Hydrogel preparation and method of use

Abstract

This specification provides a hydrogel formulation and a method for using the same. In certain embodiments, the method includes using the hydrogel formulation in the reinforcement of sutures at the suture site.

Description

[Technical Field]

[0001] cross reference This application claims the benefits of U.S. Provisional Application No. 63 / 510,321, filed on 26 June 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listings This application is filed together with an electronic sequence listing. This sequence listing is provided as a file titled 48469-709.601.xml, created on June 20, 2024, and is 11.4 kilobytes in size. The information in the electronic sequence listing is incorporated by reference in its entirety. [Overview of the project]

[0003] In certain embodiments, a method for delivering a hydrogel formulation to a suture site in a subject is described herein, the method comprising delivering the hydrogel formulation to the suture site of the subject, which is annealed in situ to form a covalently stabilized porous scaffold, to which endogenous cells infiltrate the covalently stabilized porous scaffold and form a cellular matrix on the suture site of the subject. In some embodiments, the suture was an incision. In some embodiments, the incision was a surgical incision. In some embodiments, the surgical incision was a fascial incision. In some embodiments, the fascial incision was a midline or transverse incision of the abdominal wall of the fascia. In some embodiments, the abdominal incision was the result of surgery performed on the subject, such surgery included gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. In some embodiments, delivery includes delivering the hydrogel formulation directly onto the suture after the suture has been sutured. In some embodiments, delivery includes delivering the hydrogel formulation into the suture line while the suture line is being sutured. In some embodiments, delivery includes delivering the hydrogel formulation into the suture line after the incision has been sutured. In some embodiments, delivery includes delivering the hydrogel formulation into the suture line while the suture line is being sutured and on the suture line after the suture line has been sutured. In some embodiments, the hydrogel begins to anneal on the suture line before the dermis and subcutaneous tissue of the subject is sutured. In some embodiments, the hydrogel is annealed on the suture line for at least about 10 minutes before the dermis and subcutaneous tissue of the subject is sutured. In some embodiments, delivery includes releasing the hydrogel formulation from a syringe. In some embodiments, the method further includes reinforcing the suture line at the suture line site with a cellular matrix formed on the suture line site. In some embodiments, reinforcing the suture is characterized by increasing the mechanical tensile strength of the suture compared to a reference suture at the same suture site, except that it is sutured without the delivery of the hydrogel formulation.In some embodiments, increasing the mechanical tensile strength of a suture is characterized by the formation of a certain amount or type of collagen mimicking endogenous tissue at the suture site. In some embodiments, collagen is formed at the suture site at least about 28 days after suture closure. In some embodiments, collagen is formed within and around a covalently stabilized porous scaffold. In some embodiments, the type of collagen includes type I collagen, type III collagen, or a combination thereof. In some embodiments, type I collagen is present with type III collagen in a ratio of about 10:1 or less, about 6:1 or less, or about 5:1 or less. In some embodiments, increasing the mechanical tensile strength of a suture is characterized by increasing the yield stress of the suture. In some embodiments, the yield stress is calculated from a stress-to-strain curve measured using a tensile test (e.g., with Instron). In some embodiments, the suture has a yield strength of at least about 3.0 N / mm at least about 42 days after suture closure. 2 ~Approximately 6.0 N / mm 2 This includes the yield stress. In some embodiments, increasing the mechanical tensile strength of the suture is characterized by increasing the toughness of the suture. In some embodiments, toughness is measured using a tensile test (e.g., with Instron) as the area under the stress-to-strain curve to fracture. In some embodiments, the suture is measured at least about 60.0 millijoules per cubic millimeter (mJ / mm²) at least about 42 days after suture closure. 3 ) includes toughness. In some embodiments, the suture line has a hardness of at least about 25.0 mJ / mm² at least about 42 days after suturing the suture line. 3 ~Approximately 100.0 mJ / mm 3This includes toughness. In some embodiments, increasing the mechanical tensile strength of the suture is characterized by increasing the suture recovery rate. In some embodiments, the suture recovery rate increases by about 40% or more compared to a reference suture suture sutured without delivery of the hydrogel formulation. In some embodiments, the suture recovery rate increases by at least about 20% to about 60% compared to a reference suture suture sutured without delivery of the hydrogel formulation. In some embodiments, increasing the mechanical tensile strength of the suture is characterized by increasing the yield strain of the suture. In some embodiments, the hydrogel formulation integrates with the suture site within about 14 days after suture suturing. In some embodiments, integration is characterized by new tissue formation within and around a covalently stabilized porous scaffold. In some embodiments, the cell matrix forms new tissue at the suture site in question before complete degradation of the covalently stabilized porous scaffold. In some embodiments, the new tissue is characterized by having (i) mature angiogenesis, (ii) features of the surrounding tissue at the suture site, (iii) a certain amount or type of collagen mimicking endogenous tissue at the suture site, or (iii) or a combination thereof. In some embodiments, the features of the surrounding tissue at the suture site include a cell type functionally differentiated from the surrounding tissue. In some embodiments, (i) new tissue is formed, and (ii) the covalently stabilized porous scaffold completely decomposes at least about 42 days after suture closure. In some embodiments, the new tissue is formed in addition to any tissue formed at the suture site by suture alone. In some embodiments, additional new tissue continues to form at the suture site for at least about 42 days after suture closure. In some embodiments, the new tissue is formed above (e.g., superficially) and deep within the subcutaneous tissue at the suture site. In some embodiments, the new tissue is stromal-like tissue with unaligned collagen bundles. In some embodiments, a covalently stabilized porous scaffold reinforces the suture line at the suture site of the target while minimizing the foreign body reaction of the target.In some embodiments, the foreign body reaction is characterized by causing harm to the subject. In some embodiments, the harm is characterized by causing chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof. In some embodiments, the harm is caused at the suture site. In some embodiments, a covalently stabilized porous scaffold is effective in strengthening the suture at the subject's suture site while minimizing the foreign body reaction, when the foreign body reaction is measured by detecting the amount of granuloma at the suture site using histological analysis and comparing the amount of granuloma at the suture site to a reference suture site without the hydrogel formulation. In some embodiments, a covalently stabilized porous scaffold is effective in strengthening the suture at the subject's suture site while minimizing the foreign body reaction, when the foreign body reaction is measured by detecting the amount of scar tissue at the suture site using histological analysis and comparing the amount of scar tissue at the suture site to a reference suture site without the hydrogel formulation. In some embodiments, a covalently stabilized porous scaffold is effective in strengthening the suture at a target suture site while minimizing the foreign body reaction, when the foreign body reaction is measured by detecting the amount of nodules at the suture site using histological analysis and comparing the amount of nodules at the suture site with a reference suture site without the hydrogel formulation. In some embodiments, a covalently stabilized porous scaffold is effective in strengthening the suture at a target suture site while minimizing the foreign body reaction, when the foreign body reaction is measured by detecting chronic inflammation at the suture site using histological analysis. In some embodiments, a covalently stabilized porous scaffold is effective in strengthening the suture at a target suture site while minimizing the foreign body reaction, when the foreign body reaction is measured by the presence of one or more types of macrophages at the target suture site. In some embodiments, the one or more types of macrophages include type 1 macrophages and type 2 macrophages. In some embodiments, type 1 macrophages are pro-inflammatory.In some embodiments, type 2 macrophages are pro-regenerative. In some embodiments, a covalently stabilized porous scaffold is effective in forming more type 2 macrophages than type 1 macrophages. In some embodiments, the cell matrix contains a certain amount or type of collagen that mimics endogenous tissue at the suture site. In some embodiments, collagen is formed at the suture site at least up to about 28 days after suture closure. In some embodiments, collagen is formed within and around the covalently stabilized porous scaffold. In some embodiments, the type of collagen includes type I collagen, type III collagen, or a combination thereof. In some embodiments, type I collagen is present with type III collagen in a ratio of about 10:1 or less, about 6:1 or less, or about 5:1 or less. In some embodiments, at least a portion of the suture site contains elastin after degradation of the covalently stabilized porous scaffold at the suture site. In some embodiments, the covalently stabilized porous scaffold contains a pH of about 8. In some embodiments, the covalently stabilized porous scaffold has a compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. In some embodiments, the hydrogel formulation comprises microgel particles comprising a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and cell adhesion peptides. In some embodiments, the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%. In some embodiments, the hydrogel formulation further comprises PEG-dithiol, and the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol. In some embodiments, the PEG-dithiol has a molecular weight of at least about 0.5 kilodaltons (kDa) to about 10 kDa. In some embodiments, the PEG-dithiol has a molecular weight of at least about 3.4 kDa.In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mM to about 1.0 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM.

[0004] In certain embodiments, a hydrogel system is described herein comprising microgel particles crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer, one or more K peptides and Q peptides, and a cell adhesion peptide, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%, and a PEG-dithiol, wherein the PEG-dithiol has a molecular weight of at least about 0.5 kilodaltons (kDa), and the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol. In certain embodiments, a hydrogel system is described herein comprising microgel particles crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer, one or more K peptides and Q peptides, and a cell adhesion peptide, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%, and PEG-dithiol, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol, and the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mmol (mM) to about 1.0 mM. In some embodiments, the microgel particles are present in the suspension at a volume fraction of at least 90%. In some embodiments, the PEG-dithiol and 4-arm PEG vinyl sulfone are present to provide a thiol-to-vinyl sulfone ratio of less than about 1.0. In some embodiments, the PEG-dithiol has a molecular weight of at least about 3.4 kDa. In some embodiments, the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM. In some embodiments, the microgel particles are spherical. In some embodiments, the microgel particles consist of microspheres.In some embodiments, the microgel particles include diameters ranging from 5 μm to 1000 μm, 50 μm to 1000 μm, or 70 μm to 150 μm. In some embodiments, the microgel particles have a compressive modulus of at least about 500 Pascals (Pa) before the annealing reaction. In some embodiments, the microgel particles have a compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. In some embodiments, the covalently stabilized porous scaffold includes pores with a median pore diameter of about 5 μm or more. In some embodiments, the pores include a median pore diameter of about 10 μm to about 35 μm. In some embodiments, one or more cell adhesion peptides include RGD peptides. In some embodiments, the microgel particles have a polydispersity of 0.1 or less. In some embodiments, polydispersity is calculated based on the standard deviation and mean size of the particles (e.g., PDI = (SD / mean)^2). In some embodiments, the hydrogel formulation further comprises a buffer, which includes phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or acetate buffer, or any combination thereof. In some embodiments, the annealing reaction comprises a covalent synthesis reaction. In some embodiments, the covalent synthesis reaction comprises a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the vinyl sulfone of 4-armed PEG vinyl sulfone is the Michael acceptor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the thiol of PEG-dithiol is the Michael donor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, a hydrogel formulation comprising the hydrogel system described herein in a suspension, wherein the suspension comprises a buffer, is described herein. In some embodiments, the hydrogel formulation is formulated for administration to a subject. In some embodiments, the hydrogel formulation is formulated for administration along the suture line of the subject. In some embodiments, the administration minimizes the foreign body reaction of the subject.In some embodiments, the hydrogel formulation contains a dosage volume of about 0.01 mL to about 20 mL. In some embodiments, a delivery device is described herein, comprising a body containing the hydrogel system or hydrogel formulation described herein, and an applicator in fluid communication with the body, wherein the delivery device is sterile. In some embodiments, the delivery device is a syringe or a needle.

[0005] Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. This specification is intended to supersede and / or take precedence over any publications and patents or patent applications incorporated by reference to the extent that they conflict with any disclosures contained herein. [Brief explanation of the drawing]

[0006] To better understand the features and advantages of this subject, please refer to the following detailed description and accompanying drawings, which describe exemplary embodiments. [Figure 1A] Figure 1A shows normalized yield strength versus abdominal location according to several embodiments of this specification. [Figure 1B] Figure 1B shows the normalized yield strength per rabbit according to several embodiments of this specification. [Figure 1C] Figure 1C shows the normalized yield strength under different storage conditions according to several embodiments of this specification. [Figure 2A] Figure 2A shows the zipper method for applying annealed microgel particles (MAP) according to several embodiments of this specification. [Figure 2B] Figure 2B shows a post-suture method for applying MAP according to several embodiments of this specification. [Figure 2C] Figure 2C shows a two-step method for applying MAP according to several embodiments of this specification. [Figure 2D] Figure 2D shows a one-step method for applying MAP according to some embodiments of this specification. [Figure 2E] Figure 2E is a schematic diagram of a midline incision and the application of MAP over the incision (procedure #3) in an offset dermal incision and closure according to some embodiments herein. [Figure 3A] Figure 3A shows the yield strengths measured by a linear actuarial analyzer (e.g., Instron 3342 system) 7 and 14 days after treatment for undamaged tissue, damaged tissue treated with sutures alone, and damaged tissue treated with MAP (Formulation 1 at 80%) and sutures, according to several embodiments of this specification. [Figure 3B] Figure 3B shows the ratio of yield strength of damaged tissue to undamaged tissue after treatment with suture alone or with MAP (Formulation 1 at 80%) and suture, according to several embodiments of this specification. Formulation 1 was applied intra-suture during suturing and also applied over the incision (Procedure #4). Normalization was performed for each tissue section width. [Figure 4A] Figure 4A shows representative histological images of the abdominal wall 7 days after incision and treatment with MAP (formulation 1) according to several embodiments of this specification. [Figure 4B] Figure 4B shows representative histological images of the abdominal wall 14 days after incision and treatment with MAP (Formula 1) according to several embodiments herein. Formula 1 was applied intra-suturing during suturing and also applied over the incision (Procedure #4). Formula 1 appears to be present both above and below the tissue, but not within the incision, nor directly above the incision, but rather lateral. [Figure 5A]Figure 5A shows representative histological images of the abdominal wall 14 days after incision and treatment with preparation 1 (VF=80%) according to several embodiments herein. Here, the dermis was closed offset from the midline, and preparation 1 was applied only over the incision (procedure #3). Rabbits #23 and #20. [Figure 5B] Figure 5B shows the normalized yield strengths measured by Instron 14 days after treatment for injured tissue treated with sutures alone, and injured tissue treated with MAP (Formulation 1 at 80%) and sutures, according to several embodiments of this specification. [Figure 5C] Figure 5C shows the ratio of yield strength of damaged tissue to undamaged tissue after treatment with sutures alone or with MAP (Formulation 1 at 80%) and sutures, according to several embodiments of this specification. Formulation 1 was applied over the incision (procedure #3), and the dermis was closed offset from the midline. Normalization was performed for each tissue section width. [Figure 6A] Figure 6A shows the stresses on different MAP formulations according to several embodiments of this specification. N represents the number of elongated sections tested (three elongated sections were taken per rabbit). Here, the dermis was closed offset from the midline. Normalization was performed per surface area (= width × thickness of tissue section). (Assuming all sections had the same thickness, dividing by thickness does not actually change the trend of the data between yield strength in N / mm and stress in N / mm².) [Figure 6B] Figure 6B shows representative histological images of the abdominal wall 14 days after incision and treatment with preparation 5 (VF=100%) according to several embodiments herein. Here, the dermis was closed offset from the midline, and preparation 5 was applied only over the incision (procedure #3). Rabbits #26 and #27. [Figure 6C] Figure 6C shows representative histological images of the abdominal wall 14 days after treatment with incision and preparation 4 (annealed with PRP at VF=90%) according to several embodiments herein. Here, the dermis was closed offset from the midline, and preparation 4 was applied only over the incision (procedure #3). Rabbits #36 and #37. [Figure 6D] Figure 6D shows representative histological images of the abdominal wall 14 days after treatment with incision and preparation 3 (annealed with PEG-dithiol crosslinking agent at VF=90%) according to several embodiments herein. Here, the dermis was closed offset from the midline, and preparation 4 was applied only over the incision (procedure #3). Rabbits #30 and #34. [Figure 6E] Figure 6E shows representative histological images of the abdominal wall 14 days after treatment with incision and preparation 3 (annealed with PEG-dithiol crosslinking agent at VF=90%) according to several embodiments herein. Here, the dermis was closed offset from the midline, and preparation 4 was applied inside and over the incision after suturing (procedure #5). Rabbits #31 and #33. [Figure 6F] Figure 6F shows the stresses for different MAP formulations and different application methods according to several embodiments of this specification. N represents the number of elongated pieces tested (three elongated pieces were taken per rabbit). [Figure 6G] Figure 6G shows the MAP preparation used in this experiment on the 14th day post-surgery. [Figure 7A] Figure 7A shows the yield stress over time at the abdominal incision site when formulation 3 is administered, compared to sutures alone, according to several embodiments of this specification. [Figure 7B] Figure 7B shows the 42-day recovery rate from abdominal incisions when formulation 3 is administered, compared to sutures alone, according to several embodiments of this specification. [Figure 7C] Figure 7C shows the yield stress of the incision site after application of Formulation 3 14, 28, and 42 days after surgery, according to several embodiments of this specification. [Figure 7D] Figure 7D shows the yield stress of the incision site where Formulation 3 was not applied 14, 28, and 42 days after surgery, according to several embodiments of this specification. [Figure 7E] Figure 7E shows typical stress-extension curves for abdominal incisions treated with formulation 3 compared to sutures alone, according to several embodiments of this specification. [Figure 7F] Figure 7F shows the toughness of the incision site at day 28 and day 42 in incisions to which formulation 3 was applied and in incisions to which formulation 3 was not applied, according to several embodiments of this specification. [Figure 8A] Figure 8A shows hematoxylin and eosin (H&E) histological images of the incision site 14 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number. [Figure 8B] Figure 8B shows picrosilius red images of the incision site 14 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number. [Figure 8C] Figure 8C shows detailed H&E and picrosilius red histological images of the incision site 14 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number. [Figure 8D] Figure 8D shows histological images of incisions treated with Formulation 3 14 days later, according to several embodiments of this specification. [Figure 8E] Figure 8E shows detailed H&E and picrosilius red histological images of incisions treated with Formulation 3 14 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number, as shown in Figure 8D. The arrows indicate the following features: a. cell infiltration into the scaffold, b. engraftment to the scaffold site by cell proliferation, c. virtually undamaged scaffold structure, d. neoangiogenesis of the scaffold, e. new collagen deposition on and within the scaffold. [Figure 8F] Figure 8F shows detailed H&E and picrosilius red histological images of the incision site 28 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number. [Figure 8G] Figure 8G shows histological images of incisions treated with Formulation 3 28 days later, according to several embodiments of this specification. [Figure 8H]Figure 8H shows detailed H&E and picrosilius red histological images of incisions treated with Formulation 3 28 days later, according to several embodiments herein. The numbers on each image indicate the rabbit number and the tissue section number, as shown in Figure 8G. The arrows indicate the following features: a. increased cell infiltration, b. scaffold structure immediately after degradation, c. mature angiogenesis in the tissue and undamaged scaffold, and d. marked collagen deposition around and within the scaffold. [Figure 8I] Figure 8I shows detailed H&E and picrosilius red histological images of the incision site 42 days later, according to several embodiments of this specification. The numbers on each image indicate the rabbit number and the tissue section number. [Figure 8J] Figure 8J shows histological images of incisions treated with Formulation 3 42 days later, according to several embodiments of this specification. [Figure 8K] Figure 8K shows detailed H&E and picrosilius red histological images of incisions treated with Formulation 3 42 days later, according to several embodiments herein. The numbers on each image indicate the rabbit number and the tissue section number, as shown in Figure 8J. The arrows indicate the following features: a. complete cellular infiltration into the scaffold, b. significant degradation of the scaffold with volume replaced by tissue and macrophages, c. mature angiogenesis of new tissue, d. marked collagen deposition in tissue with both fibrous and "basketweave" morphologies. [Figure 8L] Figure 8L shows histological images of the incision site over time. The numbers on each image indicate the rabbit number and the tissue section number, as per some embodiments herein. [Figure 8M] Figure 8M shows detailed histological images of the incision site over time. The numbers on each image indicate the rabbit number and the tissue section number, as per some embodiments herein. [Modes for carrying out the invention]

[0007] Midline abdominal incisions are commonly used in multiple human patient populations. Procedures associated with these incisions are called midline laparotomies. These midline laparotomies are performed on millions of patients annually and include gastrointestinal cancer treatments, hysterectomies and other ovarian cancer treatments, spinal fusion, and abdominal trauma surgery. Currently, incisions made to access the abdomen reopen in 30% of patients within 24 months post-surgery if these patients have certain comorbidities, including diabetes mellitus, a body mass index (BMI) > 25, or have undergone primary tumor resection. This reopening can lead to major complications and even death in some patients.

[0008] In certain embodiments, systems and compositions are described herein that strengthen suture lines, including midline abdominal incisions, through regenerative healing, and these systems and compositions have the potential to significantly reduce the rate of postoperative failure, morbidity, and mortality in this large patient population. In some embodiments, the systems and compositions described herein comprise one or more hydrogel formulations provided in Figure 6G. In some embodiments, the systems and compositions comprise microgel particles comprising a 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K-peptides and Q-peptides, and cell adhesion peptides. In some embodiments, the systems and compositions comprise PEG-dithiols having a molecular weight of at least about 0.5 kilodaltons to about 10 kilodaltons.

[0009] In certain embodiments, a method for delivering the hydrogel formulation described herein to a suture site of a subject is described herein. In some embodiments, the hydrogel formulation is annealed in situ to form a covalently stabilized porous scaffold. In some embodiments, endogenous cells infiltrate the covalently stabilized porous scaffold to form a cell matrix on the incision site. In some embodiments, the hydrogel formulation is delivered to the subject using an apparatus disclosed herein, such as a syringe or needle. In some embodiments, the method and / or the hydrogel formulation is optimized according to various embodiments herein for a given application (for example, to achieve a desired viscosity upon application and mechanical properties upon in situ annealing).

[0010] system Hydrogel systems are gel-like substances that may be useful for specific therapeutic applications. In therapeutic applications, hydrogel systems can strengthen suture sites when used as an aid in suture closure. For example, strengthening a suture site may be characterized by increasing the mechanical tensile strength of the suture, increasing the yield stress of the suture, increasing the toughness of the suture, increasing the recovery rate of the suture, increasing the mechanical tensile strength of the suture, or enabling collagen formation at the suture site. In some embodiments, strengthening a suture site is effective because it helps prevent suture reopening. In some embodiments, the systems and compositions described herein include one or more hydrogel formulations provided in Figure 6G.

[0011] In certain embodiments, hydrogel systems or hydrogel formulations comprising the microgel particles described herein are described herein. In some embodiments, the hydrogel system comprises poly(ethylene glycol)(PEG)-dithiol. In some embodiments, the hydrogel system comprises microgel particles comprising a crosslinked 4-arm PEG vinyl sulfone hydrogel polymer crosslinked with an MMP-degradable crosslinking agent, one or more K peptides and Q peptides, and cell adhesion peptides, 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 90%, and PEG-dithiol, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol, and the PEG-dithiol has a molecular weight of at least about 0.5 kilodaltons (kDa) to about 10 kDa, and is present in the hydrogel system at a molar concentration of at least about 0.2 mmol (mM).

[0012] Microgel particles In some embodiments, hydrogel formulations comprising a plurality of microgel particles are disclosed herein. In some embodiments, the plurality of microgel particles are formed by crosslinking together one or more reagents and raw materials according to various embodiments herein. The plurality of microgel particles may be in a slurry suitable for delivery to a subject by injection or application over a suture line. When the slurry is delivered to the subject at a tissue site, the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold in situ. The covalently stabilized porous scaffold comprises the annealed microgel particles and may be interchangeably referred to as “annealed particles,” “annealed microgel particles,” or “annealed scaffold.” In some embodiments, the covalently stabilized porous scaffold is formed such that pores are formed between the microgel particles of the covalently stabilized scaffold. In some embodiments, when the hydrogel formulation is administered to a subject, the covalently stabilized scaffold allows for the growth of a cell matrix in situ at or around the tissue site of the subject to form new tissue, even after the covalently stabilized scaffold has completely degraded. In some embodiments, the covalently stabilized scaffold allows for the in situ growth of the cell matrix in or around the target tissue site where new tissue will form before the covalently stabilized scaffold begins to degrade (e.g., degradation of the covalently stabilized scaffold is not required for the formation of the cell matrix and new tissue). In some embodiments, the new tissue formed is the endogenous tissue of the subject. In some embodiments, the new tissue is characterized as having mature angiogenesis, features of the surrounding tissue at the tissue site, or a combination thereof. In some embodiments, the features of the surrounding tissue at the tissue site include cell types that are functionally differentiated from the surrounding tissue. In some embodiments, the features of the surrounding tissue at the suture site include a certain amount or type of collagen that mimics the endogenous tissue at the suture site.

[0013] 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 include microspheres. The microgel particles may have a substantially uniform shape so as to create pores when adjacent microgel particles come into contact with each other. Other shapes of microgel particles are considered, but are not limited to, flattened, elongated, round particles, granular particles, flake particles, or 3D geometric shapes.

[0014] In some embodiments, the microparticles may have a diameter or dimensions (e.g., length, width, height, axis). In some embodiments, the microgel particles include a diameter or dimensions ranging from 0.1 micrometers (μm) to 1000 μm. In some embodiments, the microgel particles include a diameter or dimensions ranging from 5 micrometers (μm) to 1000 μm. In some embodiments, the diameter or dimensions include 50 μm to 1000 μm. In some embodiments, the diameter or dimensions include 80 μm to 140 μm. In some embodiments, the diameter or dimensions include 70 μm to 150 μm. In the globe embodiment, the diameter is approximately 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, Includes 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 μm or larger.In the globe embodiment, the diameter is approximately 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, Includes particles of 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, or 1100 μm or less. In some embodiments, the diameter or dimensions include a range of approximately 5 μm to approximately 1100 μm. In some embodiments, the diameter or dimensions include a range of approximately 10 μm to approximately 1090 μm. In some embodiments, the diameter or dimensions include a range of approximately 15 μm to approximately 1080 μm. In some embodiments, the diameter or dimensions include a range of approximately 20 μm to approximately 1070 μm. In some embodiments, the diameter or dimensions include a range of approximately 25 μm to approximately 1060 μm. In some embodiments, the diameter or dimensions include a range of approximately 30 μm to approximately 1050 μm. In some embodiments, the diameter or dimensions include a range of approximately 35 μm to approximately 1040 μm. In some embodiments, the diameter or dimensions include a range of approximately 40 μm to approximately 1030 μm. In some embodiments, the diameter or dimensions include a range of approximately 45 μm to approximately 1020 μm. In some embodiments, the diameter or dimensions include a range of approximately 50 μm to approximately 1010 μm. In some embodiments, the diameter or dimensions include a range of approximately 55 μm to approximately 1000 μm. In some embodiments, the diameter or dimensions include a range of approximately 60 μm to approximately 990 μm.In some embodiments, the diameter or dimensions include a range of approximately 65 μm to approximately 980 μm. In some embodiments, the diameter or dimensions include a range of approximately 70 μm to approximately 970 μm. In some embodiments, the diameter or dimensions include a range of approximately 75 μm to approximately 960 μm. In some embodiments, the diameter or dimensions include a range of approximately 80 μm to approximately 950 μm. In some embodiments, the diameter or dimensions include a range of approximately 85 μm to approximately 940 μm. In some embodiments, the diameter or dimensions include a range of approximately 90 μm to approximately 930 μm. In some embodiments, the diameter or dimensions include a range of approximately 95 μm to approximately 920 μm. In some embodiments, the diameter or dimensions include a range of approximately 100 μm to approximately 910 μm. In some embodiments, the diameter or dimensions include a range of approximately 110 μm to approximately 900 μm. In some embodiments, the diameter or dimensions include a range of approximately 120 μm to approximately 890 μm. In some embodiments, the diameter or dimensions include a range of approximately 130 μm to approximately 880 μm. In some embodiments, the diameter or dimensions include a range of approximately 140 μm to approximately 870 μm. In some embodiments, the diameter or dimensions include a range of approximately 150 μm to approximately 860 μm. In some embodiments, the diameter or dimensions include a range of approximately 160 μm to approximately 850 μm. In some embodiments, the diameter or dimensions include a range of approximately 170 μm to approximately 840 μm. In some embodiments, the diameter or dimensions include a range of approximately 180 μm to approximately 830 μm. In some embodiments, the diameter or dimensions include a range of approximately 190 μm to approximately 820 μm. In some embodiments, the diameter or dimensions include a range of approximately 200 μm to approximately 810 μm. In some embodiments, the diameter or dimensions include a range of approximately 210 μm to approximately 800 μm. In some embodiments, the diameter or dimensions include a range of approximately 220 μm to approximately 790 μm. In some embodiments, the diameter or dimensions include a range of approximately 230 μm to approximately 780 μm. In some embodiments, the diameter or dimensions include a range of approximately 240 μm to approximately 770 μm. In some embodiments, the diameter or dimensions include a range of approximately 250 μm to approximately 760 μm. In some embodiments, the diameter or dimensions include a range of approximately 260 μm to approximately 750 μm.In some embodiments, the diameter or dimensions include a range of approximately 270 μm to approximately 740 μm. In some embodiments, the diameter or dimensions include a range of approximately 280 μm to approximately 730 μm. In some embodiments, the diameter or dimensions include a range of approximately 290 μm to approximately 720 μm. In some embodiments, the diameter or dimensions include a range of approximately 300 μm to approximately 710 μm. In some embodiments, the diameter or dimensions include a range of approximately 310 μm to approximately 700 μm. In some embodiments, the diameter or dimensions include a range of approximately 320 μm to approximately 690 μm. In some embodiments, the diameter or dimensions include a range of approximately 330 μm to approximately 680 μm. In some embodiments, the diameter or dimensions include a range of approximately 340 μm to approximately 670 μm. In some embodiments, the diameter or dimensions include a range of approximately 350 μm to approximately 660 μm. In some embodiments, the diameter or dimensions include a range of approximately 360 μm to approximately 650 μm. In some embodiments, the diameter or dimensions include a range of approximately 370 μm to approximately 640 μm. In some embodiments, the diameter or dimensions include a range of approximately 380 μm to approximately 630 μm. In some embodiments, the diameter or dimensions include a range of approximately 390 μm to approximately 620 μm. In some embodiments, the diameter or dimensions include a range of approximately 400 μm to approximately 610 μm. In some embodiments, the diameter or dimensions include a range of approximately 410 μm to approximately 600 μm. In some embodiments, the diameter or dimensions include a range of approximately 420 μm to approximately 590 μm. In some embodiments, the diameter or dimensions include a range of approximately 430 μm to approximately 580 μm. In some embodiments, the diameter or dimensions include a range of approximately 440 μm to approximately 570 μm. In some embodiments, the diameter or dimensions include a range of approximately 450 μm to approximately 560 μm. In some embodiments, the diameter or dimensions include a range of approximately 460 μm to approximately 550 μm. In some embodiments, the diameter or dimensions include a range of approximately 470 μm to approximately 540 μm. In some embodiments, the diameter or dimensions include a range of approximately 480 μm to approximately 530 μm. In some embodiments, the diameter or dimensions include a range of approximately 490 μm to approximately 520 μm. In some embodiments, the diameter or dimensions include a range of approximately 500 μm to approximately 510 μm.

[0015] Microgel particles may have an average diameter or dimension of approximately 10 μm. Microgel particles may have an average diameter or dimension of approximately 15 μm. Microgel particles may have an average diameter or dimension of approximately 25 μm. Microgel particles may have a diameter or dimension of approximately 50 μm. Microgel particles may have an average diameter or dimension of approximately 100 μm. Microgel particles may have an average diameter or dimension of approximately 150 μm. Microgel particles may have an average diameter or dimension of approximately 200 μm. Microgel particles may have a diameter or dimension in the range of approximately 10 μm to approximately 500 μm. Microgel particles may have a diameter or dimension in the range of approximately 10 μm to approximately 200 μm. Microgel particles may have a diameter or dimension in the range of approximately 15 μm to approximately 200 μm. Microgel particles may have a diameter or dimension in the range of approximately 15 μm to approximately 150 μm. Microgel particles may have a diameter or dimensions in the range of approximately 30 μm to approximately 100 μm. Microgel particles may have an average diameter or dimensions of 10 μm. Microgel particles may have an average diameter or dimensions of 15 μm. Microgel particles may have an average diameter or dimensions of 25 μm. Microgel particles may have a diameter or dimensions of 50 μm. Microgel particles may have an average diameter or dimensions of 100 μm. Microgel particles may have an average diameter or dimensions of 150 μm.

[0016] Microgel particles may have an average diameter or average dimension of 200 μm. Microgel particles may have a diameter or dimension in the range of 10 μm to 500 μm. Microgel particles may have a diameter or dimension in the range of 10 μm to 200 μm. Microgel particles may have a diameter or dimension in the range of 15 μm to 200 μm. Microgel particles may have a diameter or dimension in the range of 15 μm to 150 μm. Microgel particles may have a diameter or dimension in the range of 30 μm to 100 μm. In some embodiments, the diameter of a microgel particle may be measured by (1) measuring the area of ​​the microgel particle, (2) solving for the radius of the microgel particle using the equation for the area of ​​a circle (i.e., A = pi * r^2), and (3) solving for the diameter by multiplying the radius by 2 (i.e., D = 2 * r).

[0017] In some embodiments, the microgel particles contain one or more cell adhesion peptides. In some embodiments, the cell adhesion peptide contains at least a portion of an extracellular matrix protein. In some embodiments, the cell adhesion peptide contains at least a portion of collagen. In some embodiments, the cell adhesion peptide contains at least a portion of fibronectin. In some embodiments, the cell adhesion peptide contains an integrin. In some embodiments, the adhesion peptide contains a ligand for a receptor expressed on the cell. In some embodiments, the adhesion peptide contains a differentiation cluster (CD) protein. In some embodiments, the adhesion peptide contains a naturally occurring peptide. In some embodiments, the adhesion peptide contains a synthetic peptide. In some embodiments, the cell adhesion peptide may be homologous to a naturally occurring peptide. In some embodiments, the cell adhesion peptide contains at least about 70% homology to a naturally occurring peptide. In some embodiments, the cell adhesion peptide is at least about 80% homologous to a naturally occurring peptide. In some embodiments, the cell adhesion peptide contains at least about 90% homology to a naturally occurring peptide. In some embodiments, the cell adhesion peptide contains at least 70% homology to naturally occurring peptides. In some embodiments, the cell adhesion peptide contains at least 80% homology to naturally occurring peptides. In some embodiments, the cell adhesion peptide contains at least 90% homology to naturally occurring peptides. In some embodiments, the cell adhesion peptide may be bound to the surface of microgel particles. In some embodiments, the cell adhesion peptide is graft-bonded to the surface of microgel particles. In some embodiments, the coupling may include one or more chemical bonds. In some embodiments, the one or more chemical bonds are one or more covalent bonds.

[0018] As a non-limiting example, the cell adhesion peptide may include the RGD peptide. In some embodiments, the RGD peptide includes RGDSPGERCG (SEQ ID NO: 1). In some embodiments, the RGD peptide includes ACDCRGDCFCG (SEQ ID NO: 2). In some embodiments, the RGD peptide includes GRGDSP (SEQ ID NO: 6). In some embodiments, the RGD peptide includes cyclo(Arg-Gly-Asp-DPhe-Val) (SEQ ID NO: 7). In some embodiments, the RGD peptide includes cyclo(Arg-Gly-Asp-DPhe-Lys)cyclo(Arg-Gly-Asp-DPhe-Cys) (SEQ ID NO: 8). In some embodiments, the RGD peptide includes KACDCRGDCFCG (SEQ ID NO: 9). In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 1. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 2. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 6. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 7. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 7.In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 7. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 8. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 9. In some embodiments, the RGD peptide comprises an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in any one of SEQ ID NOs: 1-2 or 6-9. In some embodiments, the RGD peptide is provided in whole by Moral MEG, Siahaan TJ, et al., as incorporated herein by reference. In some embodiments, the RGD peptide is modified to improve the conjugation of the RGD peptide to a substrate, such as microgel particles disclosed herein. Non-limiting examples of modifications include the addition of a cysteine ​​residue, the addition of a linker having a thiol group at one end and an amine group at the other, and the addition of a linker having a thiol group at one end and a carboxylic acid group at the other. In some embodiments, the RGD peptide includes modifications at either end (e.g., C-terminus, N-terminus). In some embodiments, the modification is located within the flanking sequence of RGD motifs within the RGD sequence.

[0019] The cell adhesion peptide may contain amino acids. In some embodiments, the cell adhesion peptide contains a K peptide. In some embodiments, one or more K peptides contain the amino acid sequence provided in Ac-FKGGERCG-NH2 (SEQ ID NO: 3). In some embodiments, the K peptide contains the amino acid sequence FKGGERCG (SEQ ID NO: 4). In some embodiments, the K peptide contains an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide contains an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide contains an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide contains an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the cell adhesion peptide includes the Q peptide. In some embodiments, the Q peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 5.In some embodiments, the Q peptide comprises an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 5.

[0020] In some embodiments, the microgel particles contain a polymer. In some embodiments, the polymer is or contains a polymer backbone. In some embodiments, the polymer backbone of the polymer consists of a main chain of polymers (e.g., a polymer that occupies a larger proportion in a substance compared to other polymers in that substance). In some embodiments, the main chain can be considered a linear chain in the polymer, to which any other chains may be pendanted. In some embodiments, the polymer is or contains a copolymer. In some embodiments, the copolymer contains polymer chains comprising two or more different monomers in substantially equal proportions. In some embodiments, the polymer is crosslinkable and capable of holding a large amount of water to form a water-insoluble hydrogel. In some embodiments, the polymer is a synthetic polymer. Non-limiting examples of polymers include poly(ethylene glycol), polyacrylamide, and polymethacrylate. In some embodiments, the polymer may include hydrophilic polymers, amphiphilic polymers, synthetic polymers, or copolymers of hydrophobic polymers and hydrophilic polymers (e.g., poly(ethylene glycol) (PEG), poly(propylene 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 include a functional group (e.g., vinyl) at at least one end of the polymer (e.g., poly(ethylene glycol) methacrylate). In some embodiments, the polymer may include a functional group (e.g., vinyl) incorporated into the polymer backbone (e.g., poly(methacrylate)). In some embodiments, the polymer may include vinyl polymers such as poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) vinyl sulfone, poly(ethylene glycol) maleimide, poly(ethylene glycol) norbornene, and poly(ethylene glycol) allyl. In some embodiments, the polymer may include polyacrylamide or polymethacrylate.In some embodiments, the polymer may include polyester, polyamide, polyurethane, or mixtures or copolymers thereof. In some embodiments, the polymer is or contains poly(ethylene) glycol (PEG). In some embodiments, the polymer consists of PEG. In some embodiments, the microgel particles include two or more polymers (e.g., polymers made from different materials). In some embodiments, the two or more polymers include PLA and PEG. In some embodiments, the two or more polymers include PMMA and PEG. In some embodiments, the two or more polymers include a polymer containing functional groups and a polymer without functional groups. In some embodiments, the microgel particles include three or more polymers, each independently made from a material selected from the group consisting of hydrophilic polymers, amphiphilic polymers, and synthetic polymers. In some embodiments, the microgel particles include three or more polymers, each independently made from a material selected from poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(propylene glycol), poly(hydroxyethyl methacrylate), or any modified version thereof. In some embodiments, if the microgel particles contain two or more polymers, the ratio of each polymer contained in the microgel particles may vary. In some embodiments, the polymers include 4-arm PEG vinyl sulfone.

[0021] In some embodiments, the microgel particles comprise a polymer including PEG or a modified version thereof. In some embodiments, the microgel particles comprise an MMP-degradable crosslinking agent. In some embodiments, the MMP-degradable crosslinking agent crosslinks the polymer within the microgel particles.

[0022] In some embodiments, the polymer (e.g., PEG vinyl sulfone) may be present in microgel particles, hydrogel formulations containing microgel particles, the resulting covalently stabilized scaffold, or any combination thereof in amounts of about 0.5% by weight (wt%) to about 50 wt%. In some embodiments, the polymer may be present in amounts of about 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% or more in microgel particles, hydrogel formulations containing microgel particles, the resulting covalently stabilized scaffold, or any combination thereof. In some embodiments, the polymer may be present in amounts of about 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt% or less. In some embodiments, the polymer may be present in amounts of approximately 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.5 wt%, 3 wt%, 4 wt%, or 5 wt% or more. In some embodiments, the polymer may be present in amounts of about 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.5 wt%, 3 wt%, 4 wt%, or 5 wt% or less. In some embodiments, the polymer may be present in amounts of about 5 wt% to about 45 wt%. In some embodiments, the polymer may be present in amounts of about 10 wt% to about 40 wt%. In some embodiments, the polymer may be present in an amount of about 15% by weight (wt%) to about 35% by weight. In some embodiments, the polymer may be present in an amount of about 20% by weight (wt%) to about 30% by weight. In some embodiments, the polymer may be present in an amount of about 0.1% by weight to about 1.5% by weight.In some embodiments, the polymer may be present in an amount of about 0.5 wt%.

[0023] In some embodiments, the polymer is modified from a polymer that is identical to the polymer except that it does not contain any modifications. In some embodiments, the modified polymer includes modified PEG, such as PEG modified to contain a thiol or a derivative thereof. Non-limiting modifications include thiolation of terminal alcohol groups on the PEG polymer. In some embodiments, the modified polymer includes PEG vinyl sulfone. In some embodiments, the modified polymer includes 4-armed PEG vinyl sulfone (4-armed PEG-VS).

[0024] In some embodiments, the molecular weight of the polymer may affect the properties of the microgel particles, the hydrogel formulation containing the microgel particles, the resulting covalently stabilized scaffold, or any combination thereof.

[0025] In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight of about 1 kilodalton (kDa) to about 60 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight of about 1 kilodalton (kDa) to 1000 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) is approximately 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, 5 20, 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 or greater. In some embodiments, the PEF contains a molecular weight of at least about 3.4 kDa.In some embodiments, the polymer (e.g., 4-arm PEG-VS) is approximately 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, 5 20, 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 or less. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 5 kDa to about 1100 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 10 kDa to about 1090 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 15 kDa to about 1080 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 20 kDa to about 1070 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 25 kDa to about 1060 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 30 kDa to about 1050 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 35 kDa to about 1040 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 40 kDa to about 1030 kDa.In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 45 kDa to about 1020 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 50 kDa to about 1010 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 55 kDa to about 1000 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 60 kDa to about 990 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 65 kDa to about 980 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 70 kDa to about 970 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 75 kDa to about 960 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 80 kDa to about 950 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 85 kDa to about 940 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 90 kDa to about 930 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 95 kDa to about 920 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 100 kDa to about 910 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 110 kDa to about 900 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 120 kDa to about 890 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight range of about 130 kDa to about 880 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight range of about 140 kDa to about 870 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) has a molecular weight range of about 150 kDa to about 860 kDa.In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 160 kDa to about 850 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 170 kDa to about 840 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 180 kDa to about 830 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 190 kDa to about 820 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 200 kDa to about 810 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 210 kDa to about 800 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 220 kDa to about 790 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 230 kDa to about 780 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 240 kDa to about 770 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 250 kDa to about 760 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 260 kDa to about 750 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 270 kDa to about 740 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 280 kDa to about 730 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 290 kDa to about 720 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 300 kDa to about 710 kDa.In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 320 kDa to about 690 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 330 kDa to about 680 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 340 kDa to about 670 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 350 kDa to about 660 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 360 kDa to about 650 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 370 kDa to about 640 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 380 kDa to about 630 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 390 kDa to about 620 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 400 kDa to about 610 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 410 kDa to about 600 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 420 kDa to about 590 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 430 kDa to about 580 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 440 kDa to about 570 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 450 kDa to about 560 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 460 kDa to about 550 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 470 kDa to about 540 kDa.In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 480 kDa to about 530 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 490 kDa to about 520 kDa. In some embodiments, the polymer (e.g., 4-arm PEG-VS) includes a molecular weight range of about 500 kDa to about 510 kDa.

[0026] In some embodiments, microgel particles can be functionalized to contain one or more functional groups. For example, microgel particles made from hydrogels may be functionalized to contain functional groups coupled to them. In some embodiments, the functional groups include hydroxyl functional groups, methyl functional groups, carbonyl functional groups, carboxyl functional groups, amino functional groups, phosphate functional groups, sulfhydryl functional groups, or combinations thereof. In some embodiments, the functional groups include alkanes, alkenes, alkynes, ethers, sulfides, amines, aldehydes, ketones, imines, nitriles, or combinations thereof. In some embodiments, the functional groups may be coupled to the microgel particles using bonding, linkage, interaction, or other coupling mechanisms. In some embodiments, the bonding is covalent. In some embodiments, the bonding is non-covalent. In some embodiments, the bonding is selected from carbon-carbon bonds, amide bonds, imine bonds, ester bonds, thioether bonds, disulfide bonds, hydrazone bonds, hydrogen bonds, and metal ligand bonds. In some embodiments, the ester bonds include cyclic boronic acid esters. In some embodiments, the linkage is selected from carbamate linkage, ester linkage, and thioether linkage. In some embodiments, the coupling is selected from oxime coupling and thiourea coupling. In some embodiments, the interaction is selected from electrostatic interaction and van der Waals interaction. In some embodiments, the functional group includes a thiol or a derivative thereof. In some embodiments, the functional group includes a matrix metalloproteinase (MMP)-sensitive peptide. Non-limiting examples of thiol derivatives include any organosulfur compound of the form R-SH (wherein R represents an alkyl or other organic substituent).In some embodiments, the thiol derivatives include methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, allyl mercaptan, butanethiol, tert-butyl mercaptan, pentanethiol, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, metallothionein, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 1-mercaptonidole, grapefruit mercaptan, furan-2-ylmethanethiol, 3-mercaptopropane-1,2-diol, 3-mercapto-1-propanesulfonic acid, 1-hexadecanethiol, pentachlorobenzenethiol, or combinations thereof. In some embodiments, the functional group includes vinyl or a derivative thereof. In some embodiments, the functional group includes vinyl sulfone (VS) or a derivative thereof. Non-limiting examples of vinyl derivatives include alkenes containing ethene, propene, butene, pentene, hexene, heptene, and octene, acrylates, methacrylates, acrylamides, methacrylamides, maleimides, norbornene, or combinations thereof. Non-limiting examples of VS derivatives include phenylvinyl sulfone, methylvinyl sulfone, ethylvinyl sulfone, or any combination thereof. In some embodiments, the functional group includes a thiol and VS, or a derivative of a thiol or VS. In some embodiments, PEG is modified to include VS or a derivative thereof to form PEG-VS. In some embodiments, PEG is modified to include a thiol or a derivative thereof to form thiolated PEG (e.g., PEG-SH, PEG-dithiol). In some embodiments, PEG-VS includes multi-armed PEG-VS. In some embodiments, multi-armed PEG-VS includes 4-armed, 6-armed, or 8-armed PEG-VS. In some embodiments, the multi-arm PEG-VS comprises a star-shaped polymer, a brush-shaped polymer, a branched polymer, a comb-shaped polymer, or a dendritic polymer PEG-VS. In some embodiments, VS comprises a vinyl sulfone. In some embodiments, PEG-VS comprises a four-arm PEG-VS.

[0027] In some embodiments, the microgel particles include functional groups that may be pH-responsive (e.g., pH-responsive microgel particles). In some embodiments, the pH-responsive microgel particles may be characterized as microgel particles that need to be in the presence of a desired pH range to induce annealing of a covalently stabilized scaffold.

[0028] The functional groups disclosed herein may include peptides. The functional groups disclosed herein may include amino acids. In some embodiments, the functional group includes a K peptide. In some embodiments, the K peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence comprising FKGGERCG (SEQ ID NO: 4). In some embodiments, the K peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the K peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the K peptide includes an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 4. In some embodiments, the functional group includes the Q peptide. In some embodiments, the Q peptide includes the amino acid sequence provided in NQEQVSPLGGERCG (SEQ ID NO: 5). In some embodiments, the Q peptide includes an amino acid sequence that is approximately 75% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide includes an amino acid sequence that is approximately 85% identical to the amino acid sequence provided in SEQ ID NO: 5. In some embodiments, the Q peptide comprises an amino acid sequence that is approximately 95% identical to the amino acid sequence provided in SEQ ID NO: 5.In some embodiments, the Q peptide comprises an amino acid sequence that is approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence provided in SEQ ID NO: 5.

[0029] The functional groups may include non-peptide polymers. Degradable functional groups may also include random sequences, Omi target sequences, and heat shock protein target sequences. The functional groups may include amino acids having D-chirality. The functional groups may also include amino acids having L-chirality. The functional groups may include hydrolyzable synthetic polymers consisting of heparin, alginic acid, poly(ethylene glycol), polyacrylamide, polymethacrylate, polycondensate copolymers, and terpolymers (other polymers such as polyester, polyamide, and polyurethane). The functional groups may be produced by synthesis or isolated naturally. The functional groups may include DNA oligonucleotides having sequences corresponding to restriction enzyme recognition sequences, CpG motifs, zinc finger motifs, CRISPR sequences or Cas-9 sequences, Talon recognition sequences, or transcription factor binding domains. The functional groups may be activated at at least two ends by reactive groups defined as chemical groups that enable the crosslinking agent to participate in the crosslinking reaction to form microgel particles (internal crosslinking within the particles) or anneal the particles together to form a covalently stabilized scaffold (internal crosslinking between particles). Examples of these functional groups include cysteine ​​amino acids, synthetic and naturally occurring thiol-containing molecules, carbene-containing groups, vinyl-containing groups, activated esters, acrylates, norborene, primary amines, hydrazides, phosphene, azides, epoxy-containing groups, SANPAH-containing groups, and diaziline-containing groups. In some embodiments, the microgel particles themselves may function as crosslinking agents. In some embodiments, the functional groups may be degradable.

[0030] In some embodiments, the microgel particles may be functionalized with acrylate, methacrylate, methacrylamide, maleimide, norbornene, or any other vinyl derivative. For example, the hydrogel formulation may further contain two or more acrylates, methacrylates, acrylamides, maleimides, norbornene, or any combination thereof.

[0031] In some embodiments, the microgel particles are drug-eluting such that the therapeutic agent disclosed herein is released in situ by the microgel particles. In some embodiments, the therapeutic agent includes an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-fibrotic agent, or an antibiotic. In some embodiments, the local anesthetic is of ester form. In some embodiments, the ester-type local anesthetic includes benzocaine, chloroprocaine, procaine, propalacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is of amide form. In some embodiments, the amide-type local anesthetic includes articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, or cincocaine, or any combination thereof. In some embodiments, the local anesthetic is lidocaine or includes it. In some embodiments, the local anesthetic consists of lidocaine. In some embodiments, the analgesic includes codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID) or a steroid. In some embodiments, the NSAID includes ibuprofen or naproxen. In some embodiments, the steroid includes a corticosteroid. In some embodiments, the antibiotic includes dicloxacillin, erythromycin, or tetracycline. In some embodiments, the antifibrotic agent includes pentoxifylline.

[0032] In some embodiments of this specification, microgel particles are characterized as having a certain degree of polydispersity. In some embodiments, the polydispersity of microgel particles is an index of the size-based heterogeneity of the microgel particles. In some embodiments, microgel particles have a polydispersity of 0.1 or less, where the polydispersity (PDI) is calculated based on the standard deviation (SD) and mean size of the microgel particles (e.g., PDI = (SD / mean)^2). In some embodiments, polydispersity is measured using the coefficient of variation (CV), which is calculated based on the standard deviation (SD) and mean size of the microgel particles (e.g., CV = SD / mean). In some embodiments, lower size-based polydispersity of microgel particles aids in the formation of a covalently stabilized scaffold. In some embodiments, lower size-based polydispersity of microgel particles aids in achieving desired mechanical properties of the covalently stabilized scaffold. In some embodiments, lower polydispersity improves the porosity of the composition (for example, greater polydispersity of particle size can lead to smaller particles being inserted into the pores of a covalently stabilized scaffold).

[0033] In some embodiments, the components of the hydrogel formulation described above can help synthesize microgel particles. In some embodiments, thiols or their derivatives and VS or its derivatives are configured to interact with each other in a certain reaction to synthesize microgel particles.

[0034] In some embodiments, the reaction involves covalent synthetic reactions. Non-limiting examples of covalent bonds include those found in carbon-carbon, amide, ester, thioether, carbamate, disulfide, oxime, thiourea, hydrazone, and imine bonds. In some embodiments, the reaction involves non-covalent synthetic reactions. Non-limiting examples of non-covalent bonds include those found in interactions such as electrostatic interactions, hydrogen bonds, cation-π, π-π stacking, metal-ligand bonds, and van der Waals interactions. In some embodiments, the method involves linking two or more microgel particles together. Non-limiting examples of linking reactions include Michael addition, amide bond coupling, "Crick" chemistry (e.g., Diels-Alder cycloaddition, Husgen 1,3-dipolar cycloaddition), reductive amination, carbamate linking, ester linking, thioether linking, disulfide bond, hydrazone linking, oxime coupling, and thiourea coupling.

[0035] In some embodiments, the reaction involves a covalent synthetic reaction. In some embodiments, the covalent synthetic reaction includes a Michael addition (e.g., thiol-em-Michael addition, aza-Michael addition, oxa-Michael addition) reaction or a pseudo-Michael addition reaction. In some embodiments, the VS of PEG-VS may undergo a covalent synthetic reaction with a thiol or its derivative. In some embodiments, the VS or its derivative and the thiol or its derivative exist in a VS-to-thiol ratio of less than about 1.0. In some embodiments, the VS or its derivative and the thiol or its derivative exist in a VS-to-thiol ratio 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 its derivative and the thiol or its derivative exist in a thiol-to-VS ratio of less than about 1.0. In some embodiments, VS or its derivatives and thiols or their derivatives are present in thiol-to-VS ratios 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, thiols or their derivatives are Michael donors in Michael addition reactions or pseudo-Michael addition reactions. In some embodiments, VS or its derivatives are Michael acceptors in Michael addition reactions or pseudo-Michael addition reactions. In some embodiments, thiols or their derivatives and VS or their derivatives are present in the hydrogel formulation in a molar ratio of about 1:1. In some embodiments, thiols or their derivatives and VS or their derivatives are present in the hydrogel formulation in molar ratios of about 0.3:1 thiol:VS to 1:1 thiol:VS. In some embodiments, thiols or their derivatives and VS or their derivatives are present in the hydrogel formulation in molar ratios of about 0.6:1 thiol:VS to 0.8:1 thiol:VS. In some embodiments, thiols or their derivatives and VS or their derivatives are present in the hydrogel formulation in a thiol:VS molar ratio of about 1:1 to about 1:2. In some embodiments, thiols or their derivatives and VS or their derivatives are present in the hydrogel formulation in a thiol:VS molar ratio of about 1:1 to about 1:1.4.In some embodiments, the molar ratio may be defined as the molar ratio of thiol (SH) to vinyl sulfone (VS) groups, where R = [SH] / [VS] = nSH / nVS, where [SH] is defined as the molar concentration of thiol, [VS] as the molar concentration of VS, nSH as the number of moles of thiol, and nVS as the number of moles of VS. In some embodiments, an excess of thiol or its derivatives, or VS or its derivatives, is present in the hydrogel formulation so that either the excess thiol or its derivatives, or VS or its derivatives, participates in the annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the vinyl sulfone of 4-armed PEG vinyl sulfone is the Michael acceptor in a Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the thiol of PEG-dithiol is the Michael donor in a Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mmol (mM).

[0036] In some embodiments, the synthesis of microgel particles may be achieved via one or more physical linkages (e.g., loops) of the polymer(s) (e.g., PEG) constituting the microgel particles. In some embodiments, the physical linkages may include weak physical interactions. In some embodiments, the weak physical interactions may include coordination bonds and ionic interactions. In some embodiments, the physical linkages assist in the reaction when synthesizing the microgel particles. In some embodiments, the synthesis of microgel particles is achieved solely by reaction. In some embodiments, the synthesis of microgel particles is achieved solely by physical linkages.

[0037] Covalently stabilized scaffolding In some embodiments, microgel particles are disclosed herein that anneal together to form a covalently stabilized scaffold. In some embodiments, the formation of the covalently stabilized scaffold occurs in situ after delivery of the microgel particles to the tissue site. In some embodiments, the hydrogel formulation comprises one or more components configured to assist the annealing reaction to form a covalently stabilized porous scaffold.

[0038] In some embodiments, one or more components that promote or induce annealing of microgel particles in a hydrogel formulation to form a covalently stabilized scaffold include annealing components, annealing agents, or combinations thereof. In some embodiments, the annealing agent includes a molecule. In some embodiments, the annealing agent includes triethanolamine. In some embodiments, the annealing agent includes an enzyme. In some embodiments, the enzyme includes thrombin. In some embodiments, the annealing agent includes a transglutaminase enzyme. A non-limiting example of the transglutaminase enzyme is factor XIII (factor XIIIa in its active form). In some embodiments, the annealing agent includes a radical initiator. In some embodiments, the annealing agent includes an electron transfer agent. Examples of additional and alternative annealing agents, non-limiting examples, include active esters and nucleophiles, catechols that crosslink upon oxidation, and other redox-sensitive molecules. In some embodiments, the annealing agent comprises a homo- or hetero-functional polymer containing a thiol, maleimide, vinyl sulfone, methacrylate, methacrylamide, or other vinyl functional group. In some embodiments, the annealing agent comprises a cyclodextrin, cucurbituryl, or calixarene. In some embodiments, the annealing component comprises a K peptide, a Q peptide, or a combination thereof. In some embodiments, the annealing component comprises a vinyl group (e.g., vinyl sulfone, methyl acrylate, acrylamide), a thiol, maleimide, or an amine. In some embodiments, the annealing component comprises a vinyl sulfone group and a dithiol group.

[0039] In some embodiments, the microgel particles do not require an annealing agent for annealing. For example, the microgel particles may contain other components (e.g., functional groups) that participate in chemical crosslinking reactions to form a covalently stabilized scaffold. In some embodiments, one or more components that promote the annealing of microgel particles in the hydrogel formulation to form a covalently stabilized scaffold include thiol derivatives, vinyl derivatives, or combinations thereof. For example, the microgel particles may consist of a polymer or copolymer modified to contain one or more vinyl derivatives and one or more thiol derivatives, with either the vinyl derivative or the thiol derivative being in excess. Such vinyl derivatives and thiol derivatives may undergo chemical crosslinking reactions to anneal the microgel particles together and form a covalently stabilized scaffold.

[0040] In some embodiments, annealing reactions, which involve annealing microgel particles together to form a covalently stabilized scaffold, include covalent synthetic reactions. Non-limiting examples of covalent bonds include those found in carbon-carbon, amide, ester, thioether, carbamate, disulfide, oxime, thiourea, hydrazone, and imine. In some embodiments, covalent synthetic reactions include Michael addition (e.g., thiol-em-Michael addition) or pseudo-Michael addition reactions. In some embodiments, thiol derivatives are Michael donors in Michael addition or pseudo-Michael addition reactions. In some embodiments, vinyl derivatives (e.g., vinyl sulfones) are Michael acceptors in Michael addition or pseudo-Michael addition reactions. In some embodiments, covalent synthetic reactions include hydroxyl (oxo-Michael addition) or amine (aza-Michael addition) reactions.

[0041] In some embodiments, one or more functional groups and components assisting the annealing reaction are configured to interact to carry out the annealing reaction. In some embodiments, the PEG-dithiol is configured to interact with the VS of PEG-VS in the annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the annealing reaction includes a covalent annealing reaction. In some embodiments, the covalent annealing reaction includes a Michael addition reaction or a pseudo-Michael addition reaction. In some embodiments, the thiol of PEG-dithiol or a derivative thereof is the Michael donor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the VS of PEG-VS is the Michael acceptor in the Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mmol (mM). In some embodiments, PEG-dithiol is present in the hydrogel system at molar concentrations of at least about 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, PEG-dithiol is present in the hydrogel system at molar concentrations of at least about 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 less than 2.0 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at molar concentrations of about 0.1 to about 2.0 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 1.9 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.3 to about 1.8 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.4 to about 1.7 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.5 to about 1.6 mM.In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.6 to about 1.5 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.7 to about 1.4 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.8 to about 1.3 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.9 to about 1.2 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 1.0 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 0.9 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 0.7 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 0.6 mM. In some embodiments, PEG-dithiol is present in the hydrogel system at a molar concentration of about 0.2 to about 0.5 mM.

[0042] In some embodiments, the hydrogel formulation further comprises PEG-divinylsulfone or a derivative thereof. In some embodiments, PEG-divinylsulfone may be an auxiliary component for the annealing reaction. In some embodiments, PEG-divinylsulfone or a derivative thereof is configured to interact with an excess thiol or a derivative thereof in the annealing reaction to form a covalently stabilized porous scaffold. In some embodiments, the divinylsulfone or a derivative thereof of PEG-divinylsulfone is a Michael acceptor in a Michael addition reaction or pseudo-Michael addition reaction. In some embodiments, the excess thiol is a Michael donor in a Michael addition reaction or pseudo-Michael addition reaction.

[0043] In some embodiments, the annealing reaction that anneals microgel particles together to form a stabilized scaffold includes non-covalent synthetic reactions. Non-limiting examples of non-covalent reactions include interactions such as electrostatic interactions, hydrogen bonds, cation-π, π-π stacking, metal-ligand bonding, van der Waals interactions, and host-guest interactions such as cyclodextrin-adamantane reactions. In some embodiments, annealing reactions involving host-guest interactions may be reversible. In some embodiments, the annealing reaction that anneals microgel particles together to form a stabilized scaffold includes covalent synthetic reactions. Non-limiting examples of covalent reactions include Michael addition, amide coupling, "Crick" chemistry (e.g., Diels-Alder cycloaddition, Husgen 1,3-dipolar cycloaddition), reductive amination, carbamate linkage, ester linkage, thioether linkage, disulfide linkage, hydrazone linkage, oxime coupling, and thiourea coupling.

[0044] In some embodiments, the molecule contains PEG. In some embodiments, the molecule contains PEG-dithiol. In some embodiments, the PEG-dithiol has a molecular weight of about 0.5 kDa to about 10 kDa. In some embodiments, the PEG-dithiol has 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 has a molecular weight 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, PEG-dithiols have molecular weights of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or less than 20 kDa. In some embodiments, PEG-dithiols have molecular weights of about 0.5 kDa to about 10.0 kDa. In some embodiments, PEG-dithiols have molecular weights of about 1.0 kDa to about 15.0 kDa. In some embodiments, PEG-dithiols have molecular weights of about 1.0 kDa to about 20.0 kDa. In some embodiments, PEG-dithiols have molecular weights of about 3.0 kDa to about 10.0 kDa. In some embodiments, PEG-dithiols have molecular weights of about 3.0 kDa to about 5.0 kDa. In some embodiments, PEG-dithiols have molecular weights of about 3.4 kDa. In some embodiments, PEG-dithiol includes linear PEG-dithiol, multi-armed PEG-dithiol, or a combination thereof. In some embodiments, PEG-thiol includes multi-armed PEG-thiol. In some embodiments, multi-armed PEG-thiol includes 4-armed, 6-armed, or 8-armed PEG-thiol. In some embodiments, multi-armed PEG-thiol includes star-shaped polymer, brush-shaped polymer, branched polymer, comb-shaped polymer, or dendritic polymer PEG-thiol.

[0045] In some embodiments, microgel particles are annealed in the presence of one or more buffers to form a covalently stabilized scaffold. Non-limiting examples of buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4), calcium chloride (CaCl2), phosphate-buffered saline (PBS) (pH 6.0-8.0), TRIS, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid buffer (MES), or combinations thereof. In some embodiments, further stimuli such as light or pH are added to catalyze the reaction.

[0046] In some embodiments, the covalently stabilized scaffold is porous. In some embodiments, the covalently stabilized scaffold includes pores having a median pore diameter of about 5 μm or more. In some embodiments, the pores include a median pore diameter of about 10 μm to about 35 μm. In some embodiments, the pores include a median pore diameter of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 μm or more. In some embodiments, the pores include a median pore diameter of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 μm or less. In some embodiments, the pores include a median pore diameter of about 5 μm to about 70 μm. In some embodiments, the pores include a median pore diameter of about 10 μm to about 65 μm. In some embodiments, the pores include a median pore diameter of about 15 μm to about 60 μm. In some embodiments, the pores include a median pore diameter of about 20 μm to about 55 μm. In some embodiments, the pores include a median pore diameter of about 25 μm to about 50 μm. In some embodiments, the pores include a median pore diameter of about 30 μm to about 45 μm. In some embodiments, the pores include a median pore diameter of about 35 μm to about 40 μm. The median pore diameter may be measured for pore sampling by a process that includes (1) measuring the area of ​​the pore, (2) solving for the radius of the pore using the equation for the area of ​​a circle (i.e., A = pi * r^2), and (3) solving for the diameter by multiplying the radius by 2 (i.e., D = 2 * r).

[0047] In some embodiments, the covalently stabilized scaffold is degradable in vivo by one or more degradation pathways. In some embodiments, one or more degradation pathways include oxidative degradation, enzymatic degradation, photodegradation, or hydrolysis. In some embodiments, the microgel particle composition is finely tuned to achieve a desired degradation profile depending on the application.

[0048] In some embodiments, the covalently stabilized scaffold is present in the tissue site for about 1–2 months before complete degradation. In some embodiments, the covalently stabilized scaffold is present in the tissue site for at least about 1 month before complete degradation. In some embodiments, the covalently stabilized scaffold is present in the tissue site for at least about 2 months before complete degradation. In some embodiments, the hydrogel formulation enables increased growth of proteins, cells, and tissues in the cell matrix. Specifically, Figures 8D–8K show that the hydrogel formulations described herein minimize xenobiotic reactions, such as enabling vascular perfusion within the pore space, deposition of new proteins (e.g., fibrous, unaligned) within the pore space, and minimizing or completely preventing the formation of multinucleated giant cells (MNGCs).

[0049] In some embodiments, the covalently stabilized scaffold (e.g., microgel particles after annealing) has a compressive modulus of 1,000 Pascals (Pa) to 100,000 Pa. In some embodiments, the covalently stabilized scaffold has a compressive modulus of at least about 1,500 Pa after annealing. In some embodiments, the covalently stabilized scaffold has a compressive modulus of at least about 6,000 Pa after annealing. In some embodiments, the covalently stabilized scaffold may be annealed when the microgel particles are in a swollen and unswollen state. In some embodiments, the covalently stabilized scaffold may be annealed when the microgel particles are swollen with water or the like. In some embodiments, the microgel particles are in an unswollen state when the covalently stabilized scaffold is annealed. In some embodiments, as discussed above, the compressive modulus of the scaffold can be adjusted by adjusting the molecular weight, substitution rate, and molar ratio of the hydrogel polymer components.

[0050] In some embodiments, the covalently stabilized scaffold has a compressive modulus of 5,000 Pascals (Pa) to 100,000 Pa in a non-swelling state. In some embodiments, the covalently stabilized scaffold, in its non-swelling state, includes a compressive modulus of approximately 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, 125,000, 130,000, 135,000, 140,000, 145,000, or 150,000 Pa or more. In some embodiments, the covalently stabilized scaffold, in its non-swelling state, includes a compressive modulus of approximately 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, 125,000, 130,000, 135,000, 140,000, 145,000, or 150,000 Pa or less. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 5,000 Pa to about 150,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 10,000 Pa to about 145,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 15,000 Pa to about 140,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 20,000 Pa to about 135,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 25,000 Pa to about 130,000 Pa in an unswelled state.In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 30,000 Pa to about 125,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 35,000 Pa to about 120,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 40,000 Pa to about 115,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 45,000 Pa to about 110,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 50,000 Pa to about 105,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 55,000 Pa to about 100,000 Pa in an unswelled state. In some embodiments, the covalently stabilized scaffold has a compressive modulus in the range of about 60,000 Pa to about 95,000 Pa in the non-swollen state. In some embodiments, the covalently stabilized scaffold has a compressive modulus in the range of about 65,000 Pa to about 90,000 Pa in the non-swollen state. In some embodiments, the covalently stabilized scaffold has a compressive modulus in the range of about 70,000 Pa to about 85,000 Pa in the non-swollen state. In some embodiments, the covalently stabilized scaffold has a compressive modulus in the range of about 75,000 Pa to about 80,000 Pa in the non-swollen state.

[0051] In some embodiments, the covalently stabilized scaffold has a compressive modulus of 1,000 Pascals (Pa) to 50,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold, in its swollen state, includes a compressive modulus of approximately 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 more. In some embodiments, the covalently stabilized scaffold, in its swollen state, includes a compressive modulus of approximately 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 less. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 5,000 Pa to about 110,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 10,000 Pa to about 105,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 15,000 Pa to about 100,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 20,000 Pa to about 95,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 25,000 Pa to about 90,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 30,000 Pa to about 85,000 Pa in a swollen state.In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 35,000 Pa to about 80,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 40,000 Pa to about 75,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 45,000 Pa to about 70,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 50,000 Pa to about 65,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffolding has a compressive modulus in the range of about 55,000 Pa to about 60,000 Pa in a swollen state.

[0052] In some embodiments, a swollen microgel particle suspension is formulated for administration by needle. In some embodiments, when the swollen microgel particle suspension is formulated for administration by needle and includes a shear rate of about 0.1 to 10 s⁻¹, it includes an apparent viscosity of about 1, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 Pascals-seconds (Pa·s) or more. In some embodiments, when a swollen microgel particle suspension is formulated for administration by needle and includes a shear rate of about 0.1 to 10 s⁻¹, it includes an apparent viscosity of about 1, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 Pa·s or less. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 1 to about 10,000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 50 to about 9,500 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 100 to about 9,000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 150 to about 8,500 Pa·s.In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 200 to about 8000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 250 to about 7500 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 300 to about 7000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 350 to about 6500 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 400 to about 6000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 450 to about 5500 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 500 to about 5000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 550 to about 4500 Pa·s. In some embodiments, when the swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 600 to about 4000 Pa·s. In some embodiments, when the swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 650 to about 3500 Pa·s.In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 700 to about 3000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 750 to about 2500 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 800 to about 2000 Pa·s. In some embodiments, when a swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 850 to about 1500 Pa·s. In some embodiments, when the swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 900 to about 1000 Pa·s. In some embodiments, when the swollen microgel particle suspension is formulated for injection and includes a shear rate of about 0.1 to 10 s⁻¹, it has an apparent viscosity of about 100 to about 1000 Pa·s.

[0053] In some embodiments, the volume fraction and compressive modulus of the microgel particles can be adjusted to achieve a desired compressive modulus for the covalently stabilized scaffold. Both the volume fraction and compressive modulus of the microgel particles influence the final compressive modulus of the covalently stabilized scaffold. As illustrated, higher volume fractions and higher compressive moduli of the microgel particles can lead to a higher compressive modulus of the covalently stabilized scaffold.

[0054] In some embodiments, the covalently stabilized scaffold has a storage modulus of 10 Pascals (Pa) to 10,000 Pa in the swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus of 10 Pa to 1,000 Pa in the swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus of approximately 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 in the swollen state. Includes storage moduli of 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 higher. In some embodiments, the covalently stabilized scaffold is approximately 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 in a swollen state. The storage modulus includes 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or less. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 5,000 Pa to about 110,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 10,000 Pa to about 105,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 15,000 Pa to about 100,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 20,000 Pa to about 95,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 25,000 Pa to about 90,000 Pa in a swollen state.In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 30,000 Pa to about 85,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 35,000 Pa to about 80,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 40,000 Pa to about 75,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 45,000 Pa to about 70,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 50,000 Pa to about 65,000 Pa in a swollen state. In some embodiments, the covalently stabilized scaffold has a storage modulus in the range of about 55,000 Pa to about 60,000 Pa in a swollen state.

[0055] In some embodiments, the microgel particles have a compressive modulus of about 1,000 Pa to about 100,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 1,000 Pa to about 50,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 1,000 Pa to about 46,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 1,000 Pa to about 75,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 1,000 Pa to about 25,000 Pa in an unswelled state. In some embodiments, the microgel particles, in their non-swollen state, include a compressive modulus of approximately 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Pa or more, or exceeding that. In some embodiments, the microgel particles have a compressive modulus of approximately 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Pa or less, or greater, in their non-swollen state. In some embodiments, the microgel particles have a compressive modulus of approximately 10,000 Pa to approximately 100,000 Pa in their non-swollen state. In some embodiments, the microgel particles have a compressive modulus of about 15,000 Pa to about 95,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 20,000 Pa to about 90,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 25,000 Pa to about 85,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 30,000 Pa to about 80,000 Pa in an unswelled state.In some embodiments, the microgel particles have a compressive modulus of about 35,000 Pa to about 75,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 40,000 Pa to about 70,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 45,000 Pa to about 65,000 Pa in an unswelled state. In some embodiments, the microgel particles have a compressive modulus of about 50,000 Pa to about 60,000 Pa in an unswelled state.

[0056] In some embodiments, covalently stabilized scaffolds have a loss modulus of approximately 1 Pascal (Pa) to 10,000 Pa in a swollen state. The loss modulus may be measured by measuring the shear modulus as described above, and by performing amplitude and frequency sweeps of shear stress in a parallel plate system. This may allow for the calculation of both the storage modulus and loss modulus of the viscoelastic material (the storage modulus and loss modulus together include the shear modulus). In some embodiments, the covalently stabilized scaffold has a loss modulus of approximately 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 more in a swollen state. In some embodiments, the covalently stabilized scaffold has a loss modulus of elasticity of approximately 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, or 11000 Pa or less in a swollen state.In some embodiments, the covalently stabilized scaffold is swollen to approximately 10 Pa to 11,000 Pa, 20 Pa to 10,500 Pa, 30 Pa to 10,000 Pa, 40 Pa to 9,500 Pa, 50 Pa to 9,000 Pa, 60 Pa to 8,500 Pa, 70 Pa to 8,000 Pa, 80 Pa to 7,500 Pa, 90 Pa to 7,000 Pa, 100 Pa to 6,500 Pa, 150 Pa to 6,000 Pa, and 200 Pa. This includes loss moduli of elasticity of Pa~5500Pa, 250Pa~5000Pa, 300Pa~4500Pa, 350Pa~4000Pa, 400Pa~3500Pa, 450Pa~3000Pa, 500Pa~2500Pa, 550Pa~2000Pa, 600Pa~1500Pa, 650Pa~1000Pa, 700Pa~950Pa, 750Pa~900Pa, or 800Pa~850Pa.

[0057] In some embodiments, the microgel particles have a compressive modulus of about 500 Pa to about 50,000 Pa in a swollen state. In some embodiments, the microgel particles have a compressive modulus of about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or greater than 50,000 Pa in a swollen state. In some embodiments, the microgel particles, in their swollen state, include compressive moduli of approximately 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or less than 50,000 Pa. In some embodiments, the microgel particles have compressive moduli of approximately 500-50,000, 1,000-45,000, 2,000-40,000, 3,000-35,000, 4,000-30,000, 5,000-25,000, 6,000-20,000, 7,000-20,000, 8,000-15,000, or 9,000-10,000 Pa in a swollen state. In some embodiments, the microgel particles have compressive moduli of approximately 500-50,000, 500-40,000, 500-30,000, 500-20,000, or 500-10,000 Pa in a swollen state.

[0058] In some embodiments, the microgel particles have an elastic modulus of about 5,000 to 20,000 Pa after annealing. In some embodiments, the microgel particles have an elastic modulus of about 10,000 to 15,000 Pa after annealing. In some embodiments, the microgel particles have an elastic modulus of about 5,000 to 15,000 Pa after annealing. In some embodiments, the microgel particles have an elastic modulus of about 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or more than 20,000 Pa after annealing. In some embodiments, the microgel particles have elastic moduli of less than approximately 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 Pa after annealing.

[0059] In some embodiments, the microgel particles have a storage modulus of about 10 Pa to about 5,000 Pa in a swollen state. In some embodiments, the microgel particles have a storage modulus of about 10, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or greater than 5,000 Pa in a swollen state. In some embodiments, the microgel particles have a storage modulus of about 10, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or less than 5,000 Pa in a swollen state. In some embodiments, the microgel particles have a storage modulus of approximately 10–5,000, 100–4,500, 500–4,000, or 1,000–3,000 Pa in a swollen state.

[0060] In some embodiments, the microgel particles have a storage modulus of about 50 Pa to about 10,000 Pa in an unswelled state. In some embodiments, the microgel particles have a storage modulus of about 50, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or more than 10,000 Pa in an unswelled state. In some embodiments, the microgel particles have a storage modulus of about 50, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or less than 10,000 Pa in an unswelled state. In some embodiments, the microgel particles have a storage modulus of approximately 50–10,000, 100–9,000, 500–8,000, 1,000–7,000, 2,000–6,000, or 3,000–5,000 Pa in their non-swollen state.

[0061] In some embodiments, the microgel particles have a loss modulus of about 0.1 Pa to about 2,000 Pa in a swollen state. In some embodiments, the microgel particles have a loss modulus of about 0.1, 0.5, 1, 50, 100, 500, 1,000, 1,500, or greater than 2,000 Pa in a swollen state. In some embodiments, the microgel particles have a loss modulus of about 0.1, 0.5, 1, 50, 100, 500, 1,000, 1,500, or less than 2,000 Pa in a swollen state. In some embodiments, the microgel particles have a loss modulus of about 0.1 to 2,000, 0.5 to 1,500, 1 to 1,000, or 50 to 500 Pa in a swollen state.

[0062] In some embodiments, the microgel particles have a loss modulus of about 1 Pa to about 5,000 Pa in the unswelled state. In some embodiments, the microgel particles have a loss modulus of about 1, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or greater than 5,000 Pa in the unswelled state. In some embodiments, the microgel particles have a loss modulus of about 1, 100, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or less than 5,000 Pa in the unswelled state. In some embodiments, the microgel particles have a loss modulus of approximately 1–5,000, 100–4,500, 500–4,000, or 1,000–3,000 Pa in a non-swollen state.

[0063] In some embodiments, the microgel particle suspension has a compressive modulus of about 100 Pa to about 20,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a compressive modulus of about 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or more than 20,000 Pa in a swollen state. In some embodiments, the microgel particle suspension, in its swollen state, includes compressive modulus less than approximately 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 Pa. In some embodiments, the microgel particle suspension, in its swollen state, contains compressive moduli of approximately 100-20,000, 500-19,000, 1,000-18,000, 2,000-17,000, 3,000-16,000, 4,000-15,000, 5,000-14,000, 6,000-13,000, 7,000-12,000, 8,000-11,000, or 9,000-10,000 Pa.

[0064] In some embodiments, the microgel particle suspension has a compressive modulus of about 500 Pa to about 50,000 Pa in its unswelled state. In some embodiments, the microgel particle suspension has a compressive modulus of about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or greater than 50,000 Pa in its unswelled state. In some embodiments, the microgel particle suspension has a compressive modulus of approximately 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or less than 50,000 Pa in its non-swollen state. In some embodiments, the microgel particle suspension has a compressive modulus of approximately 500-50,000, 1,000-45,000, 2,000-40,000, 3,000-35,000, 4,000-30,000, 5,000-25,000, 6,000-20,000, 7,000-20,000, 8,000-15,000, or 9,000-10,000 Pa in its unswelled state.

[0065] In some embodiments, the microgel particle suspension has a storage modulus of about 1 to about 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a storage modulus of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or greater than 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a storage modulus of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or less than 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a storage modulus of approximately 1 to 10,000, 10 to 9,000, 100 to 8,000, 500 to 7,000, 1,000 to 6,000, or 2,000 to 5,000 Pa in a swollen state.

[0066] In some embodiments, the microgel particle suspension has a loss modulus of about 1 to about 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a loss modulus of about 1, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or greater than 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a loss modulus of about 1, 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or less than 10,000 Pa in a swollen state. In some embodiments, the microgel particle suspension has a loss modulus of approximately 1–10,000, 100–9,000, 500–8,000, 1,000–7,000, 2,000–6,000, or 3,000–5,000 Pa in a swollen state.

[0067] In some embodiments, thiols or their derivatives and vinyl sulfones or their derivatives are present in the hydrogel formulation in a molar ratio of about 0.3 to about 0.95 thiol to VS (thiol:VS) to achieve a desired compressive modulus of about 500 Pa to about 50,000 Pa (for example, when the hydrogel formulation is formulated for injection). In some embodiments, microgel particles are present in a suspension containing microgel particles and water, with 50% to 100% of the suspension's volume fraction consisting of microgel particles to achieve a desired compressive modulus (for example, when the hydrogel formulation is formulated for injection).

[0068] In some embodiments, the covalently stabilized scaffold has an apparent viscosity of about 1 to about 10,000 Pascal seconds (Pa s) in the shear rate range of 0.1 to 10 s⁻¹. In some embodiments, the volume fraction of microgel particles and the compressive modulus of microgel particles can be adjusted to achieve a desired viscosity.

[0069] In some embodiments, the covalently stabilized scaffold has a pH of 5.0 to 9.0. In some embodiments, the covalently stabilized scaffold has a pH of about 7 to about 9. In some embodiments, the pH is about 8.0. In some embodiments, the covalently stabilized scaffold has a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In some embodiments, the covalently stabilized scaffold has a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or lower. In some embodiments, the covalently stabilized scaffold has a pH of about 4.0 to about 10.0. In some embodiments, the covalently stabilized scaffold has a pH of about 4.5 to about 9.5. In some embodiments, the covalently stabilized scaffold has a pH of about 5.0 to about 9.0. In some embodiments, the covalently stabilized scaffold has a pH of about 5.5 to about 8.5. In some embodiments, the covalently stabilized scaffold has a pH of about 6.0 to about 8.0. In some embodiments, the covalently stabilized scaffold has a pH of about 6.5 to about 7.5. In some embodiments, the covalently stabilized scaffold has a pH of about 7.0 to about 7.5. In some embodiments, the covalently stabilized scaffold has a pH of about 7.0 to about 9.0. In some embodiments, the covalently stabilized scaffold has a pH of about 7.5 to about 8.5. In some embodiments, the covalently stabilized scaffold has a pH of about 8.0 to about 8.5.

[0070] In some embodiments, the microgel particles have a pH of 5.0 to 9.0. In some embodiments, the microgel particles have a pH of 6.5 to 7.5. In some embodiments, the microgel particles have a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In some embodiments, the microgel particles have a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or lower. In some embodiments, the microgel particles have a pH of about 4.0 to about 10.0. In some embodiments, the microgel particles have a pH of about 4.5 to about 9.5. In some embodiments, the microgel particles have a pH of about 5.0 to about 9.0. In some embodiments, the microgel particles have a pH of about 5.5 to about 8.5. In some embodiments, the microgel particles have a pH of about 6.0 to about 8.0. In some embodiments, the microgel particles have a pH of about 6.5 to about 7.5. In some embodiments, the microgel particles have a pH of about 7.0 to about 7.5.

[0071] Hydrogel formulations In some embodiments, hydrogel formulations comprising the microgel particles of this disclosure and further activators (e.g., therapeutic agents), reagents, or solvents are disclosed herein.

[0072] In some embodiments, the hydrogel formulation comprises microgel particles and a therapeutic agent. In some embodiments, the therapeutic agent is not released from the microgel particles. In some embodiments, the therapeutic agent is released from the microgel particles (e.g., drug-eluting microgel particles). In some embodiments, the activator is the therapeutic agent. In some embodiments, the therapeutic agent comprises an antibiotic.

[0073] In some embodiments, the hydrogel formulation can withstand sterilization. Sterilization can be carried out by steam sterilization, filtration, microfiltration, e-beam, gamma irradiation, ethylene oxide (ETO), light, supercritical carbon dioxide, vaporized hydrogen peroxide, or any combination thereof. In some embodiments, certain components of the hydrogel formulation may be steam sterilized (e.g., autoclaved) without degrading their physical properties, such as microgel particles. However, if the hydrogel formulation contains further components such as therapeutic agents, the components of the hydrogel formulation containing therapeutic agents can be sterilized by means other than heat treatment, such as by using filtration sterilization.

[0074] In some embodiments, sterilization of hydrogel formulations is performed by autoclaving. Autoclaving can be achieved by applying a combination of heat, pressure, and moisture to the formulation to be sterilized. Many different sterilization temperatures, pressures, and cycle times can be used. For example, in some embodiments, filled syringes may be sterilized at a temperature of at least about 120°C to about 130°C or higher. Moisture may or may not be used. 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 higher. In some embodiments, the sterilization cycle may be at least about 1 minute to about 30 minutes or higher. In some embodiments, the sterilization cycle may be at least about 15 minutes to about 30 minutes or higher.

[0075] In some embodiments, the sterilization method involves the use of a gas species known to kill or remove infectious agents. In some embodiments, ethylene oxide is used as the sterilization gas to sterilize any medical device, product, or delivery device disclosed herein.

[0076] In some embodiments, the sterilization method involves the use of an irradiation source that kills or removes infectious agents. The irradiation beam targets a delivery device (e.g., a syringe) containing the hydrogel formulation, and the wavelength of the energy kills or removes the undesirable infectious agents. Non-limiting examples of useful energies include, but are not limited to, ultraviolet (UV) light, electron beam (e-beam) irradiation, gamma ray irradiation, visible light, microwaves, or any other wavelength or wavelength band that kills or removes the undesirable infectious agents, preferably without substantially altering the degradation of the hydrogel formulation.

[0077] In some embodiments, the hydrogel formulation includes reagents such as annealing agents that facilitate the annealing reaction of the hydrogel formulation to form a covalently stabilized scaffold. In some embodiments, the annealing agent includes PEG-dithiol. In some embodiments, the annealing agent includes a photoinitiator. In a non-limiting example, the photoinitiator may be eosin Y. In some embodiments, the annealing agent includes triethanolamine. In some embodiments, the annealing agent includes an enzyme. In some embodiments, the enzyme includes thrombin. In some embodiments, the annealing agent includes a transglutaminase enzyme. A non-limiting example of the transglutaminase enzyme is factor XIII (factor XIIIa in its active form). In some embodiments, the annealing agent is endogenous to the subject or tissue. For example, the enzyme (e.g., factor XIII or factor XIIIa) may be naturally present in the tissue and can participate in the in situ annealing reaction of the hydrogel formulation, provided that a suitable annealing component is present in the hydrogel formulation. 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 hydrogel formulation in situ, provided that a suitable annealing component is present. In some embodiments, factor XIII is modified before delivery to the tissue or subject. In some embodiments, the annealing agent includes a co-initiator. In some embodiments, the annealing agent includes an electron transfer agent. Examples of additional and alternative annealing agents, non-limiting examples, include active esters and nucleophiles, catechols that crosslink upon oxidation, and other redox-sensitive molecules. In some embodiments, the annealing component includes a K peptide, a Q peptide, or a combination thereof. In some embodiments, the reagent includes a stabilizer, a sterilizer, or a thermal protectant. Non-limiting examples of stabilizers include reagents, salts, and additives. Non-limiting examples of sterilizers include reagents, salts, and additives.Non-limiting examples of thermal protectants include antioxidants, glycerin, and PEG. In some embodiments, hydrogel formulations are protected during sterilization by freezing the hydrogel before and / or during final sterilization (e.g., irradiation). In some embodiments, skin filler systems are protected during sterilization by placing the material in a sealed, inert atmosphere or under a vacuum ampoule.

[0078] In some embodiments, the hydrogel formulation 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 containing water. In some embodiments, the hydrogel formulation comprises a buffer. In some embodiments, the buffer includes phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, or acetate buffer, citrate buffer, borate buffer, or a combination thereof. In some embodiments, the buffer adjusts the pH of the hydrogel formulation to a desired pH. The pH of the disclosed hydrogel formulation 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 hydrogel formulation comprises a suspension of microgel particles in an aqueous solvent containing a buffer. In some embodiments, the buffer may be a buffering agent. In some embodiments, 50% to 100% of the volume of the suspension contains microgel particles. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the volume fraction of the suspension contains microgel particles. In some embodiments, when the hydrogel is formulated for injection, the volume fraction of microgel particles is about 50% or more. In some embodiments, when the hydrogel is formulated for injection, the volume fraction of microgel particles is about 50%, 60%, 70%, 80%, or 90% or more.

[0079] In some embodiments, the hydrogel formulation is colorless. In some embodiments, the hydrogel formulation is substantially optically transparent. In some embodiments, the polydispersity of the hydrogel formulation is 0.1 or less. In some embodiments, the coefficient of variation of the hydrogel formulation is about 62.5%, 60%, 57.5%, 55%, 52.5%, 50%, 47.5%, 45%, 42.5%, 40%, 37.5%, 35%, 32.5%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, or 10% or less. In some embodiments, the coefficient of variation of the hydrogel formulation is about 62.5% or less. In some embodiments, the coefficient of variation of the hydrogel formulation is about 30% or less. In some embodiments, the skin infill system is odorless.

[0080] In some embodiments, the hydrogel formulation is formulated for administration to a subject. In some embodiments, the administration is subdermal, dermal, intradermal, or subcutaneous. In some embodiments, the administration minimizes the subject's foreign body reaction. In some embodiments, the hydrogel formulation is formulated for administration by needle. In some embodiments, the hydrogel formulation is formulated for administration by microneedle or microneedle patch. In some embodiments, the hydrogel formulation is formulated for administration by needles having a gauge of approximately 26 gauge, 27 gauge, 28 gauge, 29 gauge, or 30 gauge. For example, a hydrogel formulation formulated for a 27 gauge syringe may have an apparent viscosity of approximately 1 to 1000 pascals / seconds when measured at a shear rate of 0.1 to 10 s⁻¹. On the other hand, a hydrogel formulation formulated for a 30 gauge syringe may have an apparent viscosity of approximately 1 to 500 pascals / seconds when measured at a shear rate of 0.1 to 10 s⁻¹. Therefore, the properties of the hydrogel formulation may be fine-tuned according to the mode of administration.

[0081] In some embodiments, the dose of the hydrogel formulation includes a volume of about 0.01 mL to about 20 mL. In some embodiments, the volume includes about 0.75 ml to about 1.0 mL. In some embodiments, the volume includes about 0.5 mL to about 3.0 mL. In some embodiments, the volume includes 0.75 mL to about 2.75 mL, 1.0 mL to about 2.25 mL, 1.25 mL to about 2.0 mL, 1.0 mL to about 1.75 mL, or 1.25 mL to 1.50 mL. In some embodiments, the dose includes approximately 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, 3.0 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 or more.

[0082] In some embodiments, the hydrogel formulation is manufactured aseptically. In some embodiments, the hydrogel formulation is sterile. In some embodiments, the hydrogel formulation is formulated for sterilization by steam sterilization, filtration, microfiltration, e-beam, gamma irradiation, ethylene oxide (ETO), light, supercritical carbon dioxide, vaporized hydrogen peroxide, or any combination thereof. In some embodiments, the hydrogel formulation includes at least two separate containers, each suitable for sterilization by a different method. In some embodiments, the microgel particles are lyophilized. In some embodiments, the lyophilized microgel particles are stored in a first container that can withstand steam sterilization, and this first container is separate from a second container containing components of the hydrogel formulation that may be decomposable by steam sterilization, such as therapeutic agents. In another embodiment, the hydrogel formulation is stored in a single container that can be sterilized together. In some embodiments, the system also includes a reconstitution medium for restoring the lyophilized hydrogel formulation. In some embodiments, the reconstitution medium contains a physiologically isotonic buffer, such as phosphate-buffered saline. In some embodiments, the reconstitution medium has a pH higher than physiological pH. In some embodiments, the reconstitution medium has a pH lower than the physiological pH. In some embodiments, the reconstitution medium includes a buffer with varying buffering capacities.

[0083] delivery device In some embodiments, delivery devices configured to deliver hydrogel formulations are disclosed herein. Non-limiting examples of delivery devices are needles or microneedles (e.g., microneedle patches). In some embodiments, the delivery device comprises a body and an applicator in fluid communication with the body. In some embodiments, the body is elongated (e.g., a barrel). In some embodiments, the body of the delivery device includes an inner chamber containing the hydrogel formulation. In some embodiments, the delivery device comprises a pump or plunger configured to pressurize the hydrogel formulation contained in the body under conditions that the hydrogel formulation flows through the applicator and flows out of the applicator through the applicator outlet. In some embodiments, the body of the delivery device comprises a first chamber for microgel particles and a second chamber for an annealing agent and / or components. In some embodiments, the delivery device mixes the components of the first and second chambers. In some embodiments, the syringe is pre-filled with the hydrogel formulation. In some embodiments, the syringe is sterile. In some embodiments, the syringe is packaged separately from the hydrogel formulation, and both the syringe and the hydrogel formulation are sterile.

[0084] In some embodiments, the syringe includes a needle. In some embodiments, the needle includes a blunt needle. In some embodiments, the needle has a gauge ranging from about 10 gauge to about 20 gauge. In some embodiments, the needle has a gauge ranging from about 18 gauge.

[0085] method In some embodiments, methods for preparing or using the hydrogel formulations disclosed herein are disclosed. In some embodiments, the methods disclosed herein include delivering the hydrogel formulations disclosed herein to a subject. In some embodiments, delivery includes administering the hydrogel formulation to a subject. In some embodiments, administration includes subdermal, dermal, intradermal, or subcutaneous administration of the hydrogel formulation to a tissue site of subject. In some embodiments, administration includes administering the hydrogel formulation to an incision or suture site. In some embodiments, administering the hydrogel formulation to a tissue site of subject is effective in treating the tissue site or surrounding tissue, for example, to improve the strength of an incision or to improve healing at the suture site. In some embodiments, the methods for delivering or administering the hydrogel formulations disclosed herein are carried out while minimizing the foreign body reaction induced by the subject in response to delivery or administration. Also provided are methods for purifying the microgel particles of the hydrogel formulations disclosed herein, for example, in a water-in-oil emulsion. In some embodiments, the method includes freeze-drying the microgel particles so that they can be stored and / or distributed over a long period of time before being restored and delivered to the target at the required time.

[0086] Delivery method In some embodiments, methods for delivering the hydrogel formulations provided herein to a target are disclosed herein. In some embodiments, the hydrogel formulation is delivered to a tissue site of target. In some embodiments, the tissue site is a suture site. In some embodiments, the hydrogel formulation is delivered to a suture site. In some embodiments, the method involves delivering the hydrogel formulation to the tissue site under conditions sufficient to cause microgel particles to anneal to each other to form a covalently stabilized porous scaffold. In some embodiments, the formation of the covalently stabilized porous scaffold occurs in vivo, in situ, or both in vivo and in situ. In some embodiments, the covalently stabilized porous scaffold is formed under conditions sufficient to allow cells to grow within the pores of the covalently stabilized porous scaffold to generate a cell matrix. In some embodiments, the formed cell matrix remains in the target after the complete degradation of the covalently stabilized porous scaffold, thereby permanently filling at least a portion of the tissue site of target while minimizing the foreign body reaction of the target. The delivery methods disclosed herein may be subdermal, dermal, intradermal, or subcutaneous. In some embodiments, the delivery method includes injection using, for example, a delivery device disclosed herein (e.g., a syringe).

[0087] In some embodiments, the suture line was an incision. In some embodiments, the hydrogel formulation is delivered on the suture line after it has been sutured. In some embodiments, the hydrogel formulation is delivered within the suture line while it is being sutured. In some embodiments, the hydrogel formulation is delivered on the suture line after it has been sutured and within the suture line while it is being sutured. In some embodiments, the hydrogel begins to anneal on the suture line for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes before the dermis and subcutaneous tissue of the subject is sutured. In some embodiments, the suture line was an abdominal incision below the dermis and subcutaneous tissue of the subject. In some embodiments, the hydrogel begins to anneal on the incision for at least about 10 minutes before the dermis and subcutaneous tissue is closed. In some embodiments, the hydrogel begins to anneal over the incision for at least about 1 minute before the dermis and subcutaneous tissue close. In some embodiments, the hydrogel begins to anneal over the incision for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes before the dermis and subcutaneous tissue close. In some embodiments, the hydrogel annealing strengthens the suture line at the suture site.

[0088] In some embodiments, the method further includes reinforcing the suture at the site of the suture by a cellular matrix formed on the site of the suture. In some embodiments, reinforcing the suture is characterized by increasing the mechanical tensile strength of the suture compared to a reference suture at an otherwise identical site of the suture that is closed without delivery of the hydrogel formulation. In some embodiments, increasing the mechanical tensile strength of the suture is characterized by increasing the yield stress of the suture. The yield stress may be calculated from a stress-versus-strain curve measured using a tensile test (e.g., with Instron). A non-limiting example of this is shown in Figure 6A.

[0089] In some embodiments, the yield stress is calculated from a stress-strain curve measured using a tensile test (e.g., on an Instron 3342). In some embodiments, the suture has a yield stress of at least about 4.0 newtons per square millimeter (N / mm 2 ) measured after closure. In some embodiments, the suture has a yield stress of at least about 1.0 N / mm 2 , about 1.5 N / mm 2 , about 2.0 N / mm 2 , about 2.5 N / mm 2 , about 3.0 N / mm 2 , about 3.5 N / mm 2 , about 4.0 N / mm 2 , about 4.5 N / mm 2 , about 5.0 N / mm 2 , about 5.5 N / mm 2 , about 6.0 N / mm 2 , about 6.5 N / mm 2 , about 7.0 N / mm 2 , about 7.5 N / mm 2 , about 8.0 N / mm 2 , about 8.5 N / mm 2 , about 9.0 N / mm 2 , about 9.5 N / mm 2 , about 10.0 N / mm 2 , about 15.0 N / mm 2 , about 20.0 N / mm 2 , about 25.0 N / mm 2 , about 30.0 N / mm 2 , about 35.0 N / mm 2 , about 40.0 N / mm 2 , about 45.0 N / mm 2 , about 50.0 N / mm 2 , about 55.0 N / mm 2 , about 60.0 N / mm 2 , about 65.0 N / mm 2 , about 70.0 N / mm 2 , about 75.0 N / mm 2 , about 80.0 N / mm 2 , about 85.0 N / mm 2 , about 90.0 N / mm 2 , about 95.0 N / mm 2 , or about 100.0 N / mm2 This includes the yield strength of the suture line. In some embodiments, the suture line has a yield strength of approximately 1.0 N / mm 2 , about 1.5N / mm 2 Approximately 2.0 N / mm 2 , about 2.5N / mm 2 Approximately 3.0 N / mm 2 , about 3.5N / mm 2 Approximately 4.0 N / mm 2 , about 4.5N / mm 2 Approximately 5.0 N / mm 2 , about 5.5N / mm 2 Approximately 6.0 N / mm 2 , about 6.5N / mm 2 Approximately 7.0 N / mm 2 , about 7.5N / mm 2 Approximately 8.0 N / mm 2 , about 8.5N / mm 2 Approximately 9.0 N / mm 2 , about 9.5N / mm 2 Approximately 10.0 N / mm 2 Approximately 15.0 N / mm 2 Approximately 20.0 N / mm 2 Approximately 25.0 N / mm 2 Approximately 30.0 N / mm 2 Approximately 35.0 N / mm 2 Approximately 40.0 N / mm 2 Approximately 45.0 N / mm 2 Approximately 50.0 N / mm 2 Approximately 55.0 N / mm 2 Approximately 60.0 N / mm 2 Approximately 65.0 N / mm 2 Approximately 70.0 N / mm 2 Approximately 75.0 N / mm 2 Approximately 80.0 N / mm 2 Approximately 85.0 N / mm 2 Approximately 90.0 N / mm 2 Approximately 95.0 N / mm 2 , or approximately 100.0 N / mm 2It includes the following yield stress. 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, or 50 days after closure. In some embodiments, the yield stress is measured at least about 42 days after closure.

[0090] In some embodiments, the suture has a yield stress of at least about 3.0 N / mm 2 to about 6.0 N / mm 2 after closure. In some embodiments, the suture has a yield stress of at least about 4.0 N / mm 2 to about 5.0 N / mm 2 after closure. In some embodiments, the suture has a yield stress of at least about 3.0 N / mm 2 to about 4.0 N / mm 2 after closure. In some embodiments, the suture has a yield stress of about 3.0 N / mm 2 , about 3.5 N / mm 2 , about 4.​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​2 Approximately 60.0 N / mm 2 Approximately 65.0 N / mm 2 Approximately 70.0 N / mm 2 Approximately 75.0 N / mm 2 Approximately 80.0 N / mm 2 Approximately 85.0 N / mm 2 Approximately 90.0 N / mm 2 Approximately 95.0 N / mm 2 , or approximately 100.0 N / mm 2 Includes yield stress exceeding . In some embodiments, the suture line has a yield stress of approximately 3.0 N / mm 2 , about 3.5N / mm 2 Approximately 4.0 N / mm 2 , about 4.5N / mm 2 Approximately 5.0 N / mm 2 , about 5.5N / mm 2 Approximately 6.0 N / mm 2 , about 6.5N / mm 2 Approximately 7.0 N / mm 2 , about 7.5N / mm 2 Approximately 8.0 N / mm 2 , about 8.5N / mm 2 Approximately 9.0 N / mm 2 , about 9.5N / mm 2 Approximately 10.0 N / mm 2 Approximately 15.0 N / mm 2 Approximately 20.0 N / mm 2 Approximately 25.0 N / mm 2 Approximately 30.0 N / mm 2 Approximately 35.0 N / mm 2 Approximately 40.0 N / mm 2 Approximately 45.0 N / mm 2 Approximately 50.0 N / mm 2 Approximately 55.0 N / mm 2 Approximately 60.0 N / mm 2 Approximately 65.0 N / mm 2 Approximately 70.0 N / mm 2 Approximately 75.0 N / mm 2 Approximately 80.0 N / mm 2 Approximately 85.0 N / mm 2 Approximately 90.0 N / mm 2 Approximately 95.0 N / mm 2 , or approximately 100.0 N / mm 2This includes yield stresses less than 10. 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, or 50 days after closure. In some embodiments, the yield stress is measured at least about 42 days after closure.

[0091] In some embodiments, the increase in the mechanical tensile strength of the suture is characterized by the formation of a certain amount or type of collagen that mimics endogenous tissue at the suture site. In some embodiments, the type of collagen includes type I collagen, type III collagen, or a combination thereof. In some embodiments, type I collagen is present in a ratio of about 10:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 6:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 5:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 1:1 to about 10:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 1.5:1 to about 9.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 2:1 to about 9:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of about 2.5:1 to about 8.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 3:1 to approximately 8:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 3.5:1 to approximately 7.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 4:1 to approximately 7:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 4.5:1 to approximately 6.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 5:1 to approximately 6:1 with type III collagen. In some embodiments, collagen is formed at the site of the suture line at least 28 days after the closure of the suture line (e.g., suturing). In some embodiments, collagen is formed at the site of the suture line at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after suturing.

[0092] In some embodiments, increasing the mechanical tensile strength of the suture is characterized by increasing the toughness of the suture. Toughness may be measured using a tensile test (e.g., with Instron) as the area under the stress-to-strain curve to fracture. A non-limiting example is shown in Figure 7F. In some embodiments, the suture is made to have a tensile strength of at least about 60.0 millijoules per cubic millimeter (mJ / mm²) at least about 42 days after closure. 3 ) includes toughness. In some embodiments, the toughness is at least about 25.0 mJ / mm 3 Approximately 30.0 mJ / mm 3 Approximately 35.0 mJ / mm 3 Approximately 40.0 mJ / mm 3 Approximately 45.0 mJ / mm 3 Approximately 50.0 mJ / mm 3 Approximately 55.0 mJ / mm 3 Approximately 60.0 mJ / mm 3 Approximately 65.0 mJ / mm 3 Approximately 70.0 mJ / mm 3 Approximately 75.0 mJ / mm 3 Approximately 80.0 mJ / mm 3 Approximately 85.0 mJ / mm 3 Approximately 90.0 mJ / mm 3 , or approximately 100.0 mJ / mm 3 This includes. In some embodiments, the toughness is approximately 25.0 mJ / mm 3 Approximately 30.0 mJ / mm 3 Approximately 35.0 mJ / mm 3 Approximately 40.0 mJ / mm 3 Approximately 45.0 mJ / mm 3 Approximately 50.0 mJ / mm 3 Approximately 55.0 mJ / mm 3 Approximately 60.0 mJ / mm 3 Approximately 65.0 mJ / mm 3 Approximately 70.0 mJ / mm 3 Approximately 75.0 mJ / mm 3 Approximately 80.0 mJ / mm 3 Approximately 85.0 mJ / mm 3 Approximately 90.0 mJ / mm 3 , or approximately 100.0 mJ / mm 3This includes the following: In some embodiments, the ductility 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 ductility of the closure body is measured at least about 42 days after suturing.

[0093] In some embodiments, the suture line, after suturing, has a humidity of at least about 25.0 mJ / mm². 3 ~Approximately 100.0 mJ / mm 3 This includes toughness. In some embodiments, the toughness is at least about 25.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 30.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 35.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 40.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 45.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 50.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 55.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 60.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 65.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 70.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 75.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 80.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 Approximately 85.0 mJ / mm 3 ~Approximately 100.0 mJ / mm 3 , or approximately 90.0 mJ / mm 3 ~Approximately 100.0 mJ / mm3 This includes. In some embodiments, suture ductility 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 closure. In some embodiments, suture ductility is measured at least about 42 days after suturing.

[0094] In some embodiments, increasing the mechanical tensile strength of a suture is characterized by increasing the suture recovery rate. In some embodiments, the recovery rate is calculated as the ratio of the wound tissue tensile strength at the suture site to the non-wound tissue tensile strength from the same anatomical region. In some embodiments, the suture recovery rate increases by about 40% or more compared to a reference suture closed without delivery of the hydrogel formulation, and the recovery rate is measured after suturing. In some embodiments, the recovery rate increases by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% or more compared to a reference suture suture sutured without delivery of the hydrogel formulation. In some embodiments, the recovery rate is measured at least approximately 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 present recovery rate is measured at least approximately 42 days after suturing.

[0095] In some embodiments, the suture recovery rate is increased by at least about 20% to about 60% compared to a reference suture sutured without delivery of the hydrogel formulation, and the recovery rate is measured after suturing. In some embodiments, the recovery rate is increased by at least about 10% to about 50%, at least about 20% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, at least about 30% to about 90%, or at least about 40% to about 100%. In some embodiments, the recovery rate 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 recovery rate is measured at least about 42 days after suturing.

[0096] In some embodiments, an increase in the mechanical tensile strength of a suture is characterized by an increase in the yield strain of the suture.

[0097] In some embodiments, delivery includes releasing a hydrogel formulation from a syringe. In some embodiments, the syringe includes a needle. In some embodiments, the needle has a gauge ranging from about 10 gauge to about 20 gauge. In some embodiments, the needle has a gauge ranging from about 18 gauge. In some embodiments, the needle has a gauge ranging from about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 gauge.

[0098] 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 includes about 5.0 mL. In some embodiments, the internal volume includes about 0.5 mL to about 3.0 mL. In some embodiments, the volume includes 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 contains doses including about 0.50 mL, 0.75 mL, 1.0 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2.0 mL, 2.25 mL, 2.5 mL, 2.75 mL, 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 or more. The dose of the hydrogel formulation may depend on the administration area. In some embodiments, the internal volume is related to the inner diameter of the syringe, which affects the extrusion force required to inject the hydrogel formulation composition. In some embodiments, the inner diameter may be about 4 mm to about 9 mm. In some embodiments, the inner diameter may be about 4.5 mm to about 6.5 mm. In some embodiments, the inner diameter may be about 4.5 mm to about 8.8 mm. In some embodiments, the extrusion force required to deliver the hydrogel formulation from the syringe depends on the needle gauge.

[0099] In some embodiments, the method includes separately delivering hydrogel microparticles (e.g., as disclosed herein) and an annealing agent (e.g., as disclosed herein) to a target. In some embodiments, the method includes delivering hydrogel microparticles (e.g., as disclosed herein) and an annealing agent (e.g., as disclosed herein) together to a target. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 12 months when the hydrogel microparticles and the annealing agent are stored as a mixture in a single container. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and the annealing agent (e.g., as disclosed herein) have a shelf life of at least about 36 months when the hydrogel microparticles and the annealing agent are stored as a mixture in a single container. In some embodiments, the shelf life of the mixture of hydrogel microparticles (e.g., as disclosed herein) and annealing agent (e.g., as disclosed herein) corresponds to the amount of time the mixture is stored at room temperature (e.g., 12 months or 36 months). In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and annealing agent (e.g., as disclosed herein) have a shelf life of at least about 12 months when the hydrogel microparticles and annealing agent are stored in separate containers. In some embodiments, the hydrogel microparticles (e.g., as disclosed herein) and annealing agent (e.g., as disclosed herein) have a shelf life of at least about 36 months when the hydrogel microparticles and annealing agent are stored in separate containers. In some embodiments, the shelf life of the hydrogel microparticles (e.g., as disclosed herein) and annealing agent (e.g., as disclosed herein) when stored in separate containers corresponds to the amount of time they are stored at room temperature (e.g., 12 months or 36 months).

[0100] This specification discloses a method comprising administering the hydrogel formulation described herein to a tissue site of interest. In some embodiments, the tissue site is a surgical site of interest. In some embodiments, the tissue site is an abdominal incision. In some embodiments, the surgical site is a midline lateral abdominal incision. In some embodiments, the surgical site is a transverse abdominal incision. In some embodiments, the tissue site is any location including soft tissue. In some embodiments, the tissue site is any location including connective tissue. In some embodiments, the tissue site is any location including epithelial tissue. In some embodiments, the tissue site is any location including muscle tissue. In some embodiments, the tissue site is any location including nerve tissue. In some embodiments, the method comprises administering a certain dose of the hydrogel formulation to an interest, 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 includes a wound site of interest. In some embodiments, the wound site includes a site of a skin abrasion, delamination, incision, laceration, puncture, or a combination thereof. In some embodiments, the wound site includes a burn site. In some embodiments, the tissue site includes a scarred site (for example, a site on the skin or within body tissue where a mark remains, such as a wound, burn, or pain that has not completely healed and where fibrous connective tissue in the subject has developed). In some embodiments, the scar is a keloid, hypertrophy, contracture, adhesion, or a combination thereof.

[0101] In some embodiments, the methods disclosed herein include delivering a hydrogel formulation to a target under conditions sufficient to cause adjacent microgel particles to anneal to each other and form a stabilized scaffold. In some embodiments, the stabilized scaffold formed is covalently stabilized (e.g., covalent interactions between adjacent microgel particles facilitate annealing). In some embodiments, the stabilized scaffold is porous. In some embodiments, the method includes carrying out an annealing reaction of microgel particles to form a covalently stabilized scaffold. In some embodiments, the covalently stabilized scaffold is one of the covalently stabilized scaffolds described herein. In some embodiments, the method includes forming a covalently stabilized scaffold such that pores are formed between the microgel particles of the covalently stabilized scaffold (e.g., the covalently stabilized scaffold is porous).

[0102] In some embodiments, the hydrogel formulation integrates with the suture site within approximately 14 days after closure. In some embodiments, integration is characterized by the formation of new tissue within and around a covalently stabilized porous scaffold.

[0103] In some embodiments, the methods disclosed herein involve delivering a hydrogel formulation to a target under conditions sufficient to allow endogenous cells of the target to penetrate and grow within a covalently stabilized porous scaffold. In some embodiments, the cells form a cell matrix within the covalently stabilized porous scaffold. In some embodiments, the covalently stabilized porous scaffold remains in 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 method involves angiogenesis, deposition of extracellular matrix, or generation of proteins and enzymes in the tissue site to assist in treating the tissue site, or a combination thereof. In some embodiments, the method involves forming new tissue from the cell matrix at the injection site or tissue site. In some embodiments, the new tissue is characterized by having mature angiogenesis, features of the surrounding tissue at the tissue site, a certain amount or type of collagen mimicking endogenous tissue at the tissue site, or a combination thereof. In some embodiments, features of the surrounding tissue at the tissue site include cell types functionally differentiated 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, stem cells, and collagen fibers. In some embodiments, the new tissue is characterized as having an extracellular matrix. In some embodiments, the hydrogel formulation is partially degraded in vivo. In some embodiments, the new tissue is formed, and the covalently stabilized porous scaffold is completely degraded 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 the suture by suture alone. In some embodiments, the new tissue continues to remodel at the site of the suture for at least about 42 days after closure. In some embodiments, the new tissue is formed above (e.g., superficially) and deep (e.g., below) the subcutaneous tissue at the site of the suture. In some embodiments, the new tissue is stromal-like tissue with unaligned collagen bundles.

[0104] In some embodiments, at least a portion of the suture site contains elastin after the decomposition of the covalently stabilized porous scaffold at the suture site.

[0105] In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least 25% of the tissue site after degradation of the covalently stabilized porous scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% or more of the tissue site after degradation of the covalently stabilized porous scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 10% to about 50% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 15% to about 45% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 20% to about 40% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 25% to about 35% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 10% to about 90% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 15% to about 90% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 20% to about 85% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 25% to about 80% of the tissue site after the degradation of the covalently stabilized scaffold in the tissue site.In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 30% to about 75% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 35% to about 70% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 40% to about 65% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 45% to about 60% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, at least a portion of the tissue site containing the cell matrix comprises at least about 50% to about 55% of the tissue site after degradation of the covalently stabilized scaffold in the tissue site. In some embodiments, the method comprises growing cells within a stabilized porous scaffold within about one day after delivery. In some embodiments, the method involves forming a cell matrix within a stabilized porous scaffold within approximately 30 days after delivery. In some embodiments, the cell matrix begins to form within the scaffold within 7 days after administration.

[0106] In some embodiments, the methods for delivering the hydrogel formulations disclosed herein minimize the foreign body reaction induced by the subject in response to the hydrogel formulation. In some embodiments, a covalently stabilized porous scaffold is effective in reinforcing sutures while minimizing the foreign body reaction of the subject. In some embodiments, the foreign body reaction is characterized by chronic inflammation. In some embodiments, the foreign body reaction is characterized by granuloma formation. In some embodiments, the foreign body reaction is characterized by scar tissue formation. In some embodiments, the foreign body reaction is characterized by nodule formation. In some embodiments, the foreign body reaction is characterized by swelling, pain, or a combination thereof. In some embodiments, chronic inflammation, granuloma formation, nodule formation, swelling, pain, or a combination thereof is localized to the tissue site or its periphery. In some embodiments, chronic inflammation, granuloma formation, nodule formation, swelling, pain, or a combination thereof is localized to the suture site or its periphery. In some embodiments, the foreign body reaction is induced at a location other than the tissue site. In some embodiments, the foreign body reaction is characterized by the presence of multinucleated giant cells (MNGCs) (e.g., monocyte or macrophage fusions) in the suture line of the subject. In some embodiments, the foreign body reaction is characterized by the persistence of MNGCs over a long period of time. In some embodiments, the period includes about 1, 2, 3, or 4 weeks or longer. In some embodiments, the period includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. In some embodiments, the period includes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years or longer. In some embodiments, minimizing the foreign body reaction is characterized by avoiding any formation of MNGCs in the tissue site. In some embodiments, minimizing the foreign body reaction is characterized by the absence of MNGCs in the tissue site after a certain period following delivery of the hydrogel formulation.In some embodiments, the period after delivery of the hydrogel formulation includes 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 day. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 30 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 29 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 28 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 27 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 26 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 25 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 24 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 23 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 22 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 21 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 20 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 19 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 18 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 17 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 16 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 15 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 14 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 13 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 12 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 11 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 10 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 9 days.In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 8 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 7 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 6 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 5 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 4 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 3 days. In some embodiments, the period after delivery of the hydrogel formulation includes 1 to 2 days.

[0107] In some embodiments, the foreign body reaction is measured by the presence of one or more types of macrophages in the suture line of the subject. In some embodiments, the one or more types of macrophages include type 1 and type 2 macrophages. In some embodiments, type 1 macrophages are pro-inflammatory. In some embodiments, type 2 macrophages are pro-regenerative. In some embodiments, a covalently stabilized porous scaffold is effective in reducing the number of type 1 macrophages to less than the number of type 2 macrophages.

[0108] In some embodiments, the foreign body reaction is measured by the presence of one or more multinucleated giant cells (MNGCs) within or around the suture line in question. In some embodiments, covalently stabilized porous scaffolds are effective in reducing the number of MNGCs within or around the suture line compared to surgical meshes or tissue-derived surgical patches.

[0109] In some embodiments, chronic inflammation may be characterized as a slow, long-lasting inflammation that persists for a long period after delivery or administration of the hydrogel formulation. In some embodiments, chronic inflammation may persist for several months to several years. In some embodiments, as discussed above, an intentional xenobiotic reaction caused by the administration of a biostimulant results in chronic inflammation at the tissue site. In some embodiments, the method of delivering the hydrogel formulation described herein minimizes such chronic inflammation while permanently filling the tissue site with new tissue. In some embodiments, the granuloma may be a small area of ​​inflammation at or around the tissue site of interest. In some embodiments, the granuloma may be a small area / cluster of blood cells and other tissues in the inflammatory area. In some embodiments, as discussed above, an intentional xenobiotic reaction caused by the administration of a biostimulant results in granuloma formation at or around the tissue site. In some embodiments, the method of delivering the hydrogel formulation described herein minimizes such granuloma formation while filling the tissue site with new tissue. In some embodiments, scar tissue is characterized by fibrous tissue having a composition that is harder and more brittle than normal tissue. In some embodiments, as discussed above, an intentional xenobiotic reaction induced by the administration of a biostimulant results in scar tissue formation at the tissue site. In some embodiments, the method of delivering the hydrogel formulation described herein minimizes such scar tissue formation while filling the tissue site with new tissue. In some embodiments, the hydrogel formulations disclosed herein minimize such scar tissue formation because the new tissue formed within a covalently stabilized scaffold mimics the tissue site or the surrounding tissue. For example, type I collagen deposited in the cellular matrix within the covalently stabilized scaffold (forming the base of the new tissue) is less than scar tissue, while type III collagen is more than scar tissue. In some embodiments, nodules may be sites of abnormal tissue growth. In some embodiments, as discussed above, an intentional xenobiotic reaction induced by the administration of a biostimulant results in nodule formation at the tissue site.In some embodiments, the method of delivering the hydrogel formulation described herein minimizes nodule formation while filling the tissue site with new tissue. In some embodiments, the foreign body reaction is measured by detecting the amount of granulomas in the tissue site using histological analysis and comparing the amount of granulomas in the tissue site with reference tissue without the hydrogel formulation. In some embodiments, the foreign body reaction is measured by detecting the amount of scar tissue in the tissue site using histological analysis and comparing the amount of scar tissue in the tissue site with reference tissue without the hydrogel formulation. In some embodiments, the foreign body reaction is measured by detecting the amount of nodules in the tissue site using histological analysis and comparing the amount of nodules in the tissue site with reference tissue without the hydrogel formulation. In some embodiments, the foreign body reaction is measured by detecting chronic inflammation in the tissue site using histological analysis. In some embodiments, the foreign body reaction is measured by detecting the amount of multinucleated giant cells (MNGCs) (e.g., monocyte or macrophage fusions) present in the tissue site using histological analysis and comparing the amount of MNGCs in the tissue site with reference tissue without the hydrogel formulation.

[0110] In some embodiments, the method for delivering the hydrogel formulations disclosed herein is under conditions sufficient to deposit a certain amount or type of collagen in the cell matrix at a tissue site (e.g., a suture site) or a tissue site mimicking the surrounding endogenous tissue. In some embodiments, the method includes depositing a certain amount or type of collagen in the cell matrix at a tissue site or a tissue site mimicking the surrounding endogenous tissue. In some embodiments, the cell matrix contains a certain amount or type of collagen mimicking the endogenous tissue at the tissue site. In some embodiments, the type of collagen includes type I collagen, type III collagen, or a combination thereof. In some embodiments, type I collagen is present in a ratio of about 10:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 6:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 5:1 or less with type III collagen. In some embodiments, type I collagen is present in a ratio of about 1:1 to about 10:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 1.5:1 to approximately 9.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 2:1 to approximately 9:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 2.5:1 to approximately 8.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 3:1 to approximately 8:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 3.5:1 to approximately 7.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 4:1 to approximately 7:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 4.5:1 to approximately 6.5:1 with type III collagen. In some embodiments, type I collagen is present in a ratio of approximately 5:1 to approximately 6:1 with type III collagen. Type I collagen may be an indicator of scar tissue or foreign body reaction occurring in the subject. In some embodiments, a smaller ratio of type I collagen to type III collagen results in a more minimized foreign body reaction.In some embodiments, the method involves minimizing the ratio of type I collagen to type III collagen so that new tissue is constructed within the subject, thereby producing new tissue having the characteristics disclosed herein while avoiding the harms disclosed herein. For example, the method involves permanently filling at least a portion of the tissue site while minimizing or completely avoiding a xenobiotic reaction induced as a response to a biostimulant.

[0111] In some embodiments, collagen is formed at the site of the suture at least up to about 28 days after the site of suture closure (e.g., suturing). In some embodiments, collagen is formed at the site of the suture at least up to 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.

[0112] In some embodiments, the method for delivering the hydrogel formulation disclosed herein is under conditions sufficient to form elastin at the tissue site. In some embodiments, the method includes forming elastin at the tissue site. In some embodiments, the elastin remains at or around the tissue site after the complete decomposition of the covalently stabilized scaffold. The presence of elastin may indicate the absence of scar or scar tissue at the tissue site and can therefore be an indicator that the foreign body reaction has been minimized.

[0113] In some embodiments, the hydrogel formulation is biocompatible with the tissue of the tissue site, as determined by one or more techniques described in ISO standard 10993, the details of which are incorporated herein by reference in their entirety.

[0114] Treatment method In some embodiments, the method further includes treating the target tissue site by delivering a hydrogel formulation to the tissue site. In some embodiments, the hydrogel formulation is delivered on the suture line after the incision has been sutured. In some embodiments, the hydrogel formulation is delivered into the incision while the incision is being sutured. In some embodiments, the hydrogel formulation is delivered on the suture line after the incision has been sutured, and into the suture line while the incision is being sutured. In some embodiments, the hydrogel begins to anneal on the suture line for at least 1, 2, 3, 4, 15, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes before the target dermis and subcutaneous tissue is sutured.

[0115] surgical incision In some embodiments, the method includes delivering a microgel to a suture line. In some embodiments, the suture line was an incision. In some embodiments, the incision was a surgical incision. In some embodiments, the surgical incision was an abdominal fascial incision, an umbilical incision of the abdominal fascia, a hernia or dehiscence of a previous incision at 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 included a midline or transverse abdominal incision of the fascia. In some embodiments, the abdominal incision was below the dermis and subcutaneous tissue of the subject.

[0116] In some embodiments, the surgical incision was a fascial incision (e.g., a midline or transverse incision of the fascia). In some embodiments, the incision was the result of surgery performed on the subject, including treatment for gastrointestinal cancer, hysterectomy, ovarian cancer, spinal fusion, abdominal trauma surgery, or a combination thereof. In some embodiments, the incision was the result of surgery performed on the subject, including treatment for gastrointestinal cancer. In some embodiments, the incision was the result of surgery performed on the subject, including hysterectomy. In some embodiments, the incision was the result of surgery performed on the subject, including treatment for ovarian cancer. In some embodiments, the incision was the result of surgery performed on the subject, including spinal fusion. In some embodiments, the incision was the result of surgery performed on the subject, including abdominal trauma surgery.

[0117] In some embodiments, the surgical incision was an umbilical incision in the abdominal fascia. In some embodiments, the umbilical incision was a midline or transverse incision in the abdominal wall of the fascia. In some embodiments, the umbilical incision was below the dermis and subcutaneous tissue of the subject. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including hysterectomy. In some embodiments, the incision was the result of surgery performed on the subject, including ovarian cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including spinal fusion. In some embodiments, the incision was the result of surgery performed on the subject, including abdominal trauma surgery.

[0118] In some embodiments, the surgical incision was a laparoscopic keyhole incision in the abdominal fascia. In some embodiments, the laparoscopic keyhole incision was a midline or transverse incision in the abdominal wall of the fascia. In some embodiments, the laparoscopic keyhole incision was below the dermis and subcutaneous tissue of the subject. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including hysterectomy. In some embodiments, the incision was the result of surgery performed on the subject, including ovarian cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including spinal fusion. In some embodiments, the incision was the result of surgery performed on the subject, including abdominal trauma surgery.

[0119] In some embodiments, the surgical incision was an inguinal incision in the abdominal fascia. In some embodiments, the inguinal incision was a midline or transverse incision in the abdominal wall of the fascia. In some embodiments, the inguinal incision was below the dermis and subcutaneous tissue of the subject. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. In some embodiments, the incision was the result of surgery performed on the subject, including gastrointestinal cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including hysterectomy. In some embodiments, the incision was the result of surgery performed on the subject, including ovarian cancer treatment. In some embodiments, the incision was the result of surgery performed on the subject, including spinal fusion. In some embodiments, the incision was the result of surgery performed on the subject, including abdominal trauma surgery.

[0120] Combined treatment In some embodiments, the method involves administering one or more further agents (e.g., therapeutic agents) to a target, such as a local anesthetic (e.g., lidocaine), an analgesic, an anti-inflammatory agent, or others that can provide therapeutic benefit to the administration site. In some embodiments, the microgel particles comprise one or more further agents (e.g., drug-eluting microgel particles). In some embodiments, the microgel particles elute one or more activators in situ. In some embodiments, the hydrogel formulation is formulated with one or more activators. In some embodiments, the hydrogel formulation is not formulated with one or more activators, and one or more activators are administered separately from the hydrogel formulation. In some embodiments, the hydrogel formulation and one or more further activators are administered sequentially to the target. In some embodiments, the hydrogel formulation and one or more further activators are administered substantially simultaneously to the target.

[0121] In some embodiments, the therapeutic agent includes an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-fibrotic agent, or an antibiotic. In some embodiments, the local anesthetic is of the ester type. In some embodiments, the ester type local anesthetic includes benzocaine, chloroprocaine, procaine, propalacaine, tetracaine, amylocaine, or oxybuprocaine, or any combination thereof. In some embodiments, the local anesthetic is of the amide type. In some embodiments, the amide type local anesthetic includes articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, or cincocaine, or any combination thereof. In some embodiments, the local anesthetic is lidocaine or contains it. In some embodiments, the local anesthetic consists of lidocaine. In some embodiments, the analgesic includes codeine, fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone, or tramadol, or any combination thereof. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID) or a steroid. In some embodiments, the NSAID includes ibuprofen or naproxen. In some embodiments, the steroid includes a corticosteroid. In some embodiments, the antibiotic includes dicloxacillin, erythromycin, or tetracycline. In some embodiments, the antifibrotic agent includes pentoxifylline.

[0122] Method for producing hydrogel formulations In some embodiments, methods for producing hydrogel formulations and their components are disclosed herein. In some embodiments, the method includes synthesizing microgel particles from raw materials. In some embodiments, the method includes fine-tuning the mechanical properties of the microgel particles, hydrogel formulations, or the resulting covalently stabilized porous scaffold. In some embodiments, the method includes purifying the microgel particles. In some embodiments, the method includes formulation the microgel particles into a hydrogel formulation or preparation. In some embodiments, the method further includes sterilizing the hydrogel formulation or preparation.

[0123] Synthesis of microgel particles This specification discloses a method for producing microgel particles as disclosed herein, comprising combining raw materials (e.g., polymers, functional groups, peptides, etc.) under conditions sufficient to form individual microgel particles. In some embodiments, the conditions sufficient to form microgel particles may include an aqueous buffer having a pH in the range of 7 to 9. In non-limiting examples, the buffer may be phosphate-buffered saline (PBS), HEPES, or triethanolamine (TEOA). In some embodiments, the reaction may be stopped by adding an acid or base to stop the reaction after a certain time after mixing to create a water-in-oil emulsion. A reaction stop molecule may be added to the oil phase and diffused into the aqueous phase to stop the reaction occurring in the aqueous phase. In some embodiments, the reaction may be stopped by adding maleimide to react with the remaining thiols. In some embodiments, the reaction may be stopped by adding an oxidizing agent to oxidize the thiols.

[0124] In some embodiments, microgel particles may be synthesized using a microfluidic apparatus (e.g., one particle per channel). In some embodiments, microgel particles may be synthesized by a water-in-oil emulsion, as described in more detail herein. In some embodiments, microgel particles may be synthesized by a water-in-oil emulsion using mechanical stirring. In some embodiments, microgel particles may be synthesized by a water-in-oil emulsion using a static mixer. In some embodiments, microgel particles may be synthesized using in-line flow-through synthesis. In some embodiments, microgel particles may be synthesized using a parallel production method (multiple particles per channel, or multiple particles simultaneously per parallel channel).

[0125] In some embodiments, the method includes synthesizing microgel particles by a water-in-oil emulsion process. In some embodiments, the method begins with obtaining an oil or a mixed oil. In non-limiting examples, the oil may be a light mineral oil (LMO), a heavy mineral oil (HMO), or a fluorinated oil. In some embodiments, the mixed oil includes a surfactant. In some embodiments, different surfactants may be used. In some embodiments, the surfactant may be a nonionic surfactant. Non-limiting examples of nonionic surfactants include 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 include sodium dodecyl sulfate (SDS), sodium lauryl ether sulfate (SLES), and perfluorooctanesulfonic acid. In some embodiments, the surfactant may be a cationic surfactant. Non-limiting examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB) and hexadecylpyridinium bromide. In some embodiments, the surfactant may be an amphoteric surfactant. Non-limiting examples of amphoteric surfactants include betaine citrate, lauryl betaine, sodium, and (carboxymethyl)dimethyloleylammonium hydroxide. In some embodiments, the concentration of the surfactant may vary from 0.01 to 5 v / v%.

[0126] In some embodiments, the method includes adding a surfactant to the oil. In some embodiments, the method includes adding a surfactant to the oil before adding an aqueous solution / mixture to the oil. In some embodiments, the method includes adding a surfactant to an aqueous solution / mixture described herein. In some embodiments, it is beneficial to have a surfactant in the aqueous phase because, if the surfactant has high water solubility, it is easy to remove during purification.

[0127] In some embodiments, the oil or mixed oil may be added to the bioreactor vessel via a micron filter and stirred. In some embodiments, the bioreactor vessel contains a volume of about 100 ml to about 1 liter. In some embodiments, the bioreactor vessel contains a volume of about 1 liter to about 10 liters. In some embodiments, the bioreactor vessel contains a volume of about 10 liters to about 100 liters. In some embodiments, the bioreactor vessel contains a volume of about 100 liters to about 1000 liters. In some embodiments, the bioreactor vessel contains a volume of about 100 liters to about 10,000 liters. In some embodiments, the bioreactor vessel contains a volume of about 1 micron filter has a pore size of about 0.1 μm to about 1 μm. In some embodiments, the micron filter has a pore size of about 0.2 μm.

[0128] In some embodiments, the oil or mixed oil may be added to the static mixer via a micron filter and stirred. In some embodiments, the static mixer has a capacity of about 100 ml to about 1 liter. In some embodiments, the static mixer has a capacity of about 1 liter to about 10 liters. In some embodiments, the static mixer has a capacity of about 10 liters to about 100 liters. In some embodiments, the static mixer has a capacity of about 100 liters to about 1000 liters. In some embodiments, the static mixer has a capacity of about 100 liters to about 10,000 liters. In some embodiments, the static mixer has a capacity of about 10 liters to about 10,000 liters. In some embodiments, the static mixer has a capacity of about 1000 liters to about 10,000 liters. In some embodiments, the static mixer has a capacity of about 1000 liters to about 10,000 liters. In some embodiments, the micron filter has a pore size of about 0.1 μm to about 1 μm. In some embodiments, the micron filter has a pore size of about 0.2 μm.

[0129] In some embodiments, a method for synthesizing microgel particles includes providing one or more polymers as disclosed herein (e.g., in solution). In some embodiments, the one or more polymers include PEG. In some embodiments, PEG is provided at a molecular weight as disclosed herein.

[0130] In some embodiments, a method for synthesizing microgel particles involves modifying one or more polymers disclosed herein by attaching one or more functional groups. In some embodiments, PEG is modified by attaching thiol and vinyl sulfone functional groups. In some embodiments, a first PEG is modified with a thiol, and a second PEG is modified with a vinyl sulfone (VS).

[0131] In some embodiments, the method for synthesizing microgel particles includes mixing one or more modified polymers in a solution. In some embodiments, thiolated PEG is mixed with PEG-VS. In some embodiments, the functional groups react to form a hydrogel (e.g., a hydrogel mesh). In some embodiments, the functional groups react by a Michael addition reaction (e.g., a thiol-em-Michael addition reaction). In some embodiments, the method may include filtering the solution. In some embodiments, the solution may contain a peptide (e.g., a cell adhesion peptide as disclosed herein). In some embodiments, the solution may contain a buffer or buffering agent. In some embodiments, the solution may contain a base catalyst.

[0132] In some embodiments, methods for synthesizing microgel particles include those disclosed in U.S. Patent No. 10,912,860 and U.S. Patent No. 10,668,185, which are incorporated herein by reference in their entirety.

[0133] Fine-tuning of mechanical properties In some embodiments, methods for modifying the physical properties of microgel particles, covalently stabilized scaffolds, hydrogel formulations, or any combination thereof are disclosed herein. In some embodiments, the method of modifying the physical properties depends on the mode of delivery and the target. In some embodiments, the physical properties may be modified according to the mode of administration, desired biocompatibility, or any combination thereof. In some embodiments, the physical properties are the mechanical properties of microgel particles, covalently stabilized scaffolds, hydrogel formulations, or any combination thereof.

[0134] In some embodiments, the method includes adjusting the viscosity of the hydrogel, the decomposition rate of the covalently stabilized scaffold, the volume fraction of microgel particles, the pH of the microgel particles, the pH of the annealing agent solution, the pH of the covalently stabilized scaffold, the degree of polymer substitution, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percentage of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, further agents (e.g., therapeutic agents), the size of the microgel particles, or a combination thereof. In some embodiments, the method includes reducing the viscosity of the hydrogel, the decomposition rate of the covalently stabilized scaffold, the volume fraction of microgel particles, the pH of the microgel particles, the pH of the annealing agent, the pH of the covalently stabilized scaffold, the degree of polymer substitution, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percentage of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, further agents (e.g., therapeutic agents), or a combination thereof. In some embodiments, the method involves increasing the viscosity of the hydrogel, the decomposition rate of the covalently stabilized scaffold, the volume fraction of microgel particles, the pH of the microgel particles, the pH of the annealing agent, the pH of the covalently stabilized scaffold, the degree of polymer substitution, the compressive modulus of the covalently stabilized scaffold, the storage modulus of the covalently stabilized scaffold, the weight percentage of the polymer, the molar ratio of the functional groups, the molecular weight of the polymer, the molecular weight of the annealing agent, further agents (e.g., therapeutic agents), or combinations thereof.

[0135] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the viscosity of the hydrogel or the compressive modulus of the covalently stabilized scaffold. In some embodiments, the method includes adjusting the volume fraction of microgel particles. In some embodiments, the volume fraction of microgel particles may be about 80% to about 100%. In some embodiments, the volume fraction of microgel particles may be about 85% to about 100%. In some embodiments, the volume fraction of microgel particles may be about 90% to about 100%. In some embodiments, the volume fraction of microgel particles may be about 95% to about 100%. In some embodiments, the volume fraction of microgel particles may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, adjusting the volume fraction of microgel particles includes adjusting the percentage concentration of microgel particles in the hydrogel. Increasing the volume fraction of microgel particles may increase the viscosity of the suspension of microgel particle hydrogel that is annealed and becomes a covalently stabilized scaffold. Decreasing the volume fraction of microgel particles may decrease the viscosity of the hydrogel that is annealed and becomes a covalently stabilized scaffold. In some embodiments, the method includes adjusting the volume fraction of microgel particles in the range of 0.75 mL / mL to 1.0 mL / mL to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa*s. Increasing the volume fraction of microgel particles may increase the compressive modulus of the covalently stabilized scaffold. Decreasing the volume fraction of microgel particles may decrease the compressive modulus of the covalently stabilized scaffold. In some embodiments, the method includes adjusting the volume fraction of microgel particles in the range of 0.75 mL / mL to 0.95 mL / mL to achieve a compressive modulus of the covalently stabilized scaffold of about 1,000 Pa to about 17,000 Pa.

[0136] In some embodiments, the method includes adjusting the compressive modulus of the microgel particles. In some embodiments, adjusting the compressive modulus is achieved by adjusting the molar ratio of the crosslinking agent, polymer (e.g., copolymer), or combination thereof. Increasing the compressive modulus of the microgel particles may increase the viscosity of the hydrogel that is annealed to form a covalently stabilized scaffold. Decreasing the compressive modulus of the microgel particles may decrease the viscosity of the hydrogel that is annealed to form a covalently stabilized scaffold. In some embodiments, the method includes adjusting the compressive modulus of the microgel particles in the range of 15 kPa to 46 kPa to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa*s. Increasing the compressive modulus of the microgel particles may increase the compressive modulus of the covalently stabilized scaffold. Decreasing the compressive modulus of the microgel particles may decrease the compressive modulus of the covalently stabilized scaffold. In some embodiments, the method involves adjusting the compressive modulus of the microgel particles in the range of 15 kPa to 46 kPa to achieve a covalently stabilized scaffold compressive modulus of about 1,000 Pa to about 17,000 Pa. In some embodiments, the microgel particles have a compressive modulus of at least about 1,500 Pa after the annealing reaction. In some embodiments, the microgel particles have a compressive modulus of at least about 6,000 Pa after the annealing reaction.

[0137] In some embodiments, the method includes adjusting the volume fraction of microgel particles within the range of 0.75 mL / mL to 0.95 mL / mL (e.g., increasing or decreasing it) and adjusting the compressive modulus of the microgel particles within the range of 15 kPa to 46 kPa in order to achieve a hydrogel viscosity of 1,000 to 1,000,000 mPa*s. In some embodiments, the method includes adjusting the volume fraction of microgel particles within the range of 0.75 mL / mL to 0.95 mL / mL and adjusting the compressive modulus of the microgel particles within the range of 15 kPa to 46 kPa in order to achieve a covalently stabilized scaffold compressive modulus of about 1,000 Pa to about 17,000 Pa.

[0138] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the rate of degradation of the covalently stabilized scaffold. In some embodiments, the degradation rate is modified depending on the desired duration for which the covalently stabilized scaffold remains in the tissue site. In some embodiments, the method includes modifying the degradation pathway, modifying the polymer (e.g., copolymer) used to constitute the microgel particles, or a combination thereof, in order to modify the degradation rate. In some embodiments, the method includes modifying the degradation pathway to one or more of oxidative degradation, enzymatic degradation, or hydrolysis. In some embodiments, the method includes synthesizing microgel particles using PEG to reduce the degradation rate of the covalently stabilized scaffold. In some embodiments, the method includes synthesizing microgel particles without using PEG to increase the degradation rate.

[0139] In some embodiments, the method involves adjusting the degree of substitution of the polymer (e.g., increasing or decreasing it). In some embodiments, adjusting the degree of substitution is achieved by increasing or decreasing the amount of functional groups that will bind to the microgel particles. In some embodiments, the degree of substitution is changed by adjusting the molecular weight of the polymer (e.g., copolymer) of the microgel particles. In some embodiments, the method involves measuring the degree of substitution using an Elman assay or proton nuclear magnetic resonance (Proton-NMR).

[0140] In some embodiments, the method involves adjusting (e.g., increasing or decreasing) the compressive modulus of a covalently stabilized scaffold. In some embodiments, the compressive modulus of a covalently stabilized scaffold can be changed by adjusting the concentration of functional groups (e.g., thiols and vinyl sulfones) contained in the microgel particles. In some embodiments, the concentration of functional groups (e.g., thiols and vinyl sulfones) contained in the microgel particles can be changed by adjusting the molecular weight of the polymer(s) (e.g., copolymer(s)) of the microgel particles. The compressive modulus of a covalently stabilized scaffold can be increased by increasing the concentration of functional groups (e.g., thiolated PEG). The compressive modulus of a covalently stabilized scaffold can be decreased by decreasing the concentration of functional groups (e.g., thiolated PEG). The compressive modulus of a covalently stabilized scaffold can be increased by increasing the molecular weight of the polymer(s) (e.g., PEG), and the compressive modulus of a covalently stabilized scaffold can be decreased by decreasing the molecular weight of the polymer(s) (e.g., PEG). In some embodiments, the method involves adjusting the concentration of functional groups (e.g., thiols and vinyl sulfones) in the gelling solution to a range of about 10 mg / mL to about 45 mg / mL in order to achieve a compressive modulus of about 100 Pa to about 140,000 Pa for a covalently stabilized scaffold.

[0141] In some embodiments, the method includes adjusting (e.g., increasing or decreasing) the rate at which the covalently stabilized scaffold anneals. In some embodiments, the rate at which the covalently stabilized scaffold anneals can be changed by changing the annealing agent. In some embodiments, the rate at which the covalently stabilized scaffold anneals can be changed by adjusting the molecular weight of the annealing agent. PEG-dithiol (PEG(SH)2), 4-arm PEG-SH, and PETMA can be used to achieve a compressive modulus of about 4,000 Pa to about 7,000 Pa for a covalently stabilized scaffold about 60 minutes after annealing. In some embodiments, the method includes adjusting the pH of the annealing agent to change the rate at which the covalently stabilized scaffold anneals. The rate at which the covalently stabilized scaffold anneals can be increased by increasing the pH of the annealing agent, and the rate at which the covalently stabilized scaffold anneals can be decreased by decreasing the pH of the annealing agent. In some embodiments, the method includes using an annealing pH of 6.5 or less to delay the onset of the annealing reaction by 30 minutes or more. In some embodiments, the method includes delivering a therapeutic agent (e.g., a local anesthetic (e.g., lidocaine)) to alter the rate at which the covalently stabilized scaffold anneals. The local anesthetic can reduce the rate at which the covalently stabilized scaffold anneals and can function in hydrogel formulations having a compressive modulus in the range of about 1,500 Pa to about 18,000 Pa. In some embodiments, the method includes delivering a therapeutic agent (e.g., a local antibiotic (e.g., triclosan, vancomycin)) to achieve a compressive modulus of about 2,000 Pa to about 12,000 Pa of the covalently stabilized scaffold about 250 minutes after annealing.

[0142] Purification of microgel particles In some embodiments, the method includes purifying microgel particles. In some embodiments, the method includes simultaneously synthesizing and purifying microgel particles. In some embodiments, the method includes purifying microgel particles after synthesis. In some embodiments, the purification of microgel particles includes membrane separation of microgel particles from undesirable components. In some embodiments, different types of filtration membranes may be used (e.g., hollow fiber membranes having different pore sizes, different lumen IDs, dialysis membranes, or plate membranes). In some embodiments, the membrane separation includes tangential flow filtration (TFF). In some embodiments, the membrane separation includes ultrafiltration-diafiltration (UFDF). In some embodiments, the membrane separation includes microfiltration-diafiltration (MFDF). In some embodiments, the membrane separation includes hollow fiber-diafiltration (HFDF). TFF generally encompasses membrane filtration and separation techniques. TFF may be used herein to purify and concentrate microgel particles. TFF may include generating a feed stream of a solution of microgel particles passing parallel to the membrane surface. In some embodiments, a portion of the solution passes through the membrane (permeate), while the remainder (retaining solution) is recirculated and returned to the supply reservoir. This system is sometimes called diafiltration. This system allows molecules smaller than the membrane pores (in the permeate) to move toward and pass through the membrane, while larger molecules, such as microgel particles, may remain in the retaining solution. In some embodiments, the flow in the filtration system may be controlled by a peristaltic pump. In some embodiments, the flow in the filtration system may be controlled by a Quattroflow pump or any positive displacement pump. In some embodiments, the filtration system may be closed to the surrounding environment. In some embodiments, the filtration system may be open to the surrounding environment.

[0143] In some embodiments, the purification method may include removing excess oil from the microgel particles. In some embodiments, the purification method may include dispersing the particles in an alcohol solution. In some embodiments, the alcohol solution comprises alcohol and water, with alcohol present in the solution in a ratio of about 0.8:1 or higher. In some embodiments, the alcohol solution comprises alcohol and water, with alcohol present in the solution in a ratio of about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1 or higher. In some embodiments, the alcohol solution comprises alcohol and water, with alcohol present in the solution in a ratio of about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1 or lower. In some embodiments, the alcohol solution comprises alcohol and water, with alcohol present in the solution in a ratio of about 0.5:1 to about 1:1. In some embodiments, the alcohol solution comprises alcohol and water, with alcohol present in the solution in a ratio of about 0.6:1 to about 0.9:1. In some embodiments, the alcohol solution comprises alcohol and water, with the alcohol present in the solution in a ratio of about 0.7:1 to about 0.8:1. In some embodiments, the purification method may include dispersing and sufficiently swelling the particles (which consist mainly of water) and removing excess oil and surfactants that are not miscible with water, while ensuring that particle aggregation is not occurred. In some embodiments, the purification method may include slowly moving the particles into an aqueous buffer while preventing surfactant precipitation. In some embodiments, the migration rate is related to the flux of the filtrate passing through the membrane and may occur at a rate of about 1 to about 1000 LMH (liters / m² / h). In some embodiments, migration may occur at a rate of about 100 to about 500 LMH. In some embodiments, migration may occur at a rate of about 200 to about 300 LMH. This transition rate may be particularly important to ensure that surfactants do not precipitate on (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) swelling of the particle hydrogel mesh, which is a product of the affinity of a given hydrogel polymer backbone / crosslinking agent system to a particular solvent, and (ii) solubility of the surfactant in the continuous phase outside the particles.

[0144] In some embodiments, the method includes concentrating microgel particles in a solution or suspension. In some embodiments, the method includes pumping the microgel particles through a membrane filtration system while removing the volume of the continuous phase, continuously concentrating the microgel particles at a controlled membrane flux, and maintaining internal wall shear stress in the membrane filtration system. In some embodiments, the membrane filtration system is selected from tangential flow filtration (TFF), ultrafiltration-diafiltration (UFDF), microfiltration-diafiltration (MFDF), or hollow fiber-diafiltration (HFDF). In some embodiments, the membrane flux is 10 to 1000 L / m 2 It is controlled to / h. In some embodiments, the wall shear stress is maintained between 100s⁻¹ and 10,000s⁻¹.

[0145] Storage of microgel particles In some embodiments, methods for storing microgel particles, annealing agents, further activators, therapeutic agents, hydrogel formulations or formulations, or combinations thereof are disclosed herein. In some embodiments, the method includes storing microgel particles, annealing agents, further activators, therapeutic agents, or any combination thereof before formulation into a hydrogel formulation. In some embodiments, storage is performed before administration of the hydrogel formulation to a subject. In some embodiments, the storage method includes lyophilization, cryo-dehydration, cryohibernation, or cryopreservation, or a combination thereof.

[0146] In some embodiments, lyophilization of microgel particles, annealing agents, therapeutic agents, or combinations thereof involves the use of lyophilization protectants to preserve the functionality of the microgel particles, annealing agents, therapeutic agents, or combinations thereof. Lyophilization protectants include the addition of reagents, salts, or additives to protect the microgel particles, annealing agents, therapeutic agents, or combinations thereof during the drying process. Common lyophilization protectants include isopropanol, ethanol, glycerol, trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Furthermore, methods for increasing the transport of lyophilization protectants, annealing agents, therapeutic agents, or combinations thereof within the microgel particles in suspension can be utilized as methods to improve the viability and functionality of the microgel particles, annealing agents, therapeutic agents, or combinations thereof after lyophilization. These methods include electroporation and the addition of reagents. In some embodiments, the lyophilized microgel particles, annealing agents, therapeutic agents, or combinations thereof can be restored for delivery to the target tissue site. In some embodiments, restoration is achieved by introducing a restoration medium into lyophilized microgel particles, an annealing agent, a therapeutic agent, or a combination thereof.

[0147] In some embodiments, the microgel particles are rapidly frozen. In some embodiments, the microgel particles are rapidly frozen using liquid nitrogen. In some embodiments, the microgel particles are frozen at temperatures of at least about -100C, -110C, -120C, -130C, -140C, -150C, -160C, -170C, -180C, -190C, or -200C. In some embodiments, the microgel particles are frozen at temperatures of about -196C. In some embodiments, the microgel particles are in a solution of at least about 80%, 85%, 90%, 95%, or 100% isopropanol.

[0148] In some embodiments, freeze-drying occurs at a temperature of about -55°C. In some embodiments, freeze-drying occurs at temperatures below about -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, -85°C, -90°C, -95°C, and -100°C. In some embodiments, the volume fraction of microgel particles during freeze-drying is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less than 100%.

[0149] kit In some embodiments, kits useful for delivering the hydrogel formulations disclosed herein are disclosed herein. In some embodiments, the kits disclosed herein may be used to deliver the hydrogel formulation to a target tissue site. In some embodiments, the kit comprises the hydrogel formulation described herein and can be used to carry out the methods described herein. In some embodiments, the kit comprises hydrogel particles and an annealing agent in separate containers. In some embodiments, the kit comprises the hydrogel and the annealing agent in a single container. In some embodiments, the kit also comprises a reconstitution medium, such as those described herein, for reconstitution of a lyophilized hydrogel formulation (e.g., lyophilized hydrogel, annealing agent, or a combination thereof).

[0150] Instructions for use may be included in the kit. Optionally, the kit may also contain other useful components or other useful instruments, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring devices, bandage materials, etc. The materials and components combined in the kit may be stored in any convenient and suitable manner that preserves their operability and usefulness and provided to healthcare professionals. For example, components may be in soluble, dehydrated, or lyophilized form and may be provided at room temperature, refrigerated, or freezing temperature. Components are typically contained in suitable packaging material(s). As used herein, the term “packaging material” refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging material is constructed by a well-known method and preferably provides a sterile, contaminant-free environment. The packaging material used in the kit may be those conventionally used in gene expression assays and treatment administrations. As used herein, the term “packaging” refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, capable of holding individual kit components. Thus, for example, packaging may be a glass vial or pre-filled syringe used to contain a suitable amount of the pharmaceutical composition. The packaging material has an external label indicating the contents and / or purpose of the kit and its components.

[0151] In some embodiments, the kit includes a first container and a second container. In some embodiments, the first container does not contain an annealing agent and contains the hydrogel formulation described herein. In some embodiments, the second container contains an annealing agent that can anneal the hydrogel formulation in situ in the presence of the hydrogel formulation in the first container to form a covalently stabilized scaffold. In some embodiments, the first container is separate from the second container. In some embodiments, the first or second container is a pre-loaded syringe. In some embodiments, the first and second containers are sterilized using the method of the present disclosure. In some embodiments, the kit further includes an applicator such as a microneedle patch or a syringe. In some embodiments, the kit further includes instructions for using the hydrogel formulation, annealing agent, applicator, or any combination thereof to deliver the hydrogel formulation and annealing agent to, on or around a surgical incision as disclosed herein, and to form a covalently stabilized scaffold as disclosed herein. In some embodiments, the hydrogel formulation is provided in Figure 6G.

[0152] definition Unless otherwise defined, all technical terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in which the claimed subject matter pertains. In some embodiments, terms having a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantially different meaning from that commonly understood in the art.

[0153] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. References to "or" herein are intended to include "and / or" unless otherwise specified.

[0154] Throughout this specification, any reference to “several embodiments,” “further embodiments,” or “specific embodiments” means that any particular characteristics, structures, or features described in relation to an embodiment are included in at least one embodiment. Therefore, the phrases “in some embodiments,” “further embodiments,” or “specific embodiments” appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, any particular characteristics, structures, or features may be combined in any preferred manner in one or more embodiments.

[0155] When the term "approximately" is used herein in reference to a number, it refers to a number that is plus or minus 10 percent of that number. The range to which the term "approximately" applies refers to a range of minus 10 percent of the minimum value and plus 10 percent of the maximum value.

[0156] The term "annealing agent," as used herein, refers to a substance (real-world object) capable of inducing an annealing reaction between particles (e.g., microparticles) of this disclosure to form an annealed scaffold (e.g., a covalently stabilized scaffold). Non-limiting examples of annealing agents include molecules having two or more reactive functional groups, including eosin Y, PETMA (pentaerythritol tetrakis(2-mercaptoacetate)), factor XIII / factor XIIIa, thiols (e.g., PEG-dithiols), divinyl sulfone, or combinations thereof. The annealing agent does not have to be covalently involved in the linking of the particles described herein when the annealing reaction is induced. The annealing agent may be covalently linked to the particles of the annealed scaffold when the annealing reaction is induced.

[0157] As used herein, the term "annealing component" refers to a substrate bound to the microgel particles themselves in an annealing reaction between microgel particles (e.g., microparticles) as described herein. Non-limiting examples of annealing components include two or more reactive functional groups, including K-peptides or Q-peptides, thiols or thiol derivatives, vinyls or vinyl derivatives (e.g., vinyl sulfones), methacrylates, acrylates, amines, or combinations thereof.

[0158] When used herein, the term "biocompatibility" refers to biocompatibility as determined under the international standard ISO 10993-1, which is incorporated herein by reference in its entirety.

[0159] The terms “cell adhesive peptide” or “cell adhesion peptide,” as used interchangeably herein, refer to peptides that can initiate cell adhesion to synthetic materials such as microgel particles. A non-limiting example of a cell adhesion peptide is the RGD peptide. The cell adhesion peptides disclosed herein may be provided in their entirety 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 incorporated herein by reference.

[0160] As used herein, the term “cellular matrix” refers to a network of proteins or other molecules that surround, support, and / or give structure to cells and tissues in the body.

[0161] As used herein, the term “compressive modulus” refers to the stiffness of individual microgel particles, macroscopic hydrogels, or annealed scaffolds of microgel particles. The compressive modulus may also be measured by a compression test (fail or pass), in which a hydrogel, non-annealed scaffold (microgel particles), or annealed scaffold is compressed over a known distance and velocity on a compression plate of known cross-sectional area, during which the force applied to the compression plate is recorded by a force transducer attached to the compression plate. The compressive modulus may also be mathematically calculated from the stress / strain curve recorded during the compression test.

[0162] When used herein, the term “crosslinking agent” refers to a reagent involved in the crosslinking reaction of raw materials for forming microgel particles (e.g., fine particles) of the present disclosure. A crosslinking agent is a linker having two or more reactive functional groups (e.g., thiol, vinyl sulfone, maleimide, acrylate, methacrylate, acrylamide, methacrylamide, norbornene, amine, hydroxyl). If the crosslinking agent is in excess in the crosslinking reaction, the crosslinking agent may also be an annealing component and may be involved as an annealing agent in the interparticle annealing reaction of the present disclosure. Non-limiting examples of crosslinking agents include vinyl derivatives having two or more vinyl groups (e.g., PEG-VS), thiol derivatives having two or more thiol groups (e.g., PEG-dithiol or thiolated HA), peptides having two or more cysteine ​​(e.g., matrix metalloproteinase (MMP)-degradable crosslinking agents), or combinations thereof.

[0163] When used herein, the term "crosslinking" refers to a reaction for forming microgel particles (e.g., fine particles) of the present disclosure.

[0164] The terms “derivatives” relating to “vinyl” or “thiol” refer to vinyl-containing chemicals or thiol-containing chemicals, respectively. Non-limiting examples of vinyl derivatives include PEG-VS, PEG-acrylate, PEG-methacrylate, and PEG-maleimide. Non-limiting examples of vinyl groups include vinyl sulfone, acrylate, methacrylate, acrylamide, maleimide, and norbornene. Non-limiting thiol derivatives include PEG-dithiols, cysteine-containing peptides (e.g., matrix metalloproteinase (MMP)-degradable crosslinking agents), organosulfur compounds of any form R-SH (wherein R represents an alkyl or other organic substituent), methanethiols, ethanethiols, 1-propanethiols, 2-propanethiols, allyl mercaptans, butanethiols, tert-butyl mercaptans, pentanethiols, thiophenols, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, metallothionein, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 1-mercaptonidole, grapefruit mercaptans, furan-2-ylmethanethiol, 3-mercaptopropane-1,2-diol, 3-mercapto-1-propanesulfonic acid, 1-hexadecanethiol, pentachlorobenzenethiol, or combinations thereof.

[0165] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessment may be relative or absolute. “Detecting the presence of ~” may include determining the quantity of something that is present, in addition to determining whether something is present or not, depending on the context.

[0166] As used herein, the term "modulus of elasticity" refers to the mechanical property of a material relating to its resistance to elastic deformation when stress is applied, and may be calculated by the following equation:

[0167]

number

[0168]

number

[0169] The term "ex vivo" is used to describe events that occur outside the subject's body. Ex vivo assays are not performed on the subject. Rather, ex vivo assays are performed on a sample separate from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay, or on a tissue piece excised (removed) from the subject.

[0170] As used herein, the term "foreign body reaction" typically refers to a fibrotic reaction resulting from the graft or hydrogel preparation, characterized, for example, by chronic inflammation, granuloma formation, and / or scar tissue formation at or around the delivery site. The foreign body reaction in question can be detected by histological analysis of the tissue at or around the transplant site, by comparing the results of the histological analysis with the histology of a reference tissue that does not contain the graft or hydrogel preparation.

[0171] As used herein, the term "gel" refers to a three-dimensional network of cross-linked polymers swollen in a solvent.

[0172] As used herein, the term "HEPES" refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0173] When used herein, the term "in situ" refers to the original site of delivery or administration, remaining at the site of origin without infiltrating adjacent tissue.

[0174] The term "in vitro" is used to describe events that occur within a container holding an experimental reagent, as the reagent is separated from the biological source from which the substance is obtained. In vitro assays can include cell-based assays in which living or dead cells are used. In vitro assays can also include cell-free assays in which intact cells are not used.

[0175] The term "in vivo" is used to describe events that occur within the body of a subject.

[0176] As used herein, the terms “homologous,” “homologousity,” or “homology percentage” can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA Vol. 87: pp. 2264–2268, 1990, modified as in Proc. Natl. Acad. Sci. USA Vol. 90: pp. 5873–5877, 1993) when used herein to describe an amino acid sequence or nucleic acid sequence in comparison to a reference sequence. Such formulas are incorporated into the basic local alignment search tool (BLAST) program by Altschul et al. (J Mol Biol. 1990 Oct 5; 215(3): 403-10; Nucleic Acids Res. 1997 Sep 1; 25(17): 3389–402). The homology percentage of a sequence can be determined using the latest version of BLAST available as of the filing date of this application. The sequence identity percentage can be determined using the latest version of BLAST available as of the filing date of this application.

[0177] As used herein, the term "hydrogel" refers to a gel that is insoluble in water and capable of retaining water.

[0178] As used herein, the term "K-peptide" refers to a peptide comprising an amino acid sequence containing one or more lysine residues that can function as a substrate for an annealing agent in an annealing reaction or assist in the crosslinking of microgel particles disclosed herein.

[0179] The terms “microparticles” or “microspheres,” as used herein, are interchangeable in meaning for particles with a size of approximately 0.1 to approximately 1000 μm.

[0180] As used herein, the term "microgel particles" refers to particles composed of gel with a size of approximately 0.1 to approximately 1000 μm.

[0181] When used herein, the term "particle" refers to a single unit of a larger system, such as a hydrogel formulation or composition disclosed herein.

[0182] Where used herein, the term “identity percentage (%)” generally refers to the proportion of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence, after the sequences have been aligned to achieve the maximum percentage of identity and gaps have been introduced if necessary (for example, gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for the purpose of determining the identity percentage can be achieved in various ways known in the relevant art. The identity percentage of two sequences may be calculated by aligning the test sequence to the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same position in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0183] As used herein, the term "PEG" refers to poly(ethylene glycol).

[0184] As used herein, the term "4-arm PEG-VS" refers to 4-arm poly(ethylene glycol)-vinyl sulfone.

[0185] As used herein, the term "PLA" refers to polylactic acid or polylactide.

[0186] As used herein, the term "polydispersibility" is a measure of the heterogeneity of the particles of this disclosure (e.g., microgel particles) based on size. Polydispersibility may be measured by any of the following methods using a particle size analyzer: laser diffraction, dynamic light scattering, small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), or microscopy.

[0187] As used herein, the term "polymer" refers to a class of substances composed of macromolecules (giant molecules) made up of monomeric repeats. Non-limiting polymers include poly(ethylene glycol) (PEG), polylactic acid (PLA), collagen, collagen, poly(methyl methacrylate) (PMMA), or any combination thereof. The polymer may be synthetic, such as PEG, PLA, PMMA. The polymer may be a modified form, for example, containing one or more thiols or vinyl derivatives (e.g., PEG-dithiol, 4-arm PEG-thiol, PEG-VS, thiolated HA) disclosed herein.

[0188] As used herein, the term "pore size" refers to the size of each individual pore in a covalently stabilized scaffold, defined as the interstitial void space between particles. The pore size may be measured by approximating the void area as circular, and the diameter of each circle may be considered the pore size.

[0189] As used herein, the terms "porosity" or "void fraction" interchangeably refer to a measure of the void (e.g., "empty") space in a material, and may be expressed as the ratio of the volume of voids to the total volume from 0 to 1, or as a percentage from 0% to 100%. As an example, porosity P = void volume / total volume. The porosity may be measured using the method disclosed in "Void volume fraction of granular scaffolds; Lindsay Riley, Grace Wei, Yijun Bao, Peter Cheng, Katrina L. Wilson, Yining Liu, Yiyang Gong, Tatiana Segura; bioRxiv 2022.06.14.496197", which is hereby incorporated by reference in its entirety.

[0190] 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.

[0191] The term "Q peptide", as used herein, refers to a peptide comprising an amino acid sequence containing one or more glutamine residues that functions as a substrate for an annealing agent in an annealing reaction or that may assist in cross-linking of the microgel particles disclosed herein.

[0192] The term "RGD peptide", as used herein, refers to a peptide derived from an extracellular matrix protein having an RGD motif characterized by an amino acid sequence containing "Arg-Gly-Asp". Non-limiting extracellular matrix proteins include fibronectin, vitronectin, fibrinogen, von Willebrand factor, laminin, and collagen. The RGD peptide may be provided by Moral MEG, Siahaan TJ, et al. The RGD peptide may be modified to contain cysteine for binding. In some embodiments, the RGD peptide comprises an amino acid sequence containing RGDSPGERCG (SEQ ID NO: 1).

[0193] The term "storage modulus", as used herein, refers to the mechanical property of a viscoelastic material related to the energy stored in the viscoelastic material that represents the elastic portion. The storage modulus represents the ratio of elastic stress to strain. The storage modulus of the microgel particles may be measured in a surrogate non-porous gel formed from the same precursor solution that is used to make the microgel particles but is not emulsified in the oil phase to produce microspheres. The storage modulus may be measured by subjecting the shear modulus measurement as described above and performing an amplitude and frequency sweep of the shear stress in a parallel plate system. This enables calculation of both the storage modulus and loss modulus of the viscoelastic material (the storage modulus and loss modulus together include the shear modulus).

[0194] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed or suspected to be at high risk for a disease. In some cases, a subject may not necessarily be diagnosed or suspected to be at high risk for a disease.

[0195] The term "substitute gel" refers to a macroscopic substitute gel prepared from the same precursor solution used to produce the microgel particles disclosed herein.

[0196] When used herein, the term “tissue site” refers to a distinct location in the tissue to which the hydrogel formulations disclosed herein can be delivered.

[0197] As used herein, the terms “treatment” or “treating” are used in reference to a medical or other interventional regimen for obtaining a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits, preventive benefits, or cosmetic benefits. Therapeutic benefits may also refer to the eradication or remission of the symptom or underlying disease being treated. Similarly, therapeutic benefits can be achieved by the eradication or remission of one or more physiological symptoms associated with an underlying disease, such that improvement is observed in the subject, even though the subject may still be affected by the underlying disease. Preventive effects include delaying, preventing, or eliminating the onset of a disease or illness; delaying or eliminating the onset of symptoms of a disease or illness; slowing, stopping, or reversing the progression of a disease or illness; or any combination thereof. For preventive benefits, subjects at risk of progression of a particular disease, or who report one or more physiological symptoms of a disease, may receive treatment, even if a diagnosis of the disease has not been made.

[0198] As used herein, the term "VS" refers to vinyl sulfone.

[0199] As used herein, the term “closed” with respect to an incision or suture is interchangeable with “sutured.” As used herein, the term “closed incision” is interchangeable with “suture line.”

[0200] Numbered Embodiments Embodiment 1 is a method for delivering a hydrogel formulation to a suture site in a subject, the method comprising delivering a hydrogel formulation to the suture site of the subject, which is annealed in situ to form a covalently stabilized porous scaffold, and endogenous cells infiltrate the covalently stabilized porous scaffold to form a cell matrix on the suture site of the subject. Embodiment 2 is the method according to Embodiment 1, wherein the suture site was an incision. Embodiment 3 is the method according to Embodiment 2, wherein the incision site was a surgical incision. Embodiment 4 is the method according to Embodiment 3, wherein the surgical incision site was an abdominal fascial incision. Embodiment 5 is the method according to Embodiment 4, wherein the abdominal fascial incision site was a midline or transverse abdominal wall incision of the fascia. Embodiment 6 comprises the method of Embodiment 4 or 5, wherein the abdominal incision is the result of surgery performed on the subject, such surgery includes gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. Embodiment 7 comprises the method of any one of Embodiments 4 to 6, wherein the abdominal incision was below the dermis and subcutaneous tissue of the subject. Embodiment 8 comprises the method of Embodiment 3, wherein the surgical incision was made to repair a hernia or dehiscence of a previous incision at that location. Embodiment 9 comprises the method of Embodiment 3, wherein the surgical incision was an umbilical incision of the abdominal fascia. Embodiment 10 comprises the method of Embodiment 9, wherein the umbilical incision was a midline or transverse abdominal wall incision of the fascia. Embodiment 11 comprises the method of Embodiment 9 or 10, wherein the umbilical incision is the result of surgery performed on the subject, such surgery includes gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. Embodiment 12 included the method according to any one of Embodiments 9 to 11, wherein the umbilical incision was below the dermis and subcutaneous tissue of the subject. Embodiment 13 included the method according to Embodiment 3, wherein the surgical incision was a laparoscopic keyhole incision of the abdominal fascia. Embodiment 14 included the method according to Embodiment 13, wherein the laparoscopic keyhole incision was a midline or transverse abdominal wall incision of the fascia.Embodiment 15 comprises the method of Embodiment 13 or 14, wherein the laparoscopic keyhole incision is the result of surgery performed on the subject, such surgery includes gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. Embodiment 16 comprises the method of any one of Embodiments 13 to 15, wherein the laparoscopic keyhole incision was below the dermis and subcutaneous tissue of the subject. Embodiment 17 comprises the method of Embodiment 3, wherein the surgical incision was an inguinal incision in the abdominal fascia. Embodiment 18 comprises the method of Embodiment 17, wherein the inguinal incision was a midline or transverse abdominal wall incision in the fascia. Embodiment 19 comprises the method of Embodiment 17 or 18, wherein the inguinal incision is the result of surgery performed on the subject, such surgery includes gastrointestinal cancer treatment, hysterectomy, ovarian cancer treatment, spinal fusion, abdominal trauma surgery, or a combination thereof. Embodiment 20 comprises the method according to any one of Embodiments 17 to 19, wherein the inguinal incision was below the dermis and subcutaneous tissue of the subject. Embodiment 21 comprises the method according to any one of Embodiments 1 to 20, wherein delivery includes delivering the hydrogel formulation directly onto the suture after the suture has been sutured. Embodiment 22 comprises the method according to any one of Embodiments 1 to 21, wherein delivery includes delivering the hydrogel formulation into the suture while the suture is being sutured. Embodiment 23 comprises the method according to any one of Embodiments 1 to 22, wherein delivery includes delivering the hydrogel formulation into the suture after the incision has been sutured. Embodiment 24 comprises the method according to Embodiment 23, wherein delivery includes delivering the hydrogel formulation into the suture while the suture is being sutured and on the suture after the suture has been sutured. Embodiment 25 comprises the method of any one of Embodiments 21 to 24, wherein the hydrogel begins to anneal over the suture line before the dermal and subcutaneous tissue of the subject is sutured. Embodiment 26 comprises the method of Embodiment 25, wherein the hydrogel is annealed over the suture line for at least about 10 minutes before the dermal and subcutaneous tissue of the subject is sutured. Embodiment 27 comprises the method of any one of Embodiments 1 to 26, wherein delivery includes releasing the hydrogel formulation from a syringe.Embodiment 28 includes the method according to Embodiment 27, wherein the syringe includes a needle. Embodiment 29 includes the method according to Embodiment 28, wherein the needle is a blunt needle. Embodiment 30 includes the method according to Embodiment 28 or 29, wherein the needle has a gauge ranging from about 10 gauge to about 20 gauge. Embodiment 31 includes the method according to any one of Embodiments 28 to 30, wherein the needle has a gauge ranging from about 18 gauge. Embodiment 32 includes the method according to any one of Embodiments 1 to 31, further comprising reinforcing the suture at the suture site with a cellular matrix formed on the suture site. Embodiment 33 includes the method according to Embodiment 32, wherein reinforcing the suture is characterized by increasing the mechanical tensile strength of the suture compared to a reference suture at the same suture site as the suture site, except that it is sutured without delivery of the hydrogel formulation. Embodiment 34 includes the method according to Embodiment 33, wherein increasing the mechanical tensile strength of the suture is characterized by forming a certain amount or type of collagen mimicking endogenous tissue at the suture site. Embodiment 35 includes the method described in Embodiment 34, wherein collagen is formed at the suture site at least about 28 days after suture of the suture. Embodiment 36 includes the method described in Embodiment 34 or 35, wherein collagen is formed within and around a covalently stabilized porous scaffold. Embodiment 37 includes the method described in any one of Embodiments 34 to 36, wherein the type of collagen includes type I collagen, type III collagen, or a combination thereof. Embodiment 38 includes the method described in Embodiment 37, wherein type I collagen is present in a ratio of about 10:1 or less with respect to type III collagen. Embodiment 39 includes the method described in Embodiment 37, wherein type I collagen is present in a ratio of about 6:1 or less with respect to type III collagen. Embodiment 40 includes the method described in Embodiment 37, wherein type I collagen is present in a ratio of about 5:1 or less with respect to type III collagen. Embodiment 41 includes the method described in Embodiment 33, wherein increasing the mechanical tensile strength of the suture is characterized by increasing the yield stress of the suture. Embodiment 42 includes the method described in Embodiment 41, wherein the yield stress is calculated from a stress-to-strain curve measured using a tensile test (e.g., with an Instron).Embodiment 43 comprises the method of Embodiment 41 or 42, wherein the suture line has a yield stress of at least about 4.0 Newtons / millimeter square (N / mm2) at least about 42 days after suture closure. Embodiment 44 comprises the method of any one of Embodiments 41 to 43, wherein the suture line has a yield stress of at least about 3.0 N / mm2 to about 6.0 N / mm2 at least about 42 days after suture closure. Embodiment 45 comprises the method of Embodiment 33, wherein increasing the mechanical tensile strength of the suture line is characterized by increasing the toughness of the suture line. Embodiment 46 comprises the method of Embodiment 45, wherein toughness is measured as the area under the stress-to-strain curve to fracture using a tensile test (e.g., with Instron). Embodiment 47 comprises the method of Embodiment 45 or 46, wherein the suture line contains a toughness of at least about 60.0 millijoules (mJ / mm3) per cubic millimeter at least about 42 days after suture suturing. Embodiment 48 comprises the method of Embodiment 45 or 46, wherein the suture line contains a toughness of at least about 25.0 mJ / mm3 to about 100.0 mJ / mm3 at least about 42 days after suture suturing. Embodiment 49 comprises the method of Embodiment 33, wherein increasing the mechanical tensile strength of the suture line is characterized by increasing the recovery rate of the suture line. Embodiment 50 comprises the method of Embodiment 49, wherein the recovery rate of the suture line is increased by about 40% or more compared to a reference suture line sutured without delivery of the hydrogel formulation. Embodiment 51 comprises the method of Embodiment 50, wherein the recovery rate is measured at least about 42 days after suture suturing. Embodiment 52 includes the method described in Embodiment 49, wherein the suture recovery rate is increased by at least about 20% to about 60% compared to a reference suture sutured without delivery of the hydrogel formulation. Embodiment 53 includes the method described in Embodiment 52, wherein the recovery rate is measured at least about 42 days after suture suturing. Embodiment 54 includes the method described in Embodiment 33, wherein increasing the mechanical tensile strength of the suture is characterized by increasing the yield strain of the suture. Embodiment 55 includes the method described in any one of Embodiments 1 to 54, wherein the hydrogel formulation becomes integrated with the suture site within about 14 days after suture suturing.Embodiment 56 comprises the method of Embodiment 55, wherein integration is characterized by the formation of new tissue within and around a covalently stabilized porous scaffold. Embodiment 57 comprises the method of any one of Embodiments 1 to 56, wherein the cell matrix forms new tissue at the suture site in question before the complete degradation of the covalently stabilized porous scaffold. Embodiment 58 comprises the method of Embodiment 57, wherein the new tissue is characterized by having (i) mature angiogenesis, (ii) features of the surrounding tissue at the suture site, (iii) a certain amount or type of collagen mimicking endogenous tissue at the suture site, or (iii) a combination thereof. Embodiment 59 comprises the method of Embodiment 58, wherein the features of the surrounding tissue at the suture site include a cell type functionally differentiated from the surrounding tissue. Embodiment 60 comprises the method of Embodiment 59, wherein (i) new tissue is formed, and (ii) the covalently stabilized porous scaffold is completely degraded at least by about 42 days after suture closure. Embodiment 61 comprises the method according to any one of Embodiments 57-60, wherein new tissue is formed in addition to any tissue formed at the suture site by sutures alone. Embodiment 62 comprises the method according to any one of Embodiments 57-61, wherein additional new tissue continues to form at the suture site for at least about 42 days after suture of the suture. Embodiment 63 comprises the method according to any one of Embodiments 57-62, wherein the new tissue is formed above (e.g., superficially) the suture site and deep within the subcutaneous tissue. Embodiment 64 comprises the method according to any one of Embodiments 57-63, wherein the new tissue is interstitial-like tissue having unaligned collagen bundles. Embodiment 65 comprises the method according to any one of Embodiments 1-64, wherein a covalently stabilized porous scaffold reinforces the suture at the suture site of the subject while minimizing the foreign body reaction of the subject. Embodiment 66 comprises the method according to Embodiment 65, wherein the foreign body reaction is characterized by causing harm to the subject. Embodiment 67 comprises the method described in Embodiment 66, wherein the harm is characterized by causing chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof.Embodiment 68 comprises the method of Embodiment 67, wherein the harm is caused at the suture site. Embodiment 69 comprises the method of Embodiment 66, wherein the covalently stabilized porous scaffold strengthens the suture at the suture site while minimizing the foreign body reaction at the target suture site, when the amount of granuloma at the suture site is detected by histological analysis and the foreign body reaction is measured by comparing the amount of granuloma at the suture site with a reference suture site without the hydrogel formulation. Embodiment 70 comprises the method of Embodiment 66, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the target suture site while minimizing the foreign body reaction when the amount of scar tissue at the suture site is detected using histological analysis and the foreign body reaction is measured by comparing the amount of scar tissue at the suture site with a reference suture site that does not contain the hydrogel formulation. Embodiment 71 comprises the method of Embodiment 66, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the target suture site while minimizing the foreign body reaction when the amount of nodules at the suture site is detected using histological analysis and the foreign body reaction is measured by comparing the amount of nodules at the suture site with a reference suture site that does not contain the hydrogel formulation. Embodiment 72 includes the method of Embodiment 66, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the suture site while minimizing the foreign body reaction at the site, when the foreign body reaction is measured by detecting chronic inflammation at the suture site using histological analysis. Embodiment 73 includes the method of Embodiment 66, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the suture site while minimizing the foreign body reaction at the site, when the foreign body reaction is measured by the presence of one or more types of macrophages at the suture site. Embodiment 74 includes the method of Embodiment 73, wherein the one or more types of macrophages include type 1 macrophages and type 2 macrophages. Embodiment 75 includes the method of Embodiment 74, wherein type 1 macrophages are pro-inflammatory. Embodiment 76 includes the method of Embodiment 74, wherein type 2 macrophages are pro-regenerative. Embodiment 77 comprises the method of any one of Embodiments 74 to 76, wherein the covalently stabilized porous scaffold is effective in forming more type II macrophages than type I macrophages. Embodiment 78 comprises the method of any one of Embodiments 1 to 77, wherein the cell matrix comprises a certain amount or type of collagen that mimics endogenous tissue at the suture site.Embodiment 79 comprises the method of Embodiment 78, wherein collagen is formed at the suture site at least 28 days after suture of the suture line. Embodiment 80 comprises the method of Embodiment 78 or 79, wherein collagen is formed within and around a covalently stabilized porous scaffold. Embodiment 81 comprises the method of any one of Embodiments 78 to 80, wherein the type of collagen includes type I collagen, type III collagen, or a combination thereof. Embodiment 82 comprises the method of Embodiment 81, wherein type I collagen is present in a ratio of about 10:1 or less with respect to type III collagen. Embodiment 83 comprises the method of Embodiment 81, wherein type I collagen is present in a ratio of about 6:1 or less with respect to type III collagen. Embodiment 84 comprises the method of Embodiment 81, wherein type I collagen is present in a ratio of about 5:1 or less with respect to type III collagen. Embodiment 85 comprises the method of any one of Embodiments 1 to 84, wherein at least a portion of the suture site contains elastin after the decomposition of the covalently stabilized porous scaffold at the suture site. Embodiment 86 comprises the method of any one of Embodiments 1 to 85, wherein the hydrogel formulation is biocompatible with the tissue at the suture site as determined by one or more techniques described in ISO standard 10993. Embodiment 87 comprises the method of any one of Embodiments 1 to 86, wherein the covalently stabilized porous scaffold contains a pH of about 8. Embodiment 88 comprises the method of any one of Embodiments 1 to 87, wherein the covalently stabilized porous scaffold contains a compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. Embodiment 89 comprises the method of any one of Embodiments 1 to 88, wherein the hydrogel formulation comprises microgel particles containing a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and cell adhesion peptides. Embodiment 90 comprises the method described in Embodiment 89, wherein microgel particles are present in a suspension containing microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%.Embodiment 91 comprises the method described in Embodiment 89 or 90, wherein the hydrogel formulation further comprises PEG-dithiol, and the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol. Embodiment 92 comprises the method described in Embodiment 91, wherein the PEG-dithiol contains a molecular weight of at least about 0.5 kilodaltons (kDa) to about 10 kDa. Embodiment 93 comprises the method described in Embodiment 92, wherein the PEG-dithiol contains a molecular weight of at least about 3.4 kDa. Embodiment 94 comprises the method described in Embodiment 91, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mmol (mM) to about 1.0 mM. Embodiment 95 comprises the method described in Embodiment 94, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM. Embodiment 96 comprises a) microgel particles comprising a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and a cell adhesion peptide, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%; and b) PEG-dithiol comprising a PEG-dithiol having a molecular weight of at least about 0.5 kilodaltons (kDa), wherein the microgel particles undergo an annealing reaction and, after exposure to the PEG-dithiol, form a covalently stabilized porous scaffold.Embodiment 97 comprises a hydrogel system comprising: c) microgel particles crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer, one or more K peptides and Q peptides, and a cell adhesion peptide, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%; and d) PEG-dithiol, wherein the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol, and the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mmol (mM) to about 1.0 mM. Embodiment 98 comprises the hydrogel system described in Embodiment 96 or 97, wherein the microgel particles are present in the suspension at a volume fraction of at least 90%. Embodiment 99 comprises a hydrogel system described in any one of Embodiments 96-98, wherein PEG-dithiol and 4-armed PEG-vinyl sulfone are present to provide a thiol-to-vinyl sulfone ratio of less than about 1.0. Embodiment 100 comprises a hydrogel system described in any one of Embodiments 96-99, wherein PEG-dithiol and 4-armed PEG-vinyl sulfone are present to provide a thiol-to-vinyl sulfone ratio of about 0.6 to about 1.0. Embodiment 101 comprises a hydrogel system described in any one of Embodiments 96-100, wherein the PEG-dithiol contains a molecular weight of at least about 3.4 kDa. Embodiment 102 comprises a hydrogel system described in any one of Embodiments 96-101, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM. Embodiment 103 comprises a hydrogel system described in any one of Embodiments 96-102, wherein the microgel particles are spherical. Embodiment 104 comprises the method described in any one of Embodiments 96 to 103, wherein the microgel particles include microspheres. Embodiment 105 comprises the hydrogel system described in any one of Embodiments 96 to 104, wherein the microgel particles include diameters ranging from 5 μm to 1000 μm.Embodiment 106 includes the hydrogel system described in Embodiment 105, with a diameter ranging from 50 μm to 1000 μm. Embodiment 107 includes the hydrogel system described in Embodiment 105, with a diameter ranging from 70 μm to 150 μm. Embodiment 108 includes the hydrogel system described in any one of Embodiments 96 to 107, where the microgel particles have a compressive modulus of at least about 500 Pascals (Pa) before the annealing reaction. Embodiment 109 includes the hydrogel system described in any one of Embodiments 96 to 108, where the microgel particles have a compressive modulus of at least about 1500 Pascals (Pa) after the annealing reaction. Embodiment 110 includes the hydrogel system described in Embodiment 109, where the microgel particles have a compressive modulus of at least about 6000 Pa after the annealing reaction. Embodiment 111 comprises a hydrogel system described in any one of Embodiments 96 to 110, wherein the covalently stabilized porous scaffold contains pores with a median pore diameter of approximately 5 μm or more. Embodiment 112 comprises a hydrogel system described in any one of Embodiments 96 to 111, wherein the pores contain a median pore diameter of approximately 10 μm to approximately 35 μm. Embodiment 113 comprises a hydrogel system described in any one of Embodiments 96 to 112, wherein one or more cell adhesion peptides contain RGD peptides. Embodiment 114 comprises a hydrogel system described in any one of Embodiments 96 to 113, wherein the microgel particles contain a polydispersity of 0.1 or less. Embodiment 115 comprises a hydrogel system described in Embodiment 114, wherein the polydispersity is calculated based on the standard deviation and mean size of the particles (e.g., PDI = (SD / mean)^2). Embodiment 116 comprises a hydrogel system described in any one of Embodiments 96 to 115, wherein the hydrogel formulation further comprises a buffer, the buffer comprising a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof. Embodiment 117 comprises a hydrogel system described in any one of Embodiments 96 to 116, wherein the annealing reaction comprises a covalent synthesis reaction.Embodiment 118 comprises the hydrogel system described in Embodiment 117, wherein the covalent synthetic reaction includes a Michael addition reaction or a pseudo-Michael addition reaction. Embodiment 119 comprises the hydrogel system described in Embodiment 118, wherein the vinyl sulfone of 4-arm PEG vinyl sulfone is the Michael acceptor in the Michael addition reaction or pseudo-Michael addition reaction. Embodiment 120 comprises the hydrogel system described in Embodiment 118, wherein the thiol of PEG-dithiol is the Michael donor in the Michael addition reaction or pseudo-Michael addition reaction. Embodiment 121 comprises a hydrogel formulation comprising the hydrogel system described in any one of Embodiments 96 to 120 in a suspension, the suspension comprising a buffer. Embodiment 122 comprises the hydrogel formulation described in Embodiment 121, the hydrogel formulation being formulated for administration to a subject. Embodiment 123 comprises the hydrogel formulation described in Embodiment 122, the hydrogel formulation being formulated for administration along the suture line of a subject. Embodiment 124 comprises the hydrogel formulation described in Embodiment 122 or 123, and the administration minimizes the foreign body reaction of the subject. Embodiment 125 comprises the hydrogel formulation described in any one of Embodiments 121 to 124, and the hydrogel formulation comprises a dosage volume of about 0.01 mL to about 20 mL. Embodiment 126 is a) any of Embodiments 96 to 120. The delivery device comprises a body containing the hydrogel system described in any one of the embodiments described in any one of embodiments 122 to 126, and a) an applicator that is in fluid communication with the body, wherein the delivery device is sterile. Embodiment 127 comprises the method described in Embodiment 126, wherein the delivery device is a syringe or a needle. [Examples]

[0201] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0202] Example 1: Tensile strength of the uninjured abdominal wall method The cadaveric phase was performed on animals that were euthanized without any surgical incision. Immediately after euthanasia, the uninjured abdominal wall was excised for tensile analysis (see below). The MAP test sample was not used during this phase. Ten animals were used.

[0203] Immediately after euthanasia, the abdominal wall was excised and cut into rectangular sections, which were then horizontally cut into elongated sections (7 cm × 1 cm) for tensile strength testing using Instron. Measurements were performed using digital calipers. The tissue width of the region of interest was manually reduced using a biopsy punch to create a "dumbbell" shape with a width of 2.5–3.5 mm to ensure fracture at the linea alba in non-wounded tissue. Stress-strain curves were measured using Instron tensile testing. The yield strength of the tissue (the maximum force required to tear the tissue) and the area under the stress / strain curve to the yield point (the mechanical energy consumed by the tissue during strain) were calculated. Then, to account for variations in tissue thickness / area, the yield strength was normalized by the width of the tissue section at the biopsy point.

[0204] result The coefficient of variation for the normalized yield strength of tissues across all rabbits was 27.19% (Table 1).

[0205] [Table 1]

[0206] Figure 1A shows the yield strength versus abdominal position (normalized by tissue width). No correlation was observed between the abdominal position measured from the sternum / xiphoid process (cephalic) to the pelvis (caudal) and the yield strength (R2 = 0.0098). Based on these data, it was decided to make an incision of 5 cm at a position approximately 4 - 5 cm from the xiphoid process. Figure 1B shows the normalized average yield strength of each rabbit. The area under the curve (AUC) to the maximum yield strength was also calculated. However, a larger coefficient of variation (79.98%) was observed compared to the coefficient of variation of the yield strength (Table 2). In many tissue samples, significant elongation was observed before rupture. Therefore, it was determined that the yield strength is a more reproducible parameter for evaluation.

[0207]

Table 2

[0208] Using n = 3 rabbits in the dataset, a storage test was performed. The yield strength of rabbit sections stored at +4°C for t = 24 hours and 72 hours was compared with the yield strength of fresh tissue. It was found that the difference in yield strength between the conditions was negligible (Figure 1C).

[0209] [[ID=X16]]Example 2: Test to evaluate the use of MAP for healing of midline abdominal incisions The overall objective of this rabbit test was to evaluate the use of a MAP (Microporous Annealed Particle) product as an adjunctive closure for primary suture in the midline abdominal incisions of rabbits. In this test, the effect of MAP on the increase in mechanical tensile strength of the healing incision was assessed when compared to an incision closed with only suture.

[0210] Method: The MAP formulations used are described in Table 3.

[0211]

Table 3

[0212] Two methods for applying MAP within the suture margin were investigated: the zipper method and the post-suture method. The zipper method is shown in Figure 2A. In the zipper method, MAP was applied while suturing. In the post-suture method (Figure 2B), MAP was applied after suturing within the incision. MAP was applied using either a two-step method (Figure 2C) or a one-step method (Figure 1D). The intradermal skin was closed either on the midline or offset from the procedure. Midline closure was located directly above the midline incision of the linea alba in the subcutaneous tissue. Offset closure was located 2 cm from the midline incision of the linea alba in the subcutaneous tissue. Table 4 below summarizes the different conditions tested in this acute phase.

[0213] [Table 4]

[0214] The primary endpoint (tensile mechanics) included the tensile mechanical properties of the tissue (yield stress and yield strain under unidirectional tension). The secondary endpoint (histology) included total collagen deposition, tissue inflammation, and immune cell infiltration.

[0215] [Table 5]

[0216] [Table 6]

[0217] The animal testing was conducted at BTS in San Diego, California.

[0218] Prior to surgical procedures, the animals were accustomed to wearing an e-collar (Mae West collar). This process was multi-day, involving the gradual introduction of the e-collar while providing additional enrichment to the animals. A typical e-collar acclimatization regimen was as follows: • Day 1 - Wear E color for 1 hour • Day 2 - Wear E-color for 3 hours • Day 3 - Wear E-color for 4 hours

[0219] The Mae West collar is a long tube that sits around the rabbit's neck and fastens like a necktie using tape instead of a knot. The Mae West collar prevents the rabbit from reaching and pulling at the incision scar (and the staples or stitches holding it together for healing). The rabbit could still reach cecal pellets and groom itself in other areas.

[0220] The animals were anesthetized (chemically restrained) using ketamine / xylazine injection for survival surgical procedures, and then anesthesia was maintained using an isoflurane and oxygen gas mixture. The rabbits received prophylactic analgesia (buprenorphine).

[0221] The surgical site (ventral abdomen) of the rabbit was prepared by removing the fur using clippers, and the skin was disinfected by wiping twice in succession with iodine solution and / or chlorhexidine solution. A sterile drape was placed to ensure cleanliness of the surgical site, and the skin was disinfected again with iodine and / or chlorhexidine, and then wiped with sterile saline.

[0222] First, incisions were made in the epidermis, dermis, and subcutaneous tissue along the craniocaudal axis, either on the midline or 2 cm off-center from the midline, to create tissue flaps and expose the linea alba. The subcutaneous tissue was separated from the abdominal wall using blunt dissection. Surgical clamps were used to ensure the site remained open and that the midline of the abdominal wall (linea alba) was accessible for subsequent incisions.

[0223] The xiphoid process was gently palpated to determine its anatomical location, followed by the placement of the abdominal incision. A sterile incision and closure guide (304 stainless steel (.100″), 3.543 × 0.787, Tissue Marking Template 0.4 cm × 0.4 cm, 15 holes from the midline, 5 cm closure (Figure 2E), 0.3 cm hole. CAD file of SLDPRT) was placed directly on the abdomen. A skin pen was used to draw a line at the incision site and dots were placed at the bite locations for suture closure. The guide was then removed, leaving only the marks. Using surgical forceps, the caudal end of the incision was lifted in a tent shape, and the incision was started using a #11 blade. The incision was then advanced cranially until the incision length reached approximately 5 cm.

[0224] Using Prolene 3-0 sutures attached to an SH·26mm·1 / 2-circumference curved needle, the incision was closed caudally, starting from the cephalic end of the incision and progressing caudally using a single, uninterrupted (continuous) suture. Markings made from the incision / closure guide specified the placement of the suture bites. This resulted in a "small bite" method, where each suture bite was approximately 4 mm long and the distance between bites was approximately 4 mm. Sutures four times the length of the incision were used.

[0225] Here, different MAP formulations were applied using different application methods, as summarized in Table 4. Formulation 3 (90% VF, annealed with PEG-dithiol crosslinking agent) was applied over the incision site (Procedure #3).

[0226] After MAP application, if the dermal incision was made on the midline, the subcutaneous tissue flap was closed over the abdominal wall incision using Vicryl 3-0 sutures attached to an SH 1 / 2 circumferential 26 mm needle. If the dermal incision was made offset from the midline, the subcutaneous tissue flap was partially (unilaterally only) sutured over the abdominal wall incision using Vicryl 3-0 sutures attached to an SH 1 / 2 circumferential 26 mm needle. Finally, intradermal skin closure was performed on the subcutaneous tissue, either on the midline or offset (2 cm) from the midline incision, using Monocryl 4-0 sutures attached to a PS-2 3 / 8 circumferential 19 mm needle. Topical antibacterial agents were applied to the sutured skin. The incision site was further secured using a standard bandage. During recovery, the animals received 100 mL of subcutaneous fluid (Ringer's lactate solution). After the surgery, a Mae West collar was placed around the rabbit's neck.

[0227] Analgesics were administered every 24 hours by IM injection for a period of 48 hours after surgery. The animals were monitored daily for one week, and then weekly thereafter. The animals were euthanized at 7 and 14 days post-surgery.

[0228] Immediately after euthanasia, a tissue sample was taken from the abdominal wall (leaving subcutaneous tissue above the wound area during tissue collection) and placed in a plastic box with PBS or saline solution. The tissue was then cut into several elongated sections for tensile analysis and histology, as described below.

[0229] Tensile Testing: The slender specimens used for tensile testing were 10 mm × 60 mm, with a dumbbell-shaped core measuring 2.5–3.5 mm in diameter, ensuring that the specimens fractured at the incision site on the Instron. Non-absorbable sutures for the rib alba were cut after autopsy and before mechanical analysis. Stress-strain curves were measured using tensile testing with Instron, and then the tensile strength (yield stress) was calculated.

[0230] Histology: Elongated tissue strips for histology were 5 mm × 30 mm, fixed in 4% PFA for 24 hours in a cassette, then placed in 70% ethanol, and transported to Histowiz for analysis. The tissue was embedded in paraffin and then stained with hematoxylin and eosin (H&E) and picrosilius red (collagen stain).

[0231] result Cohort 1-1 (80%) was applied to the inside and above the incision site on days 7 and 14. The first rabbit cohort consisted of 10 rabbits (rabbits #1-10), and formulation 1, used in combination with sutures, was compared with sutures alone (control) 7 and 14 days after incision and treatment.

[0232] Figure 3A shows the yield strength measured by Instron 7 and 14 days after treatment for undamaged tissue, damaged tissue treated with sutures alone, and damaged tissue treated with MAP and sutures. Figure 3B shows the yield strength normalized to undamaged tissue for damaged tissue treated with sutures alone, and damaged tissue treated with MAP (formulation 1) and sutures. At either time point (7 and 14 days), no statistically significant difference in yield strength was observed between the case of sutures alone and the case of MAP (formulation 1) used in combination with sutures.

[0233] Histologically, formulation 1 (formulated at 80%) was applied inside the suture during suturing and also above the incision (procedure #4), but no material was observed inside the incision (Figures 4A-4B). Formulation 1 was observed above and below the tissue, indicating that some of the material migrated downward into the abdominal wall upon application. Formulation 1 was not observed directly above the incision, but was observed on the right and left sides of the incision at both 7 and 14 days after application, indicating that Formulation 1 was not sufficiently stabilized to remain in place.

[0234] Cohorts 2 and 3 - Alternative methods and alternative MAP formulations - 14 days. The objective of the second and third cohorts of rabbits (rabbits #11-37) was to optimize both the MAP formulation and the application method so that the MAP material would remain above, and ideally within, the incision site, thereby maximizing the potential for promoting new tissue that would reinforce the damaged tissue.

[0235] Instead of making the dermal incision directly above the linea alba incision, we decided to make the dermal incision at a position offset (2 cm) from the midline to minimize disturbance of the applied material during closure of the subcutaneous and dermal layers.

[0236] Overall, closing the dermis at an offset from the midline helped to retain more material on the abdominal wall. This technique was used for all subsequent surgeries.

[0237] However, in the case of formulation 1 (80%), the material was not located directly on the incision site, and no evidence was observed to indicate the formation of new tissue on the incision site (Figure 5A). Furthermore, no improvement in yield strength was observed compared to sutures alone (Figures 5B-5C).

[0238] Alternative MAP formulations To retain as much MAP as possible above the incision site, we decided to adjust several properties of the MAP formulation: • Increased the volume fraction from 80% to 90% (Formulation 5). • To achieve a higher modulus of elasticity and increase the volume fraction from 80% to 90%, MAP was annealed using a PEG-dithiol crosslinking agent (Formulation 3). • To achieve a higher modulus of elasticity, promote the production of growth factors, and increase the volume fraction from 80% to 90%, MAP is annealed using platelet-rich plasma (PRP) (Formulation 4).

[0239] Figure 6A shows the stress measured by Instron on samples collected on day 14 for different MAP formulations. Formulation 3 (MAP Chem Xlink) showed the highest tissue stress.

[0240] When formulated at 100%, formulation 1.2 showed more material on the abdominal wall (likely due to offset dermal incisions and closure) (Figure 6B), but the stress of this formulation was the same as formulation 1, which was formulated at 80% (Figure 6A). Increasing the volume fraction did not appear to have a significant effect.

[0241] In formulation 4 annealed with PRP (Figure 6C) and formulation 3 annealed with PEG-dithiol crosslinking agent (Figure 6D), a considerable amount of material was observed directly above the incision site. Furthermore, both formulations exhibited slightly higher stress (Figure 6A). Since formulation 3 exhibited the highest stress of all formulations, it was decided to continue with this formulation (formulation 3) for further investigation.

[0242] Alternative application methods Next, in addition to its application to the upper abdomen, it was decided to evaluate whether formulation 3 could be injected into the incision after closure (Procedure #5). In this procedure, formulation 3, filled in a syringe, was injected between the sutures using a needle. As shown in Figure 6E, no material was found in the incision. The stress was slightly higher in Procedure #5 than in Procedure #3 (applied to the upper abdomen only) (Figure 6F), but the difference was small. However, this procedure carries a higher risk of puncturing organs located beneath the abdominal wall.

[0243] Figure 6F shows the stress measured by Instron on samples taken on day 14 for different MAP formulations and different application methods. Formulation 3 (MAP Chem Xlink), added inside and on the incision (by steps 4 and 5), showed the highest tissue stress, followed by Formulation 3, added only to the surface. However, no statistically significant differences were observed between the groups. Figure 6G summarizes the screening results of the formulations.

[0244] conclusion In this acute phase of the rabbit study, formulation 3 was selected as an ideal candidate because, compared to sutures alone, it showed increased tensile strength after 14 days, a substantial amount of material integrated over the incision site, and did not require PRP or light.

[0245] Example 3: Abdominal midline test in rabbits The purpose of this study was to evaluate the use of MAP (microporous annealing particle) material products as auxiliary closure for primary suturing of midline abdominal incisions in rabbits. This study assessed the use of MAP in increasing the mechanical tensile strength of the healing incision compared to incisions closed with sutures alone. Histological evaluation of the incision site was also performed throughout the healing process to investigate how the material integrates with the incision site and surrounding tissue. method

[0246] [Table 7]

[0247] [Table 8]

[0248] Seven animals were used per condition. Animal studies were conducted at BTS in San Diego, California. Animals were accustomed to wearing an e-collar (Mae West collar) prior to surgical procedures. This process was multi-day and involved gradually fitting the e-collar while providing additional enrichment to the animals. A typical e-collar acclimatization regimen was performed as described in Example 2.

[0249] Throughout this study, two veterinarians performed the surgeries. Both used the same surgical techniques, except for the tucking sutures of the subcutaneous tissue flap, as will be described later.

[0250] The animals were anesthetized (chemically restrained) using ketamine / xylazine injection for survival surgical procedures, and then anesthesia was maintained using an isoflurane and oxygen gas mixture. The rabbits received prophylactic analgesia (buprenorphine).

[0251] The surgical site (ventral abdomen) of the rabbit was prepared by removing the fur using clippers, and the skin was disinfected by wiping twice in succession with iodine solution and / or chlorhexidine solution. A sterile drape was placed to ensure cleanliness of the surgical site, and the skin was disinfected again with iodine and / or chlorhexidine, and then wiped with sterile saline.

[0252] First, incisions were made in the epidermis, dermis, and subcutaneous tissue along the craniocaudal axis, off-center from the midline, to create a tissue flap and expose the linea alba (Figure 2E). Blunt dissection was used to separate the subcutaneous tissue from the abdominal wall. Surgical clamps were used to ensure the site remained open and that the midline of the abdominal wall (linea alba) was accessible for the next incision.

[0253] The xiphoid process was gently palpated to determine its anatomical location, followed by the placement of the abdominal incision. A sterile incision and closure guide was placed directly on the abdomen. A skin pen was used to draw a line at the incision site and to mark the bite for suture closure. The guide was then removed, leaving only the marks. Using surgical forceps, the caudal end of the incision was lifted in a tent shape, and the incision was initiated using a #11 blade. The incision was then advanced cranially until it reached a length of approximately 5 cm.

[0254] Using Prolene 3-0 sutures attached to an SH·26mm·1 / 2-circumference curved needle, the incision was closed starting from the cephalic end of the incision and progressing caudally using a single, uninterrupted (continuous) suture. Markings made from the incision / closure guide specified the placement of the suture bites. This resulted in a "small bite" method, where each suture bite was approximately 4 mm long and the distance between bites was approximately 4 mm. Sutures four times the length of the incision were used.

[0255] Formulation 3 was selected for all of these studies. Formulation 3 was formulated at 90% (volume fraction). Immediately before MAP application, Formulation 3 was annealed at pH 8.0 using PEG-dithiol (3.4kDa) dithiol crosslinking agent (Cf=0.2mM). MAP was applied directly to a completely closed incision site, annealed for 10 minutes, and then the next layer was sutured.

[0256] After applying MAP, tucking sutures were performed between the subcutaneous tissue and superficial fascia using Vicryl 3-0 sutures attached to an SH 1 / 2 round 26 mm needle to close the subcutaneous tissue flap above the abdominal wall incision. Finally, intradermal skin closure was performed above the subcutaneous tissue, offset from the midline incision, using Monocryl 4-0 sutures attached to a PS-2 3 / 8 round 19 mm needle. A topical antibacterial agent was applied to the sutured skin. The incision site was further secured using a standard bandage. During recovery, the animal received 100 mL of subcutaneous fluid (lactated Ringer's solution). Postoperatively, a May West collar was placed around the rabbit's neck.

[0257] Analgesics were administered every 24 hours by IM injection for a period of 48 hours after surgery. The animals were monitored daily for one week, and then weekly thereafter. The animals were euthanized at 14, 28, and 42 days.

[0258] The dermal and subcutaneous tissue incisions were off-center from the midline. A tissue flap was prepared to expose the linea alba. A 5 cm incision of the linea alba was made (starting at least 4 cm caudal to the xiphoid process, with the position of the xiphoid process determined by palpation). Next, the incision of the linea alba was closed with continuous and uninterrupted sutures (small bite technique) using a stainless steel template (304 series stainless steel (.100″), 3.543 × 0.787, Tissue Marking Template 0.4 cm × 0.4 cm, CAD file of 15 holes from the midline, 5 cm closure, 0.3 cm hole). MAP material was applied directly to the closed incision. Tucking sutures were performed between the subcutaneous tissue and superficial fascia (nodular sutures) to close the subcutaneous tissue flap over the incision site. Dermal closure (intradermal closure) was located offset from the midline incision.

[0259] Immediately after euthanasia, a tissue sample was taken from the abdominal wall (leaving subcutaneous tissue above the wound area during tissue colle...

Claims

1. A method for delivering a hydrogel formulation to a suture site in a subject, comprising delivering the hydrogel formulation to the suture site of the subject, wherein the hydrogel formulation is annealed in situ to form a covalently stabilized porous scaffold, and endogenous cells infiltrate the covalently stabilized porous scaffold to form a cell matrix on the suture site of the subject.

2. The method according to claim 1, wherein the suture line was an incision.

3. The method according to claim 2, wherein the incision was a surgical incision.

4. The method according to claim 3, wherein the surgical incision was an abdominal fascial incision.

5. The method according to claim 4, wherein the abdominal fascial incision was a midline or transverse incision of the fascia in the abdominal wall.

6. The method according to claim 4 or 5, wherein the abdominal fascial incision is the result of surgery performed on the subject, and the surgery includes treatment for gastrointestinal cancer, hysterectomy, ovarian cancer, spinal fusion, abdominal trauma surgery, or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the delivery includes delivering the hydrogel preparation directly onto the suture line after the suture line has been sutured.

8. The method according to any one of claims 1 to 7, wherein the delivery includes delivering the hydrogel preparation into the suture line while the suture line is being sutured.

9. The method according to any one of claims 1 to 8, wherein the delivery includes delivering the hydrogel preparation into the suture line after the incision has been sutured.

10. The method according to claim 9, wherein the delivery includes delivering the hydrogel preparation within the suture while the suture is being sutured, and on the suture after the suture has been sutured.

11. The method according to any one of claims 7 to 10, wherein the hydrogel begins to anneal on the suture line before the dermis and subcutaneous tissue of the subject is sutured.

12. The method according to claim 11, wherein the hydrogel is annealed on the suture line for at least about 10 minutes before the dermis and subcutaneous tissue of the subject is sutured.

13. The method according to any one of claims 1 to 12, wherein the delivery includes releasing the hydrogel formulation from a syringe.

14. The method according to any one of claims 1 to 13, further comprising reinforcing the suture line at the suture line site with the cell matrix formed on the suture line site.

15. The method according to claim 14, wherein strengthening the suture line is characterized by increasing the mechanical tensile strength of the suture line compared to a reference suture line at the same suture site except that it is sutured without the delivery of the hydrogel formulation.

16. The method according to claim 15, wherein increasing the mechanical tensile strength of the suture is characterized by forming a certain amount or type of collagen that mimics endogenous tissue at the suture site.

17. The method according to claim 16, wherein the collagen is formed at the suture site at least about 28 days after the suture of the suture line.

18. The method according to claim 16 or 17, wherein the collagen is formed within and around the covalently stabilized porous scaffold.

19. The method according to any one of claims 16 to 18, wherein the type of collagen includes type I collagen, type III collagen, or a combination thereof.

20. The method according to claim 19, wherein the type I collagen is present with the type III collagen in a ratio of approximately 10:1 or less, approximately 6:1 or less, or approximately 5:1 or less.

21. The method according to claim 15, characterized in that increasing the mechanical tensile strength of the suture line is achieved by increasing the yield stress of the suture line.

22. The method according to claim 21, wherein the yield stress is calculated from a stress-strain curve measured using a tensile test (e.g., with Instron).

23. The suture line, at least about 42 days after the suture of the suture line, has a load of at least about 3.0 N / mm ２ ~Approximately 6.0 N / mm ２ The method according to claim 21 or 22, comprising the yield stress of the following:

24. The method according to claim 15, characterized in that increasing the mechanical tensile strength of the suture line is achieved by increasing the toughness of the suture line.

25. The method according to claim 24, wherein the toughness is measured using a tensile test (e.g., with Instron) as the area under the stress-to-strain curve to fracture.

26. The suture line emits at least about 60.0 millijoules per cubic millimeter (mJ / mm²) at least about 42 days after the suture line is closed. ３ The method according to claim 24 or 25, comprising the toughness of ).

27. The suture line, at least about 42 days after the suture of the suture line, emits at least about 25.0 mJ / mm². ３ ~Approximately 100.0 mJ / mm ３ The method according to claim 24 or 25, comprising toughness.

28. The method according to claim 15, characterized in that increasing the mechanical tensile strength of the suture increases the recovery rate of the suture.

29. The method according to claim 28, wherein the recovery rate of the suture line is increased by about 40% or more compared to a reference suture line sutured without delivery of the hydrogel formulation.

30. The method according to claim 28, wherein the recovery rate of the suture line is increased by at least about 20% to about 60% compared to a reference suture line sutured without delivery of the hydrogel formulation.

31. The method according to claim 15, characterized in that increasing the mechanical tensile strength of the suture line is achieved by increasing the yield strain of the suture line.

32. The method according to any one of claims 1 to 31, wherein the hydrogel preparation becomes integrated with the suture site within approximately 14 days after suturing the suture line.

33. The method according to claim 32, wherein integration is characterized by the formation of new tissue within and around the covalently stabilized porous scaffold.

34. The method according to any one of claims 1 to 33, wherein the cell matrix forms new tissue at the suture site of the target before the complete degradation of the covalently stabilized porous scaffold.

35. The method according to claim 34, wherein the new tissue is characterized by having (i) mature angiogenesis, (ii) features of the surrounding tissue at the suture site, (iii) a certain amount or type of collagen mimicking endogenous tissue at the suture site, or (iii) a combination thereof.

36. The method according to claim 35, wherein the characteristics of the surrounding tissue at the suture line site include cell types functionally differentiated from the surrounding tissue.

37. The method according to claim 36, wherein (i) the new tissue is formed, and (ii) the covalently stabilized porous scaffold completely decomposes at least about 42 days after suturing of the suture line.

38. The method according to any one of claims 34 to 37, wherein the new tissue is formed in addition to any tissue formed at the suture line site by sutures alone.

39. The method according to any one of claims 34 to 38, wherein additional new tissue continues to form at the suture site for at least about 42 days after the suture of the suture line.

40. The method according to any one of claims 34 to 39, wherein the new tissue is formed above the suture line site (e.g., superficially) and deep within the subcutaneous tissue.

41. The method according to any one of claims 34 to 40, wherein the new tissue is an interstitial-like tissue having unaligned collagen bundles.

42. The method according to any one of claims 1 to 41, wherein the covalently stabilized porous scaffold strengthens the suture line at the suture line portion of the target while minimizing foreign body reaction of the target.

43. The method according to claim 42, wherein the foreign body reaction is characterized by causing harm to the object.

44. The method according to claim 43, wherein the harm is characterized by causing chronic inflammation, granuloma formation, scar tissue formation, adhesion formation, nodule formation, swelling, pain, or any combination thereof.

45. The method according to claim 44, wherein the harm is caused at the suture line site.

46. The method according to claim 43, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the suture site of a target while minimizing the foreign body reaction of the target, when the amount of granuloma at the suture site is detected by histological analysis and the foreign body reaction is measured by comparing the amount of granuloma at the suture site with a reference suture site that does not contain the hydrogel preparation.

47. The method according to claim 43, wherein the covalently stabilized porous scaffold is effective in strengthening the suture line at the suture line site of a target while minimizing the foreign body reaction of the target, when the amount of scar tissue at the suture line site is detected by histological analysis and the foreign body reaction is measured by comparing the amount of scar tissue at the suture line site with a reference suture line site that does not contain the hydrogel formulation.

48. The method according to claim 43, wherein the covalently stabilized porous scaffold is effective in strengthening the suture at the suture site of a target while minimizing the foreign body reaction of the target, when the amount of nodules at the suture site is detected by histological analysis and the amount of nodules at the suture site is compared with a reference suture site that does not contain the hydrogel formulation.

49. The method according to claim 43, wherein the covalently stabilized porous scaffold is effective in strengthening the suture line at the suture line site of the target while minimizing the foreign body reaction of the target, when the foreign body reaction is measured by detecting chronic inflammation at the suture line site using histological analysis.

50. The method according to claim 43, wherein the covalently stabilized porous scaffold is effective in reinforcing the suture line at the suture line site of the target while minimizing the foreign body reaction at the target, when a foreign body reaction is measured by the presence of one or more types of macrophages at the suture line site of the target.

51. The method according to claim 50, wherein the one or more types of macrophages include type 1 macrophages and type 2 macrophages.

52. The method according to claim 51, wherein the type 1 macrophage is pro-inflammatory.

53. The method according to claim 51, wherein the type 2 macrophage is regenerative.

54. The method according to any one of claims 51 to 53, wherein the covalently stabilized porous scaffold is effective in forming more type 2 macrophages than type 1 macrophages.

55. The method according to any one of claims 1 to 54, wherein the cell matrix comprises a certain amount or type of collagen that mimics the endogenous tissue at the suture site.

56. The method according to claim 55, wherein the collagen is formed at the suture site at least about 28 days after the suture of the suture line.

57. The method according to claim 55 or 56, wherein the collagen is formed within and around the covalently stabilized porous scaffold.

58. The method according to any one of claims 55 to 57, wherein the type of collagen includes type I collagen, type III collagen, or a combination thereof.

59. The method according to claim 58, wherein the type I collagen is present with the type III collagen in a ratio of approximately 10:1 or less, approximately 6:1 or less, or approximately 5:1 or less.

60. The method according to any one of claims 1 to 59, wherein at least a portion of the suture line portion contains elastin after the decomposition of the covalently stabilized porous scaffold in the suture line portion.

61. The method according to any one of claims 1 to 60, wherein the covalently stabilized porous scaffold contains a pH of about 8.

62. The method according to any one of claims 1 to 61, wherein the covalently stabilized porous scaffold has a compressive modulus of at least about 1,500 pascals (Pa) after the annealing reaction.

63. The method according to any one of claims 1 to 62, wherein the hydrogel formulation comprises a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and microgel particles comprising a cell adhesion peptide.

64. The method according to claim 63, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%.

65. The method according to claim 63 or 64, wherein the hydrogel formulation further comprises PEG-dithiol, and the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol.

66. The method according to claim 65, wherein the PEG-dithiol comprises a molecular weight of at least about 0.5 kilodaltons (kDa) to about 10 kDa.

67. The method according to claim 66, wherein the PEG-dithiol has a molecular weight of at least about 3.4 kDa.

68. The method according to claim 65, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mmol (mM) to about 1.0 mM.

69. The method according to claim 68, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM.

70. a) Microgel particles comprising a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and cell adhesion peptides, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%, b) A PEG-dithiol wherein the PEG-dithiol has a molecular weight of at least about 0.5 kilodaltons (kDa), and the microgel particles undergo an annealing reaction to form a covalently stabilized porous scaffold after exposure to the PEG-dithiol. A hydrogel system containing the above.

71. a) Microgel particles comprising a crosslinked 4-arm poly(ethylene glycol) (PEG) vinyl sulfone hydrogel polymer crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinking agent, one or more K peptides and Q peptides, and cell adhesion peptides, wherein the microgel particles are present in a suspension containing the microgel particles and water, and the microgel particles are present in the suspension at a volume fraction of at least 90%, b) PEG-dithiol wherein the microgel particles undergo an annealing reaction and, after exposure to the PEG-dithiol, form a covalently stabilized porous scaffold, and the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.02 mmol (mM) to about 1.0 mM. A hydrogel system containing the above.

72. The hydrogel system according to claim 70 or 71, wherein the microgel particles are present in the suspension at a volume fraction of at least 90%.

73. The hydrogel system according to any one of claims 70 to 72, wherein the PEG-dithiol and the 4-armed PEG vinyl sulfone are present to provide a thiol-to-vinyl sulfone ratio of less than about 1.

0.

74. The hydrogel system according to any one of claims 70 to 73, wherein the PEG-dithiol has a molecular weight of at least about 3.4 kDa.

75. The hydrogel system according to any one of claims 70 to 74, wherein the PEG-dithiol is present in the hydrogel system at a molar concentration of at least about 0.2 mM.

76. The hydrogel system according to any one of claims 70 to 75, wherein the microgel particles are spherical.

77. The method according to any one of claims 70 to 76, wherein the microgel particles include microspheres.

78. The hydrogel system according to any one of claims 70 to 77, wherein the microgel particles include diameters of 5 μm to 1000 μm, 50 μm to 1000 μm, or 70 μm to 150 μm.

79. The hydrogel system according to any one of claims 70 to 78, wherein the microgel particles have a compressive modulus of at least about 500 pascals (Pa) before the annealing reaction.

80. The hydrogel system according to any one of claims 70 to 79, wherein the microgel particles have a compressive modulus of at least about 1,500 pascals (Pa) after the annealing reaction.

81. The hydrogel system according to any one of claims 70 to 80, wherein the covalently stabilized porous scaffold includes pores with a median pore diameter of about 5 μm or more.

82. The hydrogel system according to any one of claims 70 to 81, wherein the pores include a median pore diameter of about 10 μm to about 35 μm.

83. The hydrogel system according to any one of claims 70 to 82, wherein one or more of the cell adhesion peptides comprises an RGD peptide.

84. The hydrogel system according to any one of claims 70 to 83, wherein the microgel particles have a polydispersity of 0.1 or less.

85. The hydrogel system according to claim 84, wherein the polydispersity is calculated based on the standard deviation and mean size of the particles (for example, PDI = (SD / mean)^2).

86. The hydrogel system according to any one of claims 70 to 85, wherein the hydrogel formulation further comprises a buffer, the buffer comprising a phosphate buffer, a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, or an acetate buffer, or any combination thereof.

87. The hydrogel system according to any one of claims 70 to 86, wherein the annealing reaction includes a covalent synthesis reaction.

88. The hydrogel system according to claim 87, wherein the covalent synthesis reaction includes a Michael addition reaction or a pseudo-Michael addition reaction.

89. The hydrogel system according to claim 88, wherein the vinyl sulfone of the four-armed PEG vinyl sulfone is the Michael acceptor in the Michael addition reaction or pseudo-Michael addition reaction.

90. The hydrogel system according to claim 88, wherein the thiol of the PEG-dithiol is the Michael donor in the Michael addition reaction or pseudo-Michael addition reaction.

91. A hydrogel formulation comprising a hydrogel system according to any one of claims 70 to 90 in a suspension, wherein the suspension comprises a buffer.

92. The hydrogel formulation according to claim 91, wherein the hydrogel formulation is formulated for administration to a subject.

93. The hydrogel formulation according to claim 92, wherein the hydrogel formulation is formulated for administration along the suture line of the target suture line.

94. The hydrogel formulation according to claim 91 or 92, wherein administration minimizes the foreign body reaction of the subject.

95. The hydrogel preparation according to any one of claims 91 to 94, wherein the hydrogel preparation contains a dosage volume of about 0.01 mL to about 20 mL.

96. a) A main body comprising a hydrogel system according to any one of claims 70 to 90 or a hydrogel formulation according to any one of claims 91 to 95, b) A delivery device comprising a main body and a dispensing device that is in fluid communication with the main body, wherein the delivery device is sterile.

97. The method according to claim 96, wherein the delivery device is a syringe or a needle.

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