Annealed scaffolds, delivery devices, and methods of use thereof

Abstract

Provided herein are delivery devices, annealed scaffolds, and methods for the use thereof. In certain aspects, the methods comprise use of annealed scaffolds to strengthen a suture line of a subject in need thereof.

Description

Attorney Docket No. 48469-711.601ANNEALED SCAFFOLDS, DELIVERY DEVICES, AND METHODS OF USE THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 738,340, filed on December 23, 2024; which is incorporated by reference herein.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 48469-711601. xml, created December 19, 2025, which is 11,705 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.SUMMARY

[0003] In certain aspects, described herein is a body comprising two or more microgel particles annealed to each other forming an annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm polyethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; and an applicator in fluidic communication with the body.

[0004] In some embodiments, the PEG-dithiol-annealed microgel particles are annealed via a chemical crosslinking reaction. In some embodiments, the crosslinking reaction comprises a chemical crosslinking reaction. In some embodiments, the chemical crosslinking reaction comprises a Michael addition or a pseudo-Michael addition reaction. In some embodiments, the crosslinking reaction comprises a physical crosslinking reaction. In some embodiments, the physical crosslinking reaction comprises electrostatic interactions or hydrogen bonding. In some embodiments, the crosslinking reaction comprises a physical and chemical crosslinking reaction. In some embodiments, the crosslinking reaction is reversible. In some embodiments, the crosslinking reaction is reversible for at least 5 cycles.

[0005] In some embodiments, the scaffold is self-healing. In some embodiments, the annealed scaffold is a shear-thinning fluid. In some embodiments, the annealed scaffold is loaded into a syringe. In some embodiments, the scaffold has at least 10% porosity. In some embodiments, the annealed scaffold has a median pore area of at least 20 pm2. In some embodiments, the annealed scaffold has a median pore area of about 20 pm2to 80 pm2. In some embodiments, the annealed scaffold has an average pore area of at most 1000 pm2. In someAttorney Docket No. 48469-711.601embodiments, the annealed scaffold has an average pore area of about 100 pm2to 1000 pm2. In some embodiments, the annealed scaffold has at most 20% porosity.

[0006] In some embodiments, the microgel particles are present in the suspension at a volume fraction of at least 70%. In some embodiments, the PEG-dithiol and the 4-arm PEG vinyl sulfone are present to provide a ratio of thiol to vinyl sulfone of less than about 1.0. In some embodiments, the PEG-dithiol comprises a molecular weight of at least about 3.4 kDa. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of at least about 0.2 mM.

[0007] In some embodiments, the microgel particles are spherical. In some embodiments, the microgel particles comprise microspheres. In some embodiments, the microgel particles comprise diameters comprising 5 pm to 1000 pm, between 50 pm to 1000 pm, or between 70 pm to 150 pm. In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 500 Pascals (Pa) before the annealing reaction. In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. In some embodiments, the annealed scaffold comprises pores comprising a median pore diameter of about 5 pm and above. In some embodiments, the pores comprise a median pore diameter of about 10 pm to about 35 pm.

[0008] In some embodiments, the one or more cell adhesive peptides comprises an RGD peptide. In some embodiments, the microgel particles comprise a poly dispersity of no more than 0.1. In some embodiments, the poly dispersity is calculated based on a standard deviation and mean size of the particles (e.g., PDI = (SD / mean)2). In some embodiments, the vinyl sulfone of the 4-arm PEG vinyl sulfone is a Michael acceptor in the Michael addition or pseudo -Michael addition reaction. In some embodiments, the thiol of the PEG-dithiol is a Michael donor in the Michael addition or pseudo-Mi chael addition reaction.

[0009] In some embodiments, the annealed scaffold comprises a buffer. In some embodiments, the buffer comprises: a phosphate buffer, a 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof. In some embodiments, the annealed scaffold is formulated for administration to a subject. In some embodiments, the annealed scaffold is formulated for administration at a suture line of the subject. In some embodiments, the administration minimizes a foreign body response in the subject. In some embodiments, the annealed scaffold comprises a dose volume of about 0.01 mL to about 20 mL.

[0010] In certain aspects, described herein is a kit comprising the delivery device disclosed herein and instructions for use thereof.Attorney Docket No. 48469-711.601

[0011] In certain aspects, described herein is a method of delivering an annealed scaffold to a site of injury in a subject, the method comprising providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide in a syringe; and disposing the annealed scaffold to the site of injury in the subject, wherein the annealed scaffold loses a degree of crosslinking to form an unannealed scaffold during said disposing, and wherein the unannealed scaffold re-anneals after being disposed to the site of injury in the subject. In some embodiments, the loss in a degree of crosslinking comprises a loss of covalent bonds in the annealed scaffold. In some embodiments, the loss in a degree of crosslinking comprises a loss of electrostatic bonds in the annealed scaffold. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more disulfide bonds in the annealed scaffold. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more thioether bonds in the annealed scaffold. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more disulfide bonds, thioether bonds, or a combination thereof. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more disulfide bonds in the annealed scaffold or one or more thioether bonds in the annealed scaffold, or a combination thereof. In some embodiments, the annealed scaffold loses a degree of crosslinking under shear strain to form the unannealed scaffold. In some embodiments, the degree of crosslinking is measured by elastic modulus. In some embodiments, the unannealed scaffold has an elastic modulus of at most about 90% compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of about 20% to about 90% compared to an elastic modulus of the annealed scaffold. In some embodiments, the degree of crosslinking is measured by viscosity. In some embodiments, the unannealed scaffold has a viscosity of at most about 80% compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of about 0.1% to about 80% compared to a viscosity of the annealed scaffold.

[0012] In certain aspects, described herein is a method of delivering an annealed scaffold to a site of injury in a subject, the method comprising: providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; and disposing the annealed scaffold onto the site of injury in the subject, wherein the annealed scaffold self-heals upon disposing.Attorney Docket No. 48469-711.601

[0013] In some embodiments, the site of injury is a site of a suture line. In some embodiments, the method further comprises closing the suture line in the subject. In some embodiments, said disposing step and said closing step do not require a time delay. In some embodiments, said disposing step and said closing step occur consecutively. In some embodiments, the annealed scaffold comprises one or more crosslinking reactions. In some embodiments, the one or more crosslinking reactions is covalent, electrostatic, or both.

[0014] In some embodiments, the suture line corresponds to the location of an incision. In some embodiments, the incision comprises a surgical incision. In some embodiments, the surgical incision comprises an abdominal fascial incision. In some embodiments, the abdominal fascial incision comprises an abdominal wall midline or transverse incision of the fascia. In some embodiments, the abdominal incision resulted from a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof.

[0015] In some embodiments, the disposing comprises disposing the annealed scaffold directly over the suture line after the suture line is sutured. In some embodiments, the disposing comprises disposing the annealed scaffold into the suture line while the suture line is being sutured. In some embodiments, the disposing comprises disposing the annealed scaffold into the suture line after the incision is sutured. In some embodiments, the disposing comprises dispensing the annealed scaffold into the suture line while the suture line is being sutured and on top of the suture line after the suture line is sutured. In some embodiments, the annealed scaffold is annealed when disposed on top of the suture line. In some embodiments, the disposing comprises releasing the annealed scaffold from the syringe through a static mixer. In some embodiments, the method further comprises strengthening the suture line at the suture line site by the cell matrix formed over the suture line site. In some embodiments, the strengthening the suture line is characterized by increasing a mechanical tensile strength of the suture line as compared to a reference suture line at an otherwise identical suture line site that was sutured without the delivery of the annealed scaffold. In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by the formation of an amount or a type of collagen mimicking endogenous tissue at the suture line site.

[0016] In some embodiments, the collagen is formed at the suture line site by at least about 28 days after suturing of the suture line. In some embodiments, the collagen is formed in and around the annealed scaffold. 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 with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6:1, or less than or equal to about 5:1. In some embodiments, the increasing the mechanicalAttorney Docket No. 48469-711.601tensile strength of the suture line is characterized by increasing the yield stress of the suture line. In some embodiments, the yield stress is calculated from a stress versus strain curve measured using a tensile test (e.g., on an Instron). In some embodiments, the suture line comprises a yield stress of at least about 3.0 N / mm2to about 6.0 N / mm2at least about 42 days after suturing of the suture line. In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by increasing the toughness of the suture line. In some embodiments, the toughness is measured as an area under the curve of a stress versus strain curve to fracture using a tensile test (e.g., on an Instron). In some embodiments, the suture line comprises a toughness of at least about 60.0 millijoules per millimeter cubed (mJ / mm3) at least about 42 days after suturing of the suture line. In some embodiments, the suture line comprises a toughness of at least about 25.0 mJ / mm3to about 100.0 mJ / mm3at least about 42 days after suturing of the suture line. In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by increasing a percent recovery of the suture line.

[0017] In some embodiments, the percent recovery of the suture line is greater than or equal to about 40% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold. In some embodiments, the percent recovery of the suture line is at least about 20% to about 60% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold. In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by increasing the yield strain of the suture line. In some embodiments, the annealed scaffold becomes integrated with the suture line site in less than or equal to about 14 days following suturing of the suture line. In some embodiments, integration is characterized by new tissue formation in and around the annealed scaffold. In some embodiments, a cell matrix forms new tissue at the suture line site of the subject before complete degradation of the annealed scaffold.

[0018] In some embodiments, the new tissue is characterized by having (i) mature vascularization, (ii) a characteristic of surrounding tissue at the suture line site, (iii) an amount or a type of collagen mimicking endogenous tissue at the suture line site (iii) or a combination thereof. In some embodiments, the characteristic of the surrounding tissue at the suture line site comprises functionally differentiated cell types from the surrounding tissue. In some embodiments, (i) the new tissue forms, and (ii) the annealed scaffold substantially degrades after about 50 days after suturing the suture line. In some embodiments, the new tissue is formed in addition to any tissue formed at the suture line site due to sutures alone. In some embodiments, additional new tissue continues to form at the suture line site for at least about 42 days after suturing of the suture line. In some embodiments, the new tissue is formed above (e.g., superficial to) the suture line site and deep to subcutaneous tissue. In some embodiments, theAttorney Docket No. 48469-711.601new tissue is stromal like tissue with non-aligned collagen bundles. In some embodiments, the annealed scaffold strengthens the suture line at the suture line site of the subject while minimizing a foreign body response in the subject. In some embodiments, the foreign body response is characterized by causing harm to the subject. In some embodiments, the harm is characterized by causing: chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof. In some embodiments, the harm is caused at the suture line site.

[0019] In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of granulomas at the suture line site with histological analysis and comparing the amount of granulomas at the suture line site with a reference suture line site that does not contain the annealed scaffold. In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of scar tissue at the suture line site with histological analysis and comparing the amount of scar tissue at the suture line site with a reference suture line site that does not contain the annealed scaffold. In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of nodules at the suture line site with histological analysis and comparing the amount of nodules at the suture line site with a reference suture line site that does not contain the annealed scaffold. In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting chronic inflammation at the suture line site with histological analysis. In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by a presence of one or more types of macrophages at the suture line site of the subject.

[0020] In some embodiments, the one or more types of macrophages comprise type 1 macrophages and type 2 macrophages. In some embodiments, the type 1 macrophages are pro-inflammatory. In some embodiments, the type 2 macrophages are pro-regenerative. In some embodiments, the annealed scaffold is effective to form more type 2 macrophages than type 1 macrophages. In some embodiments, the cell matrix comprises an amount or a type of collagen mimicking endogenous tissue at the suture line site. In some embodiments, the collagen is formed at the suture line site by at least about 28 days after suturing of the suture line. In someAttorney Docket No. 48469-711.601embodiments, the collagen is formed in and around the hydrogel. 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 with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6:1, or less than or equal to about 5:1.

[0021] In some embodiments, at least part of the suture line site comprises elastin following degradation of the annealed scaffold at the suture line site. In some embodiments, the annealed scaffold comprises a pH of about 6 to about 8. In some embodiments, the annealed scaffold comprises an elastic compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction.

[0022] In some embodiments, the microgel particles are present in a suspension comprising the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 70%.

[0023] In certain aspects, described herein is a method of preparing an annealed scaffold, the method comprising: providing a microgel particle mixture comprising microgel particles comprising a cross-linked 4-arm polyethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; washing the microgel particles; concentrating the microgel particle mixture; and combining PEG-dithiol to the microgel particle mixture, wherein the microgel particles undergo an annealing reaction to form the annealed scaffold.

[0024] In some embodiments, the method further comprises loading a syringe with the annealed scaffold. In some embodiments, said washing step comprises tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), hollow-fiber-diafiltration (HFDF), tangential flow depth filtration (TFDF), dialysis, direct flow filtration, or centrifugation. In some embodiments, said washing step comprises tangential flow filtration (TFF). In some embodiments, said concentrating step comprises tangential flow depth filtration (TFDF), centrifugation, evaporation, or a combination thereof. In some embodiments, said concentrating step comprises tangential flow depth filtration (TFDF). In some embodiments, said concentrating step comprises evaporation. In some embodiments, the evaporation is carried out under a flow of inert gas. In some embodiments, the evaporation is carried out under vacuum.INCORPORATION BY REFERENCE

[0025] All publications, patents, and patent applications mentioned in this specification 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. ToAttorney Docket No. 48469-711.601the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGSA better understanding of the features and advantages of the present subject matter will be obtained by reference to the following detailed description that sets forth illustrative embodiments and the accompanying drawings of which:

[0026] FIG. 1 are representative histology images of rabbit abdominal walls 14 days after incision and treatment with either sutures alone or with various formulations of Microporous Annealed Particle (MAP), according to some embodiments herein.

[0027] FIG. 2A is a scheme depicting an experimental setup demonstrating the reversibility of MAP annealing, according to some embodiments herein.

[0028] FIG 2B is a graph depicting the reversibility of MAP annealing, according to some embodiments herein.

[0029] FIG 2C. is a graph of storage modulus (G1) versus time of an annealed and nonannealed MAP formulation, according to some embodiments herein, using an amplitude sweep and alternative step strains.

[0030] FIG. 2D is a graph of loss modulus (G") versus time of an annealed and non-annealed MAP formulation, according to some embodiments herein, using amplitude sweep and alternative step strains.

[0031] FIG. 3 is a pictorial image of pre-annealed hydrogel formulation loaded into a syringe with a mixing applicator tip, according to some embodiments herein.

[0032] FIG. 4 is a flow diagram of a method for synthesizing, purifying, and concentrating microgel particles, according to some embodiments herein.

[0033] FIG. 5 is a graph of the elastic modulus of annealed MAP at various particle stiffnesses, according to some embodiments herein, as a function of volume fraction.

[0034] FIG. 6 is a graph of viscosity of annealed MAP at various particle stiffnesses at 1.0 s'1shear rate, according to some embodiments herein, as a function of volume fraction.

[0035] FIG. 7 is a graph of the storage modulus of annealed MAP at various particle stiffnesses, according to some embodiments herein, as a function of volume fraction.

[0036] FIG. 8A is a graph of the viscosity curves of annealed and non-annealed MAP (Formulation F4), according to some embodiments herein, between a shear rate of 0.1 s'1and 1000 s'1.Attorney Docket No. 48469-711.601

[0037] FIG. 8B is a graph of the storage modulus (G1) and loss modulus (G") of annealed and non-annealed MAP (Formulation F4), according to some embodiments herein, between a frequency sweep test from 1 rad / s to 100 rad / s and at a constant shear strain of 0.1%.

[0038] FIG. 8C is a graph of the storage modulus (G1) and loss modulus (G") of annealed MAP (Formulation F4), according to some embodiments herein, between an amplitude sweep test from 0.01% to 300% shear strain and at a constant angular frequency of 10 rad / s.

[0039] FIG. 8D is a graph of the storage modulus of annealed and non-annealed MAP (Formulation F4), according to some embodiments herein, during a reversible amplitude sweep test from 0.01% to 300% and at an angular frequency of 10 rad / s.

[0040] FIGS. 9A-9C depict the porosity of annealed MAP at various volume fractions, according to some embodiments herein. FIG. 9A is a bar graph of the percent porosity at various volume fractions. FIG. 9B is a bar graph of the average pore size at various volume fractions. FIG. 9C is a bar graph of the median pore size at various volume fractions.DETAILED DESCRIPTION

[0041] Midline abdominal incisions are used in millions of patients every year in gastrointestinal cancer procedures, hysterectomies and other ovarian cancer procedures, spinal fusions, and abdominal trauma surgeries. Currently, the incision made to access the abdomen re-opens in 30% of patients within a 24-month window after surgery, and this re-opening causes major complications and even death in some patients. As such, there remains a need of a self-healing scaffold to reinforce the incision line, which can efficiently and effectively be applied to a site of injury and requires minimal or no time delay before suturing the site of injury.

[0042] In certain aspects, described herein are systems and compositions, that are easily delivered to a surgical injured site, to make sites of suture lines, including midline abdominal incisions, stronger through regenerative healing and will have the potential to greatly reduce this rate of post-surgical failure and reduce morbidity and mortality in this large patient population. In some embodiments, the systems and compositions comprise microgel particles comprising a 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, a celladhesive peptide, and an annealing agent PEG-dithiol comprising a molecular weight of at least about 0.5 kilodaltons to about 10 kilodaltons.

[0043] In certain aspects, described herein are methods of delivering the annealed scaffolds described herein to a surgical site of a subject. In some embodiments, the annealed scaffold is provided pre-annealed to form an annealed scaffold. In some embodiments, the annealed scaffold is provided ready to use. In some embodiments, the annealed scaffold is delivered to a subjectAttorney Docket No. 48469-711.601with a device disclosed herein, such as a syringe or a needle or a mixing applicator tip (FIG. 3).In some embodiments, the annealed scaffold is a shear-thinning fluid. In some embodiments, the annealed scaffold is a non-Newtonian fluid. In some embodiments, the annealed scaffold is a shear-thickening fluid. In some embodiments, the annealed scaffold is a Newtonian fluid. In some embodiments, the annealed scaffold is a shear-thinning non-Newtonian fluid. In some embodiments, the annealed scaffold self-heals after delivery to a site. In some embodiments, the methods and / or annealed scaffolds are optimized accordingly to various embodiments herein for a given application (e.g., to achieve a desired viscosity during application, and viscoelastic mechanical properties upon delivery).DELIVERY DEVICEDelivery Device

[0044] In certain aspects, described herein is a delivery device comprising a body comprising two or more microgel particles annealed to each other forming an annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; and an applicator in fluidic communication with the body.

[0045] Disclosed herein, in some embodiments, are delivery devices configured to deliver the annealed scaffold to a subject. A non-limiting example of a delivery device is a needle, or a microneedle (e.g., microneedle patch). In some embodiments, the delivery device comprises a body and an applicator in fluidic communication with the body. In some embodiments, the body is elongated (e.g., a barrel). In some embodiments, the body of the delivery device comprises an inner chamber that contains the annealed scaffold. In some embodiments, the delivery device comprises a pump or a plunger configured to apply pressure to the annealed scaffold contained in the body under conditions that the annealed scaffold flows through and out of the applicator via an outlet of the applicator. In some embodiments, the syringe is prefilled with the annealed scaffold. In some embodiments the syringe is sterile. In some embodiments, the syringe is packaged separately from the annealed scaffold and both the syringe, and the hydrogel formulation are sterile.

[0046] In some embodiments, the syringe comprises a needle. In some embodiments, the needle comprises a blunt needle. In some embodiments, the needle has a gauge comprising from about 10 gauge to about 30 gauge. In some embodiments, the needle has a gauge comprising about 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-gauge.Attorney Docket No. 48469-711.601

[0047] In some embodiments, the delivery device comprises an applicator tip. In some embodiments, the applicator tip serves as a static mixer to homogenize the hydrogel formulation upon delivery described herein as a mixing applicator tip (FIG. 3). In some embodiments, the applicator tip is sterile.

[0048] In certain aspects, described herein is a delivery device comprising a body comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; and an applicator in fluidic communication with the body.

[0049] In some embodiments, the delivery device is a syringe. In some embodiments, the delivery device is a single syringe. In certain aspects, described herein is a syringe comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide.Annealed Scaffold

[0050] Disclosed herein, in some embodiments, is an annealed scaffold comprising 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 in accordance with various embodiments herein. In some embodiments, the microgel particles have the ability to reform broken bonds between particles or re-establish interm olecular interactions between particles after a strain has been applied, which is defined as self-healing. In some embodiments, the intermolecular interactions between particles involved in self-healing may be electrostatic, covalent, or a combination thereof. In some embodiments, the microgel particles undergo an annealing reaction to form an annealed scaffold. In some embodiments, the microgel particles are provided annealed in the delivery device disclosed herein. In some embodiments, the microgel particles are annealed by the addition of an annealing agent (e.g., PEG-dithiol). In some embodiments, the annealing reaction is reversible where the annealed microgel particles disassemble and remain flowable during delivery and re-assemble in situ after delivery. In some embodiments, self-healing comprises the reversibility of annealing. The annealed scaffold comprises the pre-annealed microgel particles and may be referred to interchangeably as the “pre-annealed particles,” “pre-annealed microgel particles,” or “annealed scaffold.” In some embodiments, the annealed scaffold forms in a manner such that pores form between the microgel particles of the annealed scaffold.Attorney Docket No. 48469-711.601Microgel Particles

[0051] Disclosed herein, in some embodiments, are microgel particles that anneal together to form an annealed scaffold. In some embodiments, two or more microgel particles may be annealed together via an annealing agent. In some embodiments, two or more microgel particles may be annealed together via a crosslinker. In some embodiments, the hydrogel formulation comprises one or more components configured to facilitate the annealing reaction to form the annealed scaffold.

[0052] Disclosed herein, in some embodiments, are microgel particles with the ability to reform broken bonds between particles or re-establish interm olecular interactions between particles after a strain has been applied, which is defined as “self-healing.” In some embodiments, self-healing may be characterized by reversible bond formation. In some embodiments, self-healing may be characterized by a mechanism wherein the interactions between the microgel particles are cleaved under a large strain, followed by a recovery of the interactions, wherein bonds between the hydrogel microparticles rapidly reform at small strains.

[0053] In some embodiments, the intermolecular interactions between particles involved in self-healing are electrostatic, covalent, or a combination thereof.

[0054] In some embodiments, the self-healing is intrinsic to the microgel particles. In some embodiments, the self-healing is facilitated or induced by the addition of an agent. In some embodiments, the self-healing comprises the reversibility of the annealing reaction.

[0055] 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 spheroidal. In some embodiments, the microgel particles comprise microspheres. The microgel particles may have a substantially uniform shape so as to produce pores when adjacent microgel particles are in contact with each other. Other shapes of microgel particles are contemplated, including, without limitation, oblate, prolate, round-particles, granular particles, flake particles, or 3D geometric shapes.

[0056] In some embodiments, the microgel particles comprise diameters comprising 5 pm to 1000 pm, between 50 pm to 1000 pm, or between 70 pm to 150 pm.

[0057] In some embodiments, the microparticles may have a diameter or dimension (e.g., length, width, height, axis). In some embodiments, the microgel particles comprise diameters or dimensions comprising 0.1 micrometers (pm) to 1000 pm. In some embodiments, the microgel particles comprise diameters or dimensions comprising 5 micrometers (pm) to 1000 pm. In some embodiments, the diameters or dimensions comprise between 50 pm to 1000 pm. In some embodiments, the diameters or dimensions comprise between 80 pm to 140 pm. In some embodiments, the diameters or dimensions comprise between 70 pm to 150 pm In someAttorney Docket No. 48469-711.601embodiments, the diameters comprise greater than or equal to 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, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 pm. In some embodiments, the diameters comprise less than or equal to 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, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 pm. In some embodiments, the diameters or dimensions comprise a range of from about 5 pm to about 1100 pm. In some embodiments, the diameters or dimensions comprise a range of from about 10 pm to about 1090 pm. In some embodiments, the diameters or dimensions comprise a range of from about 15 pm to about 1080 pm. In some embodiments, the diameters or dimensions comprise a range of from about 20 pm to about 1070 pm. In some embodiments, the diameters or dimensions comprise a range of from about 25 pm to about 1060 pm. In some embodiments, the diameters or dimensions comprise a range of from about 30 pm to about 1050 pm. In some embodiments, the diameters or dimensions comprise a range of from about 35 pm to about 1040 pm. In some embodiments, the diameters or dimensions comprise a range of from about 40 pm to about 1030 pm. In some embodiments, the diameters or dimensions comprise a range of from about 45 pm to about 1020 pm. In some embodiments, the diameters or dimensions comprise a range of from about 50 pm to about 1010 pm. In some embodiments, the diameters or dimensions comprise a range of from about 55 pm to about 1000 pm. In some embodiments, the diameters or dimensions comprise a range of from about 60 pm to about 990 pm. In some embodiments, the diameters or dimensions comprise a range of from about 65 pm to about 980 pm. In some embodiments, the diameters or dimensions comprise a range of from about 70 pm to about 970 pm. In some embodiments, the diameters or dimensions comprise a range of from about 75 pm to about 960 pm. In some embodiments, the diameters or dimensions comprise a range of from about 80 pm to about 950 pm. In some embodiments, the diameters or dimensions comprise a range of from about 85 pm to about 940 pm. In some embodiments, the diameters or dimensions comprise a range of from about 90 pm to about 930 pm. In some embodiments, theAttorney Docket No. 48469-711.601diameters or dimensions comprise a range of from about 95 pm to about 920 pm. In some embodiments, the diameters or dimensions comprise a range of from about 100 pm to about 910 pm. In some embodiments, the diameters or dimensions comprise a range of from about 110 pm to about 900 pm. In some embodiments, the diameters or dimensions comprise a range of from about 120 pm to about 890 pm. In some embodiments, the diameters or dimensions comprise a range of from about 130 pm to about 880 pm. In some embodiments, the diameters or dimensions comprise a range of from about 140 pm to about 870 pm. In some embodiments, the diameters or dimensions comprise a range of from about 150 pm to about 860 pm. In some embodiments, the diameters or dimensions comprise a range of from about 160 pm to about 850 pm. In some embodiments, the diameters or dimensions comprise a range of from about 170 pm to about 840 pm. In some embodiments, the diameters or dimensions comprise a range of from about 180 pm to about 830 pm. In some embodiments, the diameters or dimensions comprise a range of from about 190 pm to about 820 pm. In some embodiments, the diameters or dimensions comprise a range of from about 200 pm to about 810 pm. In some embodiments, the diameters or dimensions comprise a range of from about 210 pm to about 800 pm. In some embodiments, the diameters or dimensions comprise a range of from about 220 pm to about 790 pm. In some embodiments, the diameters or dimensions comprise a range of from about 230 pm to about 780 pm. In some embodiments, the diameters or dimensions comprise a range of from about 240 pm to about 770 pm. In some embodiments, the diameters or dimensions comprise a range of from about 250 pm to about 760 pm. In some embodiments, the diameters or dimensions comprise a range of from about 260 pm to about 750 pm. In some embodiments, the diameters or dimensions comprise a range of from about 270 pm to about 740 pm. In some embodiments, the diameters or dimensions comprise a range of from about 280 pm to about 730 pm. In some embodiments, the diameters or dimensions comprise a range of from about 290 pm to about 720 pm. In some embodiments, the diameters or dimensions comprise a range of from about 300 pm to about 710 pm. In some embodiments, the diameters or dimensions comprise a range of from about 310 pm to about 700 pm. In some embodiments, the diameters or dimensions comprise a range of from about 320 pm to about 690 pm. In some embodiments, the diameters or dimensions comprise a range of from about 330 pm to about 680 pm. In some embodiments, the diameters or dimensions comprise a range of from about 340 pm to about 670 pm. In some embodiments, the diameters or dimensions comprise a range of from about 350 pm to about 660 pm. In some embodiments, the diameters or dimensions comprise a range of from about 360 pm to about 650 pm. In some embodiments, the diameters or dimensions comprise a range of from about 370 pm to about 640 pm. In some embodiments, the diameters or dimensions comprise a range of from about 380 pm to about 630 pm. In some embodiments, theAttorney Docket No. 48469-711.601diameters or dimensions comprise a range of from about 390 pm to about 620 pm. In some embodiments, the diameters or dimensions comprise a range of from about 400 pm to about 610 pm. In some embodiments, the diameters or dimensions comprise a range of from about 410 pm to about 600 pm. In some embodiments, the diameters or dimensions comprise a range of from about 420 pm to about 590 pm. In some embodiments, the diameters or dimensions comprise a range of from about 430 pm to about 580 pm. In some embodiments, the diameters or dimensions comprise a range of from about 440 pm to about 570 pm. In some embodiments, the diameters or dimensions comprise a range of from about 450 pm to about 560 pm. In some embodiments, the diameters or dimensions comprise a range of from about 460 pm to about 550 pm. In some embodiments, the diameters or dimensions comprise a range of from about 470 pm to about 540 pm. In some embodiments, the diameters or dimensions comprise a range of from about 480 pm to about 530 pm. In some embodiments, the diameters or dimensions comprise a range of from about 490 pm to about 520 pm. In some embodiments, the diameters or dimensions comprise a range of from about 500 pm to about 510 pm.

[0058] The microgel particles may have an average diameter or dimension of about 10 pm. The microgel particles may have an average diameter or dimension of about 15 pm. The microgel particles may have an average diameter or dimension of about 25 pm. The microgel particles may have a diameter or dimension of about 50 pm. The microgel particles may have an average diameter or dimension of about 100 pm. The microgel particles may have an average diameter or dimension of about 150 pm. The microgel particles may have an average diameter or dimension of about 200 pm. The microgel particles may have a diameter or dimension within the range of about 10 pm to about 500 pm. The microgel particles may have a diameter or dimension within the range of about 10 pm to about 200 pm. The microgel particles may have a diameter or dimension within the range of about 15 pm to about 200 pm. The microgel particles may have a diameter or dimension within the range of about 15 pm to about 150 pm. The microgel particles may have a diameter or dimension within the range of about 30 pm to about 100 pm. The microgel particles may have an average diameter or dimension of 10 pm. The microgel particles may have an average diameter or dimension of 15 pm. The microgel particles may have an average diameter or dimension of 25 pm. The microgel particles may have a diameter or dimension of 50 pm. The microgel particles may have an average diameter or dimension of 100 pm. The microgel particles may have an average diameter or dimension of 150 pm.

[0059] The microgel particles may have an average diameter or dimension of 200 pm. The microgel particles may have a diameter or dimension within the range of 10 pm to 500 pm. The microgel particles may have a diameter or dimension within the range of 10 pm to 200 pm. The microgel particles may have a diameter or dimension within the range of 15 pm to 200 pm. TheAttorney Docket No. 48469-711.601microgel particles may have a diameter or dimension within the range of 15 pm to 150 pm. The microgel particles may have a diameter or dimension within the range of 30 pm to 100 pm. In some embodiments, the diameter of the microgel particle may be measured by: (1) measuring the area of a microgel particle; (2) solving for the radius of the microgel particle using the equation for the area of a circle (i.e., A = 7tr2); and (3) solving for the diameter by multiplying the radius by two (i.e., D = 2r).

[0060] In some embodiments, the microgel particles comprise one or more cell adhesive peptides. In some embodiments, the cell adhesive peptide comprises at least a portion of an extracellular matrix protein. In some embodiments, the cell adhesive peptide comprises at least a portion of a collagen. In some embodiments, the cell adhesive peptide comprises at least a portion of a fibronectin. In some embodiments, the cell adhesive peptide comprises an integrin. In some embodiments, the adhesive peptide comprises a ligand to a receptor expressed on the 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 adhesive peptide may be homologous to the naturally -occurring peptide. In some embodiments, the cell adhesive peptide comprises at least about 70% homologous to a naturally-occurring peptide. In some embodiments, the cell adhesive peptide is at least about 80% homologous to a naturally-occurring peptide. In some embodiments, the cell adhesive 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 coupled to a surface of the microgel particle. In some embodiments, the cell adhesive peptides are grafted to the surface of the microgel particle. In some embodiments, the coupling may comprise one or more chemical bonds. In some embodiments, the one or more chemical bonds is one or more covalent bonds.

[0061] In some embodiments, the cell adhesive peptide may comprise an RGD peptide. In some embodiments, the one or more cell adhesive peptides comprises 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)Attorney Docket No. 48469-711.601(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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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., which isAttorney Docket No. 48469-711.601hereby incorporated by reference in its entirety. In some embodiments, the RGD peptide is modified to improve conjugation of the RGD peptide to a substrate, such as for example, a microgel particle disclosed herein. Non-limiting examples of modifications include addition of a cysteine residue, addition of a linker with a thiol group on one end and an amine group on the other end, a linker with a thiol group on one end and a carboxylic acid group on the other end. In some embodiments, the RGD peptide comprises a modification on either terminus (e.g., C terminus, N terminus). In some embodiments, the modification is within a flanking sequence of the RGD motif within the RGD sequence.

[0062] Cell adhesive peptides may comprise an amino acid. In some embodiments, a cell adhesive peptide comprises a K peptide. In some embodiments, the one or more K peptides comprises an amino acid sequence provided in Ac-FKGGERCG-NH2 (SEQ ID NO: 3). In some embodiments, the K peptides 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 an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an amino acid sequence that is about 85% identical to an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an amino acid sequence that is about 95% identical to an amino acid sequence provided in SEQ ID NO: 3. 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 an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an amino acid sequence that is about 75% identical to an 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 an 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 an 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 an amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the cell adhesive peptide comprises a Q peptide. In some embodiments, the Q peptide comprises an amino acid sequence provided in NQEQVSPLGGERCG (SEQ ID NO: 5). In some embodiments, the Q peptide comprises an amino acid sequence that is about 75% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is about 85% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is about 95% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is about 50%, 55%,Attorney Docket No. 48469-711.60160%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence provided in SEQ ID NO: 5.

[0063] 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., within a substance, the polymer making up a larger proportion of the substance as compared to other polymers in the substance). In some embodiments, the main chain is a linear chain in the polymer to which any other chain may be regarded as being pendant. In some embodiments, the polymer is or comprises a co-polymer. In some embodiments, a co-polymer comprises a polymer chain comprising two or more different monomers in substantially equal proportions. In some embodiments, the polymer is capable of crosslinking and holding large amounts of water forming a water insoluble hydrogel. In some embodiments, the polymer is a synthetic polymer. Nonlimiting examples of polymer include polyethylene glycol) (PEG), polyacrylamide, and polymethacrylate. In some embodiments, the polymer may comprise a hydrophilic polymer, amphiphilic polymer, natural polymer, synthetic polymer, or a copolymer of hydrophobic and hydrophilic polymers (e.g., PEG, polypropylene glycol), or poly(hydroxyethyl methacrylate). In some embodiments, the polymer may be made from any synthetic polymer (e.g., PEG) capable of forming a hydrogel. In some embodiments, the polymer may comprise a functional group (e.g. vinyl) at least on one end of the polymer (e.g. poly(ethylene glycol) methacrylate). In some embodiments, the polymer may comprise a functional group (e.g. vinyl) incorporated into the polymer backbone (e.g. poly(methacrylate)). In some embodiments, the polymer may comprise a vinyl polymer, such as, for example: poly(ethylene glycol) acrylate, polyethylene 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 comprise a polyacrylamide or a polymethacrylate. In some embodiments, the polymer may comprise a polyester, a polyamide, a polyurethane, or a mixture or copolymer thereof. In some embodiments, the polymer is or comprises poly(ethylene) glycol (PEG). In some embodiments, the polymer consists of PEG. Other examples of polymers, but not limited to, include chitosan, hyaluronic acid, gelatin, polyvinyl alcohol (PVA), pullulan, starch, xanthan gum, cellulose, dextran, pectin, heparin, alginate, elastin, fibrin, poly(methacrylate) (PMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(acrylic acid) (PAA), poly(hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAM), poly(vinyl pyrrolidone) (PVP), poly(dimethylsiloxane) (PDMS), poly(vinyl chloride) (PVC), poly(urethane) (PU), poly(caprolactone) (PCL), or poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the microgel particles comprise two or more types of polymers (e.g., polymers made of differentAttorney Docket No. 48469-711.601materials). In some embodiments, the two or more types of polymers comprise PLA and PEG. In some embodiments, the two or more types of polymers comprise PMMA and PEG. In some embodiments, the two or more types of polymers comprise a polymer comprising a functional group and a polymer that does not comprise a functional group. In some embodiments, the microgel particles comprise three or more types of polymers each independently made of a material selected from the group consisting of a hydrophilic polymer, amphiphilic polymer, synthetic polymer. In some embodiments, the microgel particles comprise three or more types of polymers each independently made of a material selected from poly (ethylene glycol) (PEG), poly(lactic acid) (PLA), polypropylene glycol), poly(hydroxyethyl methacrylate), or modified versions of any of these. In some embodiments, when the microgel particles comprise two or more types of polymers, the ratio of each polymer included in the microgel particles may vary. In some embodiments, the polymer comprises a 4-arm PEG vinyl sulfone.

[0064] In some embodiments, the microgel particles comprise a polymer comprising PEG or a modified version thereof. In some embodiments, the microgel particles comprise a MMP-degradable crosslinker. In some embodiments, the MMP-degradable crosslinker crosslinks the polymer within a microgel particle.

[0065] In some embodiments, the polymer (e.g., PEG vinyl sulfone) may be present in the microgel particles, the hydrogel formulation containing the microgel particles, the resulting annealed scaffold, or any combination thereof at about 0.5% weight (% wt) to about a 50% wt. In some embodiments, the polymer may be present in the microgel particles, the hydrogel formulation containing the microgel particles, the resulting annealed scaffold, or any combination thereof at greater than or equal to about 1% wt, 5% wt, 10% wt, 15% wt, 20% wt, 25% wt, 30% wt, 35% wt, 40% wt, 45% wt, or 50% wt. In some embodiments, the polymer may be present at less than or equal to about 1% wt, 5% wt, 10% wt, 15% wt, 20% wt, 25% wt, 30% wt, 35% wt, 40% wt, 45% wt, or 50% wt. In some embodiments, the polymer may be present at greater than or equal to 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. In some embodiments, the polymer may be present at less than or equal to 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. In some embodiments, the polymer may be present at about 5% weight (% wt) to about a 45% wt. In some embodiments, the polymer may be present at about 10% weight (% wt) to about a 40% wt. In some embodiments, the polymer may be present at about 15% weight (% wt) to about a 35% wt. In some embodiments, the polymer may be present at about 20% weight (% wt) to about aAttorney Docket No. 48469-711.60130% 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.

[0066] In some embodiments, the polymer is modified relative to an otherwise identical polymer that does not contain a modification. In some embodiments, the modified polymer comprises modified PEG, such as for example PEG modified to contain a thiol or derivative thereof. Non-limiting modifications include thiolation of the terminal alcohol group on the PEG polymer. In some embodiments, the modified polymer comprises a PEG vinyl sulfone. In some embodiments, the modified polymer comprises a 4-arm PEG vinyl sulfone (4-arm PEG-VS).

[0067] In some embodiments, the molecular weights of the polymers may have an effect on the properties of the microgel particles, the hydrogel formulation containing the microgel particles, the annealed scaffold, or any combination thereof.

[0068] In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight of about 1 kilodalton (kDa) to about 60 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight of about of 1 kDa to 1000 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight of greater than or equal to 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, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa. In some embodiments, the PEG comprises a molecular weight of at least about 3.4 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight of less than or equal to 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, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 5 kDa to about 1100 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 10 kDa to about 1090 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range ofAttorney Docket No. 48469-711.601from about 15 kDa to about 1080 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 20 kDa to about 1070 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 25 kDa to about 1060 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 30 kDa to about 1050 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 35 kDa to about 1040 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 40 kDa to about 1030 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 45 kDa to about 1020 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 50 kDa to about 1010 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 55 kDa to about 1000 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 60 kDa to about 990 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 65 kDa to about 980 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 70 kDa to about 970 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 75 kDa to about 960 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 80 kDa to about 950 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 85 kDa to about 940 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 90 kDa to about 930 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 95 kDa to about 920 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 100 kDa to about 910 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 110 kDa to about 900 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 120 kDa to about 890 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 130 kDa to about 880 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 140 kDa to about 870 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 150 kDa to about 860 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 160 kDa to about 850 kDa. In someAttorney Docket No. 48469-711.601embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 170 kDa to about 840 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 180 kDa to about 830 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 190 kDa to about 820 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 200 kDa to about 810 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 210 kDa to about 800 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 220 kDa to about 790 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 230 kDa to about 780 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 240 kDa to about 770 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 250 kDa to about 760 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 260 kDa to about 750 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 270 kDa to about 740 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 280 kDa to about 730 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 290 kDa to about 720 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 300 kDa to about 710 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 310 kDa to about 700 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 320 kDa to about 690 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 330 kDa to about 680 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 340 kDa to about 670 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 350 kDa to about 660 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 360 kDa to about 650 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 370 kDa to about 640 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 380 kDa to about 630 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 390 kDa to about 620 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS)Attorney Docket No. 48469-711.601comprises a molecular weight range of from about 400 kDa to about 610 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 410 kDa to about 600 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 420 kDa to about 590 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 430 kDa to about 580 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 440 kDa to about 570 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 450 kDa to about 560 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 460 kDa to about 550 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 470 kDa to about 540 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 480 kDa to about 530 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 490 kDa to about 520 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) comprises a molecular weight range of from about 500 kDa to about 510 kDa.

[0069] In some embodiments, the microgel particles can be functionalized to comprise one or more functional groups. For example, a microgel particle made of hydrogel may be functionalized to comprise a functional group coupled thereto. In some embodiments, the functional group comprises a hydroxyl functional group, methyl functional group, carbonyl functional group, carboxyl functional group, amino functional group, phosphate functional group, sulfhydryl functional group, or a combination thereof. In some embodiments, the functional group comprises alkanes, alkenes, alkynes, ethers, sulfides, amines, aldehydes, ketones, imines, nitriles, or a combination thereof. In some embodiments, the functional group may be coupled to the microgel particle using a bond, linkage, interaction, or other coupling mechanism. In some embodiments, the bond is a covalent bond. In some embodiments, the bond is a non-covalent bond. In some embodiments, the bond is selected from a carbon-carbon bond, an amide bond, an imine bond, an ester bond, a thioether bond, a disulfide bond, a hydrazone bond, a hydrogen bond, and a metal ligand bond. In some embodiments, the ester bond comprises a cyclic boronate ester. 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 matrix metalloproteinase (MMP)-sensitive peptide. Non-limiting examples of thiol derivativesAttorney Docket No. 48469-711.601include any organosulfur compound of the form R-SH, where R represents an alkyl or other organic substituent. In some embodiments, the thiol derivatives include: methanethiol, ethanethiol, 1 -propanethiol, 2-propoanethiol, allyl mercaptan, butanethiol, tert-butyl mercaptan, pentanethiols, 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-l-propanesulfonic acid, 1 -hexadecanethiol, pentachlorobenzenethiol, or a combination thereof. In some embodiments, the functional group comprises a vinyl or a derivative thereof. In some embodiments, the functional group comprises a vinyl sulfone (VS) or a derivative thereof. Nonlimiting examples of vinyl derivatives include alkenes comprising ethenes, propenes, butenes, pentenes, hexenes, heptenes, octenes, acrylate, methacrylate, acrylamide, methacrylamide, maleimide, norbornene, or a combination 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 VS, or derivatives of either the thiol or the VS. In some embodiments, the PEG is modified to comprise the VS or the derivative thereof to form PEG-VS. In some embodiments, the PEG is modified to comprise the thiol or the 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 multiarm PEG-VS comprises, 4-arm or 6-arm or 8-arm PEG-VS. In some embodiments, the multi -arm PEG-VS comprises star-shaped polymer, brushed polymer, branched polymer, comb polymer or dendritic polymer PEG-VS. In some embodiments, the VS comprises vinyl sulfone. In some embodiments, the PEG-VS comprises 4-arm PEG-VS.

[0070] In some embodiments, the microgel particles comprise functional groups that may be pH responsive (e.g., pH responsive microgel particles). In some embodiments, a pH responsive microgel particle may be characterized as microgel particles needing to be in the presence of a desired pH range for the annealing of the annealed scaffold to occur.

[0071] Functional groups disclosed herein may comprise a peptide. Functional groups disclosed herein may comprise an amino acid. In some embodiments, a functional group comprises a K peptide. In some embodiments, the K peptide comprises an amino acid sequence that is about 85% identical to an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptides 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 an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an amino acid sequence that is about 85% identical to an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an aminoAttorney Docket No. 48469-711.601acid sequence that is about 95% identical to an amino acid sequence provided in SEQ ID NO: 3. 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 an amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide comprises an amino acid sequence that is about 75% identical to an 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 an 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 an 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 an 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 an amino acid sequence provided in NQEQVSPLGGERCG (SEQ ID NO: 5). In some embodiments, the Q peptide comprises an amino acid sequence that is about 75% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is about 85% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is about 95% identical to an amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q 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 an amino acid sequence provided in SEQ ID NO: 5.

[0072] Functional groups may be incorporated into a non-peptide polymer. In some embodiments, the non-peptide polymer is a synthetic or natural polymer. Degradable functional groups may also be random sequences, Omi target sequences, Heat-Shock Protein target sequences. The functional group may comprise an amino acid having D chirality. The functional group may comprise an amino acid having L chirality. Functional groups may comprise hydrolytically degradable synthetic polymers consisting of polyacrylamides, polymethacrylates, copolymers and terpolymers of polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes). The functional group may be synthetically manufactured or naturally isolated. In some embodiments, the functional groups may comprise heparin or alginate. In some embodiments, the functional groups may comprise polyethylene glycol). The functional group may comprise DNA oligonucleotides with sequences corresponding to: restriction enzyme recognition sequences, CpG motifs, Zinc finger motifs, CRISPR or Cas-9 sequences, Talon recognition sequences, or transcription factor-binding domains. In some embodiments, a functional group may be present on at least two ends of a microgel particle. InAttorney Docket No. 48469-711.601some embodiments, a first functional group may react with a second functional group, allowing the crosslinker to participate in the crosslinking reaction to form the microgel particles (intracrosslinking within particles) or to anneal particles together to form the annealed scaffold (intercrosslinking between particles), where these functionalities can include: cysteine amino acids, synthetic and naturally occurring thiol-containing molecules, carbene-containing groups, vinylcontaining groups, activated esters, acrylates, norborenes, primary amines, hydrazides, phosphenes, azides, epoxy-containing groups, SANPAH containing groups, and diazirine containing groups. In some embodiments, microgel particles themselves may act as crosslinkers. In some embodiments, the functional groups may be degradable.

[0073] In some embodiments, the microgel particles may be functionalized with an acrylate, methacrylate, methacrylamide, maleimide, norbornenes, or any other vinyl derivative. For example, the hydrogel formulation may further comprise two or more acrylates, methacrylates, acrylamides, maleimides, norbomenes, or any combination thereof.

[0074] In some embodiments, the microgel particles are drug eluting, such that a therapeutic agent disclosed herein is released by the microgel particles in situ. In some embodiments, the therapeutic agent comprises a pain medication, a local anesthetic, an anti-inflammatory medication, an anti-fibrotic medication, or an antibiotic. In some embodiments, the local anesthetic is ester based. In some embodiments, the ester based local anesthetic comprises benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is amide based. In some embodiments, the amide based 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 pain medication comprises codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory medication is a non-steroidal 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 medication comprises pentoxifylline.

[0075] In some embodiments herein, the microgel particles are characterized as having a degree of polydispersity. In some embodiments, the poly dispersity of the microgel particles is an indicator as to the heterogeneity of the microgel particles based on size. In some embodiments, the microgel particles comprise a poly dispersity of no more than 0.1 when the poly dispersityAttorney Docket No. 48469-711.601(PDI) is calculated based on a standard deviation (SD) and mean size of the microgel particles (e.g., PDI = (SD / mean)2). In some embodiments, the poly dispersity is measured using a coefficient of variant (CV), wherein the coefficient of variation is calculated based on a standard deviation (SD) and mean size of the microgel particles (e.g., CV = SD / mean). In some embodiments, the CV is, is about, is at most, or is at most about 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%, or any value therebetween. In some embodiments, the CV is, is about, is less than, or is less than about 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%, or any value therebetween. In some embodiments, a lower poly dispersity, based on microgel particle size, assists in forming the annealed scaffold. In some embodiments, a lower poly dispersity, based on microgel particle size, assists in achieving desired mechanical properties of the annealed scaffold. In some embodiments, a low poly dispersity improves the porosity of the composition (e.g., as the size of particles becomes more polydisperse, this may lead to smaller particles inserting into the pores of the annealed scaffold).

[0076] In some embodiments, the components of the annealed scaffold discussed above can aid in the synthesis of the microgel particles. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are configured to interact with each other in a reaction to synthesize the microgel particles.

[0077] In some embodiments, the reaction comprises a covalent synthesizing reaction. Nonlimiting examples of a covalent bond are bonds found in a carbon-carbon, amide, ester, thioether bond, carbamate, disulfide bond, oxime, thiourea, hydrazone, and imine. In some embodiments, the reaction comprises a non-covalent synthesizing reaction. Non-limiting examples of non-covalent bonds are those found in an interaction such as, electrostatic interactions, hydrogen bonding, cation-7t, 71-71 stacking, metal-ligand binding, and van der Waals interactions. In some embodiments, the methods comprise linking two or more microgel particles together. Nonlimiting 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 bonding, hydrazone bonding, oxime coupling, and thiourea coupling.

[0078] In some embodiments, the PEG-dithiol and the 4-arm PEG vinyl sulfone are present to provide a ratio of thiol to vinyl sulfone of less than about 1.0.Attorney Docket No. 48469-711.601

[0079] In some embodiments, the reaction comprises a covalent synthesizing reaction. In some embodiments, the covalent synthesizing reaction comprises a Michael addition (e.g., thiolene Michael addition, aza-Michael addition, oxa-Michael addition) or a pseudo-Mi chael addition reaction. In some embodiments, the VS of the PEG-VS may undergo a covalent synthesizing reaction with a thiol or a derivative thereof. In some embodiments, the VS or derivative thereof and the thiol or derivative thereof is present in a ratio of VS to thiol of less than about 1.0. In some embodiments, the VS or derivative thereof and the thiol or derivative thereof is present in a ratio of VS to thiol of about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. In some embodiments, the VS or derivative thereof and the thiol or derivative thereof is present in a ratio of thiol to VS of less than about 1.0. In some embodiments, the VS or derivative thereof and the thiol or derivative thereof is present in a ratio of thiol to VS of about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. In some embodiments, the thiol or the derivative thereof is a Michael donor in the Michael addition or pseudo-Mi chael addition reaction. In some embodiments, the VS or derivative thereof is a Michael acceptor in the Michael addition or pseudo -Mi chael addition reaction. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are present in the hydrogel formulation at a molar ratio of about 1 : 1. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are present in the hydrogel formulation at a molar ratio of about 0.3:1 thiol: VS to 1:1 thiol: VS. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are present in the hydrogel formulation at a molar ratio of about 0.6:1 thiol: VS to 0.8:1 thiol: VS. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are present in the hydrogel formulation at a molar ratio of about 1 : 1 to about 1 :2 thiol: VS. In some embodiments, the thiol or the derivative thereof and the VS or the derivative thereof are present in the hydrogel formulation at 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 thiols, [VS] is defined as molar concentration of VS, nSH is defined as molar number of thiols and nVS is defined as molar number of VS. In some embodiments, there is an excess of either of the thiol or the derivative thereof and the VS or the derivative thereof in the hydrogel formulation such that the excess of either of the thiol or the derivative thereof or the VS or the derivative thereof participates in the annealing reaction to form the porous annealed scaffold. In some embodiments, the vinyl sulfone of the 4-arm PEG vinyl sulfone is a Michael acceptor in the Michael addition or pseudo-Mi chael addition reaction. In some embodiments, the thiol of the PEG-dithiol is a Michael donor in the Michael addition or pseudo -Mi chael addition reaction. InAttorney Docket No. 48469-711.601some embodiments, the PEG-dithiol is present in the hydrogel system in a molar concentration of at least about 0.2 millimolar (mM).

[0080] In some embodiments, the synthesis of the microgel particles may be accomplished via one or more physical linking points of the polymer(s) (e.g., PEG) that make up microgel particles. In some embodiments, physical linking may include weak physical interactions. In some embodiments, the weak physical interactions may include coordination bonding and ionic interactions. In some embodiments, the physical linking points assist the reaction in synthesizing the microgel particles. In some embodiments, microgel particle synthesis is accomplished by the reaction alone. In some embodiments, microgel particle synthesis is accomplished by physical linking alone.

[0081] In some embodiments, the surrogate gel formed with the same precursor solution that is used to make the microgel particles but that is not emulsified in an oil phase to produce microspheres microgel particles comprises an elastic compressive modulus of 1,000 Pascals (Pa) to 1,000,000 Pa. In some embodiments, the elastic compressive modulus of the surrogate gel can be adjusted by adjusting the molecular weight, percent substitution, and molar ratios of the hydrogel polymer components. In some embodiments, the surrogate gel comprises an elastic compressive modulus of greater 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; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000; 18,000; 19,000; 20,000; 21,000; 22,000; 23,000; 24,000; 25,000; 26,000; 27,000; 28,000; 29,000; 30,000; 31,000; 32,000; 33,000; 34,000; 35,000; 36,000; 37,000; 38,000; 39,000; 40,000; 41,000; 42,000; 43,000; 44,000; 45,000; 46,000; 47,000; 48,000; 49,000; 50,000; 51,000; 52,000; 53,000; 54,000; 55,000; 56,000; 57,000; 58,000; 59,000; 60,000; 61,000; 62,000; 63,000; 64,000; 65,000; 66,000; 67,000; 68,000; 69,000; 70,000; 71,000; 72,000; 73,000; 74,000; 75,000; 76,000; 77,000; 78,000; 79,000; 80,000; 81,000; 82,000; 83,000; 84,000; 85,000; 86,000; 87,000; 88,000; 89,000; 90,000; 91,000; 92,000; 93,000; 94,000; 95,000; 96,000; 97,000; 98,000; 99,000; 100,000; 125,000; 150,000; 175,000; 200,000; 225,000; 250,000; 275,000; 300,000; 325,000; 350,000; 375,000; 400,000; 425,000; 450,000; 475,000; 500,000; 525,000; 550,000; 575,000; 600,000; 625,000; 650,000; 675,000; 700,000, 725,000; 750,000; 775,000; 800,000; 825,000; 850,000; 875,000; 900,000; 925,000; 950,000; 975,000; or 1,000,000 Pa, or any value therebetween. In some embodiments, the surrogate gel comprises an elastic compressive modulus 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; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000; 18,000; 19,000; 20,000; 21,000; 22,000; 23,000; 24,000; 25,000; 26,000; 27,000; 28,000; 29,000; 30,000; 31,000; 32,000; 33,000; 34,000; 35,000; 36,000; 37,000; 38,000; 39,000; 40,000; 41,000; 42,000; 43,000; 44,000; 45,000; 46,000; 47,000; 48,000; 49,000; 50,000; 51,000; 52,000; 53,000; 54,000; 55,000; 56,000; 57,000; 58,000; 59,000; 60,000;Attorney Docket No. 48469-711.60161,000; 62,000; 63,000; 64,000; 65,000; 66,000; 67,000; 68,000; 69,000; 70,000; 71,000; 72,000; 73,000; 74,000; 75,000; 76,000; 77,000; 78,000; 79,000; 80,000; 81,000; 82,000; 83,000; 84,000; 85,000; 86,000; 87,000; 88,000; 89,000; 90,000; 91,000; 92,000; 93,000; 94,000; 95,000; 96,000; 97,000; 98,000; 99,000; 100,000; 125,000; 150,000; 175,000; 200,000; 225,000; 250,000;275,000; 300,000; 325,000; 350,000; 375,000; 400,000; 425,000; 450,000; 475,000; 500,000; 525,000; 550,000; 575,000; 600,000; 625,000; 650,000; 675,000; 700,000, 725,000; 750,000; 775,000; 800,000; 825,000; 850,000; 875,000; 900,000; 925,000; 950,000; 975,000; or 1,000,000 Pa, or any value therebetween.

[0082] In some embodiments, the microgel particles are present in the suspension at a volume fraction of at least 80%. In some embodiments, the microgel particles are present in the suspension at a volume fraction of at least 70%.

[0083] In some embodiments, the microgel particles comprise a volume fraction (VF) of about 50% to about 100%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 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%, 98%, 99%, or 100%, or any value therebetween. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 50%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 55%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 60%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 65%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 70%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 75%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 80%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 85%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 90%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 95%. In some embodiments, the microgel particles comprise a volume fraction (VF) of about 100%. In some embodiments, the microgel particles (before annealing) comprise a volume fraction (VF) greater than the VF of pre-annealed microgel particles (after annealing).

[0084] In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 500 Pascals (Pa) before the annealing reaction.

[0085] In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 1,500 Pascals (Pa) after the annealing reaction.Attorney Docket No. 48469-711.601

[0086] In some embodiments, the microgel particles (before annealing) comprise an elastic compressive modulus of 100 Pascals (Pa) to 100,000 Pa. In some embodiments, the elastic compressive modulus of the microgel particles can be adjusted by adjusting the molecular weight, percent substitution, and molar ratios of the hydrogel polymer components. In some embodiments, the microgel particles comprise an elastic compressive modulus of greater than or equal to about 100; 200; 300; 400; 500; 600; 700; 800; 900; 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 any value therebetween. In some embodiments, the microgel particles comprise an elastic compressive modulus of less than or equal to about 100; 200; 300; 400; 500; 600; 700; 800; 900; 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 any value therebetween. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 100 Pa to about 10,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 10,000 Pa to about 105,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 15,000 Pa to about 100,000. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 20,000 Pa to about 95,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 25,000 Pa to about 90,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 30,000 Pa to about 85,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 35,000 Pa to about 80,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 40,000 Pa to about 75,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 45,000 Pa to about 70,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 50,000 Pa to about 65,000 Pa. In some embodiments, the microgel particles comprise an elastic compressive modulus in a range of about 55,000 Pa to about 60,000 Pa.

[0087] In some embodiments, the microgel particles (before annealing) comprise a storage modulus of 10 Pascals (Pa) to 10,000 Pa. In some embodiments, the microgel particles comprise a storage modulus of 10 Pa to 1,000 Pa. In some embodiments, the microgel particles comprise a storage modulus of greater than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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,Attorney Docket No. 48469-711.601950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa, or any value therebetween. In some embodiments, the microgel particles comprise a storage modulus of less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 any value therebetween. In some embodiments, the microgel particles comprise a storage modulus in a range of about 5,000 Pa to about 110,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 10,000 Pa to about 105,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 15,000 Pa to about 100,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 20,000 Pa to about 95,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 25,000 Pa to about 90,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 30,000 Pa to about 85,000 Pa. In some embodiments, the microgel particles comprise n storage modulus in a range of about 35,000 Pa to about 80,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 40,000 Pa to about 75,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 45,000 Pa to about 70,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 50,000 Pa to about 65,000 Pa. In some embodiments, the microgel particles comprise a storage modulus in a range of about 55,000 Pa to about 60,000 Pa.

[0088] In some embodiments, the microgel particles (before annealing) comprise a loss modulus of about 1 Pascals (Pa) to 10,000 Pa. Loss modulus may be measured by undergoing a measurement of shear modulus as described above and performing an amplitude and frequency sweep of shear stress in a parallel plate system. This may enable calculation of both the storage and the loss modulus of the viscoelastic material (together the storage and loss modulus comprise the shear modulus). In some embodiments, the microgel particles comprise a loss modulus that is greater than or equal to 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 any value therebetween. In some embodiments, the microgel particles comprise a loss modulus that is less than or equal to 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 any value therebetween. In some embodiments, the microgel particles comprise aAttorney Docket No. 48469-711.601loss modulus of about 10 Pa to 11,000 Pa, 20 Pa to 10500Pa, 30 Pa to 10000 Pa, 40 Pa to 9500 Pa, 50 Pa to 9000 Pa, 60 Pa to 8500 Pa, 70 Pa to 8000 Pa, 80 Pa to 7500 Pa, 90 Pa to 7000 Pa, 100 Pa to 6500 Pa, 150 Pa to 6000 Pa, 200 Pa to 5500 Pa, 250 Pa to 5000 Pa, 300 Pa to 4500 Pa, 350 Pa to 4000 Pa, 400 Pa to 3500 Pa, 450 Pa to 3000 Pa, 500 Pa to 2500 Pa, 550 Pa to 2000 Pa, 600 Pa to 1500 Pa, 650 Pa to 1000 Pa, 700 Pa to 950 Pa, 750 Pa to 900 Pa, or 800 Pa to 850 Pa.

[0089] In some embodiments, the microgel particles (before annealing) comprise an apparent viscosity of 0.1 to 100,000 Pascal-second (Pa*s) when testing using a shear rate of from about 0.1 s'1to 1,000 s'1. In some embodiments, the microgel particles comprise an apparent viscosity of greater than or equal to about 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 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; 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; 98; 99; 100; 100; 125; 150; 175; 200; 225; 250; 275; 300; 325; 350; 375; 400; 425; 450; 475; 500; 525; 550; 575; 600; 625; 650; 675; 700; 725; 750; 775; 800; 825; 850; 875; 900; 925; 950; 975; 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; 20,000; 21,000; 22,000; 23,000; 24,000; 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*s, or any value therebetween, when testing using a shear rate of from about 0.1 s'1to 1,000 s'1. In some embodiments, the volume fraction of the microgel particles and the elastic compressive modulus of the microgel particles can be adjusted to achieve a desired viscosity.

[0090] In some embodiments, the one or more components that facilitate or induce annealing of the microgel particles in the hydrogel formulation to form the annealed scaffold comprises annealing components, annealing agents, or a combination thereof. In some embodiments, the annealing agent comprises a molecule. In some embodiments, the annealing agent comprises PEG-dithiol. In some embodiments, the annealing agent comprises an enzyme. In some embodiments, the enzyme comprises thrombin. In some embodiments, the annealing agent comprises a transglutaminase enzyme. A non-limiting example of a transglutaminase enzyme Factor XIII (Factor Xllla in its active form). In some embodiments, the annealing agent comprises a radical initiator. In some embodiments, the annealing agent comprises an electron transfer agent. Examples of additional and alternative annealing agents include, by way of nonlimiting example, active esters and nucleophiles, catechols that crosslink upon oxidation, and other redox sensitive molecules. In some embodiments, the annealing agents comprise homo or heterofunctional polymers containing thiols, maleimides, vinyl sulfones, methacrylates, methacrylamides, or other vinyl functionalities. In some embodiments, the annealing agentAttorney Docket No. 48469-711.601comprises cyclodextrin, cucurbituril, or calixarenes. In some embodiments, the annealing components comprise a K peptide, a Q peptide, or a combination thereof. In some embodiments, the annealing components comprise a vinyl group (e.g. vinyl sulfone, methylacrylate, acrylamide), a thiol, a maleimide, or an amine. In some embodiments, the annealing components comprise a vinyl sulfone group and a thiol group.

[0091] In some embodiments, the microgel particles do not require an annealing agent for annealing. For example, the microgel particles may comprise other components (e.g., functional groups) that participate in a chemical crosslinking reaction to form the annealed scaffold. In some embodiments, one or more components that facilitate annealing of the microgel particles in the hydrogel formulation to form the annealed scaffold comprises a thiol derivative, a vinyl derivative, or a combination thereof. For example, microgel particles may be comprised of a polymer or copolymer that is modified to contain one or more vinyl derivatives and one or more thiol derivatives, in which either of the vinyl derivative and the thiol derivative is in excess. Such vinyl derivative and thiol derivative may undergo a chemical crosslinking reaction to anneal the microgel particles together to form the annealed scaffold.

[0092] In some embodiments, the crosslinking reaction comprises a chemical crosslinking reaction. In some embodiments, the chemical crosslinking reaction comprises a Michael addition or a pseudo-Mi chael addition reaction. In some embodiments, the chemical crosslinking reaction is a Michael addition reaction. In some embodiments, the chemical crosslinking reaction is a pseudo-Mi chael addition reaction.

[0093] In some embodiments, the crosslinking reaction comprises a physical crosslinking reaction. In some embodiments, the physical crosslinking reaction comprises electrostatic interactions or hydrogen bonding. In some embodiments, the physical crosslinking reaction comprises electrostatic interactions and hydrogen bonding. In some embodiments, the physical crosslinking reaction is electrostatic interactions. In some embodiments, the physical crosslinking reaction is hydrogen bonding.

[0094] In some embodiments, the crosslinking reaction comprises a physical and chemical crosslinking reaction.

[0095] In some embodiments, the crosslinking reaction is reversible. In some embodiments, the crosslinking reaction is reversible in response to a stimulus. In some embodiments, the stimulus is physical force. In some embodiments, the stimulus is shear strain.

[0096] In some embodiments, the crosslinking reaction is reversible for at least 5 cycles. In some embodiments, the crosslinking reaction is reversible for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more cycles. In some embodiments, the crosslinking reaction is reversible for at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or fewer cycles.Attorney Docket No. 48469-711.601

[0097] In some embodiments, the annealing reaction to anneal the microgel particles together to form the annealed scaffold comprises a covalent bond. Non-limiting examples of a covalent bond are bonds found in a carbon-carbon, amide, ester, thioether bond, carbamate, disulfide bond, oxime, thiourea, hydrazone, and imine. In some embodiments, the annealing reaction comprises a Michael addition (e.g., thiol-ene Michael addition) or a pseudo-Michael addition reaction. In some embodiments, the thiol derivative is a Michael donor in the Michael addition or pseudo-Michael addition reaction. In some embodiments, the vinyl derivative (e.g., vinyl sulfone) is a Michael acceptor in the Michael addition or pseudo-Michael addition reaction. In some embodiments, the annealing reaction comprises a hydroxyl (oxo-Michael addition) reaction or an amine (aza-Michael addition) reaction. In some embodiments, the annealing reaction comprises a “click” chemistry reaction. Non-limiting examples of “click” chemistry reactions are azide-alkyne Huisgen cycloadditions, Diels-Alder reactions, and nucleophilic ring opening reactions. In some embodiments, the annealing reaction may be reversible (FIG. 2B).

[0098] In some embodiments, the annealing reaction to anneal the microgel particles together to form an annealed scaffold comprises a non-covalent bond. Non-limiting examples of non-covalent bonds are those found in an interaction such as, electrostatic interactions, hydrogen bonding, cation-7t, 71-71 stacking, metal-ligand binding, van der Waals interactions, and host-guest interactions such as a cyclodextrin-adamantane reaction. In some embodiments, the annealing reaction comprising a host-guest interaction may be reversible.

[0099] In some embodiments, the functional groups and the one or more components assisting in the annealing reaction are configured to interact to perform the annealing reaction. In some embodiments, the PEG-dithiol is configured to interact with the vinyl sulfone (VS) of the PEG- VS in the annealing reaction to form the annealed scaffold. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of at least about 0.02 millimolar (mM). In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of at least about 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of less than about 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0 mM. In some embodiments, the PEG-dithiol is present in the hydrogel system in a molar concentration of about 0.1 to about 2.0 mM. In some embodiments, the PEG-dithiol is present in the hydrogel system in a molar concentration of about 0.2 to about 1.9 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.3 to about 1.8 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.4 to about 1.7 mM. In some embodiments, the PEG-dithiol is present inAttorney Docket No. 48469-711.601the annealed scaffold in a molar concentration of about 0.5 to about 1.6 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.6 to about 1.5 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.7 to about 1.4 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.8 to about 1.3 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.9 to about 1.2 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.2 to about 1.0 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.2 to about 0.9 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.2 to about 0.7 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.2 to about 0.6 mM. In some embodiments, the PEG-dithiol is present in the annealed scaffold in a molar concentration of about 0.2 to about 0.5 mM.

[0100] In some embodiments, the annealed scaffold further comprises PEG-divinyl sulfone or a derivative thereof. In some embodiments, the PEG-divinyl sulfone may be the components assisting in the annealing reaction. In some embodiments, the PEG-divinyl sulfone or derivative thereof is configured to interact with the excess thiol or derivative thereof in the annealing reaction to form the annealed scaffold. In some embodiments, the divinyl sulfone or derivative thereof of the PEG-divinyl sulfone is a Michael acceptor in the Michael addition or pseudoMichael addition reaction. In some embodiments, the excess thiol is a Michael donor in the Michael addition or pseudo-Mi chael addition reaction.

[0101] In some embodiments, the annealing agent comprises PEG. In some embodiments, the annealing agent comprises PEG-dithiol. In some embodiments, the PEG-dithiol comprises a molecular weight of about 0.5 kDa to about 10 kDa. In some embodiments, the PEG-dithiol comprises a molecular weight of at least about 3.4 kDa.

[0102] In some embodiments, the PEG-dithiol comprises a molecular weight of about 0.5, 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 greater than about 0.5, 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 0.5, 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 0.5 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 aAttorney Docket No. 48469-711.601molecular 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 linear PEG-dithiol, multi-arm PEG-dithiol, or a combination thereof. In some embodiments, the PEG-thiol comprises a multi -arm PEG- thiol. In some embodiments, the multiarm PEG-thiol comprises, 4-arm or 6-arm or 8-arm PEG- thiol. In some embodiments, the multiarm PEG- thiol comprises star-shaped polymer, brushed polymer, branched polymer, comb polymer or dendritic polymer PEG- thiol.

[0103] In some embodiments, the microgel particles are annealed to form the annealed scaffold in the presence of one or more buffers. Non-limiting examples of buffers include 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer (pH 7.4), calcium chloride (CaCh), phosphate buffered saline (PBS) (pH 6.0 to 8.0), TRIS, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid buffer (MES), or a combination thereof. In some embodiments, an additional stimulus is added to catalyze the reaction, such as light or pH.

[0104] In some embodiments, the annealed scaffold comprises pores comprising a median pore diameter of about 5 pm and above.

[0105] In some embodiments, the pores comprise a median pore diameter of about 10 pm to about 35 pm.

[0106] In some embodiments, the annealed scaffold is porous (FIGS. 9A-9C). By way of example, FIGS. 9A-9C depict the porosity of annealed MAP at various volume fractions. In some embodiments, percent porosity and pore size are affected by the volume fraction (VF) of MAP. In some embodiments, the annealed scaffold comprises pores having a median pore diameter comprising more than or equal to about 5 pm. In some embodiments, the pores comprise a median pore diameter of about 10 pm to about 35 pm. In some embodiments, the pores comprise a median pore diameter of greater than or equal to about 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, 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, or 70 pm, or any value therebetween. In some embodiments, the pores comprise a median pore diameter of less than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 pm. In some embodiments, the pores comprise a median pore diameter of about 5 pm to about 70 pm. In some embodiments, the pores comprise a median pore diameter of about 10 pm to about 65 pm. In some embodiments, the pores comprise a median pore diameter of about 15 pm to about 60 pm. In some embodiments, the pores comprise a median pore diameter of about 20 pm to about 55 pm. In some embodiments, the pores comprise a medianAttorney Docket No. 48469-711.601pore diameter of about 25 pm to about 50 pm. In some embodiments, the pores comprise a median pore diameter of about 30 pm to about 45 pm. In some embodiments, the pores comprise a median pore diameter of about 35 pm to about 40 pm. Median pore diameter may be measured by a process comprising, for a sampling of pores, (1) measuring the area of pore, (2) solving for the radius of the pore using the equation for the area of a circle (i.e., A = 7tr2) and (3) solving for the diameter by multiplying the radius by two (i.e., D = 2r).

[0107] In some embodiments, the annealed scaffold is degradable in vivo by one or more degradation pathways. In some embodiments, the one or more degradation pathways comprises oxidative degradation, enzymatic degradation, photodegradation, or hydrolytic degradation. In some embodiments, the composition of the microgel particles is fine tuned to achieve a desired degradation profile depending on the application.

[0108] In some embodiments, the annealed scaffold is present in the tissue site for about 1 to 4 weeks before complete degradation. In some embodiments, the annealed scaffold is present in the tissue site for about 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more before complete degradation. In some embodiments, the annealed scaffold is present in the tissue site for about 1 to 2 months before complete degradation. In some embodiments, the annealed scaffold is present in the tissue site for at least about 1 month before complete degradation. In some embodiments, the annealed scaffold is present in the tissue site for at least about 2 months before complete degradation. In some embodiments, the hydrogel formulations described herein allow for increased protein, cell, and tissue growth in the cell matrix.

[0109] In some embodiments, the annealed scaffold is self-healing. In some embodiments, the scaffold comprises reversible bonds. In some embodiments, the annealed scaffold comprises reversible covalent bonds.

[0110] In some embodiments, the annealed scaffold is a shear-thinning fluid. In some embodiments, the annealed scaffold is a shear-thickening fluid. In some embodiments, the annealed scaffold is a Newtonian fluid.

[0111] In some embodiments, the annealed scaffold is loaded into a syringe.

[0112] In some embodiments, the annealed scaffold has at least 10% porosity. In some embodiments, the annealed scaffold has at most 20% porosity.

[0113] In some embodiments, the annealed scaffold has about 10 % to about 50 % porosity. In some embodiments, the annealed scaffold has about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 35 %, about 15 % to about 40 %, about 15 % to about 45 %, about 15 % to about 50 %, about 20 % to about 25 %,Attorney Docket No. 48469-711.601about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 40 % to about 45 %, about 40 % to about 50 %, or about 45 % to about 50 % porosity. In some embodiments, the annealed scaffold has about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 % porosity. In some embodiments, the annealed scaffold has at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, or about 45 % porosity. In some embodiments, the annealed scaffold has at most about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 % porosity.

[0114] In some embodiments, the annealed scaffold has a median pore area of at least 20 2jim .

[0115] In some embodiments, the annealed scaffold has a median pore area of about 20 pm2to 80 pm2. In some embodiments, the annealed scaffold has a median pore area of about 20 pm2to about 90 pm2. In some embodiments, the annealed scaffold has a median pore area of about 20 pm2to about 30 pm2, about 20 pm2to about 40 pm2, about 20 pm2to about 50 pm2, about 20 pm2to about 60 pm2, about 20 pm2to about 70 pm2, about 20 pm2to about 80 pm2, about 20 pm2to about 90 pm2, about 30 pm2to about 40 pm2,about 30 pm2to about 50 pm2, about 30 pm2to about 60 pm2, about 30 pm2to about 70 pm2, about 30 pm2to about 80 pm2, about 30 pm2to about 90 pm2, about 40 pm2to about 50 pm2, about 40 pm2to about 60 pm2, about 40 pm2to about 70 pm2, about 40 pm2to about 80 pm2, about 40 pm2to about 90 pm2, about 50 pm2to about 60 pm2, about 50 pm2to about 70 pm2, about 50 pm2to about 80 pm2, about 50 pm2to about 90 pm2, about 60 pm2to about 70 pm2, about 60 pm2to about 80 pm2, about 60 pm2to about 90 pm2, about 70 pm2to about 80 pm2, about 70 pm2to about 90 pm2, or about 80 pm2to about 90 pm2. In some embodiments, the annealed scaffold has a median pore area of about 20 pm2, about 30 pm2, about 40 pm2, about 50 pm2, about 60 pm2, about 70 pm2, about 80 pm2, or about 90 pm2. In some embodiments, the annealed scaffold has a median pore area of at least about 20 pm2, about 30 pm2, about 40 pm2, about 50 pm2, about 60 pm2, about 70 pm2, or about 80 pm2. In some embodiments, the annealed scaffold has a median pore area of at most about 30 pm2, about 40 pm2, about 50 pm2, about 60 pm2, about 70 pm2, about 80 pm2, or about 90 pm2.

[0116] In some embodiments, the annealed scaffold has an average pore area of at most 1000 2jim .Attorney Docket No. 48469-711.601

[0117] In some embodiments, the annealed scaffold has an average pore area of about 100 pm2to 1000 pm2. In some embodiments, the annealed scaffold has an average pore area of about 100 pm2to about 1,000 pm2. In some embodiments, the annealed scaffold has an average pore area of about 100 pm2to about 200 pm2, about 100 pm2to about 300 pm2, about 100 pm2to about 400 pm2, about 100 pm2to about 500 pm2, about 100 pm2to about 600 pm2, about 100 pm2to about 700 pm2, about 100 pm2to about 800 pm2, about 100 pm2to about 900 pm2, about 100 pm2to about 1,000 pm2, about 200 pm2to about 300 pm2, about 200 pm2to about 400 pm2, about 200 pm2to about 500 pm2, about 200 pm2to about 600 pm2, about 200 pm2to about 700 pm2, about 200 pm2to about 800 pm2, about 200 pm2to about 900 pm2, about 200 pm2to about 1,000 pm2, about 300 pm2to about 400 pm2, about 300 pm2to about 500 pm2, about 300 pm2to about 600 pm2, about 300 pm2to about 700 pm2, about 300 pm2to about 800 pm2, about 300 pm2to about 900 pm2, about 300 pm2to about 1,000 pm2, about 400 pm2to about 500 pm2, about 400 pm2to about 600 pm2, about 400 pm2to about 700 pm2, about 400 pm2to about 800 pm2, about 400 pm2to about 900 pm2, about 400 pm2to about 1,000 pm2, about 500 pm2to about 600 pm2, about 500 pm2to about 700 pm2, about 500 pm2to about 800 pm2, about 500 pm2to about 900 pm2, about 500 pm2to about 1,000 pm2, about 600 pm2to about 700 pm2, about 600 pm2to about 800 pm2, about 600 pm2to about 900 pm2, about 600 pm2to about 1,000 pm2, about 700 pm2to about 800 pm2, about 700 pm2to about 900 pm2, about 700 pm2to about 1,000 pm2, about 800 pm2to about 900 pm2, about 800 pm2to about 1,000 pm2, or about 900 pm2to about 1,000 pm2. In some embodiments, the annealed scaffold has an average pore area of about 100 pm2, about 200 pm2, about 300 pm2, about 400 pm2, about 500 pm2, about 600 pm2, about 700 pm2, about 800 pm2, about 900 pm2, or about 1,000 pm2. In some embodiments, the annealed scaffold has an average pore area of at least about 100 pm2, about 200 pm2, about 300 pm2, about 400 pm2, about 500 pm2, about 600 pm2, about 700 pm2, about 800 pm2, or about 900 pm2. In some embodiments, the annealed scaffold has an average pore area of at most about 200 pm2, about 300 pm2, about 400 pm2, about 500 pm2, about 600 pm2, about 700 pm2, about 800 pm2, about 900 pm2, or about 1,000 pm2.

[0118] In some embodiments, the annealed scaffold (e.g., the pre-annealed microgel particles) comprises an elastic compressive modulus of 1,000 Pascals (Pa) to 100,000 Pa. In some embodiments, as discussed above, the elastic compressive modulus of the annealed scaffold can be adjusted by adjusting the molecular weight, percent substitution, and molar ratios of the hydrogel polymer components. In some embodiments, the annealed scaffold comprises an elastic compressive modulus of greater 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; orAttorney Docket No. 48469-711.601110,000 Pa; or any value therebetween. In some embodiments, the annealed scaffold comprises an elastic compressive modulus 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; or any value therebetween. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 5,000 Pa to about 110,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 10,000 Pa to about 105,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 15,000 Pa to about 100,000. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 20,000 Pa to about 95,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 25,000 Pa to about 90,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 30,000 Pa to about 85,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 35,000 Pa to about 80,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 40,000 Pa to about 75,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 45,000 Pa to about 70,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 50,000 Pa to about 65,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus in a range of about 55,000 Pa to about 60,000 Pa.

[0119] In some embodiments, the annealed scaffold comprises an apparent viscosity of 0.1 Pascal-second (Pa*s) to 100,000 Pa*s when testing using a shear rate of from about 0.1 s'1to 1,000 s'1. In some embodiments, the annealed scaffold comprises an apparent viscosity of greater than or equal to about 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 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; 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; 98; 99; 100; 100; 125; 150; 175; 200; 225; 250; 275; 300; 325; 350; 375; 400; 425; 450; 475; 500; 525; 550; 575; 600; 625; 650; 675; 700; 725; 750; 775; 800; 825; 850; 875; 900; 925; 950; 975; 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; 20,000; 21,000; 22,000; 23,000; 24,000; 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*s, or any value therebetween, when testing using a shear rate of from about 0.1 s'1to 1,000 s'1. In someAttorney Docket No. 48469-711.601embodiments, the volume fraction of the annealed scaffold and the elastic compressive modulus of the annealed scaffold can be adjusted to achieve a desired viscosity. In some embodiments, the volume fraction of the microgel particles and the elastic compressive modulus of the microgel particles can be adjusted to achieve a desired elastic compressive modulus for the annealed scaffold. Both volume fraction and elastic compressive modulus of the microgel particles effect the final elastic compressive modulus of the annealed scaffold. A higher volume fraction and higher microgel particle elastic compressive modulus may lead to a higher elastic compressive modulus of the annealed scaffold.

[0120] In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 50% to about 100%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 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%, 98%, 99%, or 100%, or any value therebetween. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 50%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 55%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 60%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 65%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 70%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 75%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 80%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 85%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 90%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 95%. In some embodiments, the annealed scaffold comprises a volume fraction (VF) of about 100%. In some embodiments, the annealed scaffold (after annealing) comprises a volume fraction (VF) lower than the VF of the microgel particles (before annealing).

[0121] In some embodiments, the annealed scaffold comprises a storage modulus of 10 Pascals (Pa) to 10,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus of 10 Pa to 1,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus of greater than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 any value therebetween. In some embodiments, the annealed scaffold comprises a storage modulus of less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8,Attorney Docket No. 48469-711.6019, 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 any value therebetween. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 5,000 Pa to about 110,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 10,000 Pa to about 105,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 15,000 Pa to about 100,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 20,000 Pa to about 95,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 25,000 Pa to about 90,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 30,000 Pa to about 85,000 Pa. In some embodiments, the annealed scaffold comprises n storage modulus in a range of about 35,000 Pa to about 80,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 40,000 Pa to about 75,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 45,000 Pa to about 70,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 50,000 Pa to about 65,000 Pa. In some embodiments, the annealed scaffold comprises a storage modulus in a range of about 55,000 Pa to about 60,000 Pa.

[0122] In some embodiments, the annealed scaffold comprises a loss modulus of about 1 Pascals (Pa) to 10,000 Pa. Loss modulus may be measured by undergoing a measurement of shear modulus as described above and performing an amplitude and frequency sweep of shear stress in a parallel plate system. This may enable calculation of both the storage and the loss modulus of the viscoelastic material (together the storage and loss modulus comprise the shear modulus). In some embodiments, the annealed scaffold comprises a loss modulus that is greater than or equal to 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 any value therebetween. In some embodiments, the annealed scaffold comprises a loss modulus that is less than or equal to 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 any value therebetween. In some embodiments, the annealed scaffold comprises a loss modulus of about 10 Pa to 11,000 Pa, 20 Pa to 10500Pa, 30 Pa to 10000 Pa, 40 Pa to 9500 Pa, 50 Pa to 9000 Pa, 60 Pa to 8500 Pa, 70 Pa to 8000 Pa, 80 Pa to 7500 Pa, 90 Pa to 7000 Pa, 100 Pa to 6500 Pa, 150 Pa to 6000 Pa, 200 Pa to 5500 Pa, 250 Pa to 5000 Pa, 300 Pa to 4500 Pa,Attorney Docket No. 48469-711.601350 Pa to 4000 Pa, 400 Pa to 3500 Pa, 450 Pa to 3000 Pa, 500 Pa to 2500 Pa, 550 Pa to 2000 Pa, 600 Pa to 1500 Pa, 650 Pa to 1000 Pa, 700 Pa to 950 Pa, 750 Pa to 900 Pa, or 800 Pa to 850 Pa.

[0123] In some embodiments, the thiol or the derivative thereof and the vinyl sulfone or the derivative thereof are present in the hydrogel formulation at a molar ratio of the thiols to the VS (thiokVS) of about 0.3 to about 0.95 to achieve a desired elastic compressive modulus of about 500 Pa to about 50,000 Pa. In some embodiments, the microgel particles are present in a suspension comprising the microgel particles and water and wherein a 50% to 100% volume fraction of the suspension comprises the microgel particles to achieve a desired elastic compressive modulus.

[0124] In some embodiments, the annealed scaffold comprises a pH of 5.0 to 9.0. In some embodiments, the annealed scaffold comprises a pH of about 7 to about 9. In some embodiments, the pH is about 7.5. In some embodiments, the pH is about 8.0. In some embodiments, the annealed scaffold comprises a pH of greater than or equal to 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 any value therebetween. In some embodiments, the annealed scaffold comprises a pH of less than or equal to 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 any value therebetween. In some embodiments, the annealed scaffold comprises a pH of about 4.0 to about 10.0. In some embodiments, the annealed scaffold comprises a pH of about 4.5 to about 9.5. In some embodiments, the annealed scaffold comprises a pH of about 5.0 to about 9.0. In some embodiments, the annealed scaffold comprises a pH of about 5.5 to about 8.5. In some embodiments, the annealed scaffold comprises a pH of about 6.0 to about 8.0. In some embodiments, the annealed scaffold comprises a pH of about 6.5 to about 7.5. In some embodiments, the annealed scaffold comprises a pH of about 7.0 to about 7.5. In some embodiments, the annealed scaffold comprises a pH of about 7.0 to about 9.0. In some embodiments, the annealed scaffold comprises a pH of about 7.5 to about 8.5. In some embodiments, the annealed scaffold comprises a pH of about 8.0 to about 8.5.

[0125] In some embodiments, the annealed scaffold comprises a buffer. In some embodiments, the buffer comprises: a phosphate buffer, a 4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

[0126] In some embodiments, the annealed scaffold is loaded into a single syringe.

[0127] In some embodiments, the annealed scaffold is a shear thinning fluid. In some embodiments, the annealed scaffold is a shear-thickening fluid.

[0128] In some embodiments, the annealed scaffold is formulated for administration to a subject. In some embodiments, the annealed scaffold is formulated for administration at a suture line of the subject. In some embodiments, the administration minimizes a foreign body response in the subject.Attorney Docket No. 48469-711.601

[0129] In some embodiments, the annealed scaffold 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 particle). In some embodiments, the active agent is a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibiotic.

[0130] In some embodiments, the annealed scaffold is capable of withstanding sterilization. Sterilization can be carried out by steam sterilization, filtration, microfiltration, e-beam, gamma radiation, X-rays, ethylene oxide (ETO), light, supercritical carbon dioxide, nitrogen dioxide, vaporized hydrogen peroxide or any combination thereof. In some embodiments, certain components of the annealed scaffold may be steam sterilized (e.g., autoclaved) without degradation of physical properties, such as the microgel particles. However, when the annealed scaffold contains an additional component, such as the therapeutic agent, the component of the annealed scaffold containing the therapeutic agent can be sterilized by means other than heat treatment, such as for example using filtration sterilization.

[0131] In some embodiments, sterilization of the annealed scaffold is by autoclave.Autoclaving can be accomplished by applying a mixture of heat, pressure, and moisture to a formulation in need of sterilization. Many different sterilization temperatures, pressures and cycle times can be used. As an example, in some embodiments, the filled syringes may be sterilized at a temperature of at least about 120° C. to about 130° C. or greater. In some embodiments, the filled syringes may be sterilized at a temperature of at least about 120° C. to about 130° C. or greater. Moisture may or may not be utilized. 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 more. In some embodiments, the sterilization cycle may be at least about 1 minute to about 30 minutes or more. In some embodiments, the sterilization cycle may be at least about 15 minutes to about 30 minutes or more.

[0132] In some embodiments, the method of sterilization incorporates the use of a gaseous species which is known to kill or eliminate transmissible agents. In some embodiments, ethylene oxide is used as the sterilization gas and can sterilize medical devices, products, or any of the delivery devices disclosed herein.

[0133] In some embodiments, the method of sterilization incorporates the use of an irradiation source to kill or eliminate transmissible agents. A beam of irradiation is targeted at the delivery device (e.g., syringe) containing the annealed scaffold, and the wavelength of energy kills or eliminates the unwanted transmissible agents. As a non-limiting example, energy useful includes, but is not limited to ultraviolet (UV) light, electron (e-beam) irradiation, gammaAttorney Docket No. 48469-711.601irradiation, visible light, microwaves, X-rays or any other wavelength or band of wavelengths which kills or eliminates the unwanted transmissible agents, preferably without substantially altering of degrading the annealed scaffold.

[0134] In some embodiments, the annealed scaffold comprises a reagent, such as an annealing agent that facilitates the annealing reaction of the annealed scaffold. In some embodiments, the annealing agent comprises PEG-dithiol. In some embodiments, the annealing agent comprises a photoinitiator. By way of non-limiting example, the photoinitiator may be Eosin Y. In some embodiments, the annealing agent comprises an enzyme. In some embodiments, the enzyme comprises thrombin. In some embodiments, the annealing agent comprises a transglutaminase enzyme. A non-limiting example of a transglutaminase enzyme Factor XIII (Factor Xllla in its active form). In some embodiments, the annealing agent is endogenous to the subject or the tissue. For example, an enzyme (e.g., Factor XIII or Factor Xllla) may be present naturally in the tissue, which can participate in an annealing reaction of the annealed scaffold in situ provided that the appropriate annealing components are present in the annealed scaffold. In some embodiments, the annealing agent is autologous to the subject or tissue. For example, endogenous Factor XIII may be obtained from the subject, and delivered to the subject to participate in the annealing reaction of the annealed scaffold in situ provided that the appropriate annealing components are present. In some embodiments, the Factor XIII is modified prior to delivery to the tissue or subject. In some embodiments, the annealing agent comprises a co-initiator. In some embodiments, the annealing agent comprises an electron transfer agent. Examples of additional and alternative annealing agents include, by way of nonlimiting example, include active esters and nucleophiles, catechols that crosslink upon oxidation, and other redox sensitive molecules. In some embodiments, the annealing components comprise a K peptide, a Q peptide, or a combination thereof. In some embodiments, the reagent comprises a stabilization agent, a sterilization agent, or a heat protectant. Non-limiting examples of stabilization agents include reagents, salts, and additives. Non-limiting examples of sterilization agents include reagents, salts, and additives. Non-limiting examples of heat protectants include antioxidants, glycerine, and PEG. In some embodiments, the annealed scaffold is protected during sterilization by freezing the hydrogel prior and / or during the terminal sterilization (e.g. irradiation). In some embodiments, the annealed scaffold is protected during sterilization by placing the material under a sealed inert atmosphere or under sous-vide ampule.

[0135] In some embodiments, the annealed scaffold comprises a solvent, such as one or more buffers, water, or a combination thereof. In some embodiments, the microgel particles are present in a suspension comprising water. In some embodiments, the annealed scaffold comprises a buffer. In some embodiments, the buffer comprises: a phosphate buffer, a 4-(2-hydroxyethyl)-l-Attorney Docket No. 48469-711.601piperazineethanesulfonic acid (HEPES) buffer, a phosphate buffer, or an acetate buffer, a citrate buffer, a borate buffer, or any combination thereof. In some embodiments, the buffer adjusts the pH of the annealed scaffold to a desired pH. The pH of the disclosed annealed scaffolds 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 annealed scaffold comprises a suspension of the microgel particles in an aqueous solvent, including the buffer. In some embodiments, the buffer may be a buffering agent. In some embodiments, a 50% to 100% volume fraction of the suspension comprises the microgel particles. In some embodiments, at least a 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% volume fraction of the suspension comprises the microgel particles. In some embodiments, the volume fraction of the microgel particles is greater than or equal to about 50% when the hydrogel is formulated for administration with a needle. In some embodiments, the volume fraction of the microgel particles is greater than or equal to about 50%, 60%, 70%, 80%, or 90% when the hydrogel is formulated for administration with a needle.

[0136] In some embodiments, the annealed scaffold is colorless. In some embodiments, the annealed scaffold is substantially optically clear. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.1. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.2. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.3. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.4. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.5. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.6. In some embodiments, the poly dispersity of the annealed scaffold is no more than 0.7. In some embodiments, the annealed scaffold is odorless.

[0137] In some embodiments, the annealed scaffold is formulated for administration to a subject. In some embodiments, the administration is subdermal administration, dermal administration, intradermal administration, or subcutaneous administration. In some embodiments, administration minimizes a foreign body response in the subject. In some embodiments, the annealed scaffold is formulated for administration by a needle. In some embodiments, the annealed scaffold is formulated for administration by a microneedle or microneedle patch. In some embodiments, the annealed scaffold is formulated for administration by a needle that has a gauge that is about 26-, 27-, 28-, 29-, or 30-gauge. For example, an annealed scaffold formulated for a 27-gauge syringe may have an apparent viscosity of about 1 to 1000 Pascal* seconds when measured at shear rates between 0.1 and 10 s'1. While an annealed scaffold formulated for a 30-gauge syringe may have an apparent viscosity of about 1 Pascal* seconds to 500 Pascal* seconds when measured at shear rates between 0.1 s'1and 10 s'1.Attorney Docket No. 48469-711.601Thus, the annealed scaffold properties may be fine-tuned depending on the mode of administration.

[0138] In some embodiments, the formulation comprises a dose volume of about 0.01 mL to about 20 mL.

[0139] In some embodiments, a dose of the annealed scaffold comprises a volume of about 0.01 mL to about 20 mL. In some embodiments, the volume comprises about 0.75 milliliter (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 at greater than or equal to about 0.01 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 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, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL,10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, or 20 mL, or any value therebetween.

[0140] In some embodiments, the annealed scaffold is aseptically manufactured. In some embodiments, the annealed scaffold is sterile. In some embodiments, the annealed scaffold is formulated for sterilization by steam sterilization, filtration, microfiltration, e-beam, gamma radiation, X-rays, ethylene oxide (ETO), light, supercritical carbon dioxide, nitrogen dioxide, vaporized hydrogen peroxide or any combination thereof. In some embodiments, the annealed scaffold comprises at least two separate containers, each container suitable for sterilization by different methods. In some embodiments, the microgel particles are lyophilized. In some embodiments, the lyophilized microgel particles are stored in a first container that is capable of withstanding steam sterilization, which is separate from a second container comprising components of the annealed scaffold that may be degradable by steam sterilization, such as the therapeutic agent. In another embodiment, the annealed scaffold is stored in a single container capable of being sterilized together. In some embodiments, the system also comprises a reconstitution medium to reconstitute the lyophilized annealed scaffold. 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 the physiological pH. In some embodiments, the reconstitution medium has a pH lower than the physiological pH. In some embodiments, the reconstitution medium comprises a buffer with a varying buffer capacity.METHODS

[0141] Disclosed herein, in some embodiments, are methods of delivering the annealed scaffold disclosed herein to a subject. In some embodiments, the delivering comprisingAttorney Docket No. 48469-711.601administering the annealed scaffolds to the subject. In some embodiments, administration comprises subdermal, dermal, intradermal, or subcutaneous administration of the annealed scaffold to a tissue site of the subject. In some embodiments, administration comprises administering the annealed scaffold at the site of an injury, incision, or suture. In some embodiments, administering the annealed scaffold to a tissue site of the subject is effective to treat the tissue at or surrounding the tissue site, such as for example, improving the strength of an incision or improving the healing at a site of a suture line. In some embodiments, methods of delivering or administering the annealed scaffold disclosed herein is performed while minimizing a foreign body response elicited by the subject in response to the delivering or the administering. Also provided are methods for purifying the microgel particles of the annealed scaffolds disclosed herein, such as for example, in a water-in-oil emulsion. In some embodiment, the methods comprise lyophilizing the microgel particles to that they may be stored and / or distributed over long periods of time prior to being reconstituted and delivered at the point of need to the subject. Also provided are methods to pre-anneal the microgel particles and fill the pre-annealed microgel particles in syringes.Methods of Delivery

[0142] In certain aspects, described herein is a method of delivering an annealed scaffold to a site of injury in a subject, the method comprising: providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide in a syringe; and disposing the annealed scaffold to the site of injury in the subject, wherein the annealed scaffold loses a degree of crosslinking to form an unannealed scaffold during said disposing, and wherein the unannealed scaffold re-anneals after being disposed to the site of injury in the subject.

[0143] In certain aspects, described herein is a method of delivering an annealed scaffold to a site of injury in a subject, the method comprising: providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; and disposing the annealed scaffold onto the site of injury in the subject, wherein the annealed scaffold self-heals upon disposing,

[0144] Disclosed herein, in some embodiments, are methods of delivering the annealed scaffold provided herein to a subject. In some embodiments, the annealed scaffold is delivered to a tissue site of a subject. In some embodiments, the tissue site is a site of an injury. In someAttorney Docket No. 48469-711.601embodiments, the tissue site is a site of a suture line. In some embodiments, the annealed scaffold is delivered in a pre-annealed state. In some embodiments, the annealed scaffold is delivered to a site of a suture line. In some embodiments, the annealed scaffold allows cells to grow within the pores of the porous annealed scaffold to produce a cell matrix. In some embodiments, the cell matrix that forms persists in the subject after complete degradation of the annealed scaffold, thereby permanently filling at least a part of the tissue site of the subject while minimizing a foreign body response in the subject. The methods of delivery disclosed herein may be subdermal, dermal, intradermal, subcutaneous, intramuscular, or intraoperatively to a surgical site. In some embodiments, the methods of delivery comprise injection, such as for example, using a delivery device (e.g., syringe or applicator) disclosed herein.

[0145] In some embodiments, the annealed scaffold loses a degree of crosslinking to form an unannealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises a loss of covalent bonds in the annealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises a loss of electrostatic bonds in the annealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises a loss of covalent bonds and electrostatic bonds in the annealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises the breaking of one or more disulfide bonds in the annealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises a breaking of one or more bonds in the annealed scaffold. In some embodiments, the loss of a degree of crosslinking comprises the breaking of one or more thioether bonds in the annealed scaffold. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more disulfide bonds, thioether bonds, or a combination thereof. In some embodiments, the loss in a degree of crosslinking comprises the breaking of one or more disulfide bonds in the annealed scaffold or one or more thioether bonds in the annealed scaffold, or a combination thereof.

[0146] In some embodiments, the annealed scaffold loses a degree of crosslinking in response to shear strain to form the unannealed scaffold.

[0147] In some embodiments, the degree of crosslinking is measured by elastic modulus. In some embodiments, the loss of a degree of crosslinking is measured by a decrease in elastic modulus.

[0148] In some embodiments, the unannealed scaffold has an elastic modulus of at most about 90% compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of about 20% to about 90% compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % toAttorney Docket No. 48469-711.601about 90 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 % compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of at least about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % compared to an elastic modulus of the annealed scaffold. In some embodiments, the unannealed scaffold has an elastic modulus of at most about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 % compared to an elastic modulus of the annealed scaffold. In some embodiments, the elastic modulus is measured at a shear strain of about 0.01% to about 100%. In some embodiments, the elastic modulus is measured at a shear strain of about 1% to about 100%. In some embodiments, the unannealed scaffold has an elastic modulus of at most about 90% compared to an elastic modulus of the annealed scaffold when measured at a shear strain of about 10% or more. In some embodiments, the shear strain is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0149] In some embodiments, the degree of crosslinking is measured by viscosity. In some embodiments, the loss of a degree of crosslinking is measured by a decrease in viscosity.

[0150] In some embodiments, the unannealed scaffold has a viscosity of at most about 80% compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of about 0.1 % to about 80 % compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of about 0.1 % to about 1 %, about 0.1 % to about 10 %, about 0.1 % to about 20 %, about 0.1 % to about 30 %, about 0.1 % to about 40 %, about 0.1 % to about 50 %, about 0.1 % to about 60 %, about 0.1 % to about 70 %, about 0.1 % to about 80 %, about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 1 % to about 80 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %,Attorney Docket No. 48469-711.601about 30 % to about 70 %, about 30 % to about 80 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 60 % to about 70 %, about 60 % to about 80 %, or about 70 % to about 80 % compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of about 0.1 %, about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of at least about 0.1 %, about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, or about 70 % compared to a viscosity of the annealed scaffold. In some embodiments, the unannealed scaffold has a viscosity of at most about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % compared to a viscosity of the annealed scaffold. In some embodiments, the viscosity is measured at a shear rate of about 0.1 s'1to about 1000 s'1. In some embodiments, the unannealed scaffold has a viscosity of about 0.1% to about 80% compared to a viscosity of the annealed scaffold when measured at a shear rate of about 1 s'1or higher. In some embodiments, the viscosity is measured at a shear rate of about 1 s'1, 10 s'1, 100 s'1, 500 s'1, or 1000 s'1.

[0151] In some embodiments, the unannealed scaffold re-anneals after being disposed to the site of injury in the subject. In some embodiments, re-anneals refers to the restoration of the degree of crosslinking that was lost during said disposing step. In some embodiments, re-anneals refers to the reformation of covalent bonds and / or electrostatic bonds that were broken during said disposing step. In some embodiments, re-anneals refers to self-healing. In some embodiments, re-anneals refers to a process wherein a viscosity of the unannealed scaffold increases to about the same viscosity of the annealed scaffold prior to said disposing step. In some embodiments, about the same viscosity refers to a value within 10% deviation of the viscosity of the annealed scaffold prior to said disposing step. In some embodiments, re-anneals refers to a process wherein an elastic modulus of the unannealed scaffold increases to about the same elastic modulus of the annealed scaffold prior to said disposing step. In some embodiments, about the same elastic modulus refers to a value within 10% deviation of the elastic modulus of the annealed scaffold prior to said disposing step. In some embodiments, endogenous cells infiltrate the annealed scaffold to form a cell matrix over the site of injury in the subject.

[0152] In some embodiments, the site of injury is a site of a suture line.

[0153] In some embodiments, the method further comprises closing the suture line in the subject. In some embodiments, said disposing step and said closing step do not require a time delay. In some embodiments, said closing step occurs immediately after said disposing step.

[0154] In some embodiments, said disposing step and said closing step occur consecutively.Attorney Docket No. 48469-711.601

[0155] In some embodiments, the annealed scaffold comprises one or more crosslinking reactions. In some embodiments, the one or more crosslinking reactions is covalent, electrostatic, or both.

[0156] In some embodiments, the suture line is an incision. In some embodiments, the annealed scaffold is delivered on top of the suture line after the suture line is sutured. In some embodiments, the annealed scaffold is delivered into the suture line while the suture line is sutured. In some embodiments, the annealed scaffold is delivered on top of the suture line after the suture line is sutured and into the suture line while the suture line is sutured.

[0157] In some embodiments, the incision corresponds to the location of a surgical incision.

[0158] In some embodiments, the surgical incision comprises an abdominal fascial incision.

[0159] In some embodiments, the abdominal fascial incision comprises an abdominal wall midline or transverse incision of the fascia.

[0160] In some embodiments, the abdominal incision resulted from a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof.

[0161] In some embodiments, the disposing comprises disposing the annealed scaffold directly over the suture line after the suture line is sutured. In some embodiments, the disposing comprises disposing the annealed scaffold into the suture line while the suture line is being sutured. In some embodiments, the disposing comprises disposing the annealed scaffold into the suture line after the incision is sutured. In some embodiments, the disposing comprises disposing the annealed scaffold into the suture line while the suture line is being sutured and on top of the suture line after the suture line is sutured.

[0162] In some embodiments, the annealed scaffold is annealed when disposed on top of the suture line.

[0163] In some embodiments, the disposing comprises releasing the annealed scaffold from the syringe through a static mixer.

[0164] In some embodiments, the method further comprises strengthening the suture line at the suture line site by the cell matrix formed over the suture line site.

[0165] In some embodiments, the methods further comprise reinforcing or strengthening the suture line at the site of a suture line by the cell matrix formed over the site of a suture line. In some embodiments, the strengthening the suture line is characterized by increasing a mechanical tensile strength of the suture line as compared to a reference suture line at an otherwise identical site of a suture line that was closed without the delivery of the annealed scaffold. In some embodiments, increasing the mechanical tensile strength of the suture line is characterized byAttorney Docket No. 48469-711.601increasing the yield stress of the suture line. The yield stress may be calculated from a stress versus strain curve measured using a tensile test (e.g., on an Instron).

[0166] In some embodiments, the suture line comprises a yield stress of at least about 3.0 N / mm2to about 6.0 N / mm2at least about 42 days after suturing of the suture line.

[0167] In some embodiments, the yield stress is calculated from a stress versus strain curve measured using a tensile test (e.g., on an Instron 3342). In some embodiments, the suture line comprises a yield stress of at least about 4.0 Newtons per millimeter squared (N / mm2) measured after closure. In some embodiments, the suture line comprises a yield stress at least about 1.0 N / mm2, about 1.5 N / mm2, about 2.0 N / mm2, about 2.5 N / mm2, about 3.0 N / mm2, about 3.5 N / mm2, about 4.0 N / mm2, about 4.5 N / mm2, about 5.0 N / mm2, about 5.5 N / mm2, about 6.0 N / mm2, about 6.5 N / mm2, about 7.0 N / mm2, about 7.5 N / mm2, about 8.0 N / mm2, about 8.5 N / mm2, about 9.0 N / mm2, about 9.5 N / mm2, about 10.0 N / mm2, about 15.0 N / mm2, about 20.0 N / mm2, about 25.0 N / mm2, about 30.0 N / mm2, about 35.0 N / mm2, about 40.0 N / mm2, about 45.0 N / mm2, about 50.0 N / mm2, about 55.0 N / mm2about 60.0 N / mm2, about 65.0 N / mm2about 70.0 N / mm2, about 75.0 N / mm2about 80.0 N / mm2, about 85.0 N / mm2about 90.0 N / mm2, about 95.0 N / mm2, or about 100.0 N / mm2. In some embodiments, the suture line comprises a yield stress of no more than about 1.0 N / mm2, about 1.5 N / mm2, about 2.0 N / mm2, about 2.5 N / mm2, about 3.0 N / mm2, about 3.5 N / mm2, about 4.0 N / mm2, about 4.5 N / mm2, about 5.0 N / mm2, about 5.5 N / mm2, about 6.0 N / mm2, about 6.5 N / mm2, about 7.0 N / mm2, about 7.5 N / mm2, about 8.0 N / mm2, about 8.5 N / mm2, about 9.0 N / mm2, about 9.5 N / mm2, about 10.0 N / mm2, about 15.0 N / mm2, about 20.0 N / mm2, about 25.0 N / mm2, about 30.0 N / mm2, about 35.0 N / mm2, about 40.0 N / mm2, about 45.0 N / mm2, about 50.0 N / mm2, about 55.0 N / mm2about 60.0 N / mm2, about 65.0 N / mm2about 70.0 N / mm2, about 75.0 N / mm2about 80.0 N / mm2, about 85.0 N / mm2about 90.0 N / mm2, about 95.0 N / mm2, or about 100.0 N / mm2. In some embodiments, the yield stress is measured at least about 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, or 65 days after closure. In some embodiments, the yield stress is measured at least about 42 days after closure.

[0168] In some embodiments, the suture line comprises a yield stress of at least about 3.0 N / mm2to about 6.0 N / mm2after closure. In some embodiments, the suture line comprises a yield stress of at least about 4.0 N / mm2to about 5.0 N / mm2after closure. In some embodiments, the suture line comprises a yield stress of at least about 3.0 N / mm2to about 4.0 N / mm2after closure. In some embodiments, the suture line comprises a yield stress of greater than about 3.0 N / mm2, about 3.5 N / mm2, about 4.0 N / mm2, about 4.5 N / mm2, about 5.0 N / mm2, about 5.5 N / mm2, about 6.0 N / mm2, about 6.5 N / mm2, about 7.0 N / mm2, about 7.5 N / mm2, about 8.0 N / mm2,Attorney Docket No. 48469-711.601about 8.5 N / mm2, about 9.0 N / mm2, about 9.5 N / mm2, about 10.0 N / mm2, about 15.0 N / mm2, about 20.0 N / mm2, about 25.0 N / mm2, about 30.0 N / mm2, about 35.0 N / mm2, about 40.0 N / mm2, about 45.0 N / mm2, about 50.0 N / mm2, about 55.0 N / mm2about 60.0 N / mm2, about 65.0 N / mm2about 70.0 N / mm2, about 75.0 N / mm2about 80.0 N / mm2, about 85.0 N / mm2about 90.0 N / mm2, about 95.0 N / mm2, or about 100.0 N / mm2. In some embodiments, the suture line comprises a yield stress of less than about 3.0 N / mm2, about 3.5 N / mm2, about 4.0 N / mm2, about 4.5 N / mm2, about 5.0 N / mm2, about 5.5 N / mm2, about 6.0 N / mm2, about 6.5 N / mm2, about 7.0 N / mm2, about 7.5 N / mm2, about 8.0 N / mm2, about 8.5 N / mm2, about 9.0 N / mm2, about 9.5 N / mm2, about 10.0 N / mm2, about 15.0 N / mm2, about 20.0 N / mm2, about 25.0 N / mm2, about 30.0 N / mm2, about 35.0 N / mm2, about 40.0 N / mm2, about 45.0 N / mm2, about 50.0 N / mm2, about 55.0 N / mm2about 60.0 N / mm2, about 65.0 N / mm2about 70.0 N / mm2, about 75.0 N / mm2about 80.0 N / mm2, about 85.0 N / mm2about 90.0 N / mm2, about 95.0 N / mm2, or about 100.0 N / mm2. In some embodiments, the yield stress is measured at least about 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, or 65 days after closure. In some embodiments, the yield stress is measured at least about 42 days after closure.

[0169] In some embodiments, the strengthening the suture line is characterized by increasing a mechanical tensile strength of the suture line as compared to a reference suture line at an otherwise identical suture line site that was sutured without the delivery of the hydrogel formulation.

[0170] In some embodiments, the increase of the mechanical tensile strength of the suture line is characterized by the formation of an amount or a type of collagen mimicking endogenous tissue at the suture line site. In some embodiments, the type of collagen comprises Type I collagen, Type III collagen, or a combination thereof. In some embodiments, the collagen is formed in and around the annealed scaffold.

[0171] In some embodiments, Type I collagen is present with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6:1, or less than or equal to about 5:1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of less than or equal to about 10:1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of less than or equal to about 6:1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 5:1 or less. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 1 : 1 to about 10: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 1.5:1 to about 9.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 2: 1 to about 9: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of aboutAttorney Docket No. 48469-711.6012.5:1 to about 8.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 3:1 to about 8: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 3.5:1 to about 7.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 4:1 to about 7: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 4.5:1 to about 6.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 5: 1 to about 6: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of, of about, ofatleast, or of at least about 15:1; 14:1; 13:1; 12:1; 11:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; or 1:15, or any value therebetween. In some embodiments, the collagen is formed at the site of a suture line by at least about 28 days after closure (e.g. suturing) of the site of a suture line. In some embodiments, the collagen is formed at the site of a suture line by at least about 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, 36, 37, 38, 39, or 40 days after suturing.

[0172] In some embodiments, increasing the mechanical tensile strength of the suture line is characterized by increasing the toughness of the suture line. In some embodiments, the toughness is measured as an area under the curve of a stress versus strain curve to fracture using a tensile test (e.g., on an Instron).

[0173] The toughness may be measured as an area under the curve of a stress versus strain curve to fracture using a tensile test (e.g., on an Instron). In some embodiments, the suture line comprises a toughness of at least about 60.0 millijoules per millimeter cubed (mJ / mm3) at least about 42 days after closure. In some embodiments, the toughness comprises at least about 25.0 mJ / mm3, about 30.0 mJ / mm3, about 35.0 mJ / mm3, about 40.0 mJ / mm3, about 45.0 mJ / mm3, about 50.0 mJ / mm3, about 55.0 mJ / mm3, about 60.0 mJ / mm3, about 65.0 mJ / mm3, about 70.0 mJ / mm3, about 75.0 mJ / mm3, about 80.0 mJ / mm3, about 85.0 mJ / mm3, about 90.0 mJ / mm3, or about 100.0 mJ / mm3. In some embodiments, the toughness comprises no more than about 25.0 mJ / mm3, about 30.0 mJ / mm3, about 35.0 mJ / mm3, about 40.0 mJ / mm3, about 45.0 mJ / mm3, about 50.0 mJ / mm3, about 55.0 mJ / mm3, about 60.0 mJ / mm3, about 65.0 mJ / mm3, about 70.0 mJ / mm3, about 75.0 mJ / mm3, about 80.0 mJ / mm3, about 85.0 mJ / mm3, about 90.0 mJ / mm3, or about 100.0 mJ / mm3. In some embodiments, the toughness of the suture line is measured at least about 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, or 50 days after suturing. In some embodiments, the toughness of the closure is measured at least about 42 days after suturing.

[0174] In some embodiments, the suture line comprises a toughness of at least about 25.0 mJ / mm3to about 100.0 mJ / mm3at least about 42 days after suturing of the suture line. In someAttorney Docket No. 48469-711.601embodiments, the suture line comprises a toughness of at least about 60.0 millijoules per millimeter cubed (mJ / mm3) at least about 42 days after suturing of the suture line.

[0175] In some embodiments, the suture line comprises a toughness of at least about 25.0 mJ / mm3to about 100.0 ml / mm3after suturing. In some embodiments, the toughness comprises at least about 25.0 mJ / mm3to about 100.0 mJ / mm3, about 30.0 mJ / mm3to about 100.0 mJ / mm3, about 35.0 mJ / mm3to about 100.0 mJ / mm3, about 40.0 mJ / mm3to about 100.0 mJ / mm3, about 45.0 mJ / mm3to about 100.0 mJ / mm3, about 50.0 mJ / mm3to about 100.0 mJ / mm3, about 55.0 mJ / mm3to about 100.0 mJ / mm3, about 60.0 mJ / mm3to about 100.0 mJ / mm3, about 65.0 mJ / mm3to about 100.0 mJ / mm3, about 70.0 mJ / mm3to about 100.0 mJ / mm3, about 75.0 mJ / mm3to about 100.0 mJ / mm3, about 80.0 mJ / mm3to about 100.0 mJ / mm3, about 85.0 mJ / mm3to about 100.0 mJ / mm3, or about 90.0 mJ / mm3to about 100.0 mJ / mm3. In some embodiments, the toughness of the suture line is measured at least about 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, or 65 days after closure. In some embodiments, the toughness of the suture line is measured at least about 42 days after suturing.

[0176] In some embodiments, increasing the mechanical tensile strength of the suture line is characterized by increasing a percent recovery of the suture line. In some embodiments, the percent recovery is calculated as the ratio of the wounded tissue tensile strength at the suture line site relative to unwounded tissue tensile strength from the same anatomical area. In some embodiments, the percent recovery of the suture line is greater than or equal to about 40% increased as compared to a reference suture line closed without the delivery of the annealed scaffold, wherein the percent recovery is measured after suturing. In some embodiments, the percent recovery is greater than or equal to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold. In some embodiments, the percent recovery is measured at least about 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, or 65 days after suturing. In some embodiments, the precent recovery is measured at least about 42 days after suturing.

[0177] In some embodiments, the percent recovery of the suture line is at least about 20% to about 60% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold, wherein the percent recovery is measured after suturing. In some embodiments, the percent recovery is at least about 10% to about 50% increased, at least about 20% to about 60% increased, at least about 30% to about 70% increased, at least about 40% to about 80% increased, at least about 30% to about 90% increased, or at least about 40% to aboutAttorney Docket No. 48469-711.601100% increased. In some embodiments, the percent recovery is measured at least about 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, or 50 days after suturing. In some embodiments, the percent recovery is measured at least about 42 days after suturing.

[0178] In some embodiments, the increase of the mechanical tensile strength of the suture line is characterized by increasing the yield strain of the suture line.

[0179] In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by increasing the yield stress of the suture line.

[0180] In some embodiments, the delivering comprises releasing the annealed scaffold from a syringe. In some embodiments, the syringe comprises a needle. In some embodiments, the needle has a gauge comprising about 10 gauge to about 20 gauge. In some embodiments, the needle has a gauge comprising about 8 gauge. In some embodiments, the needle has a gauge comprising about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 gauge. In some embodiments, the syringe comprises an applicator tip. In some embodiments, the applicator tip allows to mix and homogenize the annealed scaffold upon delivery.

[0181] In some embodiments, the syringe may have an internal volume of about 0.1 mL to about 10 mL. In some embodiments, the internal volume comprises about 5.0 mL. In some embodiments, the internal volume comprises about 0.5 mL to about 3.0 mL. In some embodiments, the volume comprises about 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, 0.1 mL to 5.0 mL, 5.0 mL to 10.0 mL, or 2.5 mL to 7.5 mL. In some embodiments, the syringe comprises a dose comprising greater than or equal to 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, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL,10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, or 20 mL. The dose of the annealed scaffold may depend on the area of administration. In some embodiments, the internal volume is associated with an internal diameter of the syringe which affects the extrusion force needed to inject the annealed scaffold compositions. In some embodiments, the internal diameters may be about 4 mm to about 9 mm. In some embodiments, the internal diameters may be about 4.5 mm to about 6.5 mm. In some embodiments, the internal diameters may be about 4.5 mm to about 8.8 mm. In some embodiments, the extrusion force needed to deliver the annealed scaffold from the syringe is dependent on the needle gauge.

[0182] In some embodiments, methods comprise delivering the annealed scaffold onto the subject. In some embodiments, the annealed scaffold has a shelflife of at least about 12 months. In some embodiments, the annealed scaffold has a shelflife of at least about 36 months. In some embodiments, the shelflife of the annealed scaffold corresponds to an amount of time (e.g., 12 orAttorney Docket No. 48469-711.60136 months) when the annealed scaffold is stored at room temperature. In some embodiments, the shelflife of the annealed scaffold corresponds to an amount of time (e.g., 12 or 36 months) when the mixture is stored refrigerated.

[0183] In some embodiments, the percent recovery of the suture line is greater than or equal to about 40% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold.

[0184] In some embodiments, the percent recovery of the suture line is at least about 20% to about 60% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold.

[0185] In some embodiments, the increasing the mechanical tensile strength of the suture line is characterized by increasing the yield strain of the suture line.

[0186] In some embodiments, the annealed scaffold becomes integrated with the suture line site in less than or equal to about 14 days following suturing of the suture line.

[0187] In some embodiments, integration is characterized by new tissue formation in and around the annealed scaffold.

[0188] In some embodiments, a cell matrix forms new tissue at the suture line site of the subject before complete degradation of the annealed scaffold.

[0189] In some embodiments, the new tissue is characterized by having (i) mature vascularization, (ii) a characteristic of surrounding tissue at the suture line site, (iii) an amount or a type of collagen mimicking endogenous tissue at the suture line site (iii) or a combination thereof.

[0190] In some embodiments, the characteristic of the surrounding tissue at the suture line site comprises functionally differentiated cell types from the surrounding tissue.

[0191] In some embodiments, (i) the new tissue forms, and (ii) the annealed scaffold substantially degrades after about 50 days after suturing the suture line.

[0192] In some embodiments, the new tissue is formed in addition to any tissue formed at the suture line site due to sutures alone.

[0193] In some embodiments, additional new tissue continues to form at the suture line site for at least about 42 days after suturing of the suture line.

[0194] In some embodiments, the new tissue is formed above (e.g., superficial to) the suture line site and deep to subcutaneous tissue.

[0195] In some embodiments, the new tissue is stromal like tissue with non-aligned collagen bundles.

[0196] In some embodiments, the annealed scaffold strengthens the suture line at the suture line site of the subject while minimizing a foreign body response in the subject.Attorney Docket No. 48469-711.601

[0197] In some embodiments, the foreign body response is characterized by causing harm to the subject.

[0198] In some embodiments, the harm is characterized by causing: chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof.

[0199] In some embodiments, the harm is caused at the suture line site.

[0200] In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of granulomas at the suture line site with histological analysis and comparing the amount of granulomas at the suture line site with a reference suture line site that does not contain the annealed scaffold.

[0201] In some embodiments, the annealed scaffold formulation is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of scar tissue at the suture line site with histological analysis and comparing the amount of scar tissue at the suture line site with a reference suture line site that does not contain the annealed scaffold.

[0202] In some embodiments, the annealed scaffold is effective to strengthen the suture line of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of nodules at the suture line site with histological analysis and comparing the amount of nodules at the suture line site with a reference suture line site that does not contain the annealed scaffold.

[0203] In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting chronic inflammation at the suture line site with histological analysis.

[0204] In some embodiments, the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by a presence of one or more types of macrophages at the suture line site of the subject. In some embodiments, the one or more types of macrophages comprise type 1 macrophages and type 2 macrophages. In some embodiments, the type 1 macrophages are pro-inflammatory. In some embodiments, the type 2 macrophages are pro-regenerative. In some embodiments, the annealed scaffold is effective to form more type 2 macrophages than type 1 macrophages.

[0205] In some embodiments, the cell matrix comprises an amount or a type of collagen mimicking endogenous tissue at the suture line site. In some embodiments, the collagen isAttorney Docket No. 48469-711.601formed at the suture line site by at least about 28 days after suturing of the suture line. In some embodiments, the collagen is formed in and around the annealed scaffold. 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 with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6: 1, or less than or equal to about 5:1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of, of about, of at least, or of at least about 15:1; 14:1; 13:1; 12:1; 11:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; or 1:15, or any value therebetween.

[0206] In some embodiments, at least part of the suture line site comprises elastin following degradation of the annealed scaffold at the suture line site.

[0207] Disclosed herein are methods comprising administering the annealed scaffolds described herein to a tissue site of a subject. In some embodiments, the tissue site is a surgical site of a subject. In some embodiments, the tissue site is an abdominal incision. In some embodiments, the surgical site is an abdominal midline lateral incision. In some embodiments, the surgical site is an abdominal transverse incision. In some embodiments, the tissue site is anywhere comprising soft tissue. In some embodiments, the tissue site is anywhere comprising connective tissue. In some embodiments, the tissue site is anywhere comprising epithelial tissue. In some embodiments, the tissue site is anywhere comprising muscle tissue. In some embodiments, the tissue site is anywhere comprising nervous tissue. In some embodiments, methods comprise administering a dose of the annealed scaffold to the subject, which may depend on the location and / or tissue at the tissue site as well as the intended therapeutic effect. In some embodiments, the tissue site comprises a wound site of the subject. In some embodiments, the wound site comprises a site of abrasion, avulsion, incision, laceration, puncture, or a combination thereof of the skin. In some embodiments, the wound site comprises a bum site. In some embodiments, the tissue site comprises a site of scarring (e.g., a site where a mark is left on the skin or within body tissue where a wound, bum, or sore has not healed completely and fibrous connective tissue has developed of the subject). In some embodiments, the scars are keloid, hypertrophic, contracture, adhesion, or a combination thereof.

[0208] In some embodiments, methods disclosed herein comprise delivering the annealed scaffold to the subject under conditions where the annealed scaffold is flowable and self-heals after delivery to form an annealed scaffold. In some embodiments, the annealed scaffold is porous. In some embodiments, the annealed scaffold is any of the annealed scaffolds described herein. In some embodiments, methods comprise forming the annealed scaffold in a manner suchAttorney Docket No. 48469-711.601that pores form between the microgel particles of the annealed scaffold (e.g., the annealed scaffold is porous).

[0209] In some embodiments, the annealed scaffold becomes integrated with the site of a suture line in less than or equal to about 14 days following closure. In some embodiments, integration is characterized by new tissue formation in and around the annealed scaffold.

[0210] In some embodiments, methods disclosed herein comprise delivering the annealed scaffold to the subject under conditions sufficient for endogenous cells of the subject to infiltrate and grow within the annealed scaffold. In some embodiments, the cells form a cell matrix within the annealed scaffold. In some embodiments, the annealed scaffold persists at the tissue site (e.g. incision or suture line) for a length of time sufficient for the cell matrix to grow into tissue in situ. In some embodiments, the methods comprise vascularizing, depositing extracellular matrix, or producing proteins and enzymes in the tissue site that aid in treating the tissue site, or any combination thereof. In some embodiments, the methods comprise forming new tissue from the cell matrix at the injection or tissue site. In some embodiments, the new tissue is characterized by having mature vascularization, a characteristic of surrounding tissue at the tissue site, an amount or type of collagen mimicking endogenous tissue at the tissue site, or a combination thereof. In some embodiments, the characteristic of the surrounding tissue at the tissue site comprises functionally differentiated cell types from the surrounding tissue. Non-limiting examples of functionally differentiated cell types include fibroblasts, epithelial cells, connective tissue cells, adipocytes, immune cells, mast cells, Langerhans cells, skeletal muscle cells, and stem cells. In some embodiments, the new tissue is characterized as having an extracellular matrix. In some embodiments, the annealed scaffold partially degrades in vivo. In some embodiments, the new tissue forms and the annealed scaffold completely degrades by at least about 42 days after closure. In some embodiments, the new tissue is formed in addition to any tissue formed at the site of a suture line due to sutures alone. In some embodiments, the new tissue continues to remodel at the site of a suture line for at least about 42 days after closure. In some embodiments, the new tissue is formed above (e.g., superficial to) the site of a suture line and deep to (e.g., under) subcutaneous tissue. In some embodiments, the new tissue is stromal like tissue with non-aligned collagen bundles.

[0211] In some embodiments, at least part of the site of a suture line comprises elastin following degradation of the annealed scaffold at the site of a suture line.

[0212] In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least 25% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%,Attorney Docket No. 48469-711.60180%, 85%, 90%, or 95% or greater of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 10% to about 50% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 15% to about 45% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 20% to about 40% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 25% to about 35% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 10% to about 90% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 15% to about 90% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 20% to about 85% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 25% to about 80% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 30% to about 75% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 35% to about 70% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 40% to about 65% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 45% to about 60% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, the at least part of the tissue site comprising the cell matrix comprises at least about 50% to about 55% of the tissue site following degradation of the annealed scaffold at the tissue site. In some embodiments, methods comprise growing cells within the annealed scaffold in less than or equal to about one day following the delivering. In some embodiments, methods comprise forming the cell matrix within the annealed scaffold in less than or equal to about 30 days following the delivering. In some embodiments, the cell matrix begins to form within the annealed scaffold within 7 days after administration.Attorney Docket No. 48469-711.601

[0213] In some embodiments, methods of delivering the annealed scaffold disclosed herein minimize a foreign body response elicited by the subject in response to the annealed scaffold. In some embodiments, the annealed scaffold is effective to strengthen the suture line while minimizing a foreign body response in the subject. 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 anything combination is localized to, or around, the tissue site. In some embodiments, the chronic inflammation, granuloma formation, nodule formation, swelling, pain or anything combination is localized to, or around, the site of a suture line. In some embodiments, the foreign body response is caused at a location other than the tissue site. In some embodiments, the foreign body response is characterized by a presence of multinucleate giant cells (MNGCs) (e.g., fusion of monocytes or macrophages) at the suture line of the subject. In some embodiments, the foreign body response is characterized by the persistence of MNGCs over an extended period of time. In some embodiments, the period of time comprising greater than or equal to about 1, 2, 3, or 4 weeks or more. In some embodiments, the period of time comprises greater than or equal to 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 greater than or equal to 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 as the absence of MNGCs at the tissue site after a period of time after delivering the annealed scaffold. In some embodiments, the period of time after delivering the annealed scaffold comprises 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, 1 days or less. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 30 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 29 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 28 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 27 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 26 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 25 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 24 days. In some embodiments, the period of time after delivering the annealed scaffoldAttorney Docket No. 48469-711.601comprises 1 to 23 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 22 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 21 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 20 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 19 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 18 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 17 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 16 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 15 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 14 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 13 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 12 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 11 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 10 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 9 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 8 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 7 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 6 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 5 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 4 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 3 days. In some embodiments, the period of time after delivering the annealed scaffold comprises 1 to 2 days.

[0214] In some embodiments, the foreign body response is measured by a presence of one or more types of macrophages at the suture line of the subject. In some embodiments, the one or more types of macrophages comprise Type 1 macrophages and Type 2 macrophages. In some embodiments, the Type I macrophages are pro-inflammatory. In some embodiments, the Type 2 macrophages are pro-regenerative. In some embodiments, the annealed scaffold is effective to reduce the amount of Type 1 macrophages to be less than the number of Type 2 macrophages.

[0215] In some embodiments, the foreign body response is measured by a presence of one or more Multinucleated Giant Cells (MNGCs) at the site of injection. In some embodiments, the annealed scaffold is effective to reduce the number of MNGCs in or around the suture line compared to a surgical mesh or tissue-derived surgical patch.Attorney Docket No. 48469-711.601

[0216] In some embodiments, chronic inflammation may be characterized as the slow, longterm inflammation lasting for an extended period of time after the delivery or administration of the annealed scaffold. In some embodiments, the chronic inflammation can last for a period of months to years. In some embodiments, the intentional foreign body response caused by the administration of biostimulators, as discussed above, results in chronic inflammation at the tissue site. In some embodiments, methods of delivering annealed scaffolds described herein minimize such chronic inflammation while permanently filling the tissue site with new tissue. In some embodiments, a granuloma may be a small area of inflammation at or around a tissue site of a subject. In some embodiments, the granuloma may be a small area / cluster of white blood cells and other tissue at the area of inflammation. In some embodiments, the intentional foreign body response caused by the administration of biostimulators, as discussed above, results in granuloma formation at or around the tissue site. In some embodiments, methods of delivering the annealed scaffold 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 harder and more brittle composition than normal tissue. In some embodiments, the intentional foreign body response caused by the administration of biostimulators, as discussed above, results in scar tissue formation at the tissue site. In some embodiments, methods of delivering the annealed scaffold described herein minimize such scar tissue formation while filling the tissue site with new tissue. In some embodiments, the annealed scaffold disclosed herein minimize such scar tissue formation at least because the new tissue that is formed within the annealed scaffold mimics the tissue at or surrounding the tissue site. For example, there is less Type I collagen and more Type III collagen deposited in the cell matrix within the annealed scaffold (forming the basis of the new tissue) than scar tissue. In some embodiments, the nodules may be sites of abnormal tissue growths. In some embodiments, the intentional foreign body response caused by the administration of biostimulators, as discussed above, results in nodule formation at the tissue site. In some embodiments, methods of delivering annealed scaffold described herein minimize such nodule formation while filling the tissue site with new tissue. In some embodiments, the foreign body response is measured by detecting an amount of granulomas at the tissue site with histological analysis and comparing the amount of granulomas at the tissue site with a reference tissue that does not contain the annealed scaffold. In some embodiments, the foreign body response is measured by detecting an amount of scar tissue at the tissue site with histological analysis and comparing the amount of scar tissue at the tissue site with a reference tissue that does not contain the annealed scaffold. In some embodiments, the foreign body response is measured by detecting an amount of nodules at the tissue site with histological analysis and comparing the amount of nodules at the tissue site with a reference tissue that does not containAttorney Docket No. 48469-711.601the annealed scaffold. In some embodiments, foreign body response is measured by detecting chronic inflammation at the tissue site with histological analysis. In some embodiments, the foreign body response is measured by detecting an amount of multinucleate giant cells (MNGC) (e.g., fusion of monocytes or macrophages) present at the tissue site with histological analysis and comparing the amount of MNGCs at the tissue site with a reference tissue that does not contain the annealed scaffold.

[0217] In some embodiments, methods of delivering the annealed scaffold disclosed herein under conditions sufficient to deposit an amount or type of collagen in the cell matrix at the tissue site mimicking endogenous tissue at or surrounding the tissue site (e.g. the site of a suture line). In some embodiments, methods comprise depositing an amount or type of collagen in the cell matrix at the tissue site mimicking endogenous tissue at or surrounding the tissue site. In some embodiments, the cell matrix comprises an amount or a type of collagen mimicking 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 with Type III collagen in a ratio of less than or equal to about 10: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of less than or equal to about 6:1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 5: 1 or less. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 1 : 1 to about 10: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 1.5:1 to about 9.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 2: 1 to about 9: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 2.5:1 to about 8.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 3 : 1 to about 8: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 3.5:1 to about 7.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 4: 1 to about 7: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 4.5:1 to about 6.5: 1. In some embodiments, Type I collagen is present with Type III collagen in a ratio of about 5 : 1 to about 6: 1. Type I collagen may be an indicator of scar tissue or a foreign body response having taken place in the subject. In some embodiments, the lower the ratio of Type I collagen to Type III collagen, the more the foreign body response has been minimized. In some embodiments, methods comprise minimizing the ratio of Type I collagen to Type III collagen such that new tissue can be built within a subject, making new tissue with the characteristics disclosed herein while avoiding the harms disclosed herein. For example, methods comprise permanently filling at least a part of the tissue site, while minimizing, or avoiding altogether, the foreign body response elicited as a response toAttorney Docket No. 48469-711.601biostimulators. In some embodiments, Type I collagen is present with Type III collagen in a ratio of, of about, ofatleast, or of at least about 1:1; 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; or 1:15, or any value therebetween.

[0218] In some embodiments, the collagen is formed at the suture line site by at least about 28 days after suturing of the suture line.

[0219] In some embodiments, the collagen is formed at the site of a suture line by at least about 28 days after closure (e.g. suturing) of the site of a suture line. In some embodiments, the collagen is formed at the site of a suture line by at least about 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, 36, 37, 38, 39, or 40 days after suturing.

[0220] In some embodiments, methods of delivering the annealed scaffold disclosed herein under conditions sufficient to form elastin at the tissue site. In some embodiments, methods comprise forming elastin at the tissue site. In some embodiments, the elastin persists at or around the tissue site following complete degradation of the annealed scaffold. The presence of elastin may indicate the absence of scars or scar tissue at the tissue site and may therefore be an indicator that the foreign body response has been minimized.

[0221] In some embodiments, the annealed scaffold is biocompatible with tissue at the tissue site as determined by one or more techniques described by ISO standard 10993, the contents of which pertaining to these techniques are hereby incorporated by reference in their entirety.Methods of Treatment

[0222] In some embodiments, the methods further comprise treating the tissue site of the subject by delivering the annealed scaffold to the tissue site. In some embodiments, the annealed scaffold is delivered on top of the suture line after the incision is sutured. In some embodiments, the annealed scaffold is delivered into the incision while the incision is sutured. In some embodiments, the annealed scaffold is delivered on top of the suture line after the incision is sutured and into the suture line while the incision is sutured.Surgical incision

[0223] In some embodiments, the methods comprise delivering the pre-annealed microgel particles to a suture line. In some embodiments, the suture line corresponds to the location of an incision. In some embodiments, the incision comprises a surgical incision. In some embodiments, the surgical incision comprises an abdominal fascial incision, an umbilical incision of the abdominal fascia, a hernia or dehiscence of a previous incision in that location, a laparoscopic keyhole incision of the abdominal fascia, an inguinal incision of the abdominal fascia, or a combination thereof. In some embodiments, the incision comprises a midline abdominal incisionAttorney Docket No. 48469-711.601or a transverse incision of the fascia. In some embodiments, the abdominal incision may be below the dermis and subcutis of the subject.

[0224] In some embodiments, the surgical incision may be an abdominal fascial incision (e.g. a midline or transverse incision of the fascia). In some embodiments, the incision may be a result of a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a gastro-intestinal cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a hysterectomy. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an ovarian cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a spinal fusion. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an abdominal trauma surgery.

[0225] In some embodiments, the surgical incision may be an umbilical incision of the abdominal fascia. In some embodiments, the umbilical incision may be an abdominal wall midline or transverse incision of the fascia. In some embodiments, the umbilical incision may be below the dermis and subcutis of the subject. In some embodiments, the incision may be a result of a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a gastro-intestinal cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a hysterectomy. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an ovarian cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a spinal fusion. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an abdominal trauma surgery.

[0226] In some embodiments, the surgical incision may be a laparoscopic keyhole incision of the abdominal fascia. In some embodiments, the laparoscopic keyhole incision may be an abdominal wall midline or transverse incision of the fascia. In some embodiments, the laparoscopic keyhole incision may be below the dermis and subcutis of the subject. In some embodiments, the incision may be a result of a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a gastro-intestinal cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subjectAttorney Docket No. 48469-711.601comprising a hysterectomy. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an ovarian cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a spinal fusion. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an abdominal trauma surgery.

[0227] In some embodiments, the surgical incision may be an inguinal incision of the abdominal fascia. In some embodiments, the inguinal incision may be an abdominal wall midline or transverse incision of the fascia. In some embodiments, the inguinal incision may be below the dermis and subcutis of the subject. In some embodiments, the incision may be a result of a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a gastro-intestinal cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a hysterectomy. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an ovarian cancer procedure. In some embodiments, the incision may be a result of a surgery performed on the subject comprising a spinal fusion. In some embodiments, the incision may be a result of a surgery performed on the subject comprising an abdominal trauma surgery.Combination Treatments

[0228] In some embodiments, the methods comprise administering to the subject one or more additional agent (e.g., therapeutic agent) such as local anesthetics (e.g., lidocaine), pain medications, anti-inflammatory agents, or others that can provide a therapeutic benefit at the site of administration. In some embodiments, the pre-annealed microgel particles comprise one or more additional agents (e.g., drug-eluting microgel particles). In some embodiments, the preannealed microgel particles elute the one or more active agents in situ. In some embodiments, the annealed scaffold is formulated with the one or more active agents. In some embodiments, the annealed scaffold is not formulated with the one or more active agents, and the one or more active agents is administered separately from the annealed scaffold. In some embodiments, the annealed scaffold and the one or more additional active agents is administered to the subject sequentially. In some embodiments, the annealed scaffold and the one or more additional active agents is administered to the subject substantially simultaneously.

[0229] In some embodiments, the therapeutic agent comprises a pain medication, a local anesthetic, an anti-inflammatory medication, an anti-fibrotic medication, or an antibiotic. In some embodiments the local anesthetic is ester based. In some embodiments, the ester based local anesthetic comprises benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine,Attorney Docket No. 48469-711.601or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is amide based. In some embodiments, the amide based 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 pain medication comprises codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory medication is a non-steroidal 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 medication comprises pentoxifylline.Methods of Producing an Annealed Scaffold

[0230] Disclosed herein, in some embodiments, are methods of producing the annealed scaffold disclosed herein and the components thereof. In some embodiments, the methods comprise fine-tuning the mechanical properties of the microgel particles, pre-annealing of the microgel particles resulting in the annealed scaffold. In some embodiments, the methods comprise purifying the microgel particles.Synthesizing Microsei Particles

[0231] Disclosed herein are methods of producing a microgel particle disclosed herein, which comprise combining raw materials (e.g., polymer, functional groups, peptides, etc.) under conditions sufficient for the individual microgel particles to form. In some embodiments, the conditions sufficient for microgel particles to form may comprise an aqueous buffer with pH ranging from 7 to 9. By way of non-limiting examples, the buffer may be phosphate buffered saline (PBS), HEPES, or Triethanolamine (TEOA). In some embodiments, the reaction may be quenched by the addition of an acid or a base to stop the reaction at a specified time after mixing and creating the water-in-oil emulsion. The reaction quenching molecule could be added to the oil phase and diffuse into the aqueous phase to quench the reaction occurring in that aqueous phase. In some embodiments, the reaction may be quenched by adding a maleimide to react with the remaining thiols. In some embodiments, the reaction may be quenched by adding an oxidizing agent to oxidize the thiols.

[0232] In some embodiments, microgel particles may be synthesized using a microfluidic device (e.g., one particle at a time per channel). In some embodiments, the microgel particles may be synthesized by water-in-oil emulsion as described in greater detail herein. In some embodiments, the microgel particles may be synthesized by water-in-oil emulsion withAttorney Docket No. 48469-711.601mechanical stirring. In some embodiments, the microgel particles may be synthesized by water-in-oil emulsion using a static mixer. In some embodiments, the microgel particles may be synthesized using in-line flow-through synthesis. In some embodiments, the microgel particles may be synthesized using a parallel production method (multiple particles at a time per channel or multiple channels in parallel).

[0233] In some embodiments, methods comprise synthesizing microgel particles by a water-in-oil emulsion process. In some embodiments, the methods begin with obtaining an oil or an oil mixture. By way of non-limiting example, the oil may be a light mineral oil (LMO), a heavy mineral oil (HMO) or a fluorinated oil. In some embodiments, oil mixtures comprise a surfactant. In some embodiments, different surfactants can be employed. In some embodiments, the surfactant may be a nonionic surfactant. Non-limiting examples of nonionic surfactants are Span80, Span20, Tween20, Tween40, Tween60, Tween80, and tocopheryl polyethylene glycol 1000 succinate (TPGS). In some embodiments, the surfactant may be an anionic surfactant. In some embodiments, the surfactant may be a fluorinated surfactant. Non-limiting examples of anionic surfactants are sodium dodecyl sulfate (SDS), sodium lauryl ether sulfate (SLES), and perfluorooctanesulfonate. In some embodiments, the surfactant may be a cationic surfactant. Non-limiting examples of cationic surfactants are cetyltrimethylammonium bromide (CTAB), and hexadecylpyridium bromide. In some embodiments, the surfactant may be an amphoteric surfactant. Non-limiting examples of amphoteric surfactants are betaine citrate, lauryl betaine, sodium, and (carboxymethyl) dimethyloleyl ammonium hydroxide. In some embodiments, the concentration of the surfactant may vary from 0.01 to 5% v / v.

[0234] In some embodiments, methods comprise adding the surfactant to the oil. In some embodiments, methods comprise adding the surfactant to the oil prior to the addition of an aqueous solution / mixture to the oil. In some embodiments, methods comprise adding the surfactant to an aqueous solution / mixture described herein. In some embodiments, having a surfactant in the aqueous phase is beneficial because if the surfactant has a high-water solubility, it is easy to remove during purification.

[0235] In some embodiments, the oil or oil mixture may be added to a bioreactor vessel through a micron filter and stirred. In some embodiments, the bioreactor vessel contains a volume from about 100 milliliters to about 1 liter. In some embodiments, the bioreactor vessel contains a volume from about 1 liter to about 10 liters. In some embodiments, the bioreactor vessel contains a volume from about 10 liters to about 100 liters. In some embodiments, the bioreactor vessel contains a volume from about 100 liters to about 1000 liters. In some embodiments, the bioreactor vessel contains a volume from about 100 liters to about 10,000 liters. In some embodiments, the bioreactor vessel contains a volume from about 10 liters toAttorney Docket No. 48469-711.601about 1000 liters. In some embodiments, the bioreactor vessel contains a volume from about 1000 liters to about 10,000 liters. In some embodiments, the micron filter has a pore size of about 0.1 pm to about 1 pm. In some embodiments, the micron filter has a pore size of about 0.2 pm.

[0236] In some embodiments, the oil or oil mixture may be added to a static mixer through a micron filter and stirred. In some embodiments, the volume of the static mixer is between about 100 milliliters and about 1 liter. In some embodiments, the volume of the static mixer is between about 1 liter to about 10 liters. In some embodiments, the volume of the static mixer is between about 10 liters and about 100 liters. In some embodiments, the volume of the static mixer is between about 100 liters and about 1000 liters. In some embodiments, the volume of the static mixer is between about 100 liters and about 10,000 liters. In some embodiments, the volume of the static mixer is between about 10 liters and about 1000 liters. In some embodiments, the volume of the static mixer is between about 1000 liters and about 10,000 liters. In some embodiments, the micron filter has a pore size of about 0.1 pm to about 1 pm. In some embodiments, the micron filter has a pore size of about 0.2 pm.

[0237] In some embodiments, methods of synthesizing microgel particles comprise providing one or more polymers as disclosed herein (e.g., in a solution). In some embodiments, the one or more polymers comprise PEG. In some embodiments, the PEG is provided in a molecular weight as disclosed herein.

[0238] In some embodiments, methods of synthesizing microgel particles comprise modifying the one or more polymers disclosed herein by attaching one or more functional groups. In some embodiments, the PEG is modified by attaching thiol and / or vinyl sulfone functional groups. In some embodiments, a first PEG is modified with thiol and a second PEG is modified with vinyl sulfone (VS).

[0239] In some embodiments, methods of synthesizing microgel particles comprise mixing the one or more modified polymers in a solution. In some embodiments, the thiolated PEG is mixed with the PEG-VS. In some embodiments, the functional groups react to form a hydrogel (e.g., hydrogel mesh). In some embodiments, the functional groups react by a Michael addition reaction (e.g., thiol-ene Michael addition reaction). In some embodiments, methods may comprise filtering the solution. In some embodiments, the solution may comprise a peptide (e.g., cell adhesive peptide as disclosed herein). In some embodiments, the solution may comprise a buffer or buffering agent. In some embodiments, the solution may comprise a base catalyst.

[0240] In some embodiments, methods of synthesizing microgel particles comprise the methods disclosed in United States Patent No. 10,912,860 or United States Patent No.10,668,185, which are incorporated herein by reference in its entirety.Attorney Docket No. 48469-711.601Fine-Tuning Mechanical Properties

[0241] Disclosed herein, in some embodiments, are methods of modulating the physical characteristics of the microgel particles, the annealed scaffold, the annealed scaffolds, or any combination thereof. In some embodiments, how the physical characteristics are modulated will depend on the mode of delivery, and the subject. In some embodiments, the physical characteristics may be altered depending on the, the mode of administration, the desired biocompatibility, or any combination thereof. In some embodiments, the physical characteristic is a mechanical property of the microgel particles, the annealed scaffold, or any combination thereof.

[0242] In some embodiments, methods comprise modulating the viscosity of the hydrogel, rate of degradation of the annealed 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 annealed scaffold, the degree of substitution of the polymer, the elastic compressive modulus of the annealed scaffold, the storage modulus of the annealed scaffold, the weight percent of the polymers, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, the additional agents (e.g., therapeutic agents), the size of the microgel particles or a combination thereof. In some embodiments, methods comprise reducing the viscosity of the hydrogel, rate of degradation of the annealed scaffold, the volume fraction of the microgel particles, the pH of the microgel particles, the pH of the annealing agent, the pH of the annealed scaffold, the degree of substitution of the polymer, the elastic compressive modulus of the annealed scaffold, the storage modulus of the annealed 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, the additional agents (e.g., therapeutic agents), or a combination thereof. In some embodiments, methods comprise increasing the viscosity of the hydrogel, rate of degradation of the annealed scaffold, the volume fraction of the microgel particles, the pH of the microgel particles, the pH of the annealing agent, the pH of the annealed scaffold, the degree of substitution of the polymer, the elastic compressive modulus of the annealed scaffold, the storage modulus of the annealed 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, the additional agents (e.g., therapeutic agents), or a combination thereof.

[0243] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) the viscosity of the hydrogel or the elastic compressive modulus of the annealed scaffold. In some embodiments, methods comprise modulating the volume fraction of the microgel particles. In some embodiments, the volume fraction of the microgel particles may be about 70% to aboutAttorney Docket No. 48469-711.601100%. In some embodiments, the volume fraction of the microgel particles may be about 75% to about 100%. In some embodiments, the volume fraction of the microgel particles may be about 80% to about 100%. In some embodiments, the volume fraction of the microgel particles may be about 85% to about 100%. In some embodiments, the volume fraction of the microgel particles may be about 90% to about 100%. In some embodiments, the volume fraction of the microgel particles may be about 95% to about 100%. In some embodiments, the volume fraction of the microgel particles may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%. In some embodiments, the volume fraction of the microgel particles may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, modulating the volume fraction of the microgel particles comprises modulating the percent concentration of microgel particles in the hydrogel. Increasing the volume fraction of the microgel particles can lead to a higher viscosity of the suspension of microgel particles hydrogel to be annealed into an annealed scaffold. Decreasing the volume fraction of the microgel particles can lead to a lower viscosity of the hydrogel to be annealed into an annealed scaffold. In some embodiments, methods comprise modulating the volume fraction of the microgel particles in a range of 0.70 mL / mL to 1.0 mL / mL to achieve a viscosity of the hydrogel of 1,000 to 1,000,000 mPa*s. Increasing the volume fraction of the microgel particles can lead to a higher elastic compressive modulus of the annealed scaffold. Decreasing the volume fraction of the microgel particles can lead to a lower elastic compressive modulus of the annealed scaffold. In some embodiments, methods comprise modulating the volume fraction of the microgel particles in a range of 0.75 mL / mL to 0.95 mL / mL to achieve an elastic compressive modulus of the annealed scaffold of about 1,000 Pa to about 17,000 Pa.

[0244] In some embodiments, methods comprise modulating the elastic compressive modulus of the microgel particles. In some embodiments, modulating the elastic compressive modulus is achieved by modulating the molar ratio of the crosslinkers, polymers (e.g., copolymers), or combination thereof. Increasing the elastic compressive modulus of the microgel particles can lead to a higher viscosity of the hydrogel to be annealed into an annealed scaffold. Decreasing the elastic compressive modulus of the microgel particles can lead to a lower viscosity of the hydrogel to be annealed into an annealed scaffold. In some embodiments, methods comprise modulating the elastic compressive modulus of the microgel particles in a range of 15 kPa to 46 kPa to achieve a viscosity of the hydrogel of 1,000 to 1,000,000 mPa*s. Increasing the elastic compressive modulus of the microgel particles can lead to a higher elastic compressive modulus of the annealed scaffold. Decreasing the elastic compressive modulus of the microgel particles can lead to a lower elastic compressive modulus of the annealed scaffold. In some embodiments, methods comprise modulating the elastic compressive modulus of theAttorney Docket No. 48469-711.601microgel particles in a range of 15 kPa to 46 kPa to achieve an elastic compressive modulus of the annealed scaffold of about 1,000 Pa to about 17,000 Pa. In some embodiments, the annealed scaffold comprises an elastic compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 1,500 Pa after the annealing reaction. In some embodiments, the microgel particles comprise an elastic compressive modulus of at least about 6,000 Pa after the annealing reaction.

[0245] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) the volume fraction of the microgel particles in a range of 0.75 mL / mL to 0.95 mL / mL, and modulating the elastic compressive modulus of the microgel particles in a range of 15 kPa to 46 kPa, to achieve a viscosity of the hydrogel of 1,000 to 1,000,000 mPa*s. In some embodiments, methods comprise modulating the volume fraction of the microgel particles in a range of 0.75 mL / mL to 0.95 mL / mL, and modulating the elastic compressive modulus of the microgel particles in a range of 15 kPa to 46 kPa, to achieve an elastic compressive modulus of the annealed scaffold of about 1,000 Pa to about 17,000 Pa.

[0246] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) the rate of degradation of the annealed scaffold. In some embodiments, the rate of degradation is altered depending on how long it is desired for the annealed scaffold to remain at the tissue site. In some embodiments, methods comprise altering the degradation pathway, altering the polymers (e.g., co-polymers) used to make up the microgel particles, or a combination thereof to alter the rate of degradation. In some embodiments, methods comprise altering the degradation pathways to one or more of oxidative degradation, enzymatic degradation, or hydrolytic degradation. In some embodiments, methods comprise synthesizing the microgel particles with PEG to decrease the rate of degradation of the annealed scaffold. In some embodiments, methods comprise synthesizing the microgel particles without PEG to increase the rate of degradation.

[0247] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) the degree of substitution of the polymer. In some embodiments, modulating the degree of substitution is achieved by increasing or decreasing the amount of functional groups to be coupled to the microgel particles. In some embodiments, modulating the molecular weights of the polymers (e.g., co-polymers) in the microgel particles may affect the degree of substitution. In some embodiments, methods comprise measuring the degree of substitution using Ellman’s assay, or Proton nuclear magnetic resonance (Proton-NMR).

[0248] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) the elastic compressive modulus of the annealed scaffold. In some embodiments, modulating the concentration of functional groups (e.g., thiol and vinyl sulfone) included in the microgel-n-Attorney Docket No. 48469-711.601particles can alter the elastic compressive modulus of the annealed scaffold. In some embodiments, modulating the molecular weight of the polymer(s) (e.g., co-polymer(s)) of the microgel particles can alter the concentration of functional groups (e.g., thiol and vinyl sulfone) included in the microgel particles. Increasing the concentration of the functional groups (e.g., thiolated PEG) can increase the elastic compressive modulus of the annealed scaffold.Decreasing the concentration of the functional groups (e.g., thiolated PEG) can decrease the elastic compressive modulus of the annealed scaffold. Increasing the molecular weight of the polymer(s) (e.g., PEG) can increase the elastic compressive modulus of the annealed scaffold, while decreasing the molecular weight of the polymer(s) (e.g., PEG) can decrease the elastic compressive modulus of the annealed scaffold. In some embodiments, methods comprise modulating the concentration of functional groups (e.g., thiol and vinyl sulfone) included in the gelation solution to a range of about 10 mg / mL to about 45 mg / mL to achieve an elastic compressive modulus of the annealed scaffold of about 100 Pa to about 140,000 Pa.

[0249] In some embodiments, methods comprise modulating (e.g., increasing or decreasing) how fast the annealed scaffold anneals. In some embodiments, altering the annealing agent can alter how fast the annealed scaffold anneals. In some embodiments, modulating the molecular weight of the annealing agent alters how fast the annealed scaffold anneals. For example, PEG-dithiol (PEG(SH)2), 4-ARM-PEG-SH, and PETMA can be used to achieve an elastic compressive modulus of the annealed scaffold of about 4,000 Pa to about 7,000 Pa after about 60 minutes of annealing. In some embodiments, methods comprise modulating the pH of the annealing agent to alter how fast the annealed scaffold anneals. Increasing the pH of the annealing agent can increase how fast the annealed scaffold anneals, and decreasing the pH of the annealing agent can decrease how fast the annealed scaffold anneals. In some embodiments, methods comprise using a pH of the annealing at or below 6.5 to delay the start of the annealing reaction by 30 minutes or more.Purifying Microgel Particles

[0250] In some embodiments, the methods comprise fine-tuning the mechanical properties of the microgel particles, pre-annealing of the microgel particles resulting in the annealed scaffold or annealed scaffold. In some embodiments, the methods comprise purifying the microgel particles. In some embodiments, the methods comprise concentrating the microgel particles. In some embodiments, the methods comprise annealing the microgel particles. In some embodiments, the methods comprise formulating the pre-annealed microgel particles into an annealed scaffold or formulation. In some embodiments, the methods further comprise sterilizing the annealed scaffold or formulation.Attorney Docket No. 48469-711.601

[0251] In certain aspects, described herein is a method of preparing an annealed scaffold, the method comprising: providing a microgel particle mixture comprising microgel particles comprising a cross-linked 4-arm polyethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; washing the microgel particles; concentrating the microgel particle mixture; and combining PEG-dithiol to the microgel particle mixture, wherein the microgel particles undergo an annealing reaction to form the annealed scaffold.

[0252] In some embodiments, the method further comprises loading a syringe with the annealed scaffold formulation. In some embodiments, the method further comprises loading a delivery device with the annealed scaffold.

[0253] In some embodiments, said washing step comprises tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), hollow-fiber-diafiltration (HFDF), tangential flow depth filtration (TFDF), dialysis, direct flow filtration, or centrifugation. In some embodiments, said washing step comprises tangential flow filtration (TFF).

[0254] In some embodiments, said concentrating step comprises tangential flow depth filtration (TFDF), centrifugation, evaporation, or a combination thereof. In some embodiments, said concentrating step comprises tangential flow depth filtration (TFDF). In some embodiments, said concentrating step comprises evaporation. In some embodiments, the evaporation is carried out under a flow of inert gas. In some embodiments, the evaporation is carried out under vacuum. In some embodiments, the evaporation is carried out under a flow of inert gas and under vacuum.

[0255] In some embodiments, methods comprise purifying the microgel particles. In some embodiments, methods comprise synthesizing and purifying microgel particles simultaneously. In some embodiments, methods comprise purifying microgel particles after synthesizing the microgel particles. In some embodiments, purifying microgel particles comprises performing membrane separation of the microgel particles from unwanted 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 or flat sheet membrane). In some embodiments, membrane separation comprises tangential flow filtration (TFF). In some embodiments, membrane separation comprises ultrafiltration-diafiltration (UFDF). In some embodiments, membrane separation comprises microfiltration-diafiltration (MFDF). In some embodiments, membrane separation comprises hollow-fiber-diafiltration (HFDF). In some embodiments, membrane separation comprises tangential flow depth filtration (TFDF). TFF generally comprises a membrane filtration and separation technique. TFF may be used herein toAttorney Docket No. 48469-711.601purify and concentrate microgel particles. TFF may comprise generating a feed stream of a solution of microgel particles that passes parallel to a membrane face. In some embodiments, one portion of the solution may pass through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir. This system may be referred to as diafiltration. This system may allow molecules (in the permeate) smaller than the membrane pores to move toward and through the membrane while the larger molecules, such as the 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 surrounding environment. In some embodiments, the filtration system may be open to surrounding environment.

[0256] In some embodiments, methods of purifying may comprise removing excess oil from the microgel particles. In some embodiments, methods of purifying may comprise dispersing the particles in an alcohol solution. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio greater than or equal to about 0.8:1. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio greater than or equal to about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio less than or equal to about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio of about 0.5 : 1 to about 1 : 1. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio of about 0.6:1 to about 0.9:1. In some embodiments, the alcohol solution comprises alcohol and water, and the alcohol is present in the solution at a ratio of about 0.7:1 to about 0.8:1. In some embodiments, methods of purifying may comprise removing excess oil and surfactant that are not miscible in water while keeping the particles (mainly composed of water) dispersed and sufficiently swollen and ensuring no particle aggregation. In some embodiments, methods of purifying may comprise slowly transferring the particles into an aqueous buffer while preventing the surfactant from precipitating. In some embodiments, transferring rate may be linked to the flux of filtrate passing through the membrane, and occur at a rate of about 1 to about 1000 LMH (liters / m2 / h). In some embodiments, transferring may occur at a rate of about 100 to about 500 LMH. In some embodiments, transferring may occur at a rate of about 200 to about 300 LMH. This transition rate may be particularly important to ensure that a surfactant does not precipitate on to (and within) the microgel particles, rendering the particles unsuitable for a microporous scaffold. In some embodiments, the transition rate may achieve at least one of (i) particleAttorney Docket No. 48469-711.601hydrogel mesh swelling, which is a product of the affinity for certain solvents for a given hydrogel polymer backbone / crosslinker system, and (ii) solubility of the surfactant in the continuous phase outside of the particle.

[0257] In some embodiments, methods comprise concentrating the microgel particles in a solution or suspension. In some embodiments, methods comprise: pumping the microgel particles through a membrane filtration system while a continuous phase volume is removed; continually concentrating the microgel particles at a controlled membrane flux; and maintaining a wall shear stress inside the membrane filtration system. In some embodiments, methods comprise concentrating the microgel particles by filtration (e.g., TFDF), evaporation, dialysis or a combination thereof. By way of example, FIG. 4 is a flow diagram of a method for synthesizing, purifying, and concentrating microgel particles. In certain embodiments, the microgel particles may be concentrated by combining TFDF and evaporation. In step 402, the microgel particles are synthesized. In step 404, the microgel particles are washed. In some embodiments, the microgel particles may be washed via tangential flow filtration (TFF). In step 406, the microparticles are concentrated. In some embodiments, the microgel particles may be concentrated via tangential flow depth filtration (TFDF). In step 408, the residual solvent is evaporated. In step 410, the microgel particles are loaded into a syringe. In some embodiments, the membrane filtration system is selected from tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), hollow-fiber-diafiltration (HFDF), or tangential flow depth filtration (TFDF). In some embodiments, the membrane flux is controlled between 100 and 1000 L / m2 / h. In some embodiments, the wall shear stress is maintained between 100 s'1and 10,000 s'1. In some embodiments, dialysis membranes have a molecular weight cutoff between 500 Da and 150,000 Da. In some embodiments, concentration by dialysis is driven by osmosis where water diffuses out from the microgel particles through the dialysis membrane to areas of low water concentration. In some embodiments, the areas of low water concentration comprise a solution of hydrophilic polymer at a high concentration. In some embodiments, the polymer is a PEG or a polysaccharide. In some embodiments, the polymer concentration is at least 10%w / v. In some embodiments, the polymer concentration is at least 20%w / v. In some embodiments, the polymer concentration is at least 30%w / v. In some embodiments, the polymer concentration is at least 40%w / v. In some embodiments, the polymer concentration is at least 50%w / v.

[0258] In some embodiments, the formation of the annealed scaffold is performed after microgel particle formation and during particle concentration. In some embodiments, the formation of the annealed scaffold is performed after microgel particle formation and immediately after particle concentration. In some embodiments, the formation of the annealed scaffold is performed after microgel particle formation and after filling in syringes. In someAttorney Docket No. 48469-711.601embodiments, the formation of the annealed scaffold is performed before delivery of the microgel particles to the tissue site.Microgel Particles Annealing and Reversibility

[0259] In some embodiments, methods comprise annealing the microgel particles by adding the annealing agent (e.g., PEG-dithiol) to form the annealed scaffold. In some embodiments, the annealing agent is added after microgel particle synthesis at the end of the purification step and during the concentration step. In some embodiments, the annealing agent is added immediately after particle concentration. In some embodiments, the annealing agent is added after filling the microgel particles in a delivery device (e.g., syringe). In some embodiments, adding the annealing agent decreases the volume fraction of the microgel particles from about 1.0 mL / mL to 0.95 mL / mL, from about 1.0 mL / mL to 0.90 mL / mL, from about 1.0 mL / mL to 0.85 mL / mL, from about 1.0 mL / mL to 0.80 mL / mL, from about 1.0 mL / mL to 0.70 mL / mL, from about 0.95 mL / mL to 0.90 mL / mL, from about 0.95 mL / mL to 0.85 mL / mL, from about 0.95 mL / mL to 0.80 mL / mL, from about 0.90 mL / mL to 0.85 mL / mL, from about 0.95 mL / mL to 0.80 mL / mL, from about 0.90 mL / mL to 0.85 mL / mL, from about 0.90 mL / mL to 0.80 mL / mL, from about 0.85 mL / mL to 0.80 mL / mL, from about 0.80 mL / mL to 0.75 mL / mL, or from about 0.80 mL / mL to 0.70 mL / mL. In some embodiments, the initial concentration (before addition) of the annealing agent is about 0.1 mM to about 10 mM. In some embodiments, the final concentration (after addition) of the annealing agent is about 0.01 mM to about 1 mM.

[0260] In some embodiments, methods comprise filling the pre-annealed microgel particles in a delivery device. In some embodiments, the pre-annealed microgel particles have shearthinning properties, remain flowable and can be loaded in syringes using a peristaltic pump, a Quattroflow pump or any positive displacement pump. In some embodiments, the pre-annealed microgel particles can be loaded in syringes using a positive pressurization to drive flow. In some embodiments, the pre-annealed microgel particles self-heals in the delivery device to form the annealed scaffold.

[0261] In some embodiments, methods comprise delivering the annealed scaffold to a tissue site. In some embodiments, the annealed scaffold has shear-shinning properties and remain flowable. In some embodiments, the annealed scaffold self-heals after delivery to form again the annealed scaffold. In some embodiments, the annealed scaffold loses a degree of crosslinking during delivery under shear strain. In some embodiments, the annealed scaffold loses a degree of crosslinking during delivery under increased shear strain. In some embodiments, the degree of crosslinking is measured by elastic modulus. In some embodiments, a decrease in degree of crosslinking corresponds to a decrease in elastic modulus. In some embodiments, an elastic modulus of the annealed scaffold decreases by at least 10% at a shear strain of 10% or more. InAttorney Docket No. 48469-711.601some embodiments, the degree of crosslinking is measured by viscosity. In some embodiments, a decrease in degree of crosslinking corresponds to a decrease in viscosity. In some embodiments, the annealed scaffold loses a degree of crosslinking during delivery under an increased shear rate. In some embodiments, a viscosity of the annealed scaffold decreases by at least 10% at a shear rate of 1 s'1to 1000 s'1. In some embodiments, the annealed scaffold becomes an unannealed scaffold during delivery. In some embodiments, the annealed scaffold becomes an unannealed scaffold during delivery and self-heals after delivery to form the annealed scaffold. In some embodiments, the annealing reaction is reversible to allow the microgel particles to re-assemble and form again the annealed scaffold after delivery, which is defined as self-healing. In some embodiments, the reversibility of annealing is described as the breakdown of the bonds between the microgel particles followed by creation of additional bonds between particles. In some embodiments, the reversibility of annealing is characterized first by a decrease of the elastic modulus followed by an increase of the elastic modulus (FIG. 2B). In some embodiments, the reversibility of annealing is characterized by a decrease of storage modulus at high strain and a return to its original storage modulus at low strain indicating the recovery of the interparticle interactions (FIG. 2C).Preserving Microgel Particles

[0262] Disclosed herein, in some embodiments, are methods of preserving the microgel particles, annealing agents, additional active agents, therapeutic agents, annealed scaffolds or formulations, or a combination thereof. In some embodiments, the methods comprise preserving the microgel particles, annealing agents, additional active agents, therapeutic agents, or any combination thereof before formulating into an annealed scaffold. In some embodiments, the preserving is performed prior to administration of the annealed scaffold to a subject. In some embodiments, methods of preservation comprise lyophilization, cryodehydration, cryohibemation, or cryopreservation, or a combination thereof.

[0263] In some embodiments, the lyophilization of the microgel particles, annealing agents, therapeutic agents, or a combination thereof comprises the use of lyoprotectants for retaining the functionality of the microgel particles, annealing agents, therapeutic agents, or a combination thereof. Lyoprotectant comprises addition of reagents, salts, or additives that protects the microgel particles, annealing agents, therapeutic agents, or a combination thereof during the desiccation process. Common lyoprotectants include isopropanol, ethanol, glycerol, trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods for increasing the transport of lyoprotectants inside the microgel particles, annealing agents, therapeutic agents, or a combination thereof in suspension can be utilized as a way of improving the viability and function of the microgel particles,Attorney Docket No. 48469-711.601annealing agents, therapeutic agents, or a combination thereof after lyophilization. These methods include electroporation, and the addition of reagents. In some embodiments, the lyophilized microgel particles, annealing agents, therapeutic agents, or a combination thereof, can be reconstituted for delivery to a tissue site of a subject. In some embodiments, reconstitution is accomplished by introducing a reconstitution medium to the lyophilized microgel particles, annealing agents, therapeutic agents, or a combination thereof.

[0264] In some embodiments, the microgel particles are flash frozen. In some embodiments, the microgel particles are flash frozen with liquid nitrogen. In some embodiments, the microgel particles are frozen at a temperature of at least about -100C, -110C, -120C, -130C, -140C, -150C, -160C, -170C, -180C, -190C or -200. In some embodiments, the microgel particles are frozen at a temperature of about -196C. In some embodiments, the microgel particles are in a solution of at least about 80%, 85%, 90%, 95%, or 100% isopropanol.

[0265] In some embodiments, lyophilization occurs at a temperature of about -55C. In some embodiments, lyophilization occurs at a temperature of less than about -50C, -55C, -60C, -65C, -70C, -75C, -80C, -85C, -90C, -95C, -100C. In some embodiments, the volume fraction of the microgel particles during lyophilization is less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.KITS

[0266] Disclosed herein, in some embodiments, are kits useful for delivering the annealed scaffold disclosed herein.

[0267] In certain aspects, the present disclosure provides a kit comprising the delivery device disclosed herein and instructions for use. In some embodiments, the kits disclosed herein may be used to deliver the annealed scaffold to a tissue site in a subject. In some embodiments, the kit comprises the annealed scaffold described herein, which can be used to perform the methods described herein. In some embodiments, the kit comprises the annealed scaffold in a single container. In some embodiments, the kit also comprises a reconstitution medium as described herein to reconstitute a lyophilized annealed scaffold.

[0268] Instructions for use may be included in the kit. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, mixing applicator tips, pipetting or measuring tools, bandaging materials, or other useful paraphernalia. The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typicallyAttorney Docket No. 48469-711.601contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant -free environment. The packaging materials employed in the kit may be those customarily utilized in gene expression assays and in the administration of treatments. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial or prefilled syringes used to contain suitable quantities of the pharmaceutical composition. The packaging material has an external label which indicates the contents and / or purpose of the kit and its components.

[0269] In some embodiments, the kit comprises a single container. In some embodiments, the container comprises an annealed scaffold described herein. In some embodiments, the container is a pre-loaded syringe. In some embodiments, the container is sterilized using the methods of the present disclosure. In some embodiments, the kit further comprises an applicator, such as a mixing applicator tip or a needle. In some embodiments, the kit further comprises instructions for use of the annealed scaffold, the syringe and the applicator to deliver the annealed scaffold, on or around a surgical incision disclosed herein, and form an annealed scaffold of the present disclosure.Definitions

[0270] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some embodiments, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0271] 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. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0272] Reference throughout this specification to “some embodiments,” “further embodiments,” or “a particular embodiment,” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “in some embodiments,” or “in further embodiments,” or “in a particular embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features,Attorney Docket No. 48469-711.601structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0273] The term “about,” as used herein, with reference to a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0274] The term, “annealing agent,” as used herein, refers to an entity capable of inducing the annealing reaction between particles of the present disclosure (e.g., microparticles) to form an annealed scaffold (e.g., covalently stabilized scaffold). Non-limiting examples of annealing agents include Eosin Y, PETMA (Pentaerythritol tetrakis(2-mercaptoacetate)) ], Factor XIII / FactorXIIIa, molecules with two or more reactive functional groups including thiols (e.g., PEG-dithiol), divinyl sulfone, or a combination thereof. An annealing agent may not covalently participate in the linkage of the particles described herein when the annealing reaction is induced. An annealing agent may be covalently linked to the particles of the annealed scaffold when the annealing reaction is induced.

[0275] The term, “annealing component,” as used herein, refers to a substrate in an annealing reaction between microgel particles of the present disclosure (e.g., microparticles) that is bound to the microgel particles themselves. Non-limiting examples of annealing components include K or Q peptides, two or more reactive functional groups including thiol or thiol derivatives, vinyl or vinyl derivatives (e.g., vinyl sulfone), methacrylates, acrylates, amines, or a combination thereof.

[0276] The term, “apparent viscosity,” as used herein, refers to the shear stress applied to a fluid divided by the shear rate. For a Newtonian fluid, the apparent viscosity is constant, and equal to the Newtonian viscosity of the fluid, but for non-Newtonian fluids, the apparent viscosity depends on the shear rate. The apparent viscosity is often measured using a viscometer or a rheometer.

[0277] The term “biocompatible,” as used herein, refers to biocompatibility as determined under the International Standard ISO 10993-1, which is hereby incorporated by reference in its entirety.

[0278] The term, “cell adhesive peptide,” or “cell adhesion peptide,” as used herein interchangeably refers to peptides capable of initiating cell adhesion to a synthetic material, such as a microgel particle. A non-limiting example of cell adhesive peptides is an RGD peptide. The cell adhesion peptide disclosed herein may be provided 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, which is hereby incorporated by reference in its entirety.Attorney Docket No. 48469-711.601

[0279] The term “cell matrix” as used herein refers to a network of proteins or other molecules that surround, support, and / or give structure to cells and tissues in the body.

[0280] The term, “elastic compressive modulus,” as used herein, refers to the stiffness of either individual microgel particles, macroscopic hydrogels, or annealed scaffolds of microgel particles. Elastic compressive modulus may be measured by compressive testing (failure or nonfailure) in which an anvil of known cross-sectional area is depressed into a hydrogel, nonannealed scaffold (microgel particles), or annealed scaffold at a known distance and speed, while a force transducer attached to the anvil records the force placed on the anvil. The elastic compressive modulus may be mathematically calculated from the stress / strain curves recorded during compression testing.

[0281] The term “crosslinker,” as used herein, refers to a reagent that participates in the crosslinking reaction of raw materials to form a microgel particle of the present disclosure (e.g., microparticles). A crosslinker is a linker with two or more reactive functional groups (e.g., thiol, vinyl sulfone, maleimide, acrylate, methacrylate, acrylamide, methacrylamide, norbornene, amine, hydroxyl). When a crosslinker is in excess in the crosslinker reaction, a crosslinker may also be an annealing component and participate with the annealing agent in an 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 crosslinker), or the combination thereof.

[0282] The term, “crosslinking,” as used herein, refers to a reaction to form the microgel particle of the present disclosure (e.g., microparticles). The term, “degree of crosslinking,” as used herein, refers to the crosslinking present to form the annealed scaffold (e.g., PEG-dithiol-annealed microparticles) from the microparticles of the present disclosure.

[0283] The term “derivative” in reference to a “vinyl” or a “thiol” refers to a vinyl -containing chemical entity or a thiol-containing chemical entity, respectively. Non-limiting examples of vinyl derivatives include PEG-VS, PEG-acrylate, PEG-methacrylate, PEG-maleimide. Nonlimiting examples of vinyl groups include vinyl sulfone, acrylate, methacrylate, acrylamide, maleimide, and norbornene. Non-limiting thiol derivatives include PEG-dithiol, cysteine-containing peptides (e.g., matrix metalloproteinase (MMP)-degradable crosslinker), any organosulfur compound of the form R-SH, where R represents an alkyl, or other organic substituent, methanethiol, ethanethiol, 1 -propanethiol, 2-propoanethiol, allyl mercaptan, butanethiol, tert-butyl mercaptan, pentanethiols, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, metallothionein, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 1 -mercaptoindole, grapefruit mercaptan, furan-2-ylmethanethiol, 3-Attorney Docket No. 48469-711.601mercaptopropane- 1, 2-diol, 3 -mercapto- 1 -propanesulfonic acid, 1 -hexadecanethiol, pentachlorobenzenethiol, or a combination thereof.

[0284] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These 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 it is present or absent depending on the context.

[0285] The term, “elastic modulus,” as used herein, refers to a mechanical property of a substance related to resistance of being deformed elastically when stress is applied to it, which stressmay be calculated with the following equation: , where stress is the force causing the deformation divided by the area to which the force is applied and strain is the ratio of the change in some parameter caused by the deformation to the original value of the parameter. Since >strain is a dimensionless quantity, the units of will be the same as the units of stress. Elastic modulus may be measured by mechanical testing (failure or non-failure) in which a force transducer is attached to a specimen in a manner that creates a mechanical continuum between the transducer and the specimen. The specimen may then be deformed either by compressing it, stretching it, or shearing it, and the anvil records the force placed on the anvil by the specimen as it deforms. The elastic modulus may be mathematically calculated from the stress / strain curves recorded during mechanical testing. Different types of elastic moduli may be measured based on the type of deformation of the specimen. In compressive deformation, the compressive modulus (e.g. “elastic compressive modulus”) may be calculated. In stretching, the tensile modulus is calculated. In shear deformation, the shear modulus may be calculated. The elastic modulus of microgel particles may be measured in a surrogate nonporous gel formed with the same precursor solution that is used to make the microgel particles but that is not emulsified in an oil phase to produce microspheres.

[0286] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “zw vitro” assay or on a piece of tissue that has been excised (removed) from a subject.

[0287] The term “foreign body response,” as used herein, refers to a fibrotic response typically resulting from an implant or annealed scaffold that is characterized, for example, by chronic inflammation, granuloma formation, and / or scar tissue formation, at or around the site ofAttorney Docket No. 48469-711.601delivery. A foreign body response can be detected in a subject by histological analysis of the tissue at or around the site of implantation, and comparing the results of the histological analysis with histology of a reference tissue that does not contain the implant or annealed scaffold.

[0288] The term “gel,” as used herein, refers to three-dimensional network of crosslinked polymers swollen in a solvent.

[0289] The term “HEPES,” as used herein, refers to 4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid.

[0290] The term “zzz itu.'' as used herein, refers to the original site of delivery or administration, confined to the site of original site without the invasion of neighboring tissues.

[0291] The term “zzz vitro" is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0292] The term “zzz vivo" is used to describe an event that takes place in a subject’s body.

[0293] As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can 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 a formula is incorporated into the basic local alignment search tool (BLAST) programs 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 homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Percent identity of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.

[0294] The term “hydrogel,” as used herein, refers to a gel that is water-insoluble and capable of holding water.

[0295] The term, “K peptide,” as used herein, refers to a peptide comprising an amino acid sequence comprising one or more lysine residues that serve as a substrate for an annealing agent in an annealing reaction, or which may assist in the crosslinking of the microgel particles disclosed herein.

[0296] The term “loss modulus,” as used herein, refers to a mechanical property of a viscoelastic substance related to the energy that is dissipated in the substance, representing its viscous portion. The loss modulus represents the ratio of the viscous stress to strain. The loss modulus of microgel particles may be measured in a surrogate nonporous gel formed with the same precursor solution that is used to make the microgel particles but that is not emulsified in anAttorney Docket No. 48469-711.601oil phase to produce microspheres. Loss modulus may be measured by undergoing a measurement of shear modulus as described above and performing an amplitude and frequency sweep of shear stress in a parallel plate system. This will enable calculation of both the storage and the loss modulus of the viscoelastic material (together the storage and loss modulus comprise the shear modulus).

[0297] The term “microparticle” or “microsphere,” as used herein, refer interchangeably to a particle that is about 0.1 pm and about 1000 pm in size.

[0298] The term, “microgel particle,” as used herein, refers to a particle comprised of gel that is about 0.1 pm and about 1000 pm in size.

[0299] The term “particle,” as used herein, refers to a singular unit of a larger system, such as, for example, the annealed scaffold or compositions disclosed herein.

[0300] The term “percent (%) identity,” as used herein, generally refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of 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 disregarded for comparison purposes). Alignment, for purposes of determining percent identity, may be achieved in various ways that are known in the relevant field. Percent identity of two sequences may be calculated by aligning a test sequence with 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 in the same position of 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.

[0301] The term “PEG,” as used herein, refers to poly (ethylene glycol).

[0302] The term “4-arm PEG-VS as used herein, refers to 4-arm polyethylene glycol)-vinyl sulfone.

[0303] The term “PLA,” as used herein, refers to polylactic acid or polylactide.

[0304] The term “poly dispersity,” as used herein is a measure of the heterogeneity of a particle of the present disclosure (e.g., microgel particles) based on size. Poly dispersity may be measured by any of laser diffraction using a particle size analyzer, dynamic light scattering, small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), or microscopy.

[0305] The term “polymer,” as used herein, refers to a class of substance composed of macromolecules comprised of monomer repeats. Non-limiting polymers include polyethylene glycol) (PEG), polylactic acid (PLA), collagen, collagen, poly(methylmethacrylate) (PMMA), or any combination thereof. The polymer may be synthetic, such as PEG, PLA, PMMA, and theAttorney Docket No. 48469-711.601like. The polymer may be a modified form of the polymer, such as for example to contain one or more thiol or vinyl derivatives disclosed herein (e.g., PEG-dithiol, 4-ARM PEG-thiol, PEG-VS, thiolated HA).

[0306] The term “pore size,” as used herein, refers the size of each individual pore in an annealed scaffold defined as interstitial void space between the particles. The pore size may be measured by approximating the void area to a circle, where the diameter of each circle may be considered the size of the pore.

[0307] The term “porosity” or “void fraction,” as used herein, refer interchangeably to a measure of the void (e.g. “empty”) spaces in a material, and may be a fraction of the volume of voids over the total volume, between 0 and 1 mL / mL, or may be a percentage between 0% and 100%. As an example, Porosity P = Volumevoid / VolumeTotai. Porosity may be measured using methods 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,” which is incorporated herein by reference in its entirety.

[0308] The term “precursor solution” refers to a solution of raw materials (e.g., polymers and / or peptides) used to form the microgel particles of the present disclosure.

[0309] The term “Q peptide,” as used herein, refers to a peptide comprising an amino acid sequence comprising one or more glutamine residues that serve as a substrate for an annealing agent in an annealing reaction, or which may assist in the crosslinking of the microgel particles disclosed herein.

[0310] As used herein, the term, “reversibility of annealing”, refers to the pre-annealed microgel particles being able to disassemble while being delivered and re-assemble in situ after delivery to form again the annealed scaffold. The reversibility of annealing is considered a type of self-healing.

[0311] 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 comprising “Arg-Gly-Asp”. Non-limiting extracellular matrix proteins include fibronectin, vitronectin, fibrinogen, von Willebrand Factor, laminin, and collagen. The RGD peptide may be provided in Moral MEG, Siahaan TJ., et. al. The RGD peptide may be modified for conjugation to contain a cysteine. In some embodiments, the RGD peptide comprises an amino acid sequence comprising RGDSPGERCG (SEQ ID NO: 1).

[0312] The term “self-healing,” refers to the ability of a material to reform broken bonds or re-establish interm olecular interactions after a strain has been applied. The interm olecular interactions may be electrostatic, covalent, or both. Herein, the interactions refer to the interactions between the microgel particles.Attorney Docket No. 48469-711.601

[0313] The term “storage modulus,” as used herein, refers to a mechanical property of a viscoelastic substance related to the energy that is stored in the substance, representing its elastic portion. The storage modulus represents the ratio of the elastic stress to strain. The storage modulus of microgel particles may be measured in a surrogate nonporous gel formed with the same precursor solution that is used to make the microgel particles but that is not emulsified in an oil phase to produce microspheres. Storage modulus may be measured by undergoing a measurement of shear modulus as described above and performing an amplitude and frequency sweep of shear stress in a parallel plate system. This will enable calculation of both the storage and the loss modulus of the viscoelastic material (together the storage and loss modulus comprise the shear modulus).

[0314] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0315] The term “surrogate gel” refers to a macroscopic surrogate bulk gel made from the same precursor solution used to make a microgel particles disclosed herein.

[0316] The term “tissue site,” as used herein, refers to the discrete location of a tissue where the annealed scaffold disclosed herein may be delivered.

[0317] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit, a prophylactic benefit, or an aesthetic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. 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. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.Attorney Docket No. 48469-711.601

[0318] The term “VS,” as used herein, refers to vinyl sulfone.

[0319] As used herein, the term “closed” in reference to an incision, or a suture is interchangeable with “sutured.” As used herein, the term “closed incision” is interchangeable with “suture line.”EXAMPLES

[0320] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: A 14-day rabbit study to evaluate the use of MAP on healing of abdominal midline incisions

[0321] The goal of this rabbit study was to evaluate different variations of MAP (Microporous Annealed Particle) that can be used during laparotomy. This study was conducted on an abdominal midline incision rabbit model for 14 days.Methods:

[0322] In this study, certain characteristics of MAP were tuned and evaluated, such as stiffness, volume fraction (VF), annealing modes and time of in situ annealing. The presence of MAP on top of the incision was evaluated by H&E histology 14 days after treatment. It was hypothesized that if MAP can stay on top of and integrate with the incision site, it would lead to more tissue ingrowth, resulting in stronger tissue.

[0323] A total of 7 MAP formulations were used (in conjunction of sutures) to treat an abdominal midline incision and compared to a control (sutures alone). The MAP formulations used in this 14-day rabbit study are described in Table 1, Table 2, and Table 3. Table 1 summarizes the different MAP formulations tested. Table 2 summarizes the different groups tested in 14-day rabbit study. Table 3 summarizes the particle size and elastic moduli of the formulations. Table 4 summarizes the endotoxin and bioburden of various formulations.Attorney Docket No. 48469-711.601Table 1. MAP Formulations Tested in 14-day Rabbit StudyTable 2. Groups Tested in 14-day Rabbit StudyAttorney Docket No. 48469-711.601Table 3. Particle Size and Elastic Moduli of FormulationsTable 4. Endotoxin and Bioburden of Various Formulations<

[0324] Animals (n = 4 per group) were anesthetized using a ketamine / xylazine injection followed by anesthesia maintenance using a gas mixture of isoflurane and oxygen for survival surgical procedures. Anesthesia was maintained using gas isoflurane / oxygen mixture. Rabbits received prophylactic analgesia (buprenorphine). During surgery, the animals received IV fluid (e.g., approx. 100 mL of Lactated Ringers). The rabbit surgical site (ventral abdomen) was prepared using clippers to remove hair, and skin was disinfected before making the incision. First, an incision for epidermis, dermis and subcutis was made off center from midline along the cranio-caudal axis, a flap was created to expose Linea Alba. Blunt dissection was used to separate the subcutaneous tissue from the abdominal wall. Surgical clamps were used to ensure the site remains open and the abdominal wall midline (linea alba) is accessible for the next incision procedure. The xyphoid process was palpated gently to determine its anatomical location and subsequently determine where to place the abdominal incision. A sterile incision and closure guide was placed directly on to the abdomen. A skin pen was used to draw a line where the incision should be made, and place dots where the bites for the suture closure were. The guide was then removed, leaving only the marks. Surgical tweezers were used to tent the caudal end ofAttorney Docket No. 48469-711.601the incision and a #11 blade was used to start the incision. Subsequently, the incision was advanced in the cranial direction until an incision length of approximately 5 cm is reached. Using Prolene 3-0 sutures fitted with an SH 26 mm U circle curved needle, closure of the incision began from the cranial end of the incision, moving in the caudal direction using a single, uninterrupted (continuous) suture. The marks made from the incision / closure guide defined where to put suture bites. This resulted in a ‘small bites’ method where each suture bite length is ~4 mm and the distance between bites is ~4 mm. A suture to incision length of 4X was used. The different formulations of MAP (Table 1) were applied directly on top of the sutured incision. Depending on the formulation, the subcutaneous flap would be closed immediately, 5 minutes or 10 minutes after application to let the annealing reaction occurs. In the case of Fl and F2, the subcutaneous flap was closed 5 after minutes of light exposure. The subcutaneous flap was closed over the abdominal wall incision with tacking sutures made between subcutis and superficial fascia using Vicryl 3-0 sutures fitted with an SH U circle 26 mm needle. Finally, intradermal skin closure was performed offset from midline incision over the subcutaneous tissue using Monocryl 4-0 sutures fitted with a PS-23 / 8 circle 19 mm needle. A topical antimicrobial cream was applied on the sutured skin. Standard bandaging (Telfa pad and Tegaderm transparent film) was used to further secure the incision site. A full body cotton bandage was used on top in order to protect the bandaging from falling and prevent the animal from scratching at the incision. A E-collar was placed around the rabbit’s neck after surgery. Analgesics (Baytril) were administered once daily by intramuscular (IM) injection for a 5-days period post-surgery. The animals were monitored every day for the rest of the study. Animals were euthanized after 15 ± 5 days. Immediately after euthanasia, the abdominal wall was collected (subcutis was left on top of wound area during tissue collection) and placed in 10% formalin to be fixed in a plastic container. 24 hours after fixing, the tissue was sectioned in 2 strips for histology The strips for histology were ~15 mm x 25 mm and were placed in a cassette fixed with 70% ethanol before for analysis. The tissues were embedded in paraffin and then stained with H&E. The Primary Endpoint (histology) was the assessment of the presence of MAP on top of the incision.Results

[0325] Representative histology images for H&E stain are shown in FIG. 1 for the different MAP formulations and sutures alone after 14 days. In FIG. 1, MAP was applied on top of the midline incision after closure with sutures, and the dermis and subcutaneous were closed offset from the midline immediately after MAP application. The number on each image indicates the rabbit number and the section number of the tissue.Attorney Docket No. 48469-711.601

[0326] Most MAP -treated rabbits had MAP integrated with the abdominal wall fascia above the incision site after 14 days. New tissue has grown into the scaffold and tissue has grown over the top of the scaffold.

[0327] F3, F4, F5, and F6 provided similar results where residual material was present directly on top of the midline incision for most of the sections. It did not seem to make a difference to wait 10 min or not before closing the subcutaneous flap after F3 application.

[0328] Regarding F5 or F6, both groups showed presence of residual material on top of the incision with tissue ingrowth.

[0329] Fl provided the least amount of residual material directly on top of the midline incision. Material was mostly present on the edges.

[0330] F2 provided improve results compared to Fl with a similar profile to F3.

[0331] Critically, F5 provided the greatest results, in addition to greatly streamlining the usability of the product.Example 2: Reversibility of annealing and self-healing

[0332] First, hydrogel microparticles were loaded in a syringe and pre-annealed with PEG-dithiol to a final concentration of 0.2 mM and a final volume fraction of 90%. Then, every hour for 6 hours, pre-annealed MAP was mixed back and forth with an empty syringe using a syringe connector between both syringes, at room temperature, and the elastic modulus was measured using an Instron immediately (TO) and 1 hour after dispensing into a mold.

[0333] FIG. 2A depicts the experimental setup demonstrating the reversibility of MAP annealing. As portrayed in FIG. 2, the following protocol was followed: Step 1 : 5 mL of nonannealed MAP was loaded in a syringe and mixed with crosslinker. Step 2: Two slides of hydrogels were prepared and tested for elastic modulus at TO and 1 hour. Step 3: The syringe with the hydrogel was mixed back and forth with an empty syringe using a syringe connector between both syringes. Step 4: Two slides of hydrogels were prepared and tested for elastic modulus at TO and 1 hour. Steps 3 and 4 were repeated six times.

[0334] FIG. 2B demonstrates that mixing pre-annealed MAP led to a decrease of its elastic modulus (TO data) indicating loss of crosslinks between particles. However, waiting 1 hour after mixing allows the particles to re-anneal as demonstrating by a return of the elastic modulus to its original value (about 7,221 Pa). MAP was able to re-anneal for at least 6-mixing events.

[0335] The reversibility of the interactions within MAP which is defined as self-healing was demonstrated using a dynamic strain amplitude cyclic test on a rheometer. The self-healing is characterized by the reversibility of the interactions within MAP after a strain has been applied. The sample was, in a stepwise manner, exposed to a constant deformation at a constant angularAttorney Docket No. 48469-711.601frequency (co=10 Hz). In the linear viscoelastic range, a small strain (y = 0.2%) was applied for 120 seconds. Then, a high strain (y = 500%) was applied for 60 seconds. The storage and loss moduli were measured during this dynamic strain amplitude cyclic test using a rheometer. At high strain, the storage modulus (G1) decreased indicating the breakdown of the bonds between microgel particles, i.e. the interactions are cleaved. At low strain, the storage modulus returned back to the original value indicating that MAP can recover the interactions. After breaking at large strains, the interactions between hydrogel microparticles can rapidly reform at small strains indicating the reversibility of the interactions between particles.

[0336] FIG 2C. provides a graph of storage modulus (G1) versus time of an annealed and non-annealed MAP formulation using an amplitude sweep and alternative step strains. FIG. 2D provides a graph of loss modulus (G") versus time of an annealed and non-annealed MAP formulation using amplitude sweep and alternative step strains. As seen in FIGS. 2C and 2D, when MAP is provided non-annealed, the reversibility of the interactions is only based on electrostatic interactions between microparticles. When MAP is provided pre-annealed, the reversibility of the interactions is based on both electrostatic and covalent interactions between microparticles as indicated by a greater storage modulus for pre-annealed MAP than for nonannealed MAP.Example 3: Characteristics of pre-annealed MAP

[0337] The rheological properties were characterized by a rheometer (Anton Paar, MCR102e) equipped with a 25 mm stainless steel sandblasted parallel -plate geometry. MAP samples were applied from a syringe between the plates at 1 mm gap.

[0338] The elastic modulus was measured on an Instron 3342 using a compressive test and a 2.5-N load cell. A 3-mm diameter anvil installed on the Instron as a surface test probe descends onto the test gel surface at 5 mm / min. The force (N) and extension (mm) values are recorded, and then converted to a stress / strain curve where the elastic modulus is determined.

[0339] The mechanical properties of pre-annealed MAP such as elastic modulus, viscosity and storage modulus were highly affected by the volume fraction and stiffness of the hydrogel microparticles (see, e.g., FIG. 5, FIG. 6 and FIG. 7). Pre-annealed MAP was formulated at a volume fraction varying from 70% to 100% at different stiffnesses from 15,000 to 27,000 Pa. An increase in volume fraction induces an increase in elastic modulus, viscosity and storage modulus. An increase in particle stiffness induces an increase in elastic modulus, viscosity and storage modulus.

[0340] To measure dynamic viscosity, MAP samples were subject to shear rates y (s’ ') ranging from 0.1 s’1to 1000 s’1, recording the torque imparted on the measurement system atAttorney Docket No. 48469-711.601each point. This torque is converted to a shear stress T (Pa), and then to an apparent viscosity r (Pa-s). An example of viscosity curve for non-annealed and pre-annealed MAP is presented in FIG. 8A. FIG. 8A is a graph of the viscosity curves of annealed (i.e., pre-annealed) and nonannealed MAP, between a shear rate of 0.1 s'1and 1000 s'1.

[0341] The frequency sweep experiments were performed in the LVR (y = 0.1 %) from 1 to 100 rad / s. An example of frequency sweep for non-annealed and pre-annealed MAP is presented in FIG. 8B FIG. 8B is a graph of the storage modulus (G1) and loss modulus (G") of annealed (i.e., pre-annealed) and non-annealed MAP between a frequency sweep test from 1 rad / s to 100 rad / s and at a constant shear strain of 0.1%. As demonstrated in FIG. 8B, the storage modulus of annealed MAP was greater than the storage modulus of non-annealed MAP at all angular frequencies. Further, the loss modulus of annealed MAP was lower at all angular frequencies than the loss modulus of non-annealed MAP.

[0342] The strain amplitude sweep experiments (y = 0.01-300 %) were performed at a constant angular frequency (co = 10 s'1) to confirm the linear viscoelastic region (LVR). An example of amplitude sweep for pre-annealed MAP is presented in FIG. 8C. FIG. 8C is a graph of the storage modulus (G1) and loss modulus (G") of annealed (i.e., pre-annealed) Formulation F4), between an amplitude sweep test from 0.01% to 300% shear strain and at a constant angular frequency of 10 rad / s.

[0343] An example of reversible amplitude sweep for non-annealed and pre-annealed MAP is presented in FIG. 8D. FIG. 8D is a graph of the storage modulus of annealed (i.e., preannealed) and non-annealed MAP, during a reversible amplitude sweep test from 0.01% to 300% and at an angular frequency of 10 rad / s. As demonstrated in FIG. 8D, the storage modulus of annealed MAP was greater than the storage modulus of non-annealed MAP during the reversible amplitude sweep test from 0.01% to 300%.

[0344] The porosity of pre-annealed MAP was assessed using a confocal laser scanning microscope. Percent porosity and pore size are affected by the volume fraction (VF) of MAP (FIGS. 9A-9C). As demonstrated in FIGS. 9A-9C, MAP formulations annealed at a volume fraction of 80% provided the lowest average pore area and median pore area, while MAP formulations annealed at a volume fraction of 70% provided the highest average pore area and median pore area.

[0345] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicingAttorney Docket No. 48469-711.601the invention. It is intended that the following claims define the scope of the disclosure and that methods and compositions within the scope of these claims and their equivalents be covered thereby.

Claims

1. Attorney Docket No. 48469-711.601CLAIMS WHAT IS CLAIMED IS:

1. A delivery device comprising:a. a body comprising two or more microgel particles annealed to each other forming an annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm polyethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide; andb. an applicator in fluidic communication with the body.

2. The delivery device of claim 1, wherein the PEG-dithiol-annealed microgel particles are annealed via a chemical crosslinking reaction.

3. The delivery device of claim 2, wherein the crosslinking reaction comprises a chemical crosslinking reaction.

4. The delivery device of claim 3, wherein the chemical crosslinking reaction comprises a Michael addition or a pseudo-Mi chael addition reaction.

5. The delivery device of claim 2, wherein the crosslinking reaction comprises a physical crosslinking reaction.

6. The delivery device of claim 5, wherein the physical crosslinking reaction comprises electrostatic interactions or hydrogen bonding.

7. The delivery device of claim 2, wherein the crosslinking reaction comprises a physical and chemical crosslinking reaction.

8. The delivery device of any one of claims 1-7, wherein the crosslinking reaction is reversible.

9. The delivery device of any one of claims 2-8, wherein the crosslinking reaction is reversible for at least 5 cycles.

10. The delivery device of any one of claims 1-9, wherein the annealed scaffold is self-healing.

11. The delivery device of any one of claims 1-10, wherein the annealed scaffold is a shearthinning fluid.

12. The delivery device of claim 1, wherein the annealed scaffold has at least 10% porosity.

13. The delivery device of claim 1, wherein the annealed scaffold has a median pore area of at least 20 pm2.

14. The delivery device of any one of claims 1-13, wherein the annealed scaffold has a median pore area of about 20 pm2to 80 pm2.Attorney Docket No. 48469-711.60115. The delivery device of any one of claims 1-14, wherein the annealed scaffold has an average pore area of at most 1000 pm2.

16. The delivery device of any one of claims 1-15, wherein the annealed scaffold has an average pore area of about 100 pm2to 1000 pm2.

17. The delivery device of any one of claims 1-16, wherein the annealed scaffold has at most 20% porosity.

18. The delivery device of any one of claims 1-17, wherein the microgel particles are present in the annealed scaffold at a volume fraction of at least 70%.

19. The delivery device of any one of claims 1-18, wherein the PEG-dithiol and the 4-arm PEG vinyl sulfone are present to provide a ratio of thiol to vinyl sulfone of less than about 1.

0.

20. The delivery device of any one of claims 1-19, wherein the PEG-dithiol comprises a molecular weight of at least about 3.4 kDa.

21. The delivery device of any one of claims 1-20, wherein the PEG-dithiol is present in the annealed scaffold in a molar concentration of at least about 0.2 mM.

22. The delivery device of any one of claims 1-21, wherein the microgel particles are spherical.

23. The delivery device of any one of claims 1-22, wherein the microgel particles comprise microspheres.

24. The delivery device of any one of claims 1-23, wherein the microgel particles comprise diameters comprising 5 pm to 1000 pm, between 50 pm to 1000 pm, or between 70 pm to 150 pm.

25. The delivery device of any one of claims 1-24, wherein the microgel particles comprise an elastic compressive modulus of at least about 500 Pascals (Pa) before the annealing reaction.

26. The delivery device of any one of claims 1-25, wherein the microgel particles comprise an elastic compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction.

27. The delivery device of any one of claims 1-26, wherein the annealed scaffold comprises pores comprising a median pore diameter of about 5 pm and above.

28. The delivery device of any one of claims 1-27, wherein the pores comprise a median pore diameter of about 10 pm to about 35 pm.

29. The delivery device of any one of claims 1-28, wherein the one or more cell adhesive peptides comprises an RGD peptide.

30. The delivery device of any one of claims 1-29, wherein the microgel particles comprise a poly dispersity of no more than 0.1.

31. The delivery device of claim 30, wherein the poly dispersity is calculated based on a standard deviation and mean size of the particles (e.g., PDI = (SD / mean)2).Attorney Docket No. 48469-711.60132. The delivery device of any one of claims 1-31, wherein the vinyl sulfone of the 4-arm PEG vinyl sulfone is a Michael acceptor in the Michael addition or pseudo-Michael addition reaction.

33. The delivery device of any one of claims 1-32, wherein the thiol of the PEG-dithiol is a Michael donor in the Michael addition or pseudo-Michael addition reaction.

34. The delivery device of any one of claims 1-33 wherein the annealed scaffold comprises a buffer.

35. The delivery device of claim 34, wherein the buffer comprises: a phosphate buffer, a 4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

36. The delivery device of any one of claims 1-35, wherein the annealed scaffold is formulated for administration to a subject.

37. The delivery device of claim 36, wherein the annealed scaffold is formulated for administration at a suture line of the subject.

38. The delivery device of claim 36 or 37, wherein the administration minimizes a foreign body response in the subject.

39. The delivery device of any one of claims 34-38, wherein the annealed scaffold comprises a dose volume of about 0.01 mL to about 20 mL.

40. The delivery device of any one of claims 1-39, wherein the delivery device is a syringe.

41. The delivery device of any one of claims 1-40, wherein the delivery device is a single syringe.

42. A kit comprising the delivery device of any one of claims 1-41 and instructions for use thereof.

43. A method of delivering an annealed scaffold to a site of injury in a subject, the method comprising:a. providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)- degradable crosslinker, one or more K-peptides and Q-peptides, and a celladhesive peptide in a syringe; andb. disposing the annealed scaffold to the site of injury in the subject, wherein the annealed scaffold loses a degree of crosslinking to form an unannealed scaffold during said disposing, and wherein the unannealed scaffold re-anneals after being disposed to the site of injury in the subject.Attorney Docket No. 48469-711.60144. The method of claim 43, wherein the loss in a degree of crosslinking comprises a loss of covalent bonds in the annealed scaffold.

45. The method of claim 43, wherein the loss in a degree of crosslinking comprises a loss of electrostatic bonds in the annealed scaffold.

46. The method of claim 43, wherein the loss in a degree of crosslinking comprises the breaking of one or more thioether bonds of the annealed scaffold or one or more disulfide bonds of the annealed scaffold, or a combination thereof.

47. The method of claim 43, wherein the annealed scaffold loses a degree of crosslinking under shear strain to form the unannealed scaffold.

48. The method of claim 43, wherein the degree of crosslinking is measured by elastic modulus.

49. The method of claim 48, wherein the unannealed scaffold has an elastic modulus of at most about 90% compared to an elastic modulus of the annealed scaffold.

50. The method of claim 48, wherein the unannealed scaffold has an elastic modulus of about 20% to about 90% compared to an elastic modulus of the annealed scaffold.

51. The method of claim 43, wherein the degree of crosslinking is measured by viscosity.

52. The method of claim 51, wherein the unannealed scaffold has a viscosity of at most about 80% compared to a viscosity of the annealed scaffold.

53. The method of claim 51, wherein the unannealed scaffold has a viscosity of about 0.1% to about 80% compared to a viscosity of the annealed scaffold.

54. A method of delivering an annealed scaffold to a site of injury in a subject, the method comprising:a. providing the annealed scaffold comprising PEG-dithiol-annealed microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)- degradable crosslinker, one or more K-peptides and Q-peptides, and a celladhesive peptide; andb. disposing the annealed scaffold onto the site of injury in the subject, wherein the annealed scaffold self-heals upon disposing.

55. The method of any one of claims 43-54, wherein the site of injury is a site of a suture line.

56. The method of any one of claims 43-55, further comprising closing the suture line in the subject.

57. The method of any one of claims 43- 56, wherein said disposing step and said closing step do not require a time delay.

58. The method of any one of claims 43-56, wherein said disposing step and said closing step occur consecutively.Attorney Docket No. 48469-711.60159. The method of any one of claims 43-58, wherein the annealed scaffold comprises one or more crosslinking reactions.

60. The method of claim 59, wherein the one or more crosslinking reactions is covalent, electrostatic, or both.

61. The method of claim 56, wherein the suture line corresponds to the location of an incision.

62. The method of claim 61, wherein the incision comprises a surgical incision.

63. The method of claim 62, wherein the surgical incision comprises an abdominal fascial incision.

64. The method of claim 63, wherein the abdominal fascial incision comprises an abdominal wall midline or transverse incision of the fascia.

65. The method of claim 63 or 64, wherein the abdominal incision resulted from a surgery performed on the subject comprising: a gastro-intestinal cancer procedure, a hysterectomy, an ovarian cancer procedure, a spinal fusion, an abdominal trauma surgery, or a combination thereof.

66. The method of any one of claims 43-65, wherein the disposing comprises disposing the annealed scaffold directly over the suture line after the suture line is sutured.

67. The method of any one of claims 43-66, wherein the disposing comprises disposing the annealed scaffold into the suture line while the suture line is being sutured.

68. The method of any one of claims 43-67, wherein the disposing comprises disposing the annealed scaffold into the suture line after the incision is sutured.

69. The method of claim 68, wherein the disposing comprises disposing the annealed scaffold into the suture line while the suture line is being sutured and on top of the suture line after the suture line is sutured.

70. The method of any one of claims 66-69, wherein the annealed scaffold is annealed when disposed on top of the suture line.

71. The method of any one of claims 43-70, wherein the disposing comprises releasing the annealed scaffold from the syringe through a static mixer.

72. The method of any one of claims 43-71, further comprising strengthening the suture line at the suture line site by the cell matrix formed over the suture line site.

73. The method of claim 72, wherein the strengthening the suture line is characterized by increasing a mechanical tensile strength of the suture line as compared to a reference suture line at an otherwise identical suture line site that was sutured without the delivery of the annealed scaffold.Attorney Docket No. 48469-711.60174. The method of claim 73, wherein the increasing the mechanical tensile strength of the suture line is characterized by the formation of an amount or a type of collagen mimicking endogenous tissue at the suture line site.

75. The method of claim 74, wherein the collagen is formed at the suture line site by at least about 28 days after suturing of the suture line.

76. The method of claim 74 or 75, wherein the collagen is formed in and around the annealed scaffold.

77. The method of any one of claims 74-76, wherein the type of collagen comprises Type I collagen, Type III collagen, or a combination thereof.

78. The method of claim 77, wherein Type I collagen is present with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6: 1, or less than or equal to about 5:1.

79. The method of claim 73, wherein the increasing the mechanical tensile strength of the suture line is characterized by increasing the yield stress of the suture line.

80. The method of claim 79, wherein the yield stress is calculated from a stress versus strain curve measured using a tensile test.

81. The method of claim 79 or 80, wherein the suture line comprises a yield stress of at least about 3.0 N / mm2to about 6.0 N / mm2at least about 42 days after suturing of the suture line.

82. The method of claim 73, wherein the increasing the mechanical tensile strength of the suture line is characterized by increasing the toughness of the suture line.

83. The method of claim 82, wherein the toughness is measured as an area under the curve of a stress versus strain curve to fracture using a tensile test.

84. The method of claim 82 or 83, wherein the suture line comprises a toughness of at least about 60.0 millijoules per millimeter cubed (mJ / mm3) at least about 42 days after suturing of the suture line.

85. The method of claim 82 or 83, wherein the suture line comprises a toughness of at least about 25.0 mJ / mm3to about 100.0 mJ / mm3at least about 42 days after suturing of the suture line.

86. The method of claim 73, wherein the increasing the mechanical tensile strength of the suture line is characterized by increasing a percent recovery of the suture line.

87. The method of claim 86, wherein the percent recovery of the suture line is greater than or equal to about 40% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold.

88. The method of claim 86, wherein the percent recovery of the suture line is at least about 20% to about 60% increased as compared to a reference suture line sutured without the delivery of the annealed scaffold.Attorney Docket No. 48469-711.60189. The method of claim 73, wherein the increasing the mechanical tensile strength of the suture line is characterized by increasing the yield strain of the suture line.

90. The method of any one of claims 43-89, wherein the annealed scaffold becomes integrated with the suture line site in less than or equal to about 14 days following suturing of the suture line.

91. The method of claim 90, wherein integration is characterized by new tissue formation in and around the annealed scaffold.

92. The method of any one of claims 43-91, wherein a cell matrix forms new tissue at the suture line site of the subject before complete degradation of the annealed scaffold.

93. The method of claim 92, wherein the new tissue is characterized by having (i) mature vascularization, (ii) a characteristic of surrounding tissue at the suture line site, (iii) an amount or a type of collagen mimicking endogenous tissue at the suture line site (iii) or a combination thereof.

94. The method of claim 93, wherein the characteristic of the surrounding tissue at the suture line site comprises functionally differentiated cell types from the surrounding tissue.

95. The method of claim 94, wherein: (i) the new tissue forms, and (ii) the annealed scaffold substantially degrades after about 50 days after suturing the suture line.

96. The method of any one of claims 92-95, wherein the new tissue is formed in addition to any tissue formed at the suture line site due to sutures alone.

97. The method of any one of claims 92-96, wherein additional new tissue continues to form at the suture line site for at least about 42 days after suturing of the suture line.

98. The method of any one of claims 92-97, wherein the new tissue is formed above (e.g., superficial to) the suture line site and deep to subcutaneous tissue.

99. The method of any one of claims 92-98, wherein the new tissue is stromal like tissue with non-aligned collagen bundles.

100. The method of any one of claims 43-99, wherein the annealed scaffold strengthens the suture line at the suture line site of the subject while minimizing a foreign body response in the subject.

101. The method of claim 100, wherein the foreign body response is characterized by causing harm to the subject.

102. The method of claim 101, wherein the harm is characterized by causing: chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof.

103. The method of claim 102, wherein the harm is caused at the suture line site.Attorney Docket No. 48469-711.601104. The method of claim 101, wherein the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of granulomas at the suture line site with histological analysis and comparing the amount of granulomas at the suture line site with a reference suture line site that does not contain the annealed scaffold.

105. The method of claim 101, wherein the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of scar tissue at the suture line site with histological analysis and comparing the amount of scar tissue at the suture line site with a reference suture line site that does not contain the annealed scaffold.

106. The method of claim 101, wherein the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting an amount of nodules at the suture line site with histological analysis and comparing the amount of nodules at the suture line site with a reference suture line site that does not contain the annealed scaffold.

107. The method of claim 101, wherein the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by detecting chronic inflammation at the suture line site with histological analysis.

108. The method of claim 101, wherein the annealed scaffold is effective to strengthen the suture line at the suture line site of the subject while minimizing a foreign body response in the subject when the foreign body response is measured by a presence of one or more types of macrophages at the suture line site of the subject.

109. The method of claim 108, wherein the one or more types of macrophages comprise type 1 macrophages and type 2 macrophages.

110. The method of claim 109, wherein the type 1 macrophages are pro-inflammatory.

111. The method of claim 109, wherein the type 2 macrophages are pro-regenerative.

112. The method of any one of claims 109-111, wherein the annealed scaffold is effective to form more type 2 macrophages than type 1 macrophages.

113. The method of any one of claims 92-112, wherein the cell matrix comprises an amount or a type of collagen mimicking endogenous tissue at the suture line site.

114. The method of claim 113, wherein the collagen is formed at the suture line site by at least about 28 days after suturing of the suture line.Attorney Docket No. 48469-711.601115. The method of claim 113 or 114, wherein the collagen is formed in and around the annealed scaffold.

116. The method of any one of claims 113-115, wherein the type of collagen comprises Type I collagen, Type III collagen, or a combination thereof.

117. The method of claim 116, wherein Type I collagen is present with Type III collagen in a ratio of less than or equal to about 10:1, less than or equal to about 6: 1, or less than or equal to about 5:1.

118. The method of any one of claims 43-117, wherein at least part of the suture line site comprises elastin following degradation of the annealed scaffold at the suture line site.

119. The method of any one of claims 43-118, wherein the annealed scaffold comprises a pH of about 6 to about 8.

120. The method of any one of claims 43-119, wherein the annealed scaffold comprises an elastic compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction.

121. The method of any one of claims 43-120, wherein the microgel particles are present in a suspension comprising the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 70%.

122. A method of preparing an annealed scaffold, the method comprising:a. providing a microgel particle mixture comprising microgel particles comprising a cross-linked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer cross-linked with a matrix metalloprotease (MMP)-degradable crosslinker, one or more K-peptides and Q-peptides, and a cell-adhesive peptide;b. washing the microgel particles;c. concentrating the microgel particle mixture;d. combining PEG-dithiol to the microgel particle mixture, wherein the microgel particles undergo an annealing reaction to form the annealed scaffold.

123. The method of claim 118, further comprising loading a syringe with the annealed scaffold.

124. The method of claim 122 or 123, wherein said washing step comprises tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), hollow-fiber-diafiltration (HFDF), tangential flow depth filtration (TFDF), dialysis, direct flow filtration, or centrifugation.

125. The method of claim 124, wherein said washing step comprises tangential flow filtration (TFF).

126. The method of any one of claims 122-125, wherein said concentrating step comprises tangential flow depth filtration (TFDF), centrifugation, evaporation, or a combination thereof.Attorney Docket No. 48469-711.601127. The method of claim 126, wherein said concentrating step comprises tangential flow depth filtration (TFDF).

128. The method of any one of claims 122-127, wherein said concentrating step comprises evaporation.

129. The method of claim 128, wherein the evaporation is carried out under a flow of inert gas.

130. The method of claim 128, wherein the evaporation is carried out under vacuum.

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