equation for the area of ​​a circle (i.e., A=pi*r^2), and (3) solving for the diameter by multiplying the radius by 2 (i.e., D=2*r).

[0098] In some embodiments, the covalently stabilized scaffold is degradable in vivo by one or more degradation pathways. In some embodiments, the one or more degradation pathways include oxidative degradation, enzymatic degradation, photolysis, or hydrolysis. In some embodiments, the composition of the microgel particles is fine-tuned to achieve a desired degradation profile depending on the application. For example, microgel particles containing PEG degrade slower than natural polymers such as HA. Therefore, the microgel particles disclosed herein may be copolymers of PEG and HA to take advantage of the degradation profiles of PEG and HA as well as other benefits of HA disclosed elsewhere herein. In some embodiments, the dermal filler system is enzymatically degraded by hyaluronidase. In some embodiments, hyaluronidase is injected to degrade the dermal filler system. In some embodiments, the dermal filler system is degraded by endogenous hyaluronidase. In some embodiments, hyaluronidase degrades the dermal filler system by degrading the HA in the dermal filler system. In some embodiments, it may be desirable for the injection of the dermal filler system to be reversible. The dermal filler systems described herein may be degradable after a certain period of time when contacted with hyaluronidase, as shown in Figure 31. In some embodiments, the dermal filler system is completely degraded after 6 hours at a temperature of 37°C using hyaluronidase.

[0099] In some embodiments, the covalently stabilized scaffold remains at the tissue site for more than 9 months before complete degradation. In some embodiments, the covalently stabilized scaffold remains at the tissue site for at least 18 months before complete degradation. In some embodiments, the covalently stabilized scaffold remains at the tissue site for at least 24 months before complete degradation. In some embodiments, the covalently stabilized scaffold remains at the tissue site for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months before complete degradation. As shown in Figures 16A-16D and 17, and as discussed in Example 2 below, dermal filler systems comprising HA and PEG exhibit a slower degradation rate than comparable fillers (e.g., Juvederm®) and also allow for increased protein, cell, and tissue growth in the cellular matrix. Specifically, Figure 17 shows that the dermal filler systems described herein minimize foreign body reactions, such as allowing vascular perfusion within the pore spaces, deposition of new proteins (e.g., fibrous, unaligned) within the pore spaces, and minimizing or completely preventing the formation of multinucleated giant cells (MNGCs).

[0100] In some embodiments, the covalently stabilized scaffold comprises a compressive modulus of 1,000 Pascals (Pa) to 100,000 Pa. In some embodiments, the covalently stabilized scaffold may be annealed when the microgel particles are in a swollen and non-swollen state. In some embodiments, the covalently stabilized scaffold may be annealed when the microgel particles are in a swollen state, such as with water. In some embodiments, the microgel particles are in a non-swollen state when the covalently stabilized scaffold is annealed. In some embodiments, the compressive modulus of the scaffold can be adjusted by adjusting the molecular weight, substitution rate, and molar ratio of the hydrogel polymer components, as discussed above.

[0101] In some embodiments, the covalently stabilized scaffold comprises a compressive modulus of 5,000 Pascals (Pa) to 100,000 Pa in an unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in an unswollen state of about 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, 125,000, 130,000, 135,000, 140,000, 145,000, or 150,000 Pa or greater. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in an unswollen state of less than or equal to about 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, 125,000, 130,000, 135,000, 140,000, 145,000, or 150,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 5,000 Pa to about 150,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 10,000 Pa to about 145,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 15,000 Pa to about 140,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 20,000 Pa to about 135,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 25,000 Pa to about 130,000 Pa in the unswollen state.In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 30,000 Pa to about 125,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 35,000 Pa to about 120,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 40,000 Pa to about 115,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 45,000 Pa to about 110,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 50,000 Pa to about 105,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 55,000 Pa to about 100,000 Pa in the unswollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 60,000 Pa to about 95,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 65,000 Pa to about 90,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 70,000 Pa to about 85,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the unswollen state that is in the range of about 75,000 Pa to about 80,000 Pa.

[0102] In some embodiments, the covalently stabilized scaffold comprises a compressive modulus of 1,000 Pascals (Pa) to 50,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in a swollen state of about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, or 110,000 Pa or greater. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in a swollen state of less than or equal to about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, or 110,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 5,000 Pa to about 110,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 10,000 Pa to about 105,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 15,000 Pa to about 100,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 20,000 Pa to about 95,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 25,000 Pa to about 90,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 30,000 Pa to about 85,000 Pa in a swollen state.In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 35,000 Pa to about 80,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 40,000 Pa to about 75,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 45,000 Pa to about 70,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 50,000 Pa to about 65,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a compressive modulus in the range of about 55,000 Pa to about 60,000 Pa in a swollen state.

[0103] In some embodiments, the swollen microgel particle suspension is formulated for administration with a needle. In some embodiments, the swollen microgel particle suspension, when formulated for administration with a needle, comprises an apparent viscosity of about 1, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 Pascal-seconds (Pa s) or greater. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 1, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 Pa·s or less. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 1 to about 10,000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 50 to about 9500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 100 to about 9000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 150 to about 8500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 200 to about 8000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 250 to about 7500 Pa·s.In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 300 to about 7000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 350 to about 6500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 400 to about 6000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 450 to about 5500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 500 to about 5000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 550 to about 4500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 600 to about 4000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 650 to about 3500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 700 to about 3000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 750 to about 2500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 800 to about 2000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 850 to about 1500 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 900 to about 1000 Pa·s. In some embodiments, the swollen microgel particle suspension, when formulated for needle administration, comprises an apparent viscosity of about 100 to about 1000 Pa·s.

[0104] In some embodiments, the volume fraction of microgel particles and the elastic modulus of the microgel particles can be adjusted to achieve a desired compressive modulus for the covalently stabilized scaffold. As shown in Figure 5, both the volume fraction and elastic modulus of the microgel particles affect the final compressive modulus of the covalently stabilized scaffold. As shown, a higher volume fraction and a higher microgel particle elastic modulus can lead to a higher compressive modulus of the covalently stabilized scaffold.

[0105] In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the swollen state of 50 Pascals (Pa) to 10,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the swollen state of 60 Pa to 1,000 Pa. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in a swollen state of about 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or greater. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in a swollen state of about 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or less. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 5,000 Pa to about 110,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 10,000 Pa to about 105,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 15,000 Pa to about 100,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 20,000 Pa to about 95,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 25,000 Pa to about 90,000 Pa in a swollen state.In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 30,000 Pa to about 85,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 35,000 Pa to about 80,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 40,000 Pa to about 75,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 45,000 Pa to about 70,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 50,000 Pa to about 65,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold comprises a storage modulus in the range of about 55,000 Pa to about 60,000 Pa in a swollen state.

[0106] In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 5,000 Pa to about 100,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 5,000 Pa to about 50,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 5,000 Pa to about 46,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 5,000 Pa to about 75,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 5,000 Pa to about 25,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about or greater than 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of less than or equal to or greater than about 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 10,000 Pa to about 100,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 15,000 Pa to about 95,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 20,000 Pa to about 90,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 25,000 Pa to about 85,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 30,000 Pa to about 80,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 35,000 Pa to about 75,000 Pa.In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 40,000 Pa to about 70,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 45,000 Pa to about 65,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in an unswollen state of about 50,000 Pa to about 60,000 Pa.

[0107] In some embodiments, the covalently stabilized scaffold comprises a loss modulus in the swollen state of about 1 Pascal (Pa) to 10,000 Pa. The loss modulus may be measured by performing shear modulus measurements as described above and performing a shear stress amplitude and frequency sweep in a parallel plate system, which may allow for calculation of both the storage modulus and loss modulus of the viscoelastic material (the storage modulus and loss modulus together comprise the shear modulus). In some embodiments, the covalently stabilized scaffold comprises a loss modulus in a swollen state of about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or greater. In some embodiments, the covalently stabilized scaffold comprises a loss modulus in a swollen state of about 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or less.In some embodiments, the covalently stabilized scaffold has a viscosity in the swollen state of about 10 Pa to 11,000 Pa, 20 Pa to 10,500 Pa, 30 Pa to 10,000 Pa, 40 Pa to 9,500 Pa, 50 Pa to 9,000 Pa, 60 Pa to 8,500 Pa, 70 Pa to 8,000 Pa, 80 Pa to 7,500 Pa, 90 Pa to 7,000 Pa, 100 Pa to 6,500 Pa, 150 Pa to 6,000 Pa, 200 Pa to 30,000 Pa, 350 Pa to 40,000 Pa, 400 Pa to 50,000 Pa, 500 Pa to 60,000 Pa, 600 Pa to 75,000 Pa, 700 Pa to 80,000 Pa, 800 Pa to 95,000 Pa, 900 Pa to 70,000 Pa, 100 Pa to 65,000 Pa, 150 Pa to 6,000 Pa, 200 Pa to 30,000 Pa, 350 Pa to 40,000 Pa, 400 Pa to 50,000 Pa, 500 Pa to 60,000 Pa, 600 Pa to 75,000 Pa, 700 Pa to 80,000 Pa, 800 Pa to 95,000 Pa, 900 Pa to 100,000 Pa, 1000 Pa to 125,000 Pa, 1500 Pa to 160,000 Pa, 1600 Pa to 200,000 Pa, 1700 Pa to 200,000 Pa, Including loss modulus of Pa~5500Pa, 250Pa~5000Pa, 300Pa~4500Pa, 350Pa~4000Pa, 400Pa~3500Pa, 450Pa~3000Pa, 500Pa~2500Pa, 550Pa~2000Pa, 600Pa~1500Pa, 650Pa~1000Pa, 700Pa~950Pa, 750Pa~900Pa, or 800Pa~850Pa.

[0108] In some embodiments, the microgel particles comprise a compressive modulus in a swollen state of about 500 Pa to about 50,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in a swollen state of greater than about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in a swollen state of less than about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in a swollen state of about 500-50,000, 1,000-45,000, 2,000-40,000, 3,000-35,000, 4,000-30,000, 5,000-25,000, 6,000-20,000, 7,000-20,000, 8,000-15,000, or 9,000-10,000 Pa. In some embodiments, the microgel particles comprise a compressive modulus in a swollen state of about 500 Pa-50,000, 500-40,000, 500-30,000, 500-20,000, or 500-10,000 Pa.

[0109] In some embodiments, the microgel particles comprise a storage modulus in a swollen state of about 10 Pa to about 5,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a swollen state of greater than about 10, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a swollen state of less than about 10, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a swollen state of about 10-5,000, 100-4,500, 500-4,000, or 1,000-3,000 Pa.

[0110] In some embodiments, the microgel particles comprise a storage modulus in an unswollen state of about 50 Pa to about 10,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in an unswollen state of greater than about 50, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in an unswollen state of less than about 50, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in an unswollen state of about 50-10,000, 100-9,000, 500-8,000, 1,000-7,000, 2,000-6,000, or 3,000-5,000 Pa.

[0111] In some embodiments, the microgel particles comprise a loss modulus in the swollen state of about 0.1 Pa to about 2,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in the swollen state of greater than about 0.1, 0.5, 1, 50, 100, 500, 1,000, 1,500, or 2,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in the swollen state of less than about 0.1, 0.5, 1, 50, 100, 500, 1,000, 1,500, or 2,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in the swollen state of about 0.1-2,000, 0.5-1,500, 1-1,000, or 50-500 Pa.

[0112] In some embodiments, the microgel particles comprise a loss modulus in an unswollen state of about 1 Pa to about 5,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in an unswollen state of greater than about 1, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in an unswollen state of less than about 1, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 Pa. In some embodiments, the microgel particles comprise a loss modulus in an unswollen state of about 1-5,000, 100-4,500, 500-4,000, or 1,000-3,000 Pa.

[0113] In some embodiments, the microgel particle suspension comprises a compressive modulus in a swollen state of about 100 Pa to about 20,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in a swollen state of greater than about 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in a swollen state of less than about 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in a swollen state of about 100-20,000, 500-19,000, 1,000-18,000, 2,000-17,000, 3,000-16,000, 4,000-15,000, 5,000-14,000, 6,000-13,000, 7,000-12,000, 8,000-11,000, or 9,000-10,000 Pa.

[0114] In some embodiments, the microgel particle suspension comprises a compressive modulus in an unswollen state of about 500 Pa to about 50,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in an unswollen state of greater than about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in an unswollen state of less than about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in an unswollen state of about 500-50,000, 1,000-45,000, 2,000-40,000, 3,000-35,000, 4,000-30,000, 5,000-25,000, 6,000-20,000, 7,000-20,000, 8,000-15,000, or 9,000-10,000 Pa. In some embodiments, the microgel particle suspension comprises a compressive modulus in an unswollen state of about 500 Pa-50,000, 500-40,000, 500-30,000, 500-20,000, or 500-10,000 Pa.

[0115] In some embodiments, the microgel particle suspension comprises a storage modulus in the swollen state of about 10 to about 10,000 Pa. In some embodiments, the microgel particle suspension comprises a storage modulus in the swollen state of greater than about 10, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particle suspension comprises a storage modulus in the swollen state of less than about 10, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particle suspension comprises a storage modulus in a swollen state of about 10-10,000, 100-9,000, 500-8,000, 1,000-7,000, 2,000-6,000, or 3,000-5,000 Pa.

[0116] In some embodiments, the microgel particle suspension comprises a loss modulus in the swollen state of about 1 to about 10,000 Pa. In some embodiments, the microgel particle suspension comprises a loss modulus in the swollen state of greater than about 1, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particle suspension comprises a loss modulus in the swollen state of less than about 1, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Pa. In some embodiments, the microgel particle suspension comprises a loss modulus in a swollen state of about 1-10,000, 100-9,000, 500-8,000, 1,000-7,000, 2,000-6,000, or 3,000-5,000 Pa.

[0117] In some embodiments, the thiol or derivative thereof and vinyl sulfone or derivative thereof are present in the dermal filler system at a molar ratio of thiol to VS (thiol:VS) of about 0.3 to about 0.8 to achieve a desired compressive modulus of about 500 Pa to about 50,000 Pa (e.g., when the dermal filler is formulated for administration with a needle). In some embodiments, the microgel particles are present in a suspension comprising microgel particles and water, with 50% to 100% volume fraction of the suspension comprising microgel particles to achieve a desired compressive modulus (e.g., when the dermal filler is formulated for administration with a needle).

[0118] In some embodiments, the covalently stabilized scaffold is -1 and apparent viscosities of about 1,000 to about 1,000,000 millipascal seconds (mPa s) at shear rates in the range of 1000 to about 1,000,000 mPa s. In some embodiments, the volume fraction of microgel particles and the modulus of elasticity of the microgel particles can be adjusted to achieve the desired viscosity.

[0119] In some embodiments, the covalently stabilized scaffold comprises a pH of 5.0 to 9.0. In some embodiments, the covalently stabilized scaffold comprises a pH of 6.5 to 7.5. In some embodiments, the covalently stabilized scaffold comprises a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or greater. In some embodiments, the covalently stabilized scaffold comprises a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or less. In some embodiments, the covalently stabilized scaffold comprises a pH of about 4.0 to about 10.0. In some embodiments, the covalently stabilized scaffold comprises a pH of about 4.5 to about 9.5. In some embodiments, the covalently stabilized scaffold comprises a pH of about 5.0 to about 9.0. In some embodiments, the covalently stabilized scaffold comprises a pH of about 5.5 to about 8.5. In some embodiments, the covalently stabilized scaffold comprises a pH of about 6.0 to about 8.0. In some embodiments, the covalently stabilized scaffold comprises a pH of about 6.5 to about 7.5. In some embodiments, the covalently stabilized scaffold comprises a pH of about 7.0 to about 7.5.

[0120] In some embodiments, the microgel particles comprise a pH of 5.0 to 9.0. In some embodiments, the microgel particles comprise a pH of 6.5 to 7.5. In some embodiments, the microgel particles comprise a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or greater. In some embodiments, the microgel particles comprise a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or less. In some embodiments, the microgel particles comprise a pH of about 4.0 to about 10.0. In some embodiments, the microgel particles comprise a pH of about 4.5 to about 9.5. In some embodiments, the microgel particles comprise a pH of about 5.0 to about 9.0. In some embodiments, the microgel particles comprise a pH of about 5.5 to about 8.5. In some embodiments, the microgel particles comprise a pH of about 6.0 to about 8.0. In some embodiments, the microgel particles comprise a pH of about 6.5 to about 7.5. In some embodiments, the microgel particles comprise a pH of about 7.0 to about 7.5.

[0121] In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 100 milliosmoles per kilogram (mOsmol / kg) to about 400 mOsmol / kg. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mOsmol / kg or greater. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mOsmol / kg or less. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 50 mOsmol / kg to about 500 mOsmol / kg. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 100 mOsmol / kg to about 450 mOsmol / kg. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 150 mOsmol / kg to about 400 mOsmol / kg. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 200 mOsmol / kg to about 350 mOsmol / kg. In some embodiments, the covalently stabilized scaffold comprises an osmolality of about 250 mOsmol / kg to about 300 mOsmol / kg.

[0122] Dermal filling system Disclosed herein, in some embodiments, is a dermal filler system comprising the microgel particles of the present disclosure and an additional active agent (e.g., a therapeutic agent), reagent, or solvent.

[0123] In some embodiments, the dermal filler system comprises microgel particles and a therapeutic agent. In some embodiments, the therapeutic agent is not released from the microgel particles. In some embodiments, the therapeutic agent is released from the microgel particles (e.g., drug-eluting microgel particles). In some embodiments, the active agent is a therapeutic agent. In some embodiments, the therapeutic agent comprises an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-fibrotic agent, or an antibiotic. In some embodiments, the local anesthetic is an ester type. In some embodiments, the ester type local anesthetic comprises benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is an amide type. In some embodiments, the amide type local anesthetic comprises articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, or cinchocaine, or any combination thereof. In some embodiments, the local anesthetic is or comprises lidocaine. In some embodiments, the local anesthetic consists of lidocaine. In some embodiments, the lidocaine is present in the dermal filler system at a concentration of about 1.0 mg / mL to about 5.0 mg / mL. In some embodiments, the lidocaine is present in the dermal filler system at a concentration of about 3.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 0.5 mg / mL to about 20.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 1.0 mg / mL to about 19.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 1.5 mg / mL to about 19.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 2.0 mg / mL to about 18.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 2.5 mg / mL to about 18.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 3.0 mg / mL to about 17.5 mg / mL.In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 3.5 mg / mL to about 17.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 4.0 mg / mL to about 16.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 4.5 mg / mL to about 16.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 5.0 mg / mL to about 15.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 5.5 mg / mL to about 15.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 6.0 mg / mL to about 14.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 6.5 mg / mL to about 14.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 7.0 mg / mL to about 13.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 7.5 mg / mL to about 13.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 8.0 mg / mL to about 12.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 8.5 mg / mL to about 12.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 9.0 mg / mL to about 11.5 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 9.5 mg / mL to about 11.0 mg / mL. In some embodiments, the local anesthetic is present in the dermal filler system at a concentration of about 9.5 mg / mL to about 10.5 mg / mL. In some embodiments, the analgesic comprises codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID) or a steroid. In some embodiments, the NSAID comprises ibuprofen or naproxen. In some embodiments, the steroid comprises a corticosteroid.In some embodiments, the antibiotic comprises dicloxacillin, erythromycin, or tetracycline. In some embodiments, the antifibrotic agent comprises pentoxifylline.

[0124] In some embodiments, the dermal filler formulation can withstand sterilization. Sterilization can be achieved by steam sterilization, filtration, microfiltration, e-beam, gamma irradiation, ethylene oxide (ETO), light, supercritical carbon dioxide, hydrogen peroxide vapor, or any combination thereof. In some embodiments, certain components of the dermal filler system may be steam sterilized (e.g., autoclaved) without degrading the physical properties of the microgel particles, etc. However, when the dermal filler formulation contains additional components such as a therapeutic agent, the components of the dermal filler formulation containing the therapeutic agent can be sterilized by means other than heat treatment, such as by sterile filtration.

[0125] In some embodiments, sterilization of the skin filler formulation is achieved by autoclaving. Autoclaving can be achieved by applying a combination of heat, pressure, and moisture to the formulation requiring sterilization. Many different sterilization temperatures, pressures, and cycle times can be used. For example, in some embodiments, the filled syringe may be sterilized at a temperature of at least about 120°C to about 130°C or higher. In some embodiments, the filled syringe may be sterilized at a temperature of at least about 120°C to about 130°C or higher, with or without the use of moisture. In some embodiments, pressure is applied depending on the temperature used in the sterilization process. In some embodiments, the sterilization cycle may be at least about 1 minute to about 20 minutes or longer. In some embodiments, the sterilization cycle may be at least about 1 minute to about 30 minutes or longer. In some embodiments, the sterilization cycle may be at least about 15 minutes to about 30 minutes or longer.

[0126] In some embodiments, the sterilization method incorporates the use of gas species known to kill or eliminate infectious agents, hi some embodiments, ethylene oxide can be used as a sterilizing gas to sterilize any of the medical devices, products, or delivery devices disclosed herein.

[0127] In some embodiments, the sterilization method incorporates the use of an irradiation source to kill or eliminate infectious agents. The irradiation beam is targeted at a delivery device (e.g., a syringe) containing the dermal filler formulation, and the wavelength of the energy kills or eliminates the undesirable infectious agents. Non-limiting examples of useful energy include, but are not limited to, ultraviolet (UV) light, electron beam (e-beam) irradiation, gamma irradiation, visible light, microwaves, or any other wavelength or wavelength range that kills or eliminates undesirable infectious agents, preferably without substantially altering the degradation of the dermal filler formulation.

[0128] In some embodiments, the dermal filler system includes a reagent, such as an annealing agent, that promotes the annealing reaction of the dermal filler system to form a covalently stabilized scaffold. In some embodiments, the annealing agent includes a photoinitiator. A non-limiting example of the photoinitiator may be eosin Y. In some embodiments, the annealing agent includes triethanolamine. In some embodiments, the annealing agent includes an enzyme. In some embodiments, the enzyme includes thrombin. In some embodiments, the annealing agent includes a transglutaminase enzyme. A non-limiting example of a transglutaminase enzyme is factor XIII (the active form of factor XIII). In some embodiments, the annealing agent includes a free radical transfer agent. In some embodiments, the annealing agent includes an electron transfer agent. Examples of additional and alternative annealing agents include, but are not limited to, active esters and nucleophiles, catechols that undergo crosslinking upon oxidation, and other redox-sensitive molecules. In some embodiments, the annealing component includes a K peptide, a Q peptide, or a combination thereof. In some embodiments, the reagent comprises a stabilizer, sterilizer, or thermoprotectant. Non-limiting examples of stabilizers include reagents, salts, and additives. Non-limiting examples of sterilizers include reagents, salts, and additives. Non-limiting examples of thermoprotectants include antioxidants, glycerin, and PEG. In some embodiments, the dermal filler system is protected during sterilization by freezing the dermal filler before and / or during terminal sterilization (e.g., irradiation). In some embodiments, the dermal filler system is protected during sterilization by placing the material under a sealed inert atmosphere or in a vacuum ampule.

[0129] In some embodiments, the dermal filler system includes a solvent, such as one or more buffers, water, or a combination thereof. In some embodiments, the microgel particles are present in a suspension containing water. In some embodiments, the dermal filler system includes a buffer. In some embodiments, the buffer includes phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, acetate buffer, citrate buffer, borate buffer, or any combination thereof. In some embodiments, the buffer adjusts the pH of the dermal filler system to a desired pH. The pH of the disclosed dermal filler formulations can be about 5.0 to about 8.0, or about 6.5 to about 7.5. In certain embodiments, the pH of the formulation is about 7.0 to about 7.4, or about 7.1 to about 7.3. In some embodiments, the dermal filler system includes a suspension of microgel particles in an aqueous solvent including a buffer. In some embodiments, the buffer may be a buffering agent. In some embodiments, 50% to 100% by volume of the suspension includes microgel particles. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the volume fraction of the suspension comprises microgel particles. In some embodiments, when the dermal filler is formulated for needle administration, the volume fraction of microgel particles is about 50% or more. In some embodiments, when the dermal filler is formulated for needle administration, the volume fraction of microgel particles is about 50%, 60%, 70%, 80%, or 90% or more.

[0130] In some embodiments, the dermal filler system is colorless. In some embodiments, the dermal filler system is substantially optically transparent. In some embodiments, the polydispersity of the dermal filler system is 0.1 or less. In some embodiments, the coefficient of variation of the dermal filler system is about 62.5%, 60%, 57.5%, 55%, 52.5%, 50%, 47.5%, 45%, 42.5%, 40%, 37.5%, 35%, 32.5%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, or 10% or less. In some embodiments, the coefficient of variation of the dermal filler system is about 62.5% or less. In some embodiments, the coefficient of variation of the dermal filler system is about 30% or less. In some embodiments, the dermal filler system is odorless.

[0131] In some embodiments, the dermal filler system is formulated for administration to a subject. In some embodiments, the administration is subdermal, dermal, intradermal, or subcutaneous. In some embodiments, the administration minimizes a foreign body reaction in the subject. In some embodiments, the dermal filler system is formulated for administration through a needle. In some embodiments, the dermal filler system is formulated for administration through a microneedle or microneedle patch. In some embodiments, the dermal filler system is formulated for administration through a needle having a gauge of about 26 gauge, 27 gauge, 28 gauge, 29 gauge, or 30 gauge. For example, a dermal filler system formulated for a 27 gauge syringe can be injected in the range of 0.1 to 10 seconds. -1 When measured at shear rates of 0.1 to 10 s, dermal filler systems formulated for 30-gauge syringes may have apparent viscosities of approximately 1,000 to 1,000,000 mPa·s. -1 When measured at a shear rate of 1000-500,000 mPa·s, the properties of the dermal filler system may be fine-tuned depending on the mode of administration.

[0132] In some embodiments, the dose of the dermal filler system comprises a volume of about 0.75 milliliters (mL) to about 1.0 mL. In some embodiments, the volume comprises about 0.5 mL to about 3.0 mL. In some embodiments, the volume comprises 0.75 mL to about 2.75 mL, 1.0 mL to about 2.25 mL, 1.25 mL to about 2.0 mL, 1.0 mL to about 1.75 mL, or 1.25 mL to 1.50 mL. In some embodiments, the dose comprises about 0.50 mL, 0.75 mL, 1.0 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2.0 mL, 2.25 mL, 2.5 mL, 2.75 mL, or 3.0 mL or more. The dose of the dermal filler system may depend on the area of ​​administration. For example, for the mid-face or cheek region, the dosage may be 1.0 mL to 3.0 mL, while for the subcheek region, the dosage may be 0.50 mL to 2.0 mL. In another example, the dosage for the under-eye region may be 0.50 mL to 1.0 mL. The dosage for the chin may be 1.0 mL to 3.0 mL. In some embodiments, the dosage for the lips may be 0.50 mL to 3.0 mL. In some embodiments, the administration region includes any one of the tissue sites disclosed herein, and the dosage comprises about 0.50 mL to about 3.0 mL.

[0133] In some embodiments, the dermal filler system is manufactured aseptically. In some embodiments, the dermal filler system is sterile. In some embodiments, the dermal filler is formulated for sterilization by steam sterilization, filtration, microfiltration, e-beam, gamma irradiation, ethylene oxide (ETO), light, supercritical carbon dioxide, hydrogen peroxide vapor, or any combination thereof. In some embodiments, the dermal filler system comprises at least two separate containers, each suitable for sterilization by a different method. In some embodiments, the microgel particles are freeze-dried. In some embodiments, the freeze-dried microgel particles are stored in a first container that can withstand steam sterilization, which is separate from a second container containing components of the dermal filler system that may be degradable by steam sterilization, such as a therapeutic agent. In another embodiment, the dermal filler system is stored in a single container that can be sterilized together. In some embodiments, the system also comprises a reconstitution medium that reconstitutes the freeze-dried dermal filler system. In some embodiments, the reconstitution medium comprises a physiologically isotonic buffer, such as phosphate-buffered saline. In some embodiments, the reconstitution medium has a pH higher than physiological pH. In some embodiments, the restoration medium has a pH lower than physiological pH. In some embodiments, the restoration medium comprises buffers with various buffer capacities.

[0134] delivery device Disclosed herein in some embodiments is a delivery device configured to deliver a dermal filler system to a subject. Non-limiting examples of a delivery device are needles or microneedles (e.g., microneedle patches). In some embodiments, the delivery device includes a body and an applicator in fluid communication with the body. In some embodiments, the body is elongated (e.g., barrel-shaped). In some embodiments, the body of the delivery device includes an internal chamber containing the dermal filler system. In some embodiments, the delivery device includes a pump or plunger configured to apply pressure to the dermal filler system contained in the body under conditions in which the dermal filler flows through the applicator and exits the applicator via an outlet of the applicator. In some embodiments, the body of the delivery device includes a first chamber for the microgel particles and a second chamber for the annealing agent and / or ingredients. In some embodiments, the delivery device mixes the ingredients of the first and second chambers. In some embodiments, the syringe is pre-filled with the dermal filler system. In some embodiments, the syringe is sterile. In some embodiments, the syringe is packaged separately from the dermal loading system, and both the syringe and the dermal loading system are sterile.

[0135] method In some embodiments, methods of preparing or using the dermal filler systems disclosed herein are disclosed herein. In some embodiments, the methods disclosed herein include delivering the dermal filler systems disclosed herein to a subject. In some embodiments, delivering includes administering the dermal filler system to a subject. In some embodiments, administering includes subdermal, dermal, intradermal, or subcutaneous administration of the dermal filler system to a tissue site in a subject. In some embodiments, administering the dermal filler system to a tissue site in a subject is effective to treat tissue at or around the tissue site, such as, for example, treating cancer in the tissue or improving the cosmetic quality of the tissue (e.g., reducing wrinkles or fine lines, filling the tissue, repairing the tissue, correcting skin irregularities, treating one or more skin lesions). In some embodiments, improving the cosmetic quality of the tissue includes filling at least a portion of the tissue site in a subject. In some embodiments, filling includes forming new tissue within a cellular matrix at the tissue site. In some embodiments, the new tissue has an in vivo survival time at the tissue site similar to the endogenous tissue surrounding the tissue site. In some embodiments, the methods of delivering or administering the dermal filler systems disclosed herein are performed while minimizing a foreign body reaction elicited by the subject in response to delivery or administration. Also provided are methods for purifying the microgel particles of the dermal filler systems disclosed herein, such as in a water-in-oil emulsion. In some embodiments, the methods include lyophilizing the microgel particles, thereby allowing them to be stored and / or distributed for extended periods of time before being reconstituted and delivered to a subject when needed.

[0136] Delivery method Disclosed herein in some embodiments are methods for delivering a dermal filler system provided herein to a subject. In some embodiments, the dermal filler is delivered to a tissue site in the subject. In some embodiments, the method includes delivering the dermal filler system to the tissue site under conditions sufficient to cause the microgel particles to anneal to each other and form a covalently stabilized porous scaffold. In some embodiments, the formation of the covalently stabilized porous scaffold occurs in vivo, in situ, or both in vivo and in situ. In some embodiments, the covalently stabilized porous scaffold is formed under conditions sufficient to grow cells within the pores of the covalently stabilized porous scaffold to generate a cellular matrix. In some aspects, the formed cellular matrix remains in the subject after complete degradation of the covalently stabilized porous scaffold, thereby filling at least a portion of the tissue site in the subject while minimizing a foreign body response in the subject. In some embodiments, filling includes forming new tissue within the cellular matrix at the tissue site. In some embodiments, the new tissue has an in vivo survival time at the tissue site similar to the endogenous tissue surrounding the tissue site. Delivery methods disclosed herein can be subdermal, dermal, intradermal, or subcutaneous. In some embodiments, delivery methods include injection, such as, for example, using a delivery device (e.g., a syringe) disclosed herein.

[0137] In some embodiments, delivering comprises subdermal administration of a dermal filler system to a subject. In some embodiments, delivering comprises dermal administration of a dermal filler system to a subject. In some embodiments, delivering comprises intradermal administration of a dermal filler system to a subject. In some embodiments, delivering comprises subcutaneous administration of a dermal filler system to a subject. In some embodiments, delivering comprises expelling the dermal filler formulation from a syringe or needle. In some embodiments, the needle has a gauge including about 25 gauge to about 35 gauge. In some embodiments, the needle has a gauge including about 27 gauge. In some embodiments, the needle has a gauge including about 30 gauge. In some embodiments, the needle has a gauge including about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 gauge. In some embodiments, delivering comprises exerting an extrusion force of up to 40 Newtons (N) on the dermal filler system. In some embodiments, the delivering comprises applying an extrusion force of up to 12 Newtons (N) to the dermal filler system. In some embodiments, the delivering comprises applying an extrusion force of up to 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 N.

[0138] In some embodiments, the syringe may have an internal volume of about 0.1 mL to about 3 mL. In some embodiments, the internal volume includes about 0.5 mL to about 3.0 mL. In some embodiments, the volume includes 0.75 mL to about 2.75 mL, 1.0 mL to about 2.25 mL, 1.25 mL to about 2.0 mL, 1.0 mL to about 1.75 mL, or 1.25 mL to 1.50 mL. In some embodiments, the syringe contains a dosage including about 0.50 mL, 0.75 mL, 1.0 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2.0 mL, 2.25 mL, 2.5 mL, 2.75 mL, or 3.0 mL or more. The dosage of the dermal filler system may depend on the administration area. In some embodiments, the internal volume is related to the internal diameter of the syringe, which affects the extrusion force required to inject the dermal filler composition. In some embodiments, the internal diameter may be about 4 mm to about 9 mm. In some embodiments, the inner diameter may be about 4.5 mm to about 6.5 mm. In some embodiments, the inner diameter may be about 4.5 mm to about 8.8 mm. In some embodiments, the extrusion force required to deliver the HA composition from the syringe depends on the needle gauge. In some embodiments, the composition is packaged in a 1 mL to about 3 mL syringe and injected using a 20 gauge to about 40 gauge needle.

[0139] In some embodiments, the method comprises separately delivering hydrogel microparticles (e.g., as disclosed herein) and an annealing agent (e.g., as disclosed herein) to a subject. In some embodiments, the method comprises co-delivering hydrogel microparticles (e.g., as disclosed herein) and an annealing agent (e.g., as disclosed herein) to a subject. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 18 months when the hydrogel microparticles and the annealing agent are stored as a mixture in a single container. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 36 months when the hydrogel microparticles and the annealing agent are stored as a mixture in a single container. In some embodiments, the shelf life of the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) as a mixture corresponds to the amount of time (e.g., 18 months or 36 months) that the mixture is stored at room temperature. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 18 months when the hydrogel microparticles and the annealing agent are stored in separate containers. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 36 months when the hydrogel microparticles and the annealing agent are stored in separate containers. In some embodiments, the shelf life of the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) when stored in separate containers corresponds to the amount of time (e.g., 18 months or 36 months) that the mixture is stored at room temperature.

[0140] Disclosed herein are methods comprising administering a dermal filler system described herein to a tissue site in a subject. In some embodiments, the tissue site comprises the mid-face or cheek region of a subject, the subject's cheek, the subject's mandible, or the subject's lips, or any combination thereof. In some embodiments, the mid-face region comprises the facial region including the nose, cheeks, and lips (e.g., the upper lip), and extends posteriorly to the anterior skull base. In some embodiments, in the superior-inferior direction, the mid-face region comprises the soft and bony tissue from the orbit to the oral cavity. In some embodiments, in the lateral direction, the mid-face region extends to the temporal bone. In some embodiments, the cheek region comprises the innermost and superior portions of the maxilla. In some embodiments, the cheek region forms the medial border of the orbit and borders the lateral border of the nasal bridge. In some embodiments, the cheek region comprises the area below the eye and above the mandible. In some embodiments, the cheek region extends between the nose and ear. In some embodiments, the cheek region comprises the soft tissue between the cheekbone and the mandible. In some embodiments, the tissue site comprises the forehead region. In some embodiments, the forehead region comprises the portion of the face above the eyebrows, below the hairline, and between the temples. In some embodiments, the tissue site comprises the lower facial region of the subject. In some embodiments, the lower face comprises the area between the mouth and the lowest point of the chin. In some embodiments, the tissue site comprises the chin of the subject. In some embodiments, the chin comprises the lower portion of the face located below the lower lip and including the central prominence of the jaw. In some embodiments, the tissue site comprises a skin ridge. In some embodiments, the tissue site comprises a nasolabial fold. In some embodiments, the nasolabial fold comprises a line extending from the side of the nose to the edge of the mouth. In some embodiments, the tissue site comprises the perioral line of the subject. In some embodiments, the perioral line comprises the small wrinkles in the skin around the mouth and lips. In some embodiments, the tissue site is on a limb of the subject (e.g., an arm, leg, hand, foot, etc.). In some embodiments, the tissue site is one or more digits (eg, fingers, toes) of the subject. In some embodiments, the tissue site comprises a wound site of the subject.In some embodiments, the wound site comprises a site of a skin abrasion, abrasion, incision, laceration, puncture wound, or a combination thereof. In some embodiments, the wound site comprises a burn site. In some embodiments, the tissue site comprises a scarring site (e.g., a site where a wound, burn, or sore has not completely healed and the subject has developed fibrous connective tissue, leaving a mark on the skin or within bodily tissue). In some embodiments, the scar is a keloid, hypertrophy, contracture, adhesion, or a combination thereof. In some embodiments, the scarring is the result of acne. In some embodiments, the tissue site is a surgical site on a subject. In some embodiments, the tissue site is any location comprising soft tissue. In some embodiments, the tissue site is any location comprising connective tissue. In some embodiments, the tissue site is any location comprising epithelial tissue. In some embodiments, the tissue site is any location comprising muscle tissue. In some embodiments, the tissue site is any location comprising nerve tissue. In some embodiments, the methods include administering a dosage of a dermal filler system to a subject, which may depend on the location and / or tissue at the tissue site and the intended therapeutic or cosmetic effect.

[0141] In some embodiments, the methods disclosed herein include delivering a dermal filler system to a subject under conditions sufficient to cause adjacent microgel particles to anneal to one another and form a stabilized scaffold. In some embodiments, the stabilized scaffold formed is covalently stabilized (e.g., covalent interactions between adjacent microgel particles facilitate annealing). In some embodiments, the stabilized scaffold is porous. In some embodiments, the method includes performing an annealing reaction of the microgel particles to form the covalently stabilized scaffold. In some embodiments, the covalently stabilized scaffold is any of the covalently stabilized scaffolds described herein. In some embodiments, the method includes forming the covalently stabilized scaffold such that pores are formed between the microgel particles of the covalently stabilized scaffold (e.g., the covalently stabilized scaffold is porous).

[0142] In some embodiments, the methods disclosed herein include delivering a dermal filler system to a subject under conditions sufficient for the subject's endogenous cells to infiltrate and grow into the covalently stabilized porous scaffold. In some embodiments, the cells form a cellular matrix within the covalently stabilized porous scaffold. In some embodiments, the covalently stabilized porous scaffold remains at the tissue site for a sufficient time for the cellular matrix to grow into tissue in situ. In some embodiments, the method includes vascularizing, depositing extracellular matrix, or producing proteins and enzymes at the tissue site that aid in healing the tissue site, or any combination thereof. In some embodiments, the method includes forming new tissue from the cellular matrix at the injection site or tissue site. In some embodiments, the new tissue is characterized by having mature vascularization, characteristics of the surrounding tissue at the tissue site, or a combination thereof. In some embodiments, the characteristics of the surrounding tissue at the tissue site include cell types that are functionally differentiated from the surrounding tissue. Non-limiting examples of cell types functionally differentiated from epithelial tissue include squamous cells, cuboidal cells, and columnar cells. Non-limiting examples of cell types functionally differentiated from the dermis include fibroblasts, macrophages, adipocytes, mast cells, Schwann cells, and stem cells. Non-limiting examples of cell types functionally differentiated from the epidermis include keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Non-limiting examples of cell types functionally differentiated from the dermis include fibroblasts, adipocytes, and macrophages. In some embodiments, the dermal filler completely degrades in vivo, while the new tissue formed from the cellular matrix remains. In some embodiments, the new tissue is characterized as having an extracellular matrix. In some embodiments, the dermal filler partially degrades in vivo.

[0143] In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least 25% of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% or more of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 10% to about 50% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 15% to about 45% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 20% to about 40% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 25% to about 35% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 10% to about 90% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 15% to about 90% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 20% to about 85% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least the portion of the tissue site comprising the cell matrix comprises at least about 25% to about 80% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site.In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 30% to about 75% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 35% to about 70% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 40% to about 65% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 45% to about 60% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, at least a portion of the tissue site comprising the cellular matrix comprises at least about 50% to about 55% of the tissue site after degradation of the covalently stabilized scaffold at the tissue site. In some embodiments, the method comprises growing cells within the stabilized porous scaffold about 1 day or less after delivery. In some embodiments, the method includes forming a cellular matrix within the stabilized porous scaffold about 30 days or less after delivery, hi some embodiments, the cellular matrix begins to form within the scaffold within 7 days after administration.

[0144] In some embodiments, the methods of delivering a dermal filler system disclosed herein minimize a foreign body response elicited by a subject in response to the dermal filler system. In some embodiments, the foreign body response is characterized by chronic inflammation. In some embodiments, the foreign body response is characterized by granuloma formation. In some embodiments, the foreign body response is characterized by scar tissue formation. In some embodiments, the foreign body response is characterized by nodule formation. In some embodiments, the foreign body response is characterized by swelling, pain, or any combination thereof. In some embodiments, the chronic inflammation, granuloma formation, nodule formation, swelling, pain, or any combination thereof is localized at or near the tissue site. In some embodiments, the foreign body response occurs at a location other than the tissue site. In some embodiments, the foreign body response is characterized by the presence of multinucleated giant cells (MNGCs) (e.g., fusions of monocytes or macrophages) at the tissue site of the subject. In some embodiments, the foreign body response is characterized by the persistence of MNGCs over time. In some embodiments, the period of time comprises about 1, 2, 3, or 4 weeks or more. In some embodiments, the period of time comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. In some embodiments, the period of time comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years or more. In some embodiments, minimizing the foreign body response is characterized as avoiding any formation of MNGCs at the tissue site. In some embodiments, minimizing the foreign body response is characterized by the absence of MNGCs at the tissue site after a period of time following delivery of the dermal filler system. In some embodiments, the period of time after delivery of the dermal filler system includes 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or less. In some embodiments, the period of time after delivery of the dermal filler system includes 1 to 30 days. In some embodiments, the period of time after delivery of the dermal filler system includes 1 to 29 days.In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 28 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 27 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 26 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 25 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 24 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 23 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 22 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 21 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 20 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 19 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 18 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 17 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 16 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 15 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 14 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 13 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 12 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 11 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 10 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 9 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 8 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 7 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 6 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 5 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 4 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 3 days. In some embodiments, the period of time after delivery of the dermal filler system comprises 1 to 2 days.

[0145] In some embodiments, chronic inflammation may be characterized as slow, long-term inflammation that persists over an extended period of time following delivery or administration of a dermal filler system. In some embodiments, chronic inflammation may persist for a period of months to years. In some embodiments, as discussed above, an intentional foreign body reaction caused by administration of a biostimulator results in chronic inflammation at the tissue site. In some embodiments, the methods of delivering a dermal filler system described herein minimize such chronic inflammation while filling the tissue site with new tissue. In some embodiments, filling includes forming new tissue within a cellular matrix at the tissue site. In some embodiments, the new tissue has an in vivo persistence time at the tissue site similar to the endogenous tissue surrounding the tissue site. In some embodiments, a granuloma may be a small area of ​​inflammation at or around a subject's tissue site. In some embodiments, a granuloma may be a small area / cluster of leukocytes and other tissue in an inflamed area. In some embodiments, an intentional foreign body reaction caused by administration of a biostimulator results in granuloma formation at or around the tissue site, as discussed above. In some embodiments, the methods of delivering the dermal filler systems described herein minimize such granuloma formation while filling the tissue site with new tissue. In some embodiments, scar tissue is characterized by fibrous tissue having a composition that is stiffer and more brittle than normal tissue. In some embodiments, as discussed above, an intentional foreign body reaction caused by administration of a biostimulatory agent results in scar tissue formation at the tissue site. In some embodiments, the methods of delivering the dermal filler systems described herein minimize such scar tissue formation while filling the tissue site with new tissue. In some embodiments, the dermal filler systems disclosed herein minimize such scar tissue formation because the new tissue formed within at least the covalently stabilized scaffold mimics the tissue at or surrounding the tissue site.For example, type I collagen deposited in a cellular matrix within a covalently stabilized scaffold (forming the foundation for new tissue) is less abundant than scar tissue, and type III collagen is more abundant than scar tissue. In some embodiments, nodules can be sites of abnormal tissue growth. In some embodiments, as discussed above, an intentional foreign body reaction caused by administration of a biostimulatory agent results in nodule formation at the tissue site. In some embodiments, the methods of delivering a dermal filler system described herein minimize such nodule formation while filling the tissue site with new tissue. In some embodiments, the foreign body reaction is measured by using histological analysis to detect the amount of granulomas at the tissue site and comparing the amount of granulomas at the tissue site to reference tissue not containing the dermal filler system. In some embodiments, the foreign body reaction is measured by using histological analysis to detect the amount of scar tissue at the tissue site and comparing the amount of scar tissue at the tissue site to reference tissue not containing the dermal filler system. In some embodiments, the foreign body response is measured by using histological analysis to detect the amount of nodules at the tissue site and comparing the amount of nodules at the tissue site with reference tissue that does not contain a dermal filler system. In some embodiments, the foreign body response is measured by using histological analysis to detect chronic inflammation at the tissue site. In some embodiments, the foreign body response is measured by using histological analysis to detect the amount of multinucleated giant cells (MNGCs) (e.g., fusions of monocytes or macrophages) present at the tissue site and comparing the amount of MNGCs at the tissue site with reference tissue that does not contain a dermal filler formulation.

[0146] In some embodiments, the method of delivering a dermal filler system disclosed herein is under conditions sufficient to deposit an amount or type of collagen in a cellular matrix at a tissue site that mimics endogenous tissue at or around the tissue site. In some embodiments, the method includes depositing an amount or type of collagen in a cellular matrix at or around the tissue site that mimics endogenous tissue. In some embodiments, the cellular matrix includes an amount or type of collagen that mimics endogenous tissue at the tissue site. In some embodiments, the type of collagen includes type I collagen, type III collagen, or a combination thereof. In some embodiments, type I collagen is present in a ratio of about 10:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 6:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 5:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 1:1 to about 10:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 1.5:1 to about 9.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 2:1 to about 9:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 2.5:1 to about 8.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 3:1 to about 8:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 3.5:1 to about 7.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 4:1 to about 7:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 4.5:1 to about 6.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 5:1 to about 6:1 with type III collagen. Type I collagen may be an indicator of scar tissue or a foreign body reaction occurring in a subject. In some embodiments, the lower the ratio of type I collagen to type I collagen, the greater the suppression of the foreign body reaction.In some embodiments, the method includes minimizing the ratio of type I collagen to type III collagen so that new tissue is constructed within the subject, thereby creating new tissue with the characteristics disclosed herein while avoiding the hazards disclosed herein. For example, the method includes filling at least a portion of the tissue site while minimizing or completely avoiding a foreign body reaction in response to the biostimulant. In some embodiments, the filling includes forming new tissue within a cellular matrix at the tissue site. In some embodiments, the new tissue has an in vivo survival time at the tissue site similar to the endogenous tissue surrounding the tissue site.

[0147] In some embodiments, the methods of delivering the dermal filler systems disclosed herein are under conditions sufficient to form elastin at the tissue site. In some embodiments, the methods include forming elastin at the tissue site. In some embodiments, the elastin remains at or near the tissue site after complete degradation of the covalently stabilized scaffold. The presence of elastin may indicate the absence of scar or scar tissue at the tissue site and, therefore, may be indicative of a minimized foreign body reaction.

[0148] In some embodiments, the dermal filler formulation is biocompatible with the tissue of the tissue site as determined by one or more techniques set forth in ISO Standard 10993, the contents of which relating to these techniques are incorporated herein by reference in their entirety.

[0149] Treatment method In some embodiments, the method further includes treating a tissue site in the subject by delivering a dermal filler system to the tissue site. In some embodiments, treating the tissue site includes improving the cosmetic quality of the tissue at the tissue site. In some embodiments, improving the cosmetic quality of the tissue includes tissue filling, skin filling, wrinkle removal, tissue repair, or correction of skin irregularities. In some embodiments, treating the tissue site includes treating one or more diseases or disorders of the tissue, such as, for example, skin cancer.

[0150] skin cancer In some embodiments, the method includes treating cancer. In some embodiments, the cancer includes skin cancer. In some embodiments, the skin cancer includes basal cell carcinoma, cutaneous squamous cell carcinoma, melanoma, and Merkel cell carcinoma. In some embodiments, the method includes delivering an anti-cancer therapeutic agent to the subject. In some embodiments, the dermal filler system includes an anti-cancer therapeutic agent. In some embodiments, the microgel particles of the dermal filler system include an anti-cancer therapeutic agent (e.g., drug-eluting microgel particles). In some embodiments, the microgel particles elute the anti-cancer therapeutic agent in vivo at the tissue site. In some embodiments, the dermal filler system is formulated with an anti-cancer therapeutic agent. In some embodiments, the anti-cancer therapeutic agent includes or is an anti-cancer therapeutic agent. In some embodiments, the anti-cancer therapeutic agent includes or is a biologic.In some embodiments, the anticancer therapeutic agent is cisplatin, 5-fluorouracil (5-FU), Aldara (imiquimod), cemiplimab-rwlc, Efudex (fluorouracil-topical), Erivedge (vismodegib), 5-FU (fluorouracil-topical), fluorouracil-topical, imiquimod, Libtayo (cemiplimab-rwlc), Odomzo (sonidegib), sonidegib, vismodegib, cemiplimab-rwlc, Keytruda (pembrolizumab), Libtayo, pembrolizumab, aldesleukin, binimetinib, Braftovi (encorafenib), cobimetinib, Cotellic (cobimetinib), dabrafenib, dacarbazine, DTIC-Dome (dacarbazine), encorafenib, IL-2 (aldesleukin), Imlygic (talimogene laherparepvec), interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), ipilimumab, Keytruda (pembrolizumab), Kimmtrak (Tebentafusp-tebn), Mekinist (trametinib dimethyl sulfoxide), Mektovi (binimetinib), nivolumab, nivolumab and leralimab-rmbw, Opdivo (nivolumab), Opdualag (nivolumab and leralimab-rmbw), peginterferon alfa-2b, pembrolizumab These include izumab, Proleukin (aldesleukin), recombinant interferon alfa-2b, Sylatron (peginterferon alfa-2b), Tafinlar (dabrafenib), talimogen, laherparepvec, laventafusp-tebn, trametinib dimethyl sulfoxide, vemurafenib, Yervoy (ipilimumab), Zelboraf (vemurafenib), avelumab, Bavencio (avelumab), or a combination thereof.

[0151] Disclosed herein are methods of treating skin cancer (e.g., basal cell carcinoma) in a subject, comprising administering to the subject one or more anticancer therapeutic agents and a microgel system. In some embodiments, the anticancer therapeutic agent comprises Aldara (imiquimod), cemiplimab-rwlc, Efudex (topical fluorouracil), Erivedge (vismodegib), 5-FU (topical fluorouracil), topical fluorouracil, imiquimod, Libtayo (cemiplimab-rwlc), Odomzo (sonidegib), sonidegib, vismodegib, or a combination thereof. In some embodiments, the method comprises treating skin cancer (e.g., cutaneous squamous cell carcinoma) by administering one or more anticancer therapeutic agents comprising cemiplimab-rwlc, Keytruda (pembrolizumab), Libtayo, pembrolizumab, or a combination thereof.In some embodiments, the method includes the use of aldesleukin, binimetinib, Braftovi (encorafenib), cobimetinib, Cotellic (cobimetinib), dabrafenib, dacarbazine, DTIC-Dome (dacarbazine), encorafenib, IL-2 (aldesleukin), Imlygic (talimogene laherparepvec), interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), ipilimumab, Keytruda (pembrolizumab), Kimmtrak (tebentafusp-tebn), Mekinist (trametinib dimethyl sulfoxide), Mektovi (binimetinib), nivolumab, nivolumab and leratolimab-rmbw, Opdivo (nivolumab), Opdualag (nivolumab and leratolimab-rmbw), peginterferon alfa-2b, pembrolizumab, Proleukin (aldesleukin), recombinant interferon alfa-2b, Sylatron (pegylated interferon alfa-2b), Tafinlar (dabrafenib), talimogene laherparepvec, tebentafsp-tebn, trametinib dimethyl sulfoxide, vemurafenib, Yervoy (ipilimumab), Zelboraf (vemurafenib), or combinations thereof. In some embodiments, the method includes treating skin cancer (e.g., Merkel cell carcinoma) by administering one or more anticancer therapeutic agents including avelumab, Bavencio (avelumab), Keytruda (pembrolizumab), pembrolizumab, or combinations thereof.

[0152] beauty treatment Disclosed herein are methods for treating a tissue site in a subject to improve the cosmetic quality of the tissue at or around the tissue site. In some embodiments, the method includes administering a dosage of a dermal filler system disclosed herein to the subject. In some embodiments, administering includes injecting the dermal filler system into the tissue site of the subject. In some embodiments, treating includes reducing wrinkles, filling fine lines in the skin, augmenting subdermal tissue, or any combination thereof. In some embodiments, treating includes filling moderate to severe facial wrinkles and skin ridges, such as nasolabial folds (e.g., lines extending from the sides of the nose to the edges of the mouth) and perioral lines (e.g., small wrinkles in the skin around the mouth and lips). In some embodiments, treating includes filling the lips, cheeks, chin, back of the hands, or a combination thereof. In some embodiments, treating includes restoring and correcting signs of facial fat loss (lipoatrophy) in people with human immunodeficiency virus (HIV). In some embodiments, treating includes correcting contour defects, such as wrinkles and acne scars. In some embodiments, the one or more skin lesions comprise acne scars, basal cell carcinoma, cellulitis, epidermolysis bullosa, melanoma, Merkel cell carcinoma, scars, skin biopsies, skin cancer, squamous cell carcinoma, stretch marks, or any combination thereof. For tissue or dermal filler applications for volume loss associated with aging, lipoatrophy, lipodystrophy, skin scarring, or shallow or deep wrinkles, direct injection of microgel particles into the dermis via a needle or cannula may be used to improve tissue contour, tissue loss, or tissue displacement.

[0153] Vocal cord augmentation Disclosed herein are methods for augmenting vocal cords. In some embodiments, the methods include administering a dosage of a dermal filler system disclosed herein to a subject. In some embodiments, administering includes injecting the dermal filler system into the vocal cords of the subject. In some embodiments, administering includes injecting the dermal filler system into the superficial lamina propria of the vocal cords or the vocal cord epithelium. In some embodiments, treating includes restoring the subject's voice, restoring flexibility to the superficial lamina propria, or reducing hoarseness in the subject's voice. In some embodiments, the subject has scarred and / or stiff vocal cords.

[0154] Combination therapy In some embodiments, the method includes administering to the subject one or more additional agents (e.g., therapeutic agents), such as a local anesthetic (e.g., lidocaine), an analgesic, an anti-inflammatory agent, an anti-cancer therapeutic agent, or others that can provide a therapeutic or cosmetic benefit to the administration site. In some embodiments, the microgel particles include one or more additional agents (e.g., drug-eluting microgel particles). In some embodiments, the microgel particles elute one or more active agents in situ. In some embodiments, the dermal filler system is formulated with one or more active agents. In some embodiments, the dermal filler system is not formulated with one or more active agents, and the one or more active agents are administered separately from the dermal filler system. In some embodiments, the dermal filler system and the one or more additional active agents are administered to the subject sequentially. In some embodiments, the dermal filler system and the one or more additional active agents are administered to the subject substantially simultaneously.

[0155] In some embodiments, the therapeutic agent comprises an analgesic, a local anesthetic, an anti-inflammatory, an anti-fibrotic, or an antibiotic. In some embodiments, the local anesthetic is an ester type. In some embodiments, the ester type local anesthetic comprises benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is an amide type. In some embodiments, the amide type local anesthetic comprises articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, or cinchocaine, or any combination thereof. In some embodiments, the local anesthetic is or comprises lidocaine. In some embodiments, the local anesthetic consists of lidocaine. In some embodiments, the analgesic comprises codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID) or a steroid. In some embodiments, the NSAID comprises ibuprofen or naproxen. In some embodiments, the steroid comprises a corticosteroid. In some embodiments, the antibiotic comprises dicloxacillin, erythromycin, or tetracycline. In some embodiments, the anti-fibrotic agent comprises pentoxifylline.

[0156] In some embodiments, the method comprises administering lidocaine at a concentration comprising about 1.0 milligrams per microliter (mg / mL) to about 5.0 mg / mL. In some embodiments, the method comprises administering lidocaine at a concentration comprising about 3.0 mg / mL. In some embodiments, the local anesthetic consists of lidocaine. In some embodiments, the method comprises administering lidocaine at a concentration of about 1.0 mg / mL to about 5.0 mg / mL. In some embodiments, the method comprises administering lidocaine at a concentration of about 3.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 0.5 mg / mL to about 20.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 1.0 mg / mL to about 19.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 1.5 mg / mL to about 19.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 2.0 mg / mL to about 18.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 2.5 mg / mL to about 18.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 3.0 mg / mL to about 17.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 3.5 mg / mL to about 17.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 4.0 mg / mL to about 16.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 4.5 mg / mL to about 16.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 5.0 mg / mL to about 15.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 5.5 mg / mL to about 15.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 6.0 mg / mL to about 14.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 6.5 mg / mL to about 14.0 mg / mL.In some embodiments, the method comprises administering the local anesthetic at a concentration of about 7.0 mg / mL to about 13.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 7.5 mg / mL to about 13.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 8.0 mg / mL to about 12.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 8.5 mg / mL to about 12.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 9.0 mg / mL to about 11.5 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 9.5 mg / mL to about 11.0 mg / mL. In some embodiments, the method comprises administering the local anesthetic at a concentration of about 9.5 mg / mL to about 10.5 mg / mL.

[0157] Method for Producing a Dermal Filler System In some embodiments, disclosed herein are methods for producing the dermal filler systems and components thereof disclosed herein. In some embodiments, the methods include synthesizing microgel particles from raw materials. In some embodiments, the methods include fine-tuning the mechanical properties of the microgel particles, dermal filler system, or the resulting covalently stabilized porous scaffold. In some embodiments, the methods include purifying the microgel particles. In some embodiments, the methods include formulating the microgel particles into a dermal filler system or formulation. In some embodiments, the methods further include sterilizing the dermal filler system or formulation.

[0158] Synthesis of microgel particles Disclosed herein are methods for producing the microgel particles disclosed herein, comprising combining raw materials (e.g., polymers, functional groups, peptides, etc.) under conditions sufficient to form individual microgel particles. In some embodiments, the conditions sufficient to form microgel particles may include an aqueous buffer solution having a pH ranging from 7 to 9. As non-limiting examples, the buffer solution may be phosphate-buffered saline (PBS), HEPES, or triethanolamine (TEOA). In some embodiments, the reaction may be quenched by adding an acid or base to terminate the reaction after a specific time after mixing to create a water-in-oil emulsion. A quenching molecule may be added to the oil phase and diffuse into the aqueous phase to terminate the reaction occurring in the aqueous phase. In some embodiments, the reaction may be quenched by adding a maleimide to react with any remaining thiols. In some embodiments, the reaction may be quenched by adding an oxidizing agent to oxidize the thiols.

[0159] In some embodiments, microgel particles may be synthesized using a microfluidic device (e.g., one particle per channel). In some embodiments, microgel particles may be synthesized by water-in-oil emulsion, as described in more detail herein. In some embodiments, microgel particles may be synthesized by water-in-oil emulsion using mechanical agitation. In some embodiments, microgel particles may be synthesized by water-in-oil emulsion using a static mixer. In some embodiments, microgel particles may be synthesized using in-line flow-through synthesis. In some embodiments, microgel particles may be synthesized using parallel production methods (channel-by-channel or multiple particles per parallel channel).

[0160] In some embodiments, the method includes synthesizing microgel particles by a water-in-oil emulsion process. In some embodiments, the method begins with obtaining an oil or a mixture of oils. Non-limiting examples of the oil include light mineral oil (LMO), heavy mineral oil (HMO), or a fluorinated oil. In some embodiments, the mixture of oils includes a surfactant. In some embodiments, different surfactants can be used. In some embodiments, the surfactant can be a non-ionic surfactant. Non-limiting examples of non-ionic surfactants are Span 80, Span 20, Tween 20, Tween 40, Tween 60, Tween 80, and tocopheryl polyethylene glycol 1000 succinate (TPGS). In some embodiments, the surfactant can be an anionic surfactant. In some embodiments, the surfactant can be a fluorinated surfactant. Non-limiting examples of anionic surfactants are sodium dodecyl sulfate (SDS), sodium lauryl ether sulfate (SLES), and perfluorooctane sulfonic acid. In some embodiments, the surfactant can be a cationic surfactant. Non-limiting examples of cationic surfactants are cetyltrimethylammonium bromide (CTAB) and hexadecylpyridinium bromide. In some embodiments, the surfactant may be an amphoteric surfactant. Non-limiting examples of amphoteric surfactants are citrate betaine, sodium lauryl betaine, and (carboxymethyl)dimethyloleylammonium hydroxide. In some embodiments, the concentration of the surfactant may vary from 0.01 to 5% v / v.

[0161] In some embodiments, the method comprises adding a surfactant to the oil. In some embodiments, the method comprises adding a surfactant to the oil before adding the aqueous solution / mixture to the oil. In some embodiments, the method comprises adding a surfactant to the aqueous solution / mixture described herein. In some embodiments, having a surfactant in the aqueous phase is beneficial because if the surfactant has high water solubility, it is easier to remove during purification.

[0162] In some embodiments, the oil or oil mixture may be added to the bioreactor vessel through a micron filter and stirred. In some embodiments, the bioreactor vessel contains a volume of about 100 milliliters to about 1 liter. In some embodiments, the bioreactor vessel contains a volume of about 1 liter to about 10 liters. In some embodiments, the bioreactor vessel contains a volume of about 10 liters to about 100 liters. In some embodiments, the bioreactor vessel contains a volume of about 100 liters to about 1000 liters. In some embodiments, the bioreactor vessel contains a volume of about 100 liters to about 10,000 liters. In some embodiments, the bioreactor vessel contains a volume of about 10 liters to about 1000 liters. In some embodiments, the bioreactor vessel contains a volume of about 1000 liters to about 10,000 liters. In some embodiments, the micron filter has a pore size of about 0.1 μm to about 1 μm. In some embodiments, the micron filter has a pore size of about 0.2 μm.

[0163] In some embodiments, the oil or oil mixture may be added to the static mixer through a micron filter and stirred. In some embodiments, the static mixer contains a volume of about 100 milliliters to about 1 liter. In some embodiments, the static mixer contains a volume of about 1 liter to about 10 liters. In some embodiments, the static mixer contains a volume of about 10 liters to about 100 liters. In some embodiments, the static mixer contains a volume of about 100 liters to about 1000 liters. In some embodiments, the static mixer contains a volume of about 100 liters to about 10,000 liters. In some embodiments, the static mixer contains a volume of about 10 liters to about 1000 liters. In some embodiments, the static mixer contains a volume of about 1000 liters to about 10,000 liters. In some embodiments, the micron filter has a pore size of about 0.1 μm to about 1 μm. In some embodiments, the micron filter has a pore size of about 0.2 μm.

[0164] In some embodiments, a method of synthesizing microgel particles includes providing (e.g., in solution) one or more polymers as disclosed herein. In some embodiments, the one or more polymers include PEG and HA. In some embodiments, the PEG and HA are provided at molecular weights as disclosed herein.

[0165] In some embodiments, the method for synthesizing microgel particles includes modifying one or more polymers disclosed herein by attaching one or more functional groups. In some embodiments, PEG and HA are modified by attaching thiol and vinyl sulfone functional groups. In some embodiments, HA is modified with thiol and PEG is modified with vinyl sulfone (VS).

[0166] In some embodiments, a method for synthesizing microgel particles includes mixing one or more modified polymers in a solution. In some embodiments, thiolated HA is mixed with PEG-VS. In some embodiments, the functional groups react to form a hydrogel (e.g., a hydrogel mesh). In some embodiments, the functional groups react via a Michael addition reaction (e.g., a thiol-ene Michael addition reaction). In some embodiments, the method may include filtering the solution. In some embodiments, the solution may include a peptide (e.g., a cell adhesive peptide as disclosed herein). In some embodiments, the solution may include a buffer or buffering agent. In some embodiments, the solution may include a base catalyst.

[0167] In some embodiments, methods for synthesizing microgel particles include those disclosed in US Pat. No. 10,668,185, which is incorporated herein by reference in its entirety.

[0168] Fine-tuning of mechanical properties In some embodiments, disclosed herein are methods for adjusting the physical characteristics of microgel particles, covalently stabilized scaffolds, dermal filler systems, or any combination thereof. In some embodiments, the manner in which the physical characteristics are adjusted can depend on the mode of delivery, the tissue type and location at the tissue site, the desired therapeutic or cosmetic outcome, and the subject. In some embodiments, the physical characteristics may be altered depending on the tissue site, the mode of administration, the desired biocompatibility, or any combination thereof. In some embodiments, the physical characteristics are the mechanical characteristics of the microgel particles, covalently stabilized scaffolds, dermal filler systems, or any combination thereof.

[0169] In some embodiments, the methods comprise adjusting the viscosity of the hydrogel, the degradation rate of the covalently stabilized scaffold, the volume fraction of the microgel particles, the pH of the microgel particles, the pH of the annealing agent solution, the pH of the covalently stabilized scaffold, the degree of substitution of the polymer, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percent of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, an additional agent (e.g., a therapeutic agent), or a combination thereof. In some embodiments, the methods comprise decreasing the viscosity of the hydrogel, the degradation rate of the covalently stabilized scaffold, the volume fraction of the microgel particles, the pH of the microgel particles, the pH of the annealing agent solution, the pH of the covalently stabilized scaffold, the degree of substitution of the polymer, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percent of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, an additional agent (e.g., a therapeutic agent), or a combination thereof. In some embodiments, the method comprises increasing the viscosity of the hydrogel, the degradation rate of the covalently stabilized scaffold, the volume fraction of the microgel particles, the pH of the microgel particles, the pH of the annealing agent, the pH of the covalently stabilized scaffold, the degree of substitution of the polymer, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percent of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, an additional agent (e.g., a therapeutic agent), or a combination thereof.

[0170] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the viscosity of the hydrogel or the compressive modulus of the covalently stabilized scaffold. In some embodiments, the method includes adjusting the volume fraction of microgel particles. In some embodiments, adjusting the volume fraction of microgel particles includes adjusting the percent concentration of microgel particles in the hydrogel. As shown in Figures 3A-3C and 4A-4D, increasing the volume fraction of microgel particles can lead to a higher viscosity of the suspension of microgel particle hydrogel that is annealed to form the covalently stabilized scaffold. As shown in Figures 3A-3C and 4A-4D, decreasing the volume fraction of microgel particles can lead to a lower viscosity of the hydrogel that is annealed to form the covalently stabilized scaffold. In some embodiments, the method includes adjusting the volume fraction of microgel particles in the range of 0.75 mL / mL to 0.95 mL / mL to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa·s. Increasing the volume fraction of microgel particles can lead to a higher compressive modulus of the covalently stabilized scaffold, as shown in Figure 5. Decreasing the volume fraction of microgel particles can lead to a lower compressive modulus of the covalently stabilized scaffold, as shown in Figure 5. In some embodiments, the method comprises adjusting the volume fraction of microgel particles in the range of 0.75 mL / mL to 0.95 mL / mL to achieve a compressive modulus of the covalently stabilized scaffold of about 1,000 Pa to about 17,000 Pa.

[0171] In some embodiments, the method includes adjusting the elastic modulus of the microgel particles. In some embodiments, adjusting the elastic modulus is achieved by adjusting the molar ratio of the crosslinker, polymer (e.g., copolymer), or a combination thereof. As shown in Figures 3A-3C and 4A-4D, increasing the elastic modulus of the microgel particles can lead to a higher viscosity of the hydrogel that is annealed into a covalently stabilized scaffold. As shown in Figures 3A-3C and 4A-4D, decreasing the elastic modulus of the microgel particles can lead to a lower viscosity of the hydrogel that is annealed into a covalently stabilized scaffold. In some embodiments, the method includes adjusting the elastic modulus of the microgel particles in the range of 15 kPa to 46 kPa to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa·s. As shown in Figure 5, increasing the elastic modulus of the microgel particles can lead to a higher compressive modulus of the covalently stabilized scaffold. Decreasing the elastic modulus of the microgel particles can lead to a lower compressive modulus of the covalently stabilized scaffold, as shown in Figure 5. In some embodiments, the method comprises adjusting the elastic modulus of the microgel particles in the range of 15 kPa to 46 kPa to achieve a compressive modulus of the covalently stabilized scaffold of about 1,000 Pa to about 17,000 Pa.

[0172] In some embodiments, the method comprises adjusting (e.g., increasing or decreasing) the volume fraction of the microgel particles from 0.75 mL / mL to 0.95 mL / mL and adjusting the elastic modulus of the microgel particles from 15 kPa to 46 kPa to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa·s. In some embodiments, the method comprises adjusting the volume fraction of the microgel particles from 0.75 mL / mL to 0.95 mL / mL and adjusting the elastic modulus of the microgel particles from 15 kPa to 46 kPa to achieve a compressive modulus of the covalently stabilized scaffold from about 1,000 Pa to about 17,000 Pa.

[0173] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the degradation rate of the covalently stabilized scaffold. In some embodiments, the degradation rate is altered depending on how long the covalently stabilized scaffold is desired to remain at the tissue site. In some embodiments, the method includes altering the degradation pathway, altering the polymer (e.g., copolymer) used to construct the microgel particles, or a combination thereof, to alter the degradation rate. In some embodiments, the method includes altering the degradation pathway to one or more of oxidative degradation, enzymatic degradation, or hydrolysis. In some embodiments, the method includes synthesizing the microgel particles with PEG to decrease the degradation rate of the covalently stabilized scaffold. In some embodiments, the method includes synthesizing the microgel particles without PEG to increase the degradation rate.

[0174] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the degree of substitution of the polymer. In some embodiments, adjusting the degree of substitution is achieved by increasing or decreasing the amount of functional groups that become attached to the microgel particle. In some embodiments, adjusting the molecular weight of the polymer (e.g., copolymer) of the microgel particle alters the degree of substitution, as shown in Table 3 below. In some embodiments, the method includes measuring the degree of substitution using an Ellman's assay.

[0175] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the compressive modulus of the covalently stabilized scaffold. In some embodiments, the compressive modulus of the covalently stabilized scaffold can be changed by adjusting the concentration of functional groups (e.g., thiol and vinyl sulfone) contained in the microgel particles. In some embodiments, the concentration of functional groups (e.g., thiol and vinyl sulfone) contained in the microgel particles can be changed by adjusting the molecular weight of the polymer(s) (e.g., copolymer(s)) of the microgel particles. As shown in Figures 19A-19B, increasing the concentration of functional groups (e.g., thiolated hyaluronic acid (SH-HA)) can increase the compressive modulus of the covalently stabilized scaffold. As shown in Figures 19A-19B, decreasing the concentration of functional groups (e.g., thiolated hyaluronic acid (SH-HA)) can decrease the compressive modulus of the covalently stabilized scaffold. 19A-19B, increasing the molecular weight of the polymer(s) (e.g., hyaluronic acid) can increase the compressive modulus of the covalently stabilized scaffold, and decreasing the molecular weight of the polymer(s) (e.g., hyaluronic acid) can decrease the compressive modulus of the covalently stabilized scaffold. In some embodiments, the method includes adjusting the concentration of functional groups (e.g., thiol and vinyl sulfone) in the gelling solution to a range of about 10 mg / mL to about 45 mg / mL to achieve a compressive modulus of the covalently stabilized scaffold of about 100 Pa to about 140,000 Pa.

[0176] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the rate at which the covalently stabilized scaffold anneals. In some embodiments, the rate at which the covalently stabilized scaffold anneals can be altered by changing the annealing agent. In some embodiments, the rate at which the covalently stabilized scaffold anneals can be altered by adjusting the molecular weight of the annealing agent. As shown in FIG. 21, PEG-dithiol (PEG(SH)2), 4-arm PEG-SH, and PETMA can be used to achieve a covalently stabilized scaffold compressive modulus of about 4,000 Pa to about 7,000 Pa after about 60 minutes of annealing. In some embodiments, the method includes adjusting the pH of the annealing agent to alter the rate at which the covalently stabilized scaffold anneals. As shown in FIG. 22, increasing the pH of the annealing agent can increase the rate at which the covalently stabilized scaffold anneals, and decreasing the pH of the annealing agent can decrease the rate at which the covalently stabilized scaffold anneals. In some embodiments, the method includes using an annealing pH of 6.5 or less to delay the initiation of the annealing reaction for 30 minutes or more. In some embodiments, the method includes delivering a therapeutic agent (e.g., a local anesthetic (e.g., lidocaine)) to modify the rate at which the covalently stabilized scaffold anneals. As shown in Figures 24A-24C (each of Figures 24A, 24B, and 24C shows the results for a different dermal filler system as described herein), the local anesthetic can decrease the rate at which the covalently stabilized scaffold anneals and can function in dermal filler systems having an elastic modulus ranging from about 1,500 Pa to about 18,000 Pa. In some embodiments, the method includes delivering a therapeutic agent (e.g., a local anesthetic (e.g., lidocaine)) to achieve a covalently stabilized scaffold elastic modulus of about 2,000 Pa to about 12,000 Pa after about 250 minutes of annealing.

[0177] Purification of microgel particles In some embodiments, the method includes purifying the microgel particles. In some embodiments, the method includes simultaneously synthesizing and purifying the microgel particles. In some embodiments, the method includes purifying the microgel particles after synthesizing the microgel particles. In some embodiments, purifying the microgel particles includes performing membrane separation of the microgel particles from undesired components. In some embodiments, different types of filtration membranes may be used (e.g., hollow fiber membranes with different pore sizes, different lumen IDs, dialysis membranes, or flat membranes). In some embodiments, membrane separation includes tangential flow filtration (TFF). In some embodiments, membrane separation includes ultrafiltration-diafiltration (UFDF). In some embodiments, membrane separation includes microfiltration-diafiltration (MFDF). In some embodiments, membrane separation includes hollow fiber diafiltration (HFDF). TFF generally includes membrane filtration and separation techniques. TFF may be used herein to purify and concentrate microgel particles. TFF may include generating a feed stream of a solution of microgel particles that passes parallel to the membrane plane. In some embodiments, a portion of the solution passes through the membrane (the permeate), while the remainder (the retentate) is recycled back to the feed reservoir. This system is sometimes called diafiltration. This system allows molecules (in the permeate) smaller than the pores of the membrane to migrate toward and pass through the membrane, while larger molecules, such as microgel particles, remain in the retentate. In some embodiments, the flow in the filtration system may be controlled by a peristaltic pump. In some embodiments, the flow in the filtration system may be controlled by a Quattroflow pump or any positive displacement pump. In some embodiments, the filtration system may be closed to the ambient environment. In some embodiments, the filtration system may be open to the ambient environment.

[0178] In some embodiments, the purification method may include removing excess oil from the microgel particles. In some embodiments, the purification method may include dispersing the particles in an alcohol solution. In some embodiments, the alcohol solution includes alcohol and water, and the alcohol is present in the solution in a ratio of about 0.8:1 or greater. In some embodiments, the alcohol solution includes alcohol and water, and the alcohol is present in the solution in a ratio of about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1 or greater. In some embodiments, the alcohol solution includes alcohol and water, and the alcohol is present in the solution in a ratio of about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1 or less. In some embodiments, the alcohol solution includes alcohol and water, and the alcohol is present in the solution in a ratio of about 0.5:1 to about 1:1. In some embodiments, the alcohol solution includes alcohol and water, and the alcohol is present in the solution in a ratio of about 0.6:1 to about 0.9:1. In some embodiments, the alcohol solution comprises alcohol and water, with the alcohol being present in the solution in a ratio of about 0.7:1 to about 0.8:1. In some embodiments, the purification method may include removing excess water-immiscible oil and surfactant while dispersing and fully swelling the particles (composed primarily of water) and ensuring no particle agglomeration. In some embodiments, the purification method may include slowly transferring the particles into an aqueous buffer while preventing surfactant precipitation. In some embodiments, the transfer rate is related to the flux of filtrate through the membrane and is about 1 to about 1000 LMH (liters per cubic meter). 2 / h). In some embodiments, the migration may occur at a rate of about 100 to about 500 LMH. In some embodiments, the migration may occur at a rate of about 200 to about 300 LMH. This transition rate may be particularly important to ensure that the surfactant does not precipitate on (and within) the microgel particles, rendering the particles unsuitable for microporous scaffolding. In some embodiments, the transition rate may achieve at least one of: (i) swelling of the particle-hydrogel mesh, which is a product of the affinity of a given hydrogel polymer backbone / crosslinker system for a particular solvent, and (ii) solubility of the surfactant in the continuous phase external to the particles.

[0179] In some embodiments, the method includes concentrating microgel particles in a solution or suspension. In some embodiments, the method includes pumping the microgel particles through a membrane filtration system while removing a volume of a continuous phase, and continuing to concentrate the microgel particles at a controlled membrane flux and maintaining a wall shear stress within the membrane filtration system. In some embodiments, the membrane filtration system is selected from tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), or hollow fiber diafiltration (HFDF). In some embodiments, the membrane flux is between 10 and 1000 L / m. 2 / h. In some embodiments, the wall shear stress is maintained between 100 s −1 and 10,000 s −1 .

[0180] Storage of microgel particles Disclosed herein in some embodiments are methods of storing microgel particles, annealing agents, additional active agents, therapeutic agents, dermal filler systems or formulations, or any combination thereof. In some embodiments, the methods include storing the microgel particles, annealing agents, additional active agents, therapeutic agents, or any combination thereof prior to formulation into a dermal filler system. In some embodiments, the storing occurs prior to administration of the dermal filler system to a subject. In some embodiments, the method of storing includes lyophilization, cryodehydration, cryohibernation, or cryopreservation, or a combination thereof.

[0181] In some embodiments, lyophilization of microgel particles, annealing agents, therapeutic agents, or combinations thereof involves the use of a lyoprotectant to preserve the functionality of the microgel particles, annealing agents, therapeutic agents, or combinations thereof. Lyoprotectants include the addition of reagents, salts, or additives that protect the microgel particles, annealing agents, therapeutic agents, or combinations thereof during the drying process. Common lyoprotectants include isopropanol, glycerol, trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods for increasing the transport of the lyoprotectant, annealing agents, therapeutic agents, or combinations thereof within the microgel particles in suspension can be utilized to improve the viability and functionality of the microgel particles, annealing agents, therapeutic agents, or combinations thereof after lyophilization. These methods include electroporation and the addition of reagents. In some embodiments, the lyophilized microgel particles, annealing agents, therapeutic agents, or combinations thereof can be reconstituted for delivery to a tissue site in a subject. In some embodiments, reconstitution is achieved by introducing a reconstitution medium to the lyophilized microgel particles, the annealing agent, the therapeutic agent, or a combination thereof.

[0182] In some embodiments, the microgel particles are flash-frozen. In some embodiments, the microgel particles are flash-frozen using liquid nitrogen. In some embodiments, the microgel particles are frozen at a temperature of at least about -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, or -200°C. In some embodiments, the microgel particles are frozen at a temperature of about -196°C. In some embodiments, the microgel particles are in a solution of at least about 80%, 85%, 90%, 95%, or 100% isopropanol.

[0183] In some embodiments, lyophilization occurs at a temperature of about −55 C. In some embodiments, lyophilization occurs at a temperature of less than about −50 C, −55 C, −60 C, −65 C, −70 C, −75 C, −80 C, −85 C, −90 C, −95 C, or −100 C. In some embodiments, the volume fraction of microgel particles that aids in lyophilization is less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0184] kit In some embodiments, disclosed herein are kits useful for delivering the dermal filler systems disclosed herein. In some embodiments, the kits disclosed herein may be used to deliver the dermal filler system to a tissue site in a subject. In some embodiments, the kits include the dermal filler system described herein and may be used to practice the methods described herein. In some embodiments, the kits include hydrogel particles and an annealing agent in separate containers. In some embodiments, the kits include the hydrogel and the annealing agent in a single container. In some embodiments, the kits also include a reconstitution medium, as described herein, for reconstituting a lyophilized dermal filler system (e.g., a lyophilized hydrogel, an annealing agent, or a combination thereof).

[0185] The kit may include instructions for use. Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring devices, bandages, or other useful implements. The materials and components combined in the kit may be stored and provided to the healthcare professional in any convenient and suitable manner that preserves their operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form and may be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging material(s). As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging material is constructed by well-known methods and preferably provides a sterile, contaminant-free environment. The packaging material used in the kit may be that conventionally used in gene expression assays and therapeutic administration. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., that can hold individual kit components.Thus, for example, the packaging may be a glass vial or a pre-filled syringe that is used to contain a suitable amount of pharmaceutical composition.The packaging material has an external label that indicates the contents and / or purpose of the kit and its components. [Example]

[0186] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0187] Example 1: Rheological testing of dermal filler systems Preparation of dermal filler system In this example, 14 different dermal filler systems were evaluated, including (1) two batches of aged, production-equivalent dermal filler systems and (2) 12 batches formulated to feature the device volume fraction (VF) concentration and elastic modulus (stiffness of the microgel particles) of the microgel particles themselves.

[0188] Four different bulk formulations of gel were created to obtain 12 batches with varying parameters. These distinct formulations were created by titrating the relative concentrations of the MMP2-sensitive peptide crosslinker and the PEG-VS polymer in the microgel particles. Varying this molar ratio (thiol / VS molar ratio) allowed for the deterministic selection of microgel particle stiffness ranging from 15 kPa to 46 kPa. These microgel particle stiffnesses were measured by compression testing, measuring the compressive modulus (EM) of macroscopic surrogate bulk gels made from the same mixtures used to create the hydrogel microgel particle suspensions. Table 1 shows the information for all test article lots / batches.

[0189] [Table 1]

[0190] [Table 2]

[0191] A modular compact rheometer (MCR102e, Anton Paar) was used to evaluate the rheological parameters of the materials tested in this example. The rheometer was equipped with a parallel plate testing system using 25 mm sandblasted plates. The sandblasted surface was selected to reduce slippage of any test material (data from previous tests indicated that slippage occurred when smooth plates were used). The sample chamber within the MCR102e was maintained at 20°C for all tests in this example.

[0192] [Table 3]

[0193] method The MCR102e was powered on and the sample chamber was allowed to equilibrate to 20°C. As part of the machine setup, a series of baseline measurements were taken to measure the resistance at the motor without the measurement system attached, followed by the moment of inertia of the 25mm diameter sandblasted top plate measurement system. Prior to measuring any test samples, a final baseline reference test was performed using APN415 viscosity and density reference standards.

[0194] The microgel particle batches selected for testing were equilibrated to room temperature for at least 30 minutes prior to rheometry testing. The products were protected from light until prepared for testing.

[0195] After cleaning the measurement system and lower plate, approximately 490 microliters (μL) of test article was loaded into the center of the lower plate using a 1,000 pL positive displacement pipette. The upper plate was then lowered from the upper plate to the lower plate, leaving a 1 mm gap, to establish the normal force (Fr) required to ensure a good reading during flow curve measurements. Any excess test article was removed from around the 1 mm gap between the plates. Each test included a 2-minute temperature equilibration period, which maintained the temperature at 20.00°C ± 0.05°C, as measured by the thermocouple located on the lower plate. Figure 1 shows the workflow for initiating and conducting flow curve measurements.

[0196] Apparent viscosity is 0.1s -1 ~1,000s -1 The shear rate (γ) was measured at a series of 25 observation points in the range of 10 s (at 0.1 s) -1 ) to 1s (@1,000s -1) with a logarithmic slope. This time profile was chosen to reduce noise and allow for more consistent measurements. Each product lot was measured in duplicate, and the coefficient of variation (CV) at each γ for each shear rate point was calculated. The CVs for these points were then averaged to obtain a total CV for each lot as a measure of measurement reproducibility. The measured torque T (N*m) was converted to shear stress τ (Pa) using Equation 1 below, and then converted to apparent viscosity η (Pa·s) using Equation 2 below. The apparent viscosity was then plotted against shear rate to obtain η (Pa·s) versus γ (s) -1 ) viscosity curves were generated.

[0197]

number

[0198] result The viscosity curves for each lot appear to exhibit shear thinning behavior, meaning that the apparent viscosity decreases as shear increases. On a log-log plot of viscosity η versus shear rate γ, the apparent viscosity decreases at 0.1 s -1 <γ<10s -1 We identified a linear regime between γ and γ (Figure 2A). In this linear regime on a log-log plot (Figure 2B), the Ostwald-de Waele power law equation can be used in its linear form to accurately calculate the viscosity η at a given shear rate γ (Equat...

Claims

1. 1. A method for delivering a dermal filler formulation to a tissue site in a subject, the method comprising: delivering to the tissue site a dermal filler formulation comprising a hydrogel that forms a covalently stabilized porous scaffold under conditions sufficient to anneal in vivo to form a cellular matrix within the covalently stabilized porous scaffold, wherein the cellular matrix forms new tissue at the tissue site while minimizing a foreign body reaction in the subject.

2. 10. The method of claim 1, wherein delivering comprises administering subdermally.

3. 10. The method of claim 1, wherein delivering comprises administering via dermal administration.

4. 10. The method of claim 1, wherein delivering comprises administering intradermally.

5. 10. The method of claim 1, wherein delivering comprises administering subcutaneously.

6. 10. The method of claim 1, wherein delivering comprises expelling the dermal filler formulation from a syringe or needle.

7. The method of claim 6, wherein the needle has a gauge comprising about 25 gauge to about 35 gauge.

8. The method of claim 6 , wherein the needle has a gauge comprising about 27 gauge.

9. The method of claim 6 , wherein the needle has a gauge comprising about 30 gauge.

10. 7. The method of claim 6, wherein delivering comprises exerting an extrusion force of up to 40 Newtons (N) on the dermal filler formulation.

11. The method of any one of claims 1 to 10, wherein the cell matrix comprises cells that are endogenous to the subject.

12. 12. The method of any one of claims 1 to 11, wherein at least a portion of the tissue site is permanently filled with the cell matrix following degradation of the covalently stabilized porous scaffold at the tissue site.

13. 13. The method of claim 12, wherein the cell matrix comprises at least 10% of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site.

14. 13. The method of claim 12, wherein the cell matrix comprises at least 25% of the tissue site after degradation of the covalently stabilized porous scaffold at the tissue site.

15. The method of any one of claims 1 to 14, wherein the cell matrix is ​​formed within about 30 days after delivery.

16. 16. The method of claim 15, wherein the cell matrix begins to form within the covalently stabilized porous scaffold within seven days after administration.

17. 17. The method of any one of claims 1 to 16, wherein the cell matrix forms new tissue at the tissue site in the subject prior to complete degradation of the covalently stabilized porous scaffold.

18. 18. The method of claim 17, wherein the new tissue is characterized by having (i) mature vascularization, (ii) characteristics of the surrounding tissue at the tissue site, (iii) or a combination thereof.

19. 20. The method of claim 18, wherein the characteristics of the surrounding tissue at the tissue site include cell types that are functionally differentiated from the surrounding tissue.

20. The method of any one of claims 1 to 19, wherein the tissue site is soft tissue.

21. The method of any one of claims 1 to 20, wherein the foreign body reaction is characterized by causing harm to the subject.

22. 22. The method of claim 21, wherein the harm is characterized by causing chronic inflammation, granuloma formation, scar tissue formation, nodule formation, swelling, pain, or any combination thereof.

23. 23. The method of claim 22, wherein the harm occurs at the tissue site.

24. 24. The method of any one of claims 1 to 23, wherein the cell matrix is ​​formed while minimizing the foreign body reaction in the subject, as measured by using histological analysis to detect the amount of granulomas at the tissue site and comparing the amount of granulomas at the tissue site to a reference tissue that does not contain the dermal filler formulation.

25. 24. The method of any one of claims 1 to 23, wherein the cell matrix is ​​formed while minimizing the foreign body response in the subject, as measured by using histological analysis to detect the amount of scar tissue at the tissue site and comparing the amount of scar tissue at the tissue site to a reference tissue that does not contain the dermal filler formulation.

26. 24. The method of any one of claims 1 to 23, wherein the cell matrix is ​​formed while minimizing the foreign body reaction in the subject, as measured by detecting the amount of nodules at the tissue site using histological analysis and comparing the amount of nodules at the tissue site to reference tissue that does not contain the dermal filler formulation.

27. 24. The method of any one of claims 1 to 23, wherein the cell matrix is ​​formed while minimizing the foreign body response in the subject, as measured by detecting chronic inflammation at the tissue site using histological analysis.

28. 28. The method of any one of claims 1 to 27, wherein the cell matrix comprises an amount or type of collagen that mimics endogenous tissue at the tissue site.

29. 29. The method of claim 28, wherein the type of collagen comprises type I collagen, type III collagen, or a combination thereof.

30. 30. The method of claim 29, wherein the type I collagen is present in a ratio of about 10:1 or less to the type III collagen.

31. 30. The method of claim 29, wherein the type I collagen is present in a ratio of about 6:1 or less with the type III collagen.

32. 30. The method of claim 29, wherein the type I collagen is present in a ratio of about 5:1 or less to the type III collagen.

33. 33. The method of any one of claims 1 to 32, wherein at least a portion of the tissue site comprises elastin following degradation of the covalently stabilized porous scaffold at the tissue site.

34. 34. The method of any one of claims 1 to 33, wherein the dermal filler formulation is biocompatible with tissue at the tissue site as determined by one or more techniques described in ISO standard 10993.

35. 35. The method of any one of claims 1-34, wherein the covalently stabilized porous scaffold remains at the tissue site in an amount sufficient to fill at least a portion of the tissue site for an amount of time of 9 months or more after delivery.

36. 36. The method of any one of claims 1 to 35, further comprising delivering lidocaine to the tissue site.

37. 37. The method of claim 36, wherein the lidocaine is delivered at a concentration comprising about 1.0 milligrams per microliter (mg / mL) to about 5.0 mg / mL.

38. 37. The method of claim 36, wherein the lidocaine is delivered at a concentration comprising about 3.0 mg / mL.

39. 39. The method of any one of claims 1-38, wherein the hydrogel comprises a polymer comprising hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), collagen, polymethyl methacrylate, or any combination thereof.

40. 40. The method of claim 39, wherein the polymer is a copolymer comprising HA and PEG.

41. 40. The method of claim 39, wherein the polymer is HA.

42. 40. The method of claim 39, wherein the polymer is PEG.

43. The method of any one of claims 1 to 42, wherein the dermal filler formulation further comprises vinyl or a derivative thereof.

44. 44. The method of claim 43, wherein the vinyl comprises vinyl sulfone (VS), acrylate, methacrylate, acrylamide, maleimide, norbornene, or any combination thereof.

45. 45. The method of any one of claims 1 to 44, wherein the dermal filler formulation further comprises a thiol or a derivative thereof.

46. 46. ​​The method of claim 45, wherein the thiol or derivative thereof comprises thiolated HA.

47. 46. ​​The method of claim 45, wherein the thiol or derivative thereof comprises two or more thiols.

48. 46. ​​The method of claim 45, wherein the thiol or derivative thereof comprises polyethylene glycol (PEG)-dithiol or derivative thereof.

49. 49. The method of claim 48, wherein the hydrogel and the PEG-dithiol or derivative thereof are delivered to the subject separately.

50. 49. The method of claim 48, wherein the hydrogel and the PEG-dithiol or derivative thereof are delivered to the subject together.

51. 51. The method of any one of claims 48-50, wherein the hydrogel and the PEG-dithiol or derivative thereof have a shelf life of at least about 18 months when the hydrogel and the PEG-dithiol or derivative thereof are stored as a mixture in a single container.

52. 51. The method of any one of claims 48-50, wherein the hydrogel and the PEG-dithiol or derivative thereof have a shelf life of at least about 36 months when the hydrogel and the PEG-dithiol or derivative thereof are stored as a mixture in a single container at room temperature.

53. 53. The method of any one of claims 1 to 52, wherein the dermal filler formulation is lyophilized.

54. 54. The method of claim 53, further comprising reconstituting the dermal filler formulation prior to delivering the dermal filler formulation to the tissue site.

55. 46. ​​The method of claim 45, wherein either the thiol or its derivative and the vinyl sulfone or its derivative are present in the dermal filler formulation in excess of the other.

56. 46. ​​The method of claim 45, wherein the thiol or its derivative and vinyl sulfone or its derivative are present in the dermal filler formulation in a 1:1 molar ratio.

57. 57. The method of any one of claims 1-56, wherein the tissue site comprises: (1) the mid-face or cheek region of the subject; (2) the cheek of the subject; (3) the chin of the subject; or (4) the lip of the subject; or (5) any combination thereof.

58. 58. The method of any one of claims 1 to 57, further comprising treating the tissue site of the subject by delivering the dermal filler formulation to the tissue site.

59. 59. The method of claim 58, wherein treating the tissue site comprises tissue filling, skin filling, wrinkle removal, cosmetically improving the skin surrounding the tissue site, tissue repair, correcting skin irregularities, treating one or more skin lesions, or any combination thereof.

60. 60. The method of claim 59, wherein tissue filling comprises building new tissue formation, generating new tissue formation, or stimulating new tissue formation, or any combination thereof.

61. 60. The method of claim 59, wherein the one or more skin lesions comprise acne scars, basal cell carcinoma, cellulitis, epidermolysis bullosa, melanoma, Merkel cell carcinoma, scars, skin biopsy, skin cancer, squamous cell carcinoma, stretch marks, or any combination thereof.

62. 60. The method of claim 58, wherein treating the tissue site is accomplished by a single delivery of the dermal filler formulation to the tissue site of the subject.

63. 60. The method of claim 58, wherein treating the tissue site is accomplished by delivering the dermal filler formulation to the tissue site of the subject twice.

64. 60. The method of claim 58, wherein treating the tissue site is accomplished by delivering the dermal filler formulation to the tissue site of the subject three times.

65. 59. The method of claim 58, wherein the covalently stabilized porous scaffold comprises a compressive modulus of about 1,000 Pascals (Pa) to about 100,000 Pa, when the modulus is measured using a compression test (e.g., with an Instron).

66. 60. The method of claim 58, wherein the covalently stabilized porous scaffold comprises a storage modulus of about 50 Pascals (Pa) to about 10,000 Pa, as measured using a rheometer.

67. 67. The method of any one of claims 1 to 66, wherein the covalently stabilized porous scaffold comprises a plurality of pores having a median diameter comprising about 5 micrometers (μm) to about 1000 μm.

68. 68. The method of any one of claims 1 to 67, wherein the dermal filler formulation further comprises a buffer.

69. 69. The method of claim 68, wherein the buffer comprises a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

70. 1. A dermal filler system comprising: a) microgel particles comprising a hydrogel polymer and a thiol or a derivative thereof, wherein the hydrogel polymer comprises hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), or a combination thereof; b) vinyl sulfone (VS) or a derivative thereof, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold, and wherein either the thiol or its derivative and the vinyl sulfone or its derivative are present in the dermal filler system in excess of the other.

1. A dermal filler system comprising:

71. 1. A dermal filler system comprising: a) a dermal filler formulation comprising microgel particles, the microgel particles comprising a hydrogel polymer and a thiol or a derivative thereof, the hydrogel polymer comprising hyaluronic acid (HA), poly(ethylene glycol) (PEG), polylactic acid (PLA), or a combination thereof; b) vinyl sulfone (VS) or a derivative thereof, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold comprising an elastic modulus of about 1,000 Pascals (Pa) to about 100,000 Pa; 1. A dermal filler system comprising:

72. 72. The dermal filler system of claim 70 or 71, wherein the microgel particles are spherical.

73. 73. The dermal filler system according to any one of claims 70 to 72, wherein the microgel particles comprise microspheres.

74. 74. The dermal filler system according to any one of claims 70 to 73, wherein the microgel particles comprise a diameter comprised between 5 μm and 1000 μm.

75. 75. The dermal filler system of claim 74, wherein the diameter is comprised between 50 μm and 1000 μm.

76. 75. The dermal filler system of any one of claims 74, wherein the diameter is comprised between 80 μm and 140 μm.

77. The dermal filler system of any one of claims 70 to 76, wherein the covalently stabilized porous scaffold comprises pores with a median pore size of about 5 μm or greater.

78. 78. The dermal filler system of claim 77, wherein the pores comprise a median pore size of about 10 μm to about 35 μm.

79. The dermal filler system according to any one of claims 70 to 77, wherein the microgel particles further comprise one or more cell adhesive peptides.

80. 80. The dermal filler system of claim 79, wherein the one or more cell adhesive peptides comprise an RGD peptide.

81. The dermal filler system of claim 80, wherein the RGD peptide comprises an amino acid sequence as provided in any one of SEQ ID NOs: 1-2 or 6-9.

82. The dermal filler system of claim 80, wherein the RGD peptide comprises an amino acid sequence that is about 75% identical to the amino acid sequence provided in any one of SEQ ID NOs: 1-3.

83. The dermal filler system according to any one of claims 70 to 82, wherein the microgel particles further comprise one or more K peptides.

84. 84. The dermal filler system of claim 83, wherein the one or more K peptides comprise an amino acid sequence as provided in any one of SEQ ID NOs:

3.

85. The dermal filler system according to any one of claims 70 to 84, wherein the microgel particles further comprise one or more Q peptides.

86. 84. The dermal filler system of claim 83, wherein the one or more Q peptides comprise an amino acid sequence as provided in any one of SEQ ID NOs:

4.

87. The dermal filler system of any one of claims 70 to 86, wherein the hydrogel polymer comprises a polydispersity of 0.1 or less.

88. The dermal filler system of claim 87, wherein the polydispersity is calculated based on the standard deviation and mean size of the microgel particles (e.g., PDI = (SD / mean)^2).

89. 89. The dermal filler system according to any one of claims 70 to 88, wherein the dermal filler further comprises a buffer solution, wherein the buffer solution comprises a phosphate buffer solution, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, or an acetate buffer solution, or any combination thereof.

90. The dermal filler system according to any one of claims 70 to 89, further comprising lidocaine.

91. 91. The dermal filler system of claim 90, wherein the lidocaine is present in the dermal filler system at a concentration of about 1.0 mg / mL to about 5.0 mg / mL.

92. 92. The dermal filler system of claim 91, wherein the lidocaine is present in the dermal filler system at a concentration of about 3.0 mg / mL.

93. The dermal filler system according to any one of claims 70 to 92, wherein the hydrogel polymer comprises HA and PEG.

94. 94. The dermal filler system of claim 93, wherein the hydrogel is a copolymer of HA and PEG having approximately the same molecular weight as each of HA and PEG.

95. 95. The dermal filler system of claim 93 or 94, wherein the HA comprises a molecular weight of 1 kilodalton (kDa) to 1 megadalton (1 MDa).

96. 96. The dermal filler system of claim 95, wherein the HA comprises a molecular weight of 10 kDa to 250 kDa (e.g., 10, 40, 50, 150, and 250 kDa).

97. 97. The dermal filler system of any one of claims 93 to 96, wherein the PEG comprises a molecular weight of 1 kilodalton (kDa) to 5 kDa.

98. The dermal filler system according to any one of claims 93 to 97, wherein the hydrogel polymer comprises a thiol or a derivative thereof, or a VS or a derivative thereof, or a combination thereof.

99. 99. The dermal filler system of claim 98, wherein the HA is modified to include a thiol or a derivative thereof to form a thiolated HA.

100. The dermal filler system of any one of claims 98 to 97, wherein the PEG is modified to include VS or a derivative thereof to form PEG-VS.

101. The dermal filler system of claim 100, wherein the PEG-VS comprises a multi-arm PEG-VS.

102. The dermal filler system of claim 101, wherein the multi-arm PEG-VS comprises a 4-arm or 8-arm PEG-VS.

103. The dermal filler system of claim 101, wherein the VS comprises divinyl sulfone.

104. The dermal filler system according to any one of claims 93 to 103, wherein the thiol or its derivative and the VS or its derivative are configured to interact with each other in a reaction to synthesize the microgel particles.

105. The dermal filler system of claim 104, wherein the reaction comprises a covalent synthesis reaction.

106. The dermal filler system of claim 105, wherein the covalent synthesis reaction comprises a Michael addition (e.g., a thiol-ene Michael addition) reaction or a pseudo-Michael addition reaction.

107. The dermal filler system of claim 106, wherein the thiol or derivative thereof is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

108. The dermal filler system of claim 106, wherein the VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

109. The dermal filler system according to any one of claims 93 to 108, wherein the thiol or derivative thereof and the VS or derivative thereof are present in the dermal filler system in a molar ratio of about 1:

1.

110. The dermal filler system according to any one of claims 93 to 108, wherein an excess of the thiol or derivative thereof and the VS or derivative thereof is present in the dermal filler system such that the excess of the thiol or derivative thereof and the VS or derivative thereof participates in an annealing reaction to form the covalently stabilized porous scaffold.

111. The dermal filler system of any one of claims 93 to 110, further comprising two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof.

112. The dermal filler system according to any one of claims 93 to 110, further comprising a molecule comprising two or more thiols or derivatives thereof.

113. The dermal filler system of claim 112, wherein the molecule comprises PEG.

114. The dermal filler system of claim 113, wherein the molecule comprises PEG-dithiol.

115. The dermal filler system of claim 114, wherein the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa.

116. The dermal filler system of claim 114, wherein the PEG-dithiol comprises a molecular weight of about 3.4 kDa.

117. The dermal filler system of any one of claims 114 to 116, wherein the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof.

118. The dermal filler system of claim 117, wherein the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol.

119. The dermal filler system of any one of claims 114 to 118, wherein the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

120. The dermal filler system of claim 119, wherein the annealing reaction comprises a covalent annealing reaction.

121. The dermal filler system of claim 120, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

122. The dermal filler system of claim 121, wherein the thiol or derivative thereof of the PEG-dithiol is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

123. The dermal filler system of claim 121, wherein the excess VS is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

124. The dermal filler system according to any one of claims 93 to 110, further comprising PEG-divinylsulfone or a derivative thereof.

125. The dermal filler system of claim 124, wherein the PEG-divinylsulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

126. The dermal filler system of claim 119, wherein the annealing reaction comprises a covalent annealing reaction.

127. The dermal filler system of claim 120, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

128. The dermal filler system of claim 121, wherein the divinyl sulfone or a derivative thereof of the PEG-divinyl sulfone is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

129. 122. The dermal filler system of claim 121, wherein the excess thiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

130. The dermal filler system according to any one of claims 70 to 92, wherein the hydrogel polymer comprises HA.

131. 131. The dermal filler system of claim 130, wherein the HA comprises a molecular weight of 1 kilodalton (kDa) to 1000 kDa.

132. 132. The dermal filler system of any one of claims 130 to 131, wherein the HA comprises a molecular weight of about 10 kDa to about 250 kDa.

133. 133. The dermal filler system of claim 132, wherein the molecular weight comprises about 10, 40, 50, 150, or 250 kDa.

134. The dermal filler system according to any one of claims 130 to 133, further comprising glutaraldehyde or a derivative thereof, divinyl sulfone or a derivative thereof, 1,4-butanediol diglycidyl ether (BDDE) or a derivative thereof, or any combination thereof, configured to interact in a cross-linking reaction to synthesize the microgel particles.

135. The dermal filler system of any one of claims 130 to 133, wherein the HA is modified to include a thiol or a derivative thereof to form a thiolated HA.

136. The dermal filler system of claim 135, wherein the HA is modified to include the VS or a derivative thereof to form HA-VS.

137. The dermal filler system of claim 136, wherein the thiol or its derivative and the VS or its derivative are configured to interact in a cross-linking reaction to synthesize the microgel particles.

138. The dermal filler system of claim 137, wherein the crosslinking reaction comprises a covalent synthesis reaction.

139. The dermal filler system of claim 138, wherein the covalent synthesis reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

140. The dermal filler system of claim 139, wherein the thiol or derivative thereof is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

141. The dermal filler system of claim 139, wherein the VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

142. The dermal filler system of any one of claims 137 to 141, wherein the thiol or derivative thereof and the VS or derivative thereof are present in the dermal filler system in a molar ratio of about 1:

1.

143. A dermal filler system according to any one of claims 137 to 141, wherein the thiol or derivative thereof and the VS or derivative thereof are present in excess of each other in the dermal filler system, such that the excess of the thiol or derivative thereof or the VS or derivative thereof participates in an annealing reaction to form a covalently stabilized porous scaffold.

144. The dermal filler system of any one of claims 130 to 143, further comprising two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof.

145. The dermal filler system according to any one of claims 130 to 143, further comprising a molecule comprising two or more thiols or derivatives thereof.

146. The dermal filler system of claim 145, wherein the molecule comprises PEG.

147. The dermal filler system of claim 146, wherein the molecule comprises PEG-dithiol.

148. The dermal filler system of claim 147, wherein the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa.

149. The dermal filler system of claim 148, wherein the PEG-dithiol has a molecular weight of about 3.4 kDa.

150. The dermal filler system of any one of claims 147 to 149, wherein the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof.

151. The dermal filler system of claim 150, wherein the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol.

152. 152. The dermal filler system of any one of claims 146 to 151, wherein the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

153. The dermal filler system of claim 152, wherein the annealing reaction comprises a covalent annealing reaction.

154. The dermal filler system of claim 153, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

155. The dermal filler system of claim 154, wherein the thiol or derivative thereof of the PEG-dithiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

156. The dermal filler system of claim 154, wherein the excess VS is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

157. The dermal filler system of any one of claims 130 to 143, further comprising PEG-divinylsulfone or a derivative thereof.

158. The dermal filler system of claim 157, wherein the PEG-divinylsulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

159. The dermal filler system of claim 158, wherein the annealing reaction comprises a covalent annealing reaction.

160. The dermal filler system of claim 159, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

161. The dermal filler system of claim 160, wherein the divinyl sulfone or a derivative thereof of the PEG-divinyl sulfone is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

162. The dermal filler system of claim 160, wherein the excess thiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

163. The dermal filler system according to any one of claims 70 to 92, wherein the hydrogel polymer comprises PEG.

164. The dermal filler system of claim 163, wherein the PEG has a molecular weight of 1 kilodalton (kDa) to 1000 kDa.

165. The dermal filler system of any one of claims 163 to 164, wherein the hydrogel polymer further comprises the thiol or a derivative thereof, the VS or a derivative thereof, or a combination thereof.

166. The dermal filler system of claim 165, wherein the PEG comprises the thiol or a derivative thereof to form a PEG-dithiol.

167. The dermal filler system of claim 165, wherein the PEG comprises the VS or a derivative thereof to form PEG-VS.

168. The dermal filler system of claim 167, wherein the PEG-VS group includes a multi-arm PEG-VS.

169. The dermal filler system of claim 167, wherein the multi-arm PEG-VS comprises a 4-arm or 8-arm PEG-VS.

170. The dermal filler system of any one of claims 167 to 169, wherein the VS comprises divinyl sulfone.

171. The dermal filler system according to any one of claims 165 to 170, wherein the thiol or its derivative and the VS or its derivative are configured to interact with each other in a reaction to synthesize the microgel particles.

172. The dermal filler system of claim 171, wherein the reaction comprises a covalent synthesis reaction.

173. The dermal filler system of claim 172, wherein the covalent synthesis reaction comprises a Michael addition (e.g., a thiol-ene Michael addition) reaction or a pseudo-Michael addition reaction.

174. The dermal filler system of claim 173, wherein the thiol or its derivative is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

175. The dermal filler system of claim 173, wherein the VS or a derivative thereof is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

176. The dermal filler system of any one of claims 165 to 175, wherein the thiol or derivative thereof and the VS or derivative thereof are present in the dermal filler system in a molar ratio of about 1:

1.

177. The dermal filler system of any one of claims 165 to 176, wherein an excess of the thiol or derivative thereof and the VS or derivative thereof is present in the dermal filler system such that the excess of the thiol or derivative thereof and the VS or derivative thereof participates in an annealing reaction to form the covalently stabilized porous scaffold.

178. The dermal filler system of any one of claims 163 to 177, further comprising two or more acrylates, methacrylates, acrylamides, maleimides, norbornenes, or any combination thereof.

179. The dermal filler system of any one of claims 163 to 177, further comprising a molecule comprising two or more thiols or derivatives thereof.

180. 180. The dermal filler system of claim 179, wherein the molecule comprises PEG.

181. The dermal filler system of claim 180, wherein the molecule comprises PEG-dithiol.

182. The dermal filler system of claim 181, wherein the PEG-dithiol comprises a molecular weight of about 1.0 kDa to about 5.0 kDa.

183. The dermal filler system of claim 181, wherein the PEG-dithiol has a molecular weight of about 3.4 kDa.

184. The dermal filler system of any one of claims 181 to 183, wherein the PEG-dithiol comprises a linear PEG-dithiol, a multi-arm PEG-dithiol, or a combination thereof.

185. The dermal filler system of claim 183, wherein the multi-arm PEG-dithiol comprises a 4-arm or 8-arm PEG-dithiol.

186. The dermal filler system of any one of claims 181 to 185, wherein the PEG-dithiol is configured to interact with excess VS or a derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

187. The dermal filler system of claim 186, wherein the annealing reaction comprises a covalent annealing reaction.

188. The dermal filler system of claim 187, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

189. The dermal filler system of claim 188, wherein the thiol or derivative thereof of the PEG-dithiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

190. The dermal filler system of claim 188, wherein the excess VS is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

191. The dermal filler system according to any one of claims 163 to 190, further comprising PEG-divinylsulfone or a derivative thereof.

192. The dermal filler system of claim 191, wherein the PEG-divinylsulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in an annealing reaction to form a covalently stabilized porous scaffold.

193. The dermal filler system of claim 192, wherein the annealing reaction comprises a covalent annealing reaction.

194. The dermal filler system of claim 193, wherein the covalent annealing reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction.

195. The dermal filler system of claim 194, wherein the divinyl sulfone or a derivative thereof of the PEG-divinyl sulfone is a Michael acceptor in the Michael addition reaction or the pseudo-Michael addition reaction.

196. The dermal filler system of claim 194, wherein the excess thiol is a Michael donor in the Michael addition reaction or the pseudo-Michael addition reaction.

197. The dermal filler system of any one of claims 70 to 196, wherein the covalently stabilized porous scaffold is degradable in vivo by one or more degradation pathways.

198. The dermal filler system of claim 194, wherein the one or more degradation pathways include oxidative degradation, enzymatic degradation, photodegradation, or hydrolysis.

199. 200. The dermal filler system of any one of claims 70 to 198, wherein the covalently stabilized porous scaffold remains at the tissue site for at least 18 months before complete degradation.

200. 200. The dermal filler system of any one of claims 70 to 198, wherein the covalently stabilized porous scaffold remains at the tissue site for at least 24 months before complete degradation.

201. The dermal filler system according to any one of claims 70 to 200, wherein the microgel particles are present in a suspension comprising the microgel particles and water.

202. The dermal filler system of claim 201, wherein a volume fraction of the suspension comprises the microgel particles at 50% to 100%.

203. The dermal filler system of claim 201, wherein when the dermal filler is formulated for administration with a needle, the volume fraction of the microgel particles is greater than or equal to about 50%.

204. 204. The dermal filler system of any one of claims 70 to 203, wherein the covalently stabilized porous scaffold comprises a compressive modulus of 1,000 Pascals (Pa) to 50,000 Pa.

205. The dermal filler system of claim 201, wherein the covalently stabilized porous scaffold comprises a compressive modulus of 5,000 Pascals (Pa) to 100,000 Pa in an unswollen state.

206. The dermal filler system of claim 201, wherein the covalently stabilized porous scaffold comprises a compressive modulus of 1,000 Pascals (Pa) to 50,000 Pa in a swollen state.

207. 207. The dermal filler system of any one of claims 70 to 206, wherein the covalently stabilized porous scaffold comprises a storage modulus of 50 Pascals (Pa) to 10,000 Pa.

208. The dermal filler system of claim 207, wherein the covalently stabilized porous scaffold comprises a storage modulus of 60 Pa to 1,000 Pa.

209. The dermal filler system of any one of claims 70 to 208, wherein the covalently stabilized porous scaffold comprises a loss modulus of about 10 Pascals (Pa) to 10,000 Pa.

210. 207. The dermal filler system of any one of claims 204 to 206, wherein the covalently stabilized porous scaffold comprises a compressive modulus of about 50 Pa or greater when the dermal filler is formulated for administration with a needle.

211. The dermal filler system of claim 210, wherein the thiol or derivative thereof and the vinyl sulfone or derivative thereof are present in the dermal filler system in a molar ratio of about 0.3 to about 0.8 to achieve a compressive modulus.

212. The dermal filler system according to any one of claims 207 to 208, wherein the microgel particles are present in a suspension comprising the microgel particles and water, and a volume fraction of the suspension comprises the microgel particles from 50% to 100% to achieve a compressive modulus.

213. The dermal filler system of any one of claims 70 to 212, wherein the covalently stabilized scaffold comprises an apparent viscosity of 1,000 to about 1,000,000 mPa·s.

214. 214. The dermal filler system of any one of claims 70 to 213, wherein the covalently stabilized porous scaffold comprises a pH of 5.0 to 9.

0.

215. 214. The dermal filler system of any one of claims 70 to 213, wherein the covalently stabilized porous scaffold comprises a pH of 6.5 to 7.

5.

216. 216. The dermal filler system of any one of claims 70 to 215, wherein the covalently stabilized porous scaffold comprises an osmolality of about 100 milliosmoles per kilogram (mOsmol / kg) to about 400 mOsmol / kg.

217. The dermal filler system of any one of claims 70 to 216, wherein the hydrogel polymer comprises a degree of substitution per monomer of about 5% to about 20%.

218. The dermal filler system of claim 217, wherein the hydrogel polymer comprises modified HA.

219. The dermal filler system of any one of claims 70 to 218, wherein the dermal filler system is freeze-dried.

220. The dermal filler system of any one of claims 10 to 218, wherein the microgel particles comprise an elastic modulus of about 10 kPa to about 100 kPa.

221. The dermal filler system of any one of claims 10 to 218, wherein the microgel particles comprise an elastic modulus of about 15 kPa to about 50 kPa.

222. 222. A cosmetic formulation comprising the dermal filler system of any one of claims 70 to 221 in a suspension, wherein the suspension also comprises a buffer and a molecule comprising two or more thiols or derivatives thereof, two or more vinyls or derivatives thereof, or a combination thereof.

223. The cosmetic preparation of claim 222, wherein the buffer comprises a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

224. The cosmetic preparation of claim 222, wherein the molecule comprises PEG-dithiol.

225. The cosmetic preparation of claim 222, wherein the cosmetic preparation is formulated for administration to a subject.

226. The cosmetic preparation of claim 225, wherein the administration is subdermal, dermal, intradermal, or subcutaneous.

227. A cosmetic preparation according to any one of claims 225 to 226, wherein administration minimizes a foreign body reaction in the subject.

228. The cosmetic formulation of any one of claims 225 to 226, wherein the cosmetic formulation comprises a dosage volume of about 0.75 mL to about 1.0 mL.

229. A cosmetic preparation according to any one of claims 225 to 228, wherein the cosmetic preparation is sterile.

230. The cosmetic preparation of any one of claims 225 to 228, further comprising lidocaine.

231. 1. A delivery device comprising: (a) a body comprising the dermal filler system according to any one of claims 70 to 221 or the cosmetic preparation according to any one of claims 222 to 230; (b) an applicator in fluid communication with the body; wherein the delivery device is sterile.

232. 232. The delivery device of claim 231, wherein the delivery device is a syringe or a needle.

233. 232. The delivery device of claim 231, wherein the delivery device is a microneedle patch.

234. 230. A method of freeze-drying a dermal filler system according to any one of claims 70 to 221 or a cosmetic formulation according to any one of claims 222 to 230, the method comprising freeze-drying the dermal filler system or the cosmetic formulation to form a powder.

235. The method of claim 234, further comprising reconstituting the freeze-dried dermal filler system or cosmetic formulation for delivery to a subject.

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