Collagen-hydrogel composite

Abstract

The present disclosure provides formulations and methods for producing hyaluronic acid and collagen microfiber composites that are biocompatible, induce minimal swelling, and provide durable volumization. When implanted in the body, the gel formulations can induce cellular infiltration, angiogenesis, and adipogenesis.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 485,240, filed February 15, 2023, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates generally to composite materials comprising hydrogels and collagen.

[0003] The present disclosure also relates generally to compositions and methods for restoring lost soft tissue volume while promoting soft tissue regeneration, as well as the application of dermal fillers, reconstructive therapies, and cellular therapies. [Background technology]

[0004] Hydrogels are popular scaffold matrices for tissue restoration in the field of soft tissue repair due to their biomimetic characteristics that closely resemble the tissue microenvironment and their ability to be tailored to the needs of various adipose tissues, such as volume and porosity. (Young, DA, et al., Biomaterialia, 2011. 7(3): pp. 1040-1049; and Varma, DM, et al., Acta Biomaterialia, 2014. 10(12): pp. 4996-5004.)

[0005] Among various hydrogel products, hyaluronic acid (HA) is widely used as a filler and foaming agent for soft tissue augmentation due to its biocompatibility as an endogenous polysaccharide, its moderate biodegradability, and physical properties similar to those of soft tissues such as adipose tissue, fibrous tissue, and nerves. See Tezel, A. et al., Journal of Cosmetic and Laser Therapy, 2008. 10(1): pp. 35-42, and Wang, F., et al., In vivo stimulation of de novo collagen production caused by cross-linked hyaluronic acid dermal filler injections in photodamaged human skin. Archives of dermatology, 2007. 143(2): pp. 155-163.

[0006] Studies disclosed in US 2020 / 0069846A1, Yao et al. Small 2022, 2202309, and Henn et al. Plast. Reconstr. Surg. 2022, 149: 433e present robust therapeutic systems constructed by chemically bonding hyaluronic acid (HA) to polycaprolactone (PCL) nanofibers, which impart higher porosity and cell permeability to the material while maintaining a storage modulus comparable to that of adipose tissue. See Li, X., et al., Science Translational Medicine, 2019.11(490): p.eaau6210, and Li, X., et al., Biomaterials, 2020.245: p.119978. Summary of the Invention [Problem to be solved by the invention]

[0007] Although collagen, a bioactive and biostimulating component, has been widely used as a filler and wound dressing, it still suffers from drawbacks such as volume retention and resulting inflammatory responses. Therefore, what is needed is a system for a new approach to soft tissue engineering that has one or more desirable characteristics, such as improved mechanical properties, biocompatibility, improved injectability, feasibility of terminal sterilization, and reduced cost. [Means for solving the problem]

[0008] Disclosed herein are methods and compositions for producing nanofiber-hydrogel composites comprising hyaluronic acid (HA), collagen fibers, and a divinyl sulfone crosslinker. In various embodiments, the HA is covalently linked to the collagen fibers by the crosslinker to form a composite network.

[0009] In various embodiments, the nanofiber-hydrogel composite is suitable for incorporation into the tissue of a human subject by injection of the composite into the tissue.

[0010] In various embodiments, the nanofiber-hydrogel composites provide native, autoclavable, one-step crosslinking of both the fiber and hydrogel phases, eliminating the need for PEG-based crosslinkers.

[0011] In one embodiment, the high thermal stability of the nanofiber-hydrogel composite provides shelf stability at ambient temperatures and compatibility with autoclave-based terminal sterilization.

[0012] In various other aspects, the nanofiber-hydrogel composites provide several advantageous biotropic or biocompatible characteristics, for example, they exhibit monocyte recruitment and / or polarization or allow cell infiltration.

[0013] In various embodiments, the nanofiber-hydrogel composite provides a biostimulatory effect selected from tissue remodeling, host cell infiltration, cell adhesion, cell migration, angiogenic response, adipogenic response, macrophage polarization towards a pro-healing phenotype, and regenerative response.

[0014] In various embodiments, the nanofiber-hydrogel composites exhibit favorable biocompatibility selected from one or more of the following properties: cell adhesion, cell migration, durable soft tissue remodeling, angiogenesis, a stimulatory effect by retaining infiltrating macrophages conditioned with the nanofiber-hydrogel composite toward a pro-regenerative M2 phenotype, neovascularization, adipose tissue formation, vasculogenesis, and accelerated tissue remodeling.

[0015] In various aspects, methods are disclosed for producing nanofiber-hydrogel composites comprising collagen nanofibers, a hyaluronic acid (HA) network, and a crosslinker, including crosslinking of both the fibers and the HA network. In some embodiments, reaction conditions include a temperature of 30°C, a reaction time of 2 hours, an HA concentration of 2.5% w / v, a collagen fiber concentration of 4.5%, and a DVS concentration of 3.1% w / v.

[0016] In various embodiments, the hydrogel material is present in a composite in a functional network.

[0017] In various embodiments, the ratio of fiber to anhydrous hydrogel material is from about 1.8 to about 2.8.

[0018] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by the divinyl sulfone, the hyaluronic acid being present in an amount ranging from about 5 mg / mL to about 15 mg / mL, and the collagen being present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0019] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by the divinyl sulfone, the hyaluronic acid being present in an amount ranging from about 5 mg / mL to about 15 mg / mL, the collagen being present in an amount ranging from about 10 mg / mL to about 40 mg / mL, and the divinyl sulfone being present in an amount ranging from about 3:1 to about 5:1 (molar ratio of DVS to hyaluronic acid subunits) prior to crosslinking.

[0020] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by the divinyl sulfone, the hyaluronic acid being present in an amount ranging from about 5 mg / mL to about 15 mg / mL, and the collagen being present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0021] In one embodiment, the nanofiber-hydrogel composite comprises the product of a process comprising reacting a hyaluronic acid material, collagen fibers, and divinyl sulfone, whereby the hyaluronic acid is covalently bonded to the collagen fibers by the divinyl sulfone, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL, and the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit in a ratio ranging from about 0.5:1 to about 5:1.

[0022] In one embodiment, the present disclosure provides a method for making a nanofiber-hydrogel composite, comprising the step of reacting a hyaluronic acid material, collagen fibers, and divinyl sulfone, whereby the hyaluronic acid is covalently bonded to the collagen fibers by the divinyl sulfone, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL, and the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit in a ratio ranging from about 0.5:1 to about 5:1.

[0023] Additionally, a kit containing the nanofiber-hydrogel composite is disclosed. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1A shows a schematic diagram of the preparation of divinyl sulfone (DVS) cross-linked hyaluronic acid (HA) hydrogel and nanofiber-hydrogel composite (NHC), micronization of the material, and subcutaneous injection into the back of a Sprague Dawley (SD) rat. [Figure 1B] FIG. 1B is an illustration showing HA (double line) and collagen fibers (thick solid line) cross-linked using DVS. [Figure 1C] FIG. 1C shows a non-limiting example of DVS crosslinking to alternative hydroxyl groups on HA. [Figure 2] FIG. 2 shows a graph depicting the % swelling of implanted gels on day 1 of the animal study (Example 6), as measured by MRI. [Figure 3] FIG. 3 shows a graph depicting the % swelling of the implanted gels at day 7 of the animal study (Example 6) along with a commercial comparison group (Voluma XC), as measured by MRI. [Figure 4] FIG. 4 shows a graph illustrating the swelling capacity of the hyaluronic acid gel (without fibers) prepared in Example 2, demonstrating an inverse correlation between the initial HA concentration and the swelling ratio after gelation. [Figure 5]FIG. 5 shows a graph depicting the final hyaluronic acid concentration of the hyaluronic acid gels (without fibers) made in Example 2, illustrating the effect of the initial HA concentration (during gelation) on the final HA concentration after gelation. [Figure 6] FIG. 6 shows a graph illustrating the final hyaluronic acid concentration of the hyaluronic acid gels (without fibers) prepared in Example 2, showing the effect of DVS concentration (during gelation) on the final HA concentration after gelation, with the initial HA concentration fixed at 30 mg / mL. [Figure 7] Figure 7 shows a graph depicting the final hyaluronic acid concentration of the hyaluronic acid gels (without fibers) made in Example 2, and illustrates the effect of DVS concentration (during gelation) on the final HA concentration after gelation. The overall correlation is weaker than when the HA concentration is fixed (e.g., in Figure 6) or other correlations (e.g., Figure 5). [Figure 8] Figure 8 shows a graph depicting the swelling capacity of the hyaluronic acid composite gel (with collagen fibers) prepared in Example 2, demonstrating an inverse correlation between the initial collagen concentration (before gelation) and the swelling ratio for the gel after gelation. Fibers covalently bound to the hydrogel phase reduce the swelling capacity of the resulting composite in a dose-response manner. This correlation is also evidence of interfacial bonding between the fiber and hydrogel phases, as the presence of fibers influences the swelling behavior of the hydrogel phase. [Figure 9] Figure 9 shows a graph depicting the swelling capacity of the hyaluronic acid composite gels (with collagen fibers) made in Example 2, demonstrating the correlation between the initial HA concentration (before gelation) and the swelling ratio for the gels after gelation. The correlation is much weaker than that for the fiber-free group (Figure 4), indicating that collagen fiber concentration has a significant impact on the swelling behavior of the gels during fabrication and is an important variable for achieving the target final HA concentration and, therefore, swelling profile in vivo. [Figure 10] Figure 10 shows a graph illustrating the swelling capacity of the hyaluronic acid composite gels (with collagen fibers) prepared in Example 2, demonstrating the correlation between DVS crosslink concentration (before gelation) and the swelling ratio for the gels after gelation. The ratio of DVS to HA subunits is inversely correlated with the swelling ratio. [Figure 11]Figure 11 shows a graph showing the final gel (in the finished syringe) concentrations of the hyaluronic acid composite gels (with collagen fibers) made in Example 2, demonstrating the correlation between collagen fiber concentration (before gelation) and HA concentration in the gel after gelation. Increased collagen content is associated with a lower swelling ratio during manufacture and therefore a higher final HA content (less degradation during manufacture after gelation). The correlation is stronger than that for the initial HA concentration (Figure 12). [Figure 12] Figure 12 shows a graph showing the final gel (in the finished syringe) concentrations of the hyaluronic acid composite gels (with collagen fibers) prepared in Example 2, demonstrating the correlation between the initial HA concentration (before gelation) and the HA concentration in the gel after gelation. Increasing HA content is weakly correlated with higher final HA content. [Figure 13] Figure 13 shows a graph depicting the final gel (in the finished syringe) concentrations of the hyaluronic acid composite gels (with collagen fibers) made in Example 2, demonstrating the correlation between the initial relative DVS crosslinker concentration (before gelation) and the HA concentration in the gel after gelation. Increased DVS content is associated with higher final HA content. [Figure 14] Figure 14 shows (A) a graph depicting the relative volumization (measured by MRI) profiles of M1-M4 formulations prepared in Example 4 and tested up to 63 days in Example 6. Values ​​are normalized to measurements taken immediately after injection. Voluma doubled in volume, while the four composite gels maintained their volume without initial swelling. [Figure 15] Figure 15 shows representative photographic images of four implanted gels from the groups during 7 days of explantation in a rat subcutaneous implantation model. All four test implants (M1-M4) and the commercial comparison group (Voluma XC) were well tolerated. 1 = M1, 2 = M2, 3 = M3, 4 = M4, 5 = Voluma XC. All images are to the same scale; scale bar = 1 cm. [Figure 16]Figure 16 shows representative photographic images of four implanted gels from the groups during 30 days of explantation in a rat subcutaneous implantation model. All four test implants (M1-M4) and the commercial comparison group (Voluma XC) were well tolerated. T1 = M1, T2 = M2, T3 = M3, T4 = M4, Voluma = Voluma XC. All images are to the same scale. Scale bar = 1 cm. [Figure 17] Figure 17 shows histology images of Group M1 from Example 6 on day 7. Masson's Trichrome staining (top) and H&E staining (center, bottom). The embedded gel maintained its integrity and shape, was clearly distinguishable from the surrounding tissue, and induced cellular infiltration from the periphery to the inside. Scale bar = 2.5 mm (top left, center left), 250 microns (top right), 100 microns (center right, bottom). [Figure 18] Figure 18 shows histology images of group M2 on day 7 from Example 6. Masson's Trichrome staining (top) and H&E staining (bottom). Cell infiltration is clearly visible. Scale bar = 2.5 mm (left column), 250 microns (right column). [Figure 19] Figure 19 shows histology images of group M3 on day 7. Areas of cellular infiltration are present in the area around the gel, although not as frequent as in groups M1 and M2. Masson's Trichrome staining (top) and H&E staining (bottom). Scale bar = 2.5 mm (left column), 250 microns (right column). [Figure 20] Figure 20 shows histology images of group M4 on day 7. Areas of cellular infiltration are rare compared to groups M1 and M2, but are present in the area around the gel. Masson's Trichrome staining (top) and H&E staining (bottom). Scale bar = 2.5 mm (left column), 250 microns (right column). [Figure 21] Figure 21 shows histology images of the control group (Voluma XC) on day 7. There is no obvious cell infiltration into the gel. Masson's Trichrome staining (top) and H&E staining (bottom). Scale bar = 2.5 mm (left column), 250 microns (right column). [Figure 22]Figure 22 shows histology images (Masson's Trichrome staining) of group M1 at day 30. The implants are infiltrated by cells, accompanied by collagen deposition and numerous functional blood vessels. Scale bars are 2.5 mm (left) and 250 microns (right). [Figure 23] Figure 23 shows histology images (Masson's Trichrome staining) of group M1 on day 30. The implants are infiltrated by cells, accompanied by collagen deposition and numerous functional blood vessels. The scale bar is 100 microns. [Figure 24] Figure 24 shows histology images (H&E staining) of group M1 on day 30. The implants are infiltrated by cells, accompanied by collagen deposition and numerous functional blood vessels. Scale bars are 500 microns (left) and 100 microns (right). [Figure 25] Figure 25 shows histology images of group M2 at day 30. Top row: Masson's Trichrome staining, bottom row: H&E staining. The implants are infiltrated by cells, accompanied by collagen deposition and numerous functional blood vessels. Scale bars are 2.5 mm (left column) and 250 microns (right column). [Figure 26] Figure 26 shows histology images of group M3 on day 30. Top row: Masson's trichrome staining, bottom row: H&E staining. The implants are infiltrated by cells, with numerous functional blood vessels. The cell and blood vessel densities are lower than in groups M1 and M2, and a significant area of ​​the implant has not yet been infiltrated by cells. The embedded gels well maintained their original bolus shape (little spreading). Scale bars are 2.5 mm (left column) and 250 microns (right column). [Figure 27]Figure 27 shows histology images of group M4 on day 30. Top row: Masson's Trichrome staining, bottom row: H&E staining. By day 30, the implants were infiltrated by cells only at the periphery. Blood vessels had not formed within the implanted material by this time point. The cell density within the implant was lower than in groups M1, M2, and M3, and most areas of the implant had not yet been infiltrated by cells. The implanted gel maintained its original bolus shape well (little spreading). Scale bars are 2.5 mm (left column) and 250 microns (right column). [Figure 28] Figure 28 shows histology images of the control group (Voluma XC) at day 30. Top row: Masson's Trichrome staining, bottom row: H&E staining. The implants are intact but have no obvious cellular infiltration within the embedded gel and no blood vessels. The gel is surrounded by a thin collagenous band. Scale bars are 2.5 mm (left column) and 250 microns (right column). [Figure 29] Figure 29 shows photographic images of four implanted gels from the M1 group during explantation after 63 days in a rat subcutaneous implantation model. All four implants were well tolerated, integrated into the surrounding tissue, and had an appearance similar to that of native tissue. Scale bar = 1 cm. [Figure 30] Figure 30 shows H&E histology images of group M1 on day 63 in a rat subcutaneous implant model. The gel maintains volumization while undergoing remodeling, accompanied by cell infiltration and angiogenesis. Active adipogenesis is ongoing around the gel (marked by the solid border area), generating new adipose tissue. Scale bar = 2.5 mm. [Figure 31] Figure 31 shows a magnified image of the section from Figure 30 of an H&E histology slide from the M1 group at POD 63, showing areas of active adipogenesis and resulting new adipose tissue, along with examples of blood vessels. Scale bar = 100 microns. Symbols: * = newly generated adipose tissue, + = active adipose tissue production, # = intact gel and fibers, ^ = blood vessels. [Figure 32]Figure 32 shows H&E histology images of group M2 on day 63 in a rat subcutaneous implant model. The gel maintains volumization while undergoing remodeling, accompanied by cell infiltration and angiogenesis. Active adipogenesis is ongoing around the gel, generating new adipose tissue. Scale bar = 2.5 mm. [Figure 33] Figure 33 shows H&E histology images of group M3 at day 63 in a rat subcutaneous implant model. The gel maintains volume while undergoing remodeling, accompanied by cellular infiltration and angiogenesis. The gel maintains its shape and thickness over time. The rate of cellular infiltration is reduced compared to groups M1 and M2 at this time point, with partial progressive cellular remodeling in the left half of the implant image and little infiltration in the right half of the implant image. Scale bar = 2.5 mm. [Figure 34] Figure 34 shows a higher magnification image of the same histology slide as Figure 33, of the M3 group on POD63. Note the progressive cellular infiltration and angiogenesis. Scale bar = 250 microns. [Figure 35] Figure 35(A) shows H&E histology images of Group M4 on day 63 in a rat subcutaneous implant model. The gel maintains its volume while undergoing remodeling, accompanied by cellular infiltration and angiogenesis. The gel maintains its shape and thickness over time. The rate of cellular infiltration is reduced compared to Groups M1 and M2 at this time point, with partial progressive cellular remodeling in the right lobe of the implant image and almost no infiltration in the left lobe of the implant image. Scale bar = 2.5 mm. (B) Photomicrograph (H&E stain) of Group M4 histology slide on day 63. Scale bar = 250 micrometers. While the degree of cellular infiltration is much lower for Group M4 than for Group M1, cells retain their natural spindle morphology and can still be seen gradually growing into the implant site. Most of the gel maintains its integrity while awaiting cellular infiltration. The gel bolus maintained its bolus shape well. In areas with more extensive ingrowth, blood vessels have formed within the bulk of the gel (black arrows) and smaller white spherical structures are visible. Scale bars are 100 microns (top) and 250 microns (bottom). [Figure 36] FIG. 36 shows a graph of a representative trace from an injection force test (on an Instron universal testing system) for Group M1 gel produced in Example 4 and tested in the animal study of Example 6, injected through a 27 gauge needle at a volumetric rate of 0.25 cc / min. [Figure 37] FIG. 37 shows a graph of a representative trace from an injection force test (on an Instron universal testing system) for Group M2 gel produced in Example 4 and tested in the animal study in Example 6, injected through a 27 gauge needle at a volumetric rate of 0.25 cc / min. [Figure 38] FIG. 38 shows a graph of a representative trace from an injection force test (on an Instron universal testing system) for Group M3 gel produced in Example 4 and tested in the animal study of Example 6, injected through a 27 gauge needle at a volumetric rate of 0.25 cc / min. [Figure 39] FIG. 39 shows a graph of a representative trace from an injection force test (on an Instron universal testing system) for Group M4 gel produced in Example 4 and tested in the animal study in Example 6, injected through a 27 gauge needle at a volumetric rate of 0.25 cc / min. [Figure 40] 40 shows a graph of representative traces of storage modulus (G') for the test groups generated in Example 4 and tested in the animal study of Example 6. Rheology testing according to Example 3 on an oscillatory rheometer (AR2000ex, TA Instruments) at 25°C using a parallel plate geometry with a 25 mm diameter top plate, a 900 micron gap, and a frequency of 1 Hz. Groups M1 (open circles), M2 (filled circles), M3 (open squares), M4 (filled squares). [Figure 41]Figure 41 shows a graph of representative traces of loss modulus (G'') for the test groups generated in Example 4 and tested in the animal study of Example 6. Rheology testing according to Example 3 on an oscillatory rheometer (AR2000ex, TA Instruments) at 25°C using a parallel plate geometry with a 25 mm diameter top plate, a 900 micron gap, and a frequency of 1 Hz. Groups M1 (open circles), M2 (filled circles), M3 (open squares), M4 (filled squares). [Figure 42] Figure 42 shows a graph of representative traces of tan-delta values ​​(tan-δ) for the test groups generated in Example 4 and tested in the animal study of Example 6. Rheology testing according to Example 3 on an oscillatory rheometer (AR2000ex, TA Instruments) at 25°C using a parallel plate geometry with a 25 mm diameter top plate, a 900 micron gap, and a frequency of 1 Hz. Groups M1 (open circles), M2 (filled circles), M3 (open squares), M4 (filled squares). [Figure 43] Figure 43 shows an SEM image of porcine collagen with an average diameter of 0.57 microns + / - 0.32 microns. It was dissolved in HFIP at 7% and spun into fibers by spinning at 0.3 ml / hr at 11 kV voltage. Scale bar = 30 micrometers. [Figure 44] Figure 44 shows an SEM image of porcine gelatin (Sigma) spun to produce a ribbon morphology. Scale bar = 80 micrometers. [Figure 45] Figure 45 shows SEM images of fibers spun from recombinant collagen (Vecollan / Evonik). The images show cylindrical or ribbon morphologies. Scale bar = 100 micrometers. [Figure 46] Figure 46 shows a line plot of volumetric data measured by MRI demonstrating maintenance of volume over time after implantation in a rat subcutaneous model. The top graph shows the average measured volume in cubic centimeters for each group up to 385 days, with error bars indicating standard deviation. [Figure 47]Figure 47(A) shows the relative volumes as a percentage of the day 7 volume for each individual implant. This more closely demonstrates the volume profile that patients would experience and illustrates the significant swelling expected for the Voluma XC® group. Figure 47(B) shows the relative volumes for only the M1 and Voluma XC® groups to more clearly highlight the improvements from the disclosed invention. [Figure 48] Figure 48 shows a photomicrograph of the M1 group histology slide at 20 weeks. There is extensive cellular ingrowth into the gel, adipose tissue growing around the implanted gel, and significant areas of adipose tissue growing within pockets in the panniculus carnosus layer. The scale bar is 2.5 mm. [Figure 49] Figure 49 shows photomicrographs (H&E stain) of M1 histology slides at 20 weeks. Large (>1 mm x 1 mm) areas of vascularized adipose tissue have developed within the embedded gel. Scale bars are 2.5 mm (A), 250 microns (B). [Figure 50] Figure 50 shows photomicrographs (H&E stain) of M2 histology slides at 26 weeks, with extensive cellular ingrowth, adipose tissue formation, and many adipocytes present within the gel in addition to the surrounding tissue. Scale bars are 2.5 mm (A), 500 microns (B). [Figure 51] Figure 51 shows a photomicrograph (H&E stain) of a Voluma XC® histology slide at week 20. Scale bar is 1 mm. [Figure 52] Figure 52 shows a photomicrograph (H&E stain) of an M1 histology slide at 40 weeks. The image is from a different section of the same tissue, and there is only a small amount of residual gel at this location. The gel is partially replaced by vascularized adipose tissue. Scale bars are 2.5 mm (A), 500 microns (B). [Figure 53]Figure 53 shows photomicrographs (H&E stain) of M2 histology slides at 40 weeks. The top image shows a tissue slice with intact gel blebs, but also extensive adipose tissue surrounding it and in the cutaneous muscle layer. The bottom image is from a different slice of the same tissue, with adipocytes present within and around the gel. Scale bars are 2.5 mm (A), 500 microns (B). [Figure 54] Figure 54(A) shows a photomicrograph (H&E stain) of an M3 histology slide at 40 weeks. The interior of the gel has some cellular ingrowth through its full thickness, but is still primarily acellular at this location. There is some adipose tissue to the left of the gel. The scale bar is 1 mm. Figure 54(B) shows a photomicrograph (H&E stain) of an M4 histology slide at 40 weeks. The interior of the gel is still primarily acellular at this point, but there are cells at the periphery of the gel and adipose tissue to the right of the gel. The scale bar is 2.5 mm. [Figure 55] Figure 55 shows photographs of gels M1, M2, M3, and M4 during explantation at 15 months. The gels maintained their integrity and shape over this time frame. [Figure 56] Figure 56 shows a photomicrograph (Masson's Trichrome stain) of a Voluma XC® histology slide at 26 weeks. The nerve pocket (B) in the cutaneous muscle layer (C) adjacent to the embedded gel (A) is devoid of adipocytes. The scale bar is 500 microns. [Figure 57] Figure 57 shows a photomicrograph (Masson's trichrome stain) of M1(A) at 26 weeks. The scale bar is 500 microns. Figure 57(a) shows a tissue section transverse to the muscle fibers of the cutaneous muscularis. Figure 57(b) shows a tissue section longitudinal to the muscle fibers of the cutaneous muscularis. Circle A shows a nerve pocket with adipocytes. Circle B shows M2. Circle C shows the cutaneous muscularis. [Figure 58] Figure 58 shows a photomicrograph (H&E stain) of M2 at 20 weeks. Scale bar is 500 microns. DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the terminology used in connection with, and techniques of, biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated.

[0026] Incorporation by Reference All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0027] definition The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0028] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0029] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0030] As used herein, "about" or "approximately" can mean within a margin of error, plus or minus less than 1%, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30%, or more than 30%, depending on the circumstances known or known to one of ordinary skill in the art.

[0031] As used herein, a "subject" or "subjects" or "individual" may include, but is not limited to, mammals, such as humans or non-human mammals, such as domestic, agricultural, or wild animals, as well as birds and aquatic animals.

[0032] As used herein, a "scaffold composite" or "composite" includes any bonding, e.g., covalent bonding, between two components: polymer fibers and hydrogel material. The scaffold composite contains the polymer fibers and hydrogel material in a "functional network," meaning that the interaction between the components provides a chemical, biochemical, biophysical, physical, or physiological benefit. Furthermore, the functional network may include additional components, including cells, biological materials (e.g., polypeptides, nucleic acids, lipids, carbohydrates), therapeutic compounds, synthetic molecules, etc. In certain embodiments, the scaffold composite promotes tissue growth and cellular infiltration when implanted into a target tissue present in a human subject.

[0033] As used herein, the term "hydrogel" is a type of "gel" and refers to a water-swellable polymeric matrix consisting of a three-dimensional network of macromolecules (e.g., hydrophilic polymers, hydrophobic polymers, mixtures thereof) held together by covalent or non-covalent crosslinks that can absorb a significant amount of water (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% per non-water molecular unit) to form an elastic gel. The polymeric matrix can be formed from any suitable synthetic or naturally occurring polymeric material. As used herein, the term "gel" refers to a solid, three-dimensional network that spans the volume of a liquid medium and traps it through surface tension effects.

[0034] As used herein, the term "nanofiber" describes individual units of fibrous material with a high specific surface area, typically made of units with a high aspect ratio (length substantially greater than width) that have at least one feature on the nanometer scale (diameters in the range of approximately 1 to 1000 nanometers). The term nanofiber can also include, but is not limited to, fibers with diameters of several thousand nanometers (single digit microns), since such materials are produced using similar techniques and have similar characteristics. As a non-limiting example, "nanofibers" produced by electrospinning generally have diameters in the range of 50 nm to 10 microns. Nanofibers can be produced by methods such as electrospinning, melt spinning, drawing, phase separation, or self-assembly. Nanofibers are typically composed of polymeric synthetic or natural materials.

[0035] As used herein, the term "microfiber" describes individual units of fibrous material having a high specific surface area, which are made up of individual units having a high aspect ratio (length substantially greater than width) with at least one feature that is typically on the micrometer scale (having a diameter in the range of about 1 to 100 microns).

[0036] As used herein, the term "crosslinked" refers to a composition containing intramolecular and / or intermolecular crosslinks, whether caused by covalent or non-covalent bonds, which may be direct or may involve a crosslinking agent. "Non-covalent" bonds include both hydrogen bonds and electrostatic (ionic) bonds.

[0037] As used herein, the term "storage modulus" is used to define a measure of the elastic component of dynamic modulus, which indicates how a material responds to deformation or stress. In one embodiment, "deformation" refers to the change in shape or size of an object due to an applied force.

[0038] As used herein, the term "shear modulus," also known as the modulus of rigidity, represented by (G'), is defined as the ratio of shear stress to shear strain. In some embodiments, "shear stress" refers to the component of stress that is coplanar with the cross-section of the material. In some embodiments, "shear strain" refers to the force normal to the cross-section of the material.

[0039] As used herein, the term "loss modulus" is also known as the modulus of viscosity and is represented by (G''). The loss modulus is the inelastic part of the dynamic (or complex) modulus. The dynamic modulus is the ratio of stress to strain.

[0040] Tan delta (Tan-δ; loss tangent) is the rheological loss modulus divided by the storage modulus. A lower tan delta number equates to a more "solid-like" as opposed to "liquid-like" material.

[0041] As used herein, the term "POD" refers to post-operative date.

[0042] As used herein, the term "concentration" in the context of divinyl sulfone (DVS) refers to the concentration of DVS calculated as the ratio of DVS molecules to hydroxyl groups in HA.

[0043] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those skilled in the art. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0044] Fiber dimensions The fibers can have one or more of a variety of morphologies. In certain embodiments, the fibers can be cylindrical or ribbon-shaped. In various embodiments, the fibers include mixed morphologies. In various embodiments, the polymer fibers include a cylindrical morphology. In various embodiments, the polymer fibers include a ribbon morphology. In various embodiments, the polymer fibers include both a cylindrical morphology and a ribbon morphology.

[0045] Cylindrical fibers do not necessarily have a perfectly round cross section and include, but are not limited to, fibers that include similar cross-sectional widths and heights.

[0046] In various embodiments, the ribbon-shaped fibers comprise a cross-sectional width that is greater than its cross-sectional height. In various embodiments, the ribbon-shaped fibers comprise a cross-sectional width that is about 10% to about 90% greater than its cross-sectional height. In various embodiments, the ribbon-shaped fibers comprise a cross-sectional width that is about 1% to about 10% greater than its cross-sectional height. In various embodiments, the ribbon-shaped fibers comprise a cross-sectional width that is about 10% to about 50% greater than its cross-sectional height. In various embodiments, the ribbon-shaped fibers comprise a cross-sectional width that is about 25% to about 75% greater than its cross-sectional height.

[0047] In various embodiments, the polymer fibers have an average diameter of less than about 100 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 20 μm, less than about 10 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, less than about 600 μm, less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, less than about 50 μm, less than about 10 μm, less than about 5 μm, or less than about 1 μm.

[0048] In various embodiments, the polymer fibers have an average diameter of less than about 10 μm, less than about 5 μm, less than about 4 μm, less than about 3 μm, less than about 2 μm, less than about 1 μm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 10 nm, less than about 5 nm, or less than about 1 nm.

[0049] In various embodiments, the polymer fibers have an average diameter ranging from about 100 μm to 1000 μm, from about 10 μm to 100 μm, or from about 10 μm to 50 μm. In certain embodiments, the fibers have an average diameter ranging from about 1 μm to 1000 μm, from about 1 μm to 500 μm, or from about 1 μm to 100 μm.

[0050] In various embodiments, the polymer fibers have an average diameter ranging from about 10 nm to 5 μm, about 100 nm to 5 μm, hi certain embodiments, the fibers have an average diameter ranging from about 1 nm to 1,000 nm, about 1 nm to 500 nm, or about 1 nm to 100 nm.

[0051] In various embodiments, the polymer fibers have an average diameter of about 10 nm to about 10,000 nm, e.g., about 100 nm to about 8,000 nm, or about 150 nm to about 5,000 nm, or about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, or 8,000 nm.

[0052] In various embodiments, the polymer fibers (e.g., microfibers) have an average length of greater than about 1 μm, greater than about 5 μm, greater than about 10 μm, greater than about 20 μm, greater than about 30 μm, greater than about 40 μm, greater than about 50 μm, greater than about 60 μm, greater than about 70 μm, greater than about 80 μm, greater than about 90 μm, greater than about 100 μm, greater than about 200 μm, greater than about 300 μm, greater than about 400 μm, greater than about 500 μm, greater than about 600 μm, greater than about 700 μm, greater than about 800 μm, greater than about 900 μm, or about 1 mm. In certain embodiments, the fibers (e.g., microfibers) have lengths ranging from about 1 to 1,000 μm, from about 10 to 500 μm, or from about 100 to 500 μm.

[0053] The diameter and length of the polymer fibers may be determined using light microscopy (including fluorescence microscopy) or electron microscopy.

[0054] In various embodiments, the polymer fibers can have aspect ratios ranging from at least about 10 to at least about 10,000. Because the fiber diameter is so small, the fibers are understood to have a high surface area per unit of mass. This high surface area to mass ratio allows the fiber-forming solution or liquid to transform from a liquid or solvated fiber-forming material into solid fibers in a fraction of a second.

[0055] Fiber to Hydrogel Ratio The ratio of polymer fibers to hydrogel material can be determined by any means known in the art. The ratio may be reported on a component weight basis. For example, in various embodiments, the ratio of polymer fibers to hydrogel material ranges from about 1:100 to about 100:1. In various embodiments, the ratio of polymer fibers to hydrogel material ranges from about 1:50 to about 50:1, from about 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:3 to about 3:1 on a component weight basis.

[0056] The ratio of polymer fiber to hydrogel material may also be provided on a concentration basis, e.g., as a given weight of polymer fiber per volume of hydrogel material, e.g., a concentration of about 10 mg / mL to about 40 mg / mL.

[0057] Collagen fiber composition Any suitable form of collagen can be used. In various embodiments, the collagen is human type collagen. The collagen can be fibrillar or non-fibrillar. In various embodiments, the collagen is fibrillar collagen. In various embodiments, the fibrillar collagen is type I, type II, type III, type V, or type XI collagen. In various embodiments, the collagen is non-fibrillar collagen. In various embodiments, the non-fibrillar collagen is FACIT (fibril-associated collagen with interrupted triple helices) (types IX, XII, XIV, XIX, or XXI) collagen. In various embodiments, the non-fibrillar collagen is MACIT (membrane-associated collagen with interrupted triple helices) (types XIII or XVII) collagen. In various embodiments, the non-fibrillar collagen is short chain (types VIII or X). In various embodiments, the non-fibrillar collagen is multiplexin (multiple triple helical domains with interruptions) (types XV or XVIII) collagen. In various embodiments, the non-fibrillar collagen is microfibril-forming (type VI) collagen. In various embodiments, the non-fibrillar collagen is anchoring fibrillar (type VII) collagen. In various embodiments, the collagen is synthetic collagen.

[0058] In various embodiments, the collagen is type I collagen.

[0059] In various embodiments, the collagen is type II collagen.

[0060] In various embodiments, the collagen is type III collagen.

[0061] In various embodiments, the collagen is type IV collagen.

[0062] In various embodiments, the collagen nanofibers comprise bovine collagen nanofiber fragments of type I. In various embodiments, the collagen nanofibers comprise porcine, ovine, chicken, fish, or recombinant forms of collagen.

[0063] In various embodiments, the collagen nanofibers comprise bovine collagen type I. In various embodiments, the collagen nanofibers comprise bovine atelocollagen type I.

[0064] In various embodiments, the collagen nanofibers comprise porcine type I. In various embodiments, the collagen nanofibers comprise porcine type I atelocollagen.

[0065] In various embodiments, the collagen nanofibers comprise collagen from a non-animal, recombinant source. In various embodiments, the collagen is recombinant human type 3 collagen by Bloomage. In various embodiments, the collagen is Demulcent SFA by Jland Biotech. In various embodiments, the collagen is Vecollan by Evonik. Vecollan can be made by a fermentation-based process. The recombinant collagen can comprise a single alpha chain or multiple alpha chains, with or without hydroxyproline residues. In various embodiments, the recombinant collagen can comprise a single alpha chain. In various embodiments, the recombinant collagen can comprise multiple alpha chains. In various embodiments, the recombinant collagen can comprise hydroxyproline residues. In various embodiments, the recombinant collagen can be free of hydroxyproline residues.

[0066] In various embodiments, the collagen nanofibers comprise gelatin. In various embodiments, the gelatin is denatured collagen. The gelatin can be sourced from a variety of sources, including, but not limited to, bovine, porcine, fish, and chicken sources.

[0067] In various embodiments, collagen forms include those with and without triple helix secondary structure (gelatin).

[0068] In various embodiments, collagen includes forms with and without telopeptides. In various embodiments, collagen forms include telopeptides. In various embodiments, collagen forms do not include telopeptides. In various embodiments, collagen is atelocollagen. Atelocollagen is a less immunogenic derivative of collagen obtained by removal of N- and C-terminal telopeptide components. Terminal telopeptide components are known to induce antigenicity in humans. Atelocollagen can be prepared from type I collagen of calf dermis by pepsin treatment.

[0069] Once spun, fibers obtained from alternative sources can be processed as previously described for bovine collagen and used to form fiber-hydrogel composites. Other forms of collagen or gelatin, such as other types of collagen, such as type III, or recombinant "collagen-like" peptides from bacterial sequences, are applicable.

[0070] Depending on the raw material source, the resulting fibers may have less secondary structure when spun than previously described collagens (e.g., gelatin is not triple helical) or may have different concentrations of cross-linking moieties due to variations in peptide sequence. Such fibers may require more intensive cross-linking before contact with water. In addition to, or instead of, the EDC / NHS crosslinking previously described, the fibers can be crosslinked with DVS or BDDE, can be crosslinked with aldehyde-based crosslinkers such as vapor-phase glutaraldehyde treatment, can be treated with the Maillard reaction, such as with D-ribose and heat, dehydrothermal treatment, genipin and transglutaminase, irradiation (UV, gamma, or e-beam), plasma treatment, or other methods known in the art (such as those described in [Ehrmann A. Non-Toxic Crosslinking of Electrospun Gelatin Nanofibers for Tissue Engineering and Biomedicine—A Review. Polymers (Basel). 2021 Jun 15;13(12):1973]).

[0071] Once rendered water insoluble, the fibers can be mechanically processed into a dispersed form and covalently gelled into composites using the methods described in this application.

[0072] In various embodiments, the collagen nanofibers are electrospun, melt spun, blow spun, and / or cryomilled. In one particular embodiment, the collagen nanofibers are electrospun.

[0073] Cross-linking of collagen to hyaluronic acid As provided herein, one form of interaction for composites containing polymer fibers and hydrogels involves the use of a crosslinking agent, such as DVS (divinyl sulfone), typically present in an amount effective to introduce bonds between the polymer fibers and the hydrogel material, e.g., to induce crosslinking between collagen fibers and hyaluronic acid. In a specific embodiment, interfacial bonds are generated between the polymer fibers and the hydrogel material to form a composite network. For example, nanofibers are introduced into an HA network while crosslinking with DVS to generate interfacial bonds between the HA network and the nanofibers.

[0074] Compound Creation The method for producing a collagen hydrogel composite may further include a step of swelling the collagen hydrogel composite. In certain embodiments, the swelling step is carried out by incubating the collagen hydrogel composite in an aqueous buffer solution (e.g., PBS). In certain embodiments, the buffer solution may be used at a volume greater than 10, 50, 100, 200, 300, 400, 500, or 1000 times the volume of the collagen hydrogel composite. In certain embodiments, the swelling is carried out for more than 1 hour, more than 2 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 15 hours, more than 20 hours, more than 24 hours, more than 30 hours, more than 40 hours, more than 50 hours, more than 60 hours, or more than 72 hours (continuous or discontinuous). Preferably, the swelling is carried out at room temperature for 24 to 72 hours (continuous or discontinuous).

[0075] The method of producing a collagen hydrogel composite may further include microparticulating the collagen hydrogel composite into microbeads. In certain embodiments, the microbeads have an average diameter ranging from about 1 μm to about 1000 μm, about 10 μm to about 1000 μm, about 20 μm to about 1000 μm, about 30 μm to about 1000 μm, about 40 μm to about 1000 μm, about 50 μm to about 1000 μm, about 50 μm to about 500 μm, about 50 μm to about 400 μm, or about 100 μm to about 500 μm. As described above, the collagen hydrogel composite may be formed into a microparticulate formulation (e.g., microbeads or microgels), allowing for the use of higher concentrations of each component and improved stability. In certain embodiments, a micronization system may be used in which a preformed collagen hydrogel composite is physically modulated, such as by forcing it through one, two, three, or more mesh screens to create a population of non-spherical beads that are relatively similar in shape and size. In certain embodiments, the processing may include mechanical crushing or mechanical sorting, preferably by applying shear using a mesh. This two-screen system allows for precise control over the size of the beads, thus allowing the user to modulate the size as needed. Such microbeads (e.g., non-spherical) are disclosed in US 2020 / 0069846. For example, a collagen hydrogel composite is micronized by applying mechanical shear through a mesh with a defined size range of 50 to 400 μm.

[0076] In certain embodiments, the collagen hydrogel composites produced herein can be in biphasic or monophasic gels. For example, the collagen hydrogel composites are biphasic gels with similar bead sizes created by forcing the gel through a screen. Alternatively, the fiber-hydrogel composites are monophasic gels with a continuous distribution of gel bead sizes created by homogenizing or mechanically disrupting the gel during the crosslinking reaction.

[0077] The methods described herein may include an autoclaving step. In various embodiments, the autoclave temperature is about 100°C to about 150°C. In various embodiments, the autoclave temperature is about 110°C to about 140°C. In various embodiments, the autoclave temperature is about 110°C to about 130°C. In various embodiments, the autoclave temperature is about 115°C to about 125°C. In various embodiments, the autoclave temperature is about 118°C. In various embodiments, the autoclave temperature is about 121°C. In various embodiments, the autoclave temperature is about 138°C. In various embodiments, the autoclave time is about 1 minute to about 1 hour. In various embodiments, the autoclave time is about 5 minutes to about 45 minutes. In various embodiments, the autoclave time is about 5 minutes to about 30 minutes. In various embodiments, the autoclave time is about 5 minutes, about 10 minutes, about 20 minutes, or about 30 minutes.

[0078] The method for producing a collagen hydrogel composite may further include sterilizing the collagen hydrogel composite. Any sterilization method or process known in the art may be used without limitation. For example, sterilizing the collagen hydrogel composite may include sterilizing by autoclaving at 121°C for about 5 to 30 minutes. In various embodiments, the method for producing a collagen hydrogel composite includes an autoclaving step. In various embodiments, the method for producing a collagen hydrogel composite includes an autoclaving step at 121°C for about 5 to 30 minutes. In various embodiments, the method for producing a collagen hydrogel composite includes an autoclaving step at 121°C for about 5 to 30 minutes.

[0079] In various embodiments, the collagen hydrogel composite is part of a packaged composite. In various embodiments, the packaged composite is sterilized. In various embodiments, the method of producing the packaged composite includes a step of sterilization. For example, sterilizing the packaged composite can include sterilizing by autoclaving at 121°C for about 5-30 minutes. In various embodiments, the method of producing the packaged composite includes a step of autoclaving. In various embodiments, the method of producing the packaged composite includes a step of autoclaving at 121°C for about 5-30 minutes. In various embodiments, the method of producing the packaged composite includes a step of autoclaving at 121°C for about 5-30 minutes.

[0080] The method of producing the collagen hydrogel composite may further include processing the collagen hydrogel composite into a sheet or an injectable fluid.

[0081] In certain embodiments, the method may further include incubating infiltrating macrophages in the collagen hydrogel composite. In certain embodiments, the collagen hydrogel composite containing collagen nanofibers may condition a population of M2 phenotype infiltrating macrophages.

[0082] Medical application In various aspects, the collagen hydrogel composites may be applied in medical applications including, but not limited to, skin cosmetic, reconstruction, fat grafting, and wound care applications.

[0083] In various aspects, the collagen hydrogel composites exhibit minimal swelling, minimal tissue irritation, durable volumization, and / or robust neotissue generation suitable for the target tissue without fibrosis or foreign body reaction.

[0084] In various aspects, the collagen hydrogel composite, when implanted, injected, or placed in a target tissue or wound in vivo, should completely degrade or resorb over a time frame of 6 to 24 months.

[0085] Methods of treating a subject using the collagen-hydrogel composite are provided.

[0086] In one aspect, the present disclosure provides a method for forming adipose tissue in a subject. The method includes administering to the subject a collagen-hydrogel composite described herein. The collagen-hydrogel composite induces long-term in vivo retention of the collagen-hydrogel composite or enhanced host cell infiltration in the subject. In certain embodiments, the collagen-hydrogel composite includes cells or growth factors. In certain embodiments, the collagen-hydrogel composite does not include cells or growth factors.

[0087] In certain embodiments, the subject exhibits neovasculature promoted by M2 macrophage polarization. In certain embodiments, the subject exhibits about a 2.5-fold increase in a-SMA+ cells at the administration site. In certain embodiments, the subject exhibits localization of endothelial cells and CD163+ M2 macrophages at the administration site. In certain embodiments, the subject exhibits about a 1.5-fold increase in CD68+ pan-macrophages at the administration site by day 7 post-surgery. In certain embodiments, the subject exhibits about a 2-fold increase in CD68+ pan-macrophages at the administration site by day 14 post-surgery.

[0088] In certain aspects, the present disclosure also provides a method of delivering cells or tissues in a subject, the method comprising encapsulating one or more cells or tissues in a collagen-hydrogel composite described herein to form a suspension, and applying the suspension to a target site in the subject.

[0089] In another aspect, the present disclosure also provides a method for delivering adipose tissue to a subject. The method includes encapsulating one or more adipose tissues in a collagen-hydrogel composite described herein to form a suspension, and applying the suspension to a target site in the subject. In certain embodiments, the one or more adipose tissues preferably comprise adipose-derived stem cells, adipocytes, or a combination thereof.

[0090] In certain aspects, the present disclosure also provides a method of delivering a pharmaceutical agent in a subject, the method comprising combining the pharmaceutical agent with a collagen-hydrogel composite described herein to form a mixture, and applying the mixture to an intended delivery site.

[0091] In some aspects, the present disclosure also provides a method for delivering cells or tissues in a subject, the method comprising encapsulating one or more cells or tissues in a collagen-hydrogel composite described herein to form a flowable formulation, and applying the flowable formulation to a target site in the subject.

[0092] In certain aspects, the present disclosure also provides a method of delivering cells or tissues in a subject, the method comprising encapsulating one or more cells or tissues in a collagen-hydrogel composite described herein to form a mat, and applying the mat to a target site in the subject.

[0093] In certain aspects, the present disclosure also provides a method of delivering cells or tissues in a subject, the method comprising encapsulating one or more cells or tissues in a collagen-hydrogel composite described herein to form an injectable, and applying the injectable to a target site in the subject.

[0094] As discussed above, the collagen-hydrogel composites described herein can be advantageously used in many tissue repair situations, as well as in other applications, such as providing coatings on catheters and other surgical devices and implants. The collagen-hydrogel composites can also be used to deliver the active agents described herein, such as antibiotics, growth factors, and immunosuppressants. For example, the present disclosure provides methods for healing soft tissue defects, including applying a collagen-hydrogel composite to the soft tissue defect.

[0095] It is understood that the advantageous properties of the collagen-hydrogel composites described herein include: I) the ability to provide easy characterization and quality control; 2) the ability to integrate with existing tissue matrices; 3) the ability to be directly incorporated into newly formed matrices; 4) the ability to directly encapsulate cells and bioactive factors; 5) the ability to maintain biocompatibility; 6) the ability to control bioresorption; 7) the ability to be easily cast into complex anatomical shapes due to high structural rigidity resulting from the nanostructure; 8) the ability to exhibit mechanical properties of natural tissues such as articular cartilage; 9) the ability to crosslink the fiber and hydrogel phases in a single step; and 10) the ability to retain mechanical properties after terminal sterilization by autoclaving.

[0096] In another embodiment, the collagen-hydrogel composite can be used to repair cartilage tissue.

[0097] Current biologically based surgical procedures for cartilage repair include autologous chondrocyte implantation, drilling, abrasion chondroplasty, microfracture, and mosaic arthroplasty. All of these procedures treat only localized articular cartilage damage and do not treat cartilage-denuded joint surfaces, such as those seen in severe osteoarthritis and rheumatoid arthritis. These procedures also use either cartilage tissue plugs harvested from the patient or expanded chondrocytes to fill the cartilage defect. These tissues or chondrocytes are expected to fill the defect by integrating with the existing cartilage matrix and synthesizing an entirely new material, such as newly synthesized hyaline cartilage, that possesses the biomechanical properties of normal cartilage. However, all such procedures promote the formation of repair tissue (fibrocartilage) rather than true hyaline cartilage, and involve additional mechanical damage to the fibrocartilage, which is thought to predispose the joint to osteoarthritis. Furthermore, the availability of endogenous cartilage as a repair material is very limited and presents its own risks and morbidity to the patient. As is evident from the foregoing discussion, the resulting collagen hydrogel compositions disclosed herein represent practical materials for promising new therapies in patients suffering from cartilage degenerative diseases.

[0098] As described herein, collagen-hydrogel composites can be prepared with a variety of properties suitable for implanting or augmenting a number of synthetic tissues, as well as other clinical applications. As previously described, collagen-hydrogel composites can be used to repair cartilage defects resulting from either injury or disease. Injury-induced defects that can be so repaired may be sports- or accident-related and may involve only the superficial cartilage layer or may involve the underlying subchondral bone. Disease-induced defects that can be repaired using the compositions described herein include those resulting from osteoarthritis and rheumatoid arthritis. Whether resulting from injury or disease, such defects can be in either mature or growth plate cartilage. Formulations of hydrogels for synthetic growth plate cartilage may require the inclusion of a non-substituting scaffolding material to allow for controlled bioresorption of the biomaterial during growth.

[0099] Another area in which the collagen-hydrogel composites described herein may be useful is the repair, reconstruction, or augmentation of cartilaginous and soft tissues of the head and neck. The availability of biomaterials for soft tissue augmentation and head and neck reconstruction remains a fundamental challenge in the fields of plastic and reconstructive surgery. Considerable research and investment has been devoted to developing materials with suitable biocompatibility and longevity. The results of this research have not been encouraging. When placed in immunocompetent animals, the structural integrity of collagen-hydrogel composites has been shown to be compromised due to scaffold resorption. Furthermore, while traditional synthetic materials offer excellent longevity, they present certain unavoidable pitfalls. For example, silicone raises concerns about safety and long-term immune-related effects. The synthetic polymers PTFE (Gore-Tex) and Silastic offer less tissue reactivity but do not provide tissue integration and may represent a long-term risk of foreign body infection and extrusion. Collagen-hydrogel composites are useful for preparing synthetic soft tissue scaffold materials for head and neck augmentation or soft tissue defect repair. In particular, collagen hydrogel compositions that are non-inflammatory, non-immunogenic, and can be prepared to have an appropriate degree of viscoelasticity (see description herein) can be used as effective implantable scaffold materials.

[0100] Furthermore, collagen-hydrogel composites can be used as novel, biocompatible, and biocompliant materials for preparing cartilage implants, which are frequently used in head and neck reconstructive procedures to repair cartilage or bony defects secondary to, for example, trauma or congenital anomalies. Applications specific to the ear include otoplasty and auricular reconstruction, which are often performed to repair cartilage defects due to trauma, neoplasia (i.e., squamous cell carcinoma, basal cell carcinoma, and melanoma), and congenital defects, such as microtia. Applications specific to the nose include cosmetic and reconstructive procedures of the nose and nasal septum. Dorsal hump augmentation, tips, shields, and spreader grafts are frequently used in cosmetic rhinoplasty. Nasal reconstruction following trauma, neoplasia, autoimmune diseases such as Wegener's granulomatosis, or congenital defects requires cartilage for repair. Septal perforations are difficult to treat and often result in failed treatment.

[0101] Because autologous or donor cartilage is often unavailable, cartilage grafts would be ideal for these applications. Throat-specific applications include laryngotracheal reconstruction, which typically requires the harvesting of rib cartilage in children, which is not without morbidity. Auricular and septal cartilage are often insufficient for these applications. Synthetic cartilaginous materials prepared from the hydrogels disclosed herein can be synthesized to suit each of the aforementioned applications by adjusting hydrogel synthesis parameters, such as reagent concentration, substitution, and crosslinking rate. Laryngotracheal reconstruction is typically performed for airway narrowing due to subglottic and tracheal stenosis. The etiology can be traumatic (i.e., intubation trauma or tracheotomy) or idiopathic. Other possibilities include chin and cheek augmentation and use in lower eyelid eversion repair, in addition to many craniofacial applications. Note that these applications may not require cartilage with the stringent mechanical properties of articular cartilage. The inclusion of cell populations or bioactive agents may also be desirable.

[0102] The collagen-hydrogel composite described herein can be used for nasal cavity repair and narrowing, usually after overly aggressive surgical resection, to prevent chronic accumulation of fluid in the nasal passages, which leads to infection and crust formation. Another promising application is in laryngotracheal reconstruction in both children and adults as a result of laryngotracheal injury, for example, due to intubation during surgical procedures such as cardiovascular surgery. The collagen-hydrogel composite described herein can also be used to provide a cricoid cartilage replacement to protect the carotid artery after cancer resection in the neck. The collagen-hydrogel composite can be placed between the carotid artery and the skin as a protective barrier against loss of the skin barrier. As a protective coating during neuronal regrowth of resected nerves. Often, fibrous tissue forms faster than neuronal regrowth, preventing its eventual formation. Placing nerve endings within a pre-cast tube of the collagen-hydrogel composite can eliminate fibrous tissue formation from the regrowth site.

[0103] The collagen-hydrogel composite can also be used to repair soft tissue defects in any internal or external organ. For example, the collagen-hydrogel composite can be used for chin and cheek augmentation and lower eyelid eversion repair, in addition to many craniofacial applications. For cosmetic and reconstructive purposes in areas other than the head and neck, for example, for breast augmentation, for body sculpting, as a breast implant, or as a wound sealant to fill voids left after lymph node removal (e.g., due to cancer) in the breast or neck, sealing lymphatic vessels and reducing uncontrolled drainage to the excision site (which can lead to infection and other complications).

[0104] In addition to the uses described above, the collagen-hydrogel composites described herein can be used in other tissue engineering applications to generate synthetic orthopedic tissues, including, but not limited to, bone, tendons, ligaments, menisci, and intervertebral discs, using strategies and methods similar to those described above for the synthesis of artificial forms of cartilage. The collagen-hydrogel composites can also be used to create synthetic non-orthopedic tissues, including, but not limited to, vocal cords, vitreous humor, heart valves, liver, pancreas, and kidneys, using strategies and methods similar to those described above for the synthesis of artificial forms of cartilage.

[0105] Another area in which the collagen-hydrogel composites disclosed herein can be used is in gastrointestinal applications where it is necessary to treat or prevent the formation of scar tissue or strictures in the abdominal or gastrointestinal tract. Several products, generally referred to as "hydrogels," are already in various stages of clinical and FDA approval that are designed or intended to be useful in the treatment and prevention of scarring and / or stricture formation. The collagen-hydrogel composites disclosed herein are superior to other known hydrogels in that those disclosed herein may contain nanostructures that can provide support, shape, and strength to the hydrogel material. The collagen-hydrogel composites disclosed herein can be used in similar applications to those in which known hydrogels are used or intended to be used, such as for the treatment of strictures or scarring in the gastrointestinal tract. Treatment includes injection of the collagen-hydrogel composite at the site of a suspected stricture to prevent scarring, or at the site of a stricture that exists after therapy to dilate a narrowed GI tract to prevent the stricture from recurring.

[0106] The collagen-hydrogel composites described herein can also be used to treat esophageal strictures. Esophageal strictures are a common complication of gastroesophageal reflux disease (GERD). GERD is caused by acid, bile, and other harmful stomach contents backing up into the esophagus, damaging the cells lining the esophagus. Approximately 7-23% of GERD patients develop esophageal strictures, or fibrous scarring of the esophagus. Esophageal scarring can also be caused by ablation therapy, which is used to treat Barrett's esophagus. A major complication of such ablation therapy is that the ablation lesion extends too deeply into the esophageal wall, resulting in scarring or stricture of the esophagus. Esophageal strictures interfere with normal swallowing and are a significant cause of patient morbidity. The materials described herein can be used to treat or prevent esophageal strictures resulting from GERD, Barrett's esophagus, and esophageal ablation therapy.

[0107] The collagen-hydrogel composite may also be used to treat Crohn's disease, which causes strictures or scarring that block or narrow the lumen of the intestine, interfering with normal bowel function. For example, the collagen-hydrogel composite may be useful for treating or preventing such strictures.

[0108] The collagen-hydrogel composite can also be used in methods for treating primary sclerosing cholangitis (PSC). PSC is a rare disease of the liver's bile ducts. Bile ducts form a branching network within the liver and exit the liver through two major branches that merge into a common bile duct, which drains bile from the liver and gallbladder into the duodenum. Although bile ducts are very narrow in diameter, typically only 2 mm at their largest and most distal portions, they must normally drain many liters of bile from the liver into the duodenum every day. Blockage of these ducts can lead to a serious condition known as jaundice, which causes the accumulation of many toxins, especially hemoglobin breakdown products, in the body. PSC is a scarring or stricture disease of the bile ducts within the liver and the extrahepatic bile ducts that connect the liver to the small intestine. Bile duct strictures in PSC can be treated or prevented using the collagen hydrogel composition of the present invention.

[0109] The collagen-hydrogel composite can also be used to treat chronic pancreatitis. Chronic pancreatitis is a chronic inflammatory disease of the pancreas that can be exacerbated by scarring or narrowing of the pancreatic duct. These narrowings block the drainage of pancreatic juice, which normally must leave the pancreas through a duct system or drainage channel into the small intestine. Pancreatic juice contains many digestive enzymes and other components important for normal digestion and nutrient absorption. Blockage or narrowing of the pancreatic duct due to chronic pancreatitis can lead to serious complications, such as the pancreas becoming autodigestive and forming life-threatening abdominal infections and / or abscesses. Pancreatic narrowing in chronic pancreatitis can be treated or prevented using the hydrogel of the present invention.

[0110] The collagen-hydrogel composite can also be used to treat gallstone-induced strictures of the bile duct and pancreatic duct. Gallstones are a very common disorder, and their main complication is the formation of strictures of the bile duct and pancreatic duct, which can be treated or prevented using hydrogels. For the treatment of ischemic bowel disease. The intestine is prone to scarring or stricture formation when blood supply is impaired. Impaired blood flow is called ischemia and can be caused by many pathologies, such as cardiovascular disease, atherosclerosis, hypertension, hypovolemia, renal or liver disease-induced hypoalbuminemia, vasculitis, drug-induced diseases, and many others. The end result of all of these pathologies can lead to intestinal strictures, which block the intestine and prevent its normal function. The collagen hydrogel composite of the present invention can be used to treat or prevent ischemic bowel strictures.

[0111] The collagen-hydrogel composite can also be used to treat radiation-induced intestinal strictures. Cancer radiation therapy is associated with many morbidities, the most important of which is the formation of intestinal strictures. The collagen-hydrogel composite can be used to treat or prevent radiation-induced intestinal strictures.

[0112] In addition to creating synthetic tissue or repairing natural tissue, the collagen-hydrogel composites disclosed herein can also be used to provide coatings for non-biological structures or devices to be used in surgery or otherwise for in vivo implantation, such as surgical instruments or ceramic or metallic prosthetic devices. Such coatings would provide a barrier between the non-biological device material and living tissue. Roles of collagen-hydrogel composites as barriers for non-biological devices include, but are not limited to: I) preventing the absorption of macromolecules and / or cells onto the surface of non-biological devices, which can lead to protein fouling or thrombus formation on the device surface; 2) presenting a non-toxic, non-inflammatory, non-immunogenic, biologically compatible surface for devices fabricated from materials that are otherwise not biologically compatible; 3) being compatible with device function, such as glucose diffusion to a glucose sensor, mechanical force transmission to a pressure sensor, or endothelialization of a vascular graft or stent; 4) enhancing device function, such as providing a charge barrier to the size barriers present in MEMS-based artificial nephrons; 5) incorporating viable cell populations into non-biological devices, trapped within an aqueous, physiologically compatible environment; and 6) including drugs or bioactive factors, such as growth factors, antivirals, antibiotics, or adhesion molecules, designed to promote vascularization, epithelialization, or endothelialization of the device.

[0113] Based on the foregoing, collagen-hydrogel composites can be used to provide non-allergenic coatings for various implantable devices, including implantable glucose sensors for diabetes management. Additionally, collagen-hydrogel composites can be used to provide: a charge barrier for the development of MEMS-based artificial nephrons; an aqueous, physiologically compatible environment in which implanted kidney cells, such as podocytes, can be incorporated into MEMS-based artificial nephron designs; and coatings for implantable MEMS devices designed for various purposes, including, but not limited to, for drug delivery, mechanical sensing, and as biodetection systems.

[0114] The disclosed collagen-hydrogel composites, particularly those containing HA, may also be covalently attached to silicon-based devices, for example, by first covalently attaching the primary amines of tyramine to the silicon surface to provide a hydroxyphenyl-coated surface chemistry.

[0115] This can use the same chemistry as that used to attach DNA modified with free amines to silicon surfaces. The HA-based collagen-hydrogel composite is then covalently attached to the hydroxyphenyl-coated surface using the same peroxidase-driven chemistry used in the preferred crosslinking mode described above.

[0116] Collagen-hydrogel composites can also be used to coat non-biologic cardiovascular devices, such as catheters, stents, and vascular grafts. These would include devices made from materials not traditionally used due to their bioincompatibility, but with design features superior to currently used devices. Bioactive factors can be incorporated into the hydrogel to promote endothelialization or epithelialization of the hydrogel and, therefore, of the implanted device.

[0117] The composite, which retains 85% of its initial volume for over one year, produces vascularized adipose tissue, making it an excellent candidate for dermal filling applications and body sculpting.

[0118] Composites exhibiting more gel spreading for greater diffusion efficiency may be desired for superficial placement within the body. Lower cross-linking and collagen concentration, and the resulting larger pore size, allow for more rapid cell migration and diffusion for therapeutic applications for cell or drug delivery, or to increase cell homing and interaction within the implanted gel.

[0119] Composites that exhibit slower cell infiltration, maintain their original shape, and resist spreading. These formulations are excellent candidates for use when implant characteristics such as slow degradation and geometric stability are desirable. Examples include implanting devices in areas of high tissue tension or weight bearing locations, such as supraperiosteal placement in the body, or for applications where a more permanent plug is desired, such as fistula repair, hernia repair, or coating of permanent implants.

[0120] An ideal soft tissue filler would initially immediately fill the defect site with 100% of the target volume, have mechanical properties similar to the surrounding soft tissue, avoid filler migration or flow, and induce no pain or irritation. The filler should not induce swelling and, moreover, should maintain nearly 100% of its volume indefinitely, while, paradoxically, completely degrading without causing scarring or adverse events (e.g., nodules or granulomas). The formulations disclosed herein come much closer to achieving this "ideal" profile than any current product, such as the Voluma XC® control included in this study.

[0121] Development of HA DVS cross-linked formulation One advantage of the present disclosure is that it limits swelling after treatment.

[0122] In various embodiments, the method for producing the collagen hydrogel composite has the advantage of obtaining a gel with good mechanical properties and a hyaluronic acid concentration of approximately 7-12 mg / mL to limit swelling after treatment.

[0123] By varying the HA concentration and the ratio of DVS to HA, various embodiments vary how much the HA gel swells during the manufacturing process (swelling ratio), and also vary the HA concentration in the final device.

[0124] In general, the higher the HA concentration during gelation, the more tightly crosslinked the resulting gel will be, and therefore the lower the swelling ratio. However, the higher the initial HA concentration during gelation, the greater the swelling ratio required during purification to reduce the HA concentration to the target range. At a gelation HA concentration of 21 mg / mL, the gel swelled 4.88-fold, reducing the final device HA concentration to 4.3 mg / mL, which is below the target. These experiments demonstrated that gelation HA concentrations of 27 or 30 mg / mL were able to produce final gels after purification with the target window of 7-12 mg / mL (experimental concentrations of 7.8 mg / mL to 10.7 mg / mL were obtained).

[0125] At a fixed initial gelling HA concentration of 30 mg / mL, DVS ratios of 3.22, 4.5, and 5.0 produced gels that fell within the targeted final HA concentration range (Figure 6). Increasing DVS concentration had a gradual decrease in swelling ratio and, therefore, final gel HA concentration (increasing crosslink concentration results in increased crosslink density and decreased swelling). Over this relatively wide range of DVS ratios (3.22-5.0), HA gelling concentration is a sensitive variable for final gel properties. The graph in Figure 7 shows a weaker correlation between DVS ratio and final HA concentration when a wider range of HA gelling concentrations (21-30 mg / mL) was used.

[0126] Based on HA experiments, various embodiments of the present disclosure include hyaluronic acid concentrations ranging from about 27 to about 30 mg / mL at gelation, with a ratio of DVS to HA subunits of 3.22 to 5x (or a DVS to HA hydroxyl molar ratio of 0.8:1 to 1.25:1).

[0127] Collagen content had an unexpectedly large effect on gel swelling ratio, necessitating further evaluation of gelation conditions to achieve the target final gel properties. On the other hand, HA gelation concentration had a much smaller direct effect on swelling ratio in the presence of collagen fibers. Without being bound by theory, fibers may significantly limit the ability of hydrogels to swell during fabrication because they are the second most abundant solid phase covalently attached to hyaluronic acid gels to form composites. Because collagen content limited the amount of gel swelling during purification, HA needed to be at a lower concentration during gelation to achieve the target final concentration.

[0128] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by the divinyl sulfone, the hyaluronic acid being present in an amount ranging from about 5 mg / mL to about 15 mg / mL, and the collagen being present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0129] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by the divinyl sulfone, the hyaluronic acid being present in an amount ranging from about 5 mg / mL to about 15 mg / mL, the collagen being present in an amount ranging from about 10 mg / mL to about 40 mg / mL, and the divinyl sulfone being present in an amount ranging from about 3:1 to about 5:1 prior to crosslinking.

[0130] In one embodiment, the nanofiber-hydrogel composite comprises a hydrogel material comprising hyaluronic acid, a nanofiber material comprising collagen, and a crosslinker comprising divinyl sulfone, wherein the hyaluronic acid is covalently bonded to the collagen by divinyl sulfone, the amount of crosslinking (an HA subunit bound to another subunit of HA or to collagen via DVS) is in the range of about 0.1% to about 30%, the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL, and the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0131] In one embodiment, the nanofiber-hydrogel composite comprises the product of a process comprising reacting a hyaluronic acid material, collagen fibers, and divinyl sulfone, whereby the hyaluronic acid is covalently bonded to the collagen fibers by the divinyl sulfone, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL, and the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit in a ratio ranging from about 0.5:1 to about 5:1.

[0132] In one embodiment, the present disclosure provides a method for making a nanofiber-hydrogel composite, comprising the step of reacting a hyaluronic acid material, collagen fibers, and divinyl sulfone, whereby the hyaluronic acid is covalently bonded to the collagen fibers by the divinyl sulfone, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL, and the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit in a ratio ranging from about 0.5:1 to about 5:1.

[0133] Swelling of the composite The composites of the present disclosure may swell during manufacturing (in vitro) or after administration to a subject (in vivo), such as by injection, implantation, or application. For clarity, "swelling ratio" refers to swelling during manufacturing, while "swelling percentage" (swell%) refers to swelling of the final device in vivo. Swelling ratio is the multiple of the increase in volume from the initial volume. Swelling ratio can be expressed as a rational number with the integer 1 as the denominator. Swelling % is the percentage increase in volume of the implant in the body or physiologically equivalent conditions.

[0134] Swelling ratio - during manufacturing In various embodiments, the composite swells a certain amount during the manufacturing process.

[0135] In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 4.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 1.5 to about 4.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 1.0 to about 3.0.

[0136] In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 2.0 to about 3.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 1.0 to about 2.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 1.0.

[0137] In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 2.0 to about 3.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 2.2 to about 3.0. In various embodiments, the composite comprises a swelling ratio during manufacture ranging from about 2.3 to about 2.8.

[0138] In various embodiments, the composite comprises a swelling ratio of about 2.3 during manufacture.

[0139] In various embodiments, the composite comprises a swelling ratio of about 2.4 during manufacture.

[0140] In various embodiments, the composite comprises a swelling ratio of about 2.6 during manufacture.

[0141] In various embodiments, the composite comprises a swelling ratio of about 2.8 during manufacture.

[0142] Swelling after treatment (in vivo) In various embodiments, the composite swells a certain amount when placed in vivo.

[0143] In various embodiments, the composite comprises a % swelling in vivo of up to 100%.

[0144] In various embodiments, the composite comprises a % swelling in vivo of up to 75%. In various embodiments, the composite comprises a % swelling in vivo of up to 50%. In various embodiments, the composite comprises a % swelling in vivo of up to 40%. In various embodiments, the composite comprises a % swelling in vivo of up to 30%. In various embodiments, the composite comprises a % swelling in vivo of up to 20%. In various embodiments, the composite comprises a % swelling in vivo of up to 15%. In various embodiments, the composite comprises a % swelling in vivo of up to 12%. In various embodiments, the composite comprises a % swelling in vivo of up to 10%.

[0145] In various embodiments, the composite comprises a % swelling in vivo of up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0146] In various embodiments, the composite comprises a % swelling in vivo ranging from about 0% to about 100%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 1% to about 100%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 1% to about 50%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 1% to about 20%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 1% to about 10%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 10% to about 20%. In various embodiments, the composite comprises a % swelling in vivo ranging from about 10% to about 15%.

[0147] In various embodiments, the composite comprises a % swelling in vivo of about 50%, 40%, 30%, 20%, or 10%.

[0148] In various embodiments, the composite comprises a % swelling in vivo of about 10%, 11%, 12%, 13%, 14%, or 15%.

[0149] In various embodiments, the composite comprises a % swelling in vivo of about 20%.

[0150] In various embodiments, the composite comprises a % swelling in vivo of about 12%.

[0151] In one embodiment, swelling is reduced by 86% compared to Voluma; 14% vs. 100%.

[0152] In one embodiment, swelling is reduced by 80% compared to Voluma. 20% vs. 100%.

[0153] In one embodiment, swelling is reduced by 88% compared to Voluma. 12% vs. 100%.

[0154] In one embodiment, swelling is reduced by 84% compared to Voluma. 16% vs. 100%.

[0155] In various embodiments, the composite comprises a swelling ratio in the range of about 0.1 to about 3.0 when placed in vivo.

[0156] In various embodiments, the composite comprises a swelling ratio ranging from about 1.0 to about 3.0, from about 2.0 to about 3.0, from about 1.0 to about 2.0, or from about 0.1 to about 1.0.

[0157] In various embodiments, the composite comprises a swelling ratio of about 2.3, about 2.4, about 2.6, or about 2.8. The composite comprises a swelling percentage in or on the body (in vivo) ranging from about 12% to about 20%.

[0158] Exemplary data is provided in FIG.

[0159] hyaluronic acid As used herein, "hyaluronic acid" or "HA" is commonly referred to as a non-sulfated glycosaminoglycan. HA is a polymer of disaccharide monomers. The disaccharide monomers of hyaluronic acid may be referred to as "hyaluronic acid subunits" or "HA subunits." HA subunits contain D-glucuronic acid and N-acetylglucosamine. HA subunits are linked to each other by alternating beta-1,4 and beta-1,3 glycosidic bonds.

[0160] The IUPAC name for hyaluronic acid is (1→4)-(2-acetamido-2-deoxy-D-gluco)-(1→3)-D-glucuronoglycan. A diagram of the chemical structure of hyaluronic acid is shown in Figure 1. An exemplary representation of divinyl-sulfone cross-linked hyaluronic acid is shown in Figure 2.

[0161] In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 1 MDa to about 2 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 1 MDa to about 1.5 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 1.5 MDa to about 2 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 500 kDa to about 1 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 500 kDa to about 500 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 100 kDa to about 500 kDa.

[0162] In various embodiments, the hyaluronic acid comprises a molecular weight of about 100 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 200 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 300 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 400 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 500 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 600 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 700 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 800 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 900 kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.1 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.2 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.3 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.4 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.5 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.6 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.7 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.8 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.9 MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 2 MDa.

[0163] Hyaluronic acid concentration Hyaluronic acid concentration at gelation In various embodiments, the gelling conditions of the composite include hyaluronic acid present in an amount ranging from about 20 mg / mL to about 30 mg / mL, from about 20 mg / mL to about 25 mg / mL, or from about 23 mg / mL to about 25 mg / mL.

[0164] In various embodiments, the gelling conditions of the composite include hyaluronic acid present in an amount of about 23 mg / mL or about 25 mg / mL.

[0165] Concentration of hyaluronic acid in the composite (finished device / post-gelation) In various embodiments, the composition comprises hyaluronic acid present in an amount ranging from about 5 mg / mL to about 10 mg / mL, or from about 9 mg / mL to about 10 mg / mL.

[0166] In various embodiments, the composite comprises hyaluronic acid present in an amount of about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 10.5 mL, about 11 mL, about 11.5 mL, about 12 mL, or about 12.5 mL.

[0167] Collagen concentration Collagen fiber concentration during gelation In various embodiments, the gelling conditions of the composite include collagen fibers present in an amount ranging from about 30 mg / mL to about 100 mg / mL, from about 40 mg / mL to about 70 mg / mL, or from about 45 mg / mL to about 65 mg / mL.

[0168] In various embodiments, the gelling conditions of the composite include collagen fibers present in an amount of about 45 mg / mL or about 65 mg / mL.

[0169] Concentration of collagen fibers in the composite In various embodiments, the composite comprises collagen present in an amount ranging from about 10 mg / mL to about 40 mg / mL, from about 15 mg / mL to about 30 mg / mL, from about 20 mg / mL to about 30 mg / mL, or from about 25 mg / mL to about 30 mg / mL.

[0170] In various embodiments, the composite comprises collagen present in an amount of about 15 mg / mL, about 30 mg / mL, or about 25 mg / mL.

[0171] DVS concentration In various embodiments, the gelling conditions for the composite include divinyl sulfone present in an amount ranging from about 1:1 to about 5:1 divinyl sulfone molecules per hyaluronic acid subunit, from about 2:1 to about 5:1 divinyl sulfone molecules per hyaluronic acid subunit, from about 3:1 to about 5:1 divinyl sulfone molecules per hyaluronic acid subunit, from about 3.5:1 to about 4:1 divinyl sulfone molecules per hyaluronic acid subunit.

[0172] In various embodiments, the gelling conditions of the composite include divinyl sulfone present in an amount of about 3.5:1 divinyl sulfone molecules per hyaluronic acid subunit, about 4:1 divinyl sulfone molecules per hyaluronic acid subunit, or about 4.5:1 divinyl sulfone molecules per hyaluronic acid subunit.

[0173] In various embodiments, the gelling conditions for the composite include divinyl sulfone present in an amount of about 230 mM divinyl sulfone, about 240 mM divinyl sulfone, about 260 mM divinyl sulfone, or about 270 mM divinyl sulfone.

[0174] pore A scaffold composite is generally not a uniform solid material. Instead, it contains a plurality of pores present on or within the surface of the scaffold composite. The presence, size, distribution, frequency, and other parameters of the pores can be modulated during production of the scaffold composite. The average pore size can be from about less than 1 micron up to 100 microns, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 microns. The average pore size can be narrowly tailored, e.g., so that at least 40%, e.g., 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of the pores are of a desired size or within a desired size range.

[0175] In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 100 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 75 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 50 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 25 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 10 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 5 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 1 micron to about 2 microns.

[0176] In various embodiments, the scaffold composite comprises an average pore size ranging from about 10 microns to about 100 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 25 microns to about 100 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 50 microns to about 100 microns. In various embodiments, the scaffold composite comprises an average pore size ranging from about 75 microns to about 100 microns.

[0177] In various embodiments, the scaffold composite comprises an average pore size of about 1 micron. In various embodiments, the scaffold composite comprises an average pore size of about 2 microns. In various embodiments, the scaffold composite comprises an average pore size of about 3 microns. In various embodiments, the scaffold composite comprises an average pore size of about 4 microns. In various embodiments, the scaffold composite comprises an average pore size of about 5 microns. In various embodiments, the scaffold composite comprises an average pore size of about 6 microns. In various embodiments, the scaffold composite comprises an average pore size of about 7 microns. In various embodiments, the scaffold composite comprises an average pore size of about 8 microns. In various embodiments, the scaffold composite comprises an average pore size of about 9 microns. In various embodiments, the scaffold composite comprises an average pore size of about 10 microns.

[0178] In various embodiments, the scaffold composite comprises an average pore size of about 15 microns. In various embodiments, the scaffold composite comprises an average pore size of about 20 microns. In various embodiments, the scaffold composite comprises an average pore size of about 25 microns. In various embodiments, the scaffold composite comprises an average pore size of about 30 microns. In various embodiments, the scaffold composite comprises an average pore size of about 40 microns. In various embodiments, the scaffold composite comprises an average pore size of about 50 microns. In various embodiments, the scaffold composite comprises an average pore size of about 60 microns. In various embodiments, the scaffold composite comprises an average pore size of about 70 microns. In various embodiments, the scaffold composite comprises an average pore size of about 75 microns. In various embodiments, the scaffold composite comprises an average pore size of about 80 microns. In various embodiments, the scaffold composite comprises an average pore size of about 90 microns. In various embodiments, the scaffold composite comprises an average pore size of about 100 microns.

[0179] Pore ​​size uniformity In various embodiments, the pore size is substantially uniform. In various embodiments, the pore size is within a narrow size distribution.

[0180] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 100 microns of each other.

[0181] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 10 microns of each other.

[0182] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 1 micron of each other.

[0183] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 100 microns of the average pore size.

[0184] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 10 microns of the average pore size.

[0185] In various embodiments, the pores comprise a substantially uniform size distribution, with at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores being within about 1 micron of the average pore size.

[0186] The pores may also be provided at a sufficient density that two or more pores touch each other and overlap in volume, forming tunnels between them. These tunnels may be substantially devoid of composite material forming the pore scaffold. The tunnels may be predominantly circular in shape or may be more irregular in shape. The shortest distance between the ends of these tunnels may be from less than about 1 micron up to 100 microns, for example, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 microns.

[0187] Reduced toxicity In various aspects, the present disclosure describes a method for reducing the toxicity of divinyl sulfone-crosslinked materials containing pendant vinyl groups, the method comprising reacting the pendant vinyl sulfone groups with a quenching agent, thereby converting the pendant vinyl sulfone groups to a less toxic material. The present disclosure also reduces the toxicity of any unreacted, free-floating DVS that may remain in the gel after processing. Without being bound by theory, the compositions of the present invention are converted to less toxic compositions because the material is converted to groups that lack or have limited reactivity for crosslinking or alkylating proteins and biological components in vivo. In various embodiments, a "less toxic material" is a material that lacks or has limited reactivity for crosslinking or alkylating proteins and biological components in vivo. In various embodiments, "reducing toxicity" refers to limiting the reactivity of the material for crosslinking or alkylating proteins and biological components in vivo.

[0188] Quenching Agent In various embodiments, the quenching agent comprises one or more nucleophiles or Lewis bases. Non-limiting examples of nucleophiles include thiols (also known as sulfhydryl groups), amines, hydroxyls, or halogens. In various embodiments, the quenching agent comprises a molecule comprising a thiol (also known as sulfhydryl groups), amines, hydroxyls, or halogens. In various embodiments, the halogen is fluorine, chlorine, bromine, or iodine.

[0189] In various embodiments, the quenching agent comprises one or more amine groups. In various embodiments, the quenching agent comprises one or more thiol groups. In various embodiments, the quenching agent comprises one or more hydroxyl groups. In various embodiments, the quenching agent comprises any combination of amine groups, thiol groups, or hydroxyl groups.

[0190] In various embodiments, the quenching agent comprises two or more nucleophiles. The two or more nucleophiles can be the same or different. In various embodiments, the quenching agent comprises an amine and a thiol.

[0191] In various embodiments, the quenching agent comprises two or more amines.

[0192] In various embodiments, the quenching agent comprises two or more thiols.

[0193] In various embodiments, the quenching agent comprises two or more hydroxyls.

[0194] In various embodiments, the quenching agent comprises an amine and a thiol.

[0195] In various embodiments, the quenching agent comprises an amine and a hydroxyl.

[0196] In various embodiments, the quenching agent comprises a thiol and a hydroxyl.

[0197] In various embodiments, the quenching agent comprises a polyamine molecule. In various embodiments, the quenching agent comprises a polythiol molecule. In various embodiments, the quenching agent comprises a polyhydroxyl molecule.

[0198] In various embodiments, the quenching agent comprises two or more nucleophiles.

[0199] In various embodiments, the quenching agent comprises two or more nucleophiles, and at least one nucleophile comprises an amine.

[0200] In various embodiments, the quenching agent comprises two or more nucleophiles, at least one of the nucleophiles comprises an amine, and the amine remains available for subsequent reactions after the quenching reaction.

[0201] In various embodiments, the amine in the primary amine after the quenching reaction.

[0202] In various embodiments, the quenching reaction forms a quenched pendant group, wherein the quenched pendant group comprises an amine, a thiol, or a hydroxyl. In various embodiments, the quenched pendant group comprises an amine. In various embodiments, the quenched pendant group comprises a primary amine. In various embodiments, the quenched pendant group comprises a thiol. In various embodiments, the quenched pendant group comprises a primary thiol. In various embodiments, the quenched pendant group comprises a hydroxyl. In various embodiments, the quenched pendant group comprises a primary hydroxyl. Exemplary quenching agents include, but are not limited to, cysteamine, ethylenediamine (EDA), and amino acids.

[0203] In various embodiments, amino acids include, but are not limited to, cysteine ​​(with a reactive thiol group), lysine (with a primary amine), arginine, histidine, glycine, glutamic or aspartic acid (anionic), leucine (increases hydrophobicity), and asparagine (amide), methionine, serine (hydroxyl), and threonine (hydroxyl).

[0204] In various embodiments, amino acids include, but are not limited to, essential amino acids, non-essential amino acids, and amino acid derivatives.

[0205] A "non-essential" amino acid residue, as used herein, is an amino acid residue present in the wild-type sequence of a polypeptide that can be altered without abolishing or substantially altering an essential biological or biochemical activity of the polypeptide (e.g., receptor binding or activation).

[0206] An "essential" amino acid residue, as used herein, is an amino acid residue present in the wild-type sequence of a polypeptide that, when altered, results in abrogating or substantially reducing an essential biological or biochemical activity of the polypeptide (e.g., receptor binding or activation).

[0207] In various embodiments, the amino acid comprises any isomer of the amino acid. In various embodiments, the amino acid or amino acid analog is racemic. In various embodiments, the L-isomer of the amino acid is used. In various embodiments, the D-isomer of the amino acid is used. In various embodiments, the amino acid comprises a chiral center that is in the R or S configuration. In various embodiments, the chiral center is in the R configuration. In various embodiments, the chiral center is in the S configuration.

[0208] In various embodiments, the amino acid includes, but is not limited to, an amino acid comprising an unblocked nucleophilic side chain. In various embodiments, the amino acid includes, but is not limited to, an amino acid comprising an amino group of a β-amino acid substituted with a protecting group. Amino protecting groups include, but are not limited to, tert-butoxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), and toluenesulfonyl (tosyl, Ts). In various embodiments, the amino acid comprises an amino group substituted with a protecting group selected from the group consisting of tert-butoxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), and toluenesulfonyl (tosyl, Ts). In various embodiments, the carboxylic acid functionality of the β-amino acid is protected. In various embodiments, the carboxylic acid is protected as an ester derivative. In various embodiments, the nucleophilic side chain is protected. In various embodiments, a salt of the amino acid analog is used. In various embodiments, the salt is a pharmaceutically acceptable salt.

[0209] In various embodiments, the amino acid comprises a capping group. The term "capping group" refers to a chemical moiety that occurs at either the carboxy- or amino-terminus of a polypeptide chain. Carboxy-terminal capping groups include unmodified carboxylic acids (i.e., -COOH) or substituted carboxylic acids. For example, the carboxy-terminus can be substituted with an amino group to create a carboxamide at the C-terminus. Various substituents include, but are not limited to, primary amines and secondary amines, such as, but not limited to, pegylated secondary amines. Amino-terminal capping groups include unmodified amines (i.e., -NH2) or substituted amines. For example, the amino-terminus can be substituted with an acyl group to create a carboxamide at the N-terminus. Various substituents include, but are not limited to, substituted acyl groups, such as, for example, C1-C6 carbonyl, C7-C8 carbonyl, C9-C10 carbonyl, C11-C12 carbonyl, C12-C14 carbonyl, C13-C15 carbonyl, C14-C16 carbonyl, C15-C16 carbonyl, C16-C18 carbonyl, C17-C18 carbonyl, C18-C19 carbonyl, C19-C20 carbonyl, C19-C21 carbonyl, C19-C22 carbonyl, C19-C23 carbonyl, C19-C24 carbonyl, C19-C25 carbonyl, C19-C26 carbonyl, C19-C27 carbonyl, C19-C28 carbonyl, C19-C29 carbonyl, C20-C29 carbonyl, C21-C25 carbonyl, C22-C25 carbonyl, C23-C24 carbonyl, C24-C25 carbonyl, C25-C26 carbonyl, C25-C27 carbonyl, 30 Polypeptides can also be used as quenching agents.

[0210] In various embodiments, the quenching agent comprises a polypeptide. In various embodiments, the polypeptide comprises a bioactive sequence.

[0211] In various embodiments, the bioactive sequence comprises cell recognition or cell adhesion activity. In various embodiments, the bioactive sequence comprises a sequence present in a cell adhesion domain of a biological polymer. In various embodiments, the bioactive sequence comprises a sequence present in a cell adhesion domain of a biological polymer including fibronectin, vitronectin, laminin, or collagen. In various embodiments, the bioactive sequence comprises Arg-Gly-Asp (RGD). In various embodiments, the bioactive sequence comprises Arg-Gly-Asp-Ser (RGDS), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Thr (RGDT), and Ile-Lys-Val-Ala-Val (IKVAV).

[0212] In various embodiments, the quenching agent comprises an amine (also referred to as an amine quenching agent). In various embodiments, the quenching agent includes, but is not limited to, an amine quenching agent.

[0213] In various embodiments, the quenching agent comprises a polyamine, including but not limited to ethylenediamine (EDA), polyethyleneamine, putrescine, spermine, thermospermine, and spermidine.

[0214] In various embodiments, the quenching agent comprises a thiol, hi various embodiments, the thiol includes, but is not limited to, propane-1,3-dithiol and dimercaprol.

[0215] In various embodiments, the quenching agent comprises an aminothiol. As referred to herein, an aminothiol is a molecule containing both an amine functional group and a thiol functional group. In various embodiments, the quenching agent includes, but is not limited to, cysteamine, ((aminopropyl)amino)ethanethiol, 3-(methylamino)-2-((methylamino)methyl)propane-1-thiol, or 3-amino-2-(aminomethyl)propane-1-thiol.

[0216] The advantage of using amino acids, amines, polyamines, or their derivatives as quenching agents is the resulting ability to promote cell adhesion and migration. Without being bound by theory, the incorporation of amino acids such as lysine or amine-containing molecules such as EDA may result in amines present on the polymer backbone, thereby promoting cell adhesion and migration. Cell adhesion and migration may be desirable for cell-permeable gels.

[0217] pH of the quenching reaction Without being bound by theory, functional groups with higher reactivity allow for faster reaction rates. For example, a lower reaction time is an example of a lower reaction rate factor, which may allow for the use of a wider range of pH conditions. In various embodiments, the quenching agent comprises a highly reactive functional group. Without being bound by theory, the quenching agent is more reactive when it comprises a functional group that deprotonates more easily or at a lower pH.

[0218] In various embodiments, the quenching agent is any suitable agent capable of reacting as a nucleophile with a vinyl group during a Michael addition reaction. In various embodiments, the quenching agent is any suitable agent capable of reacting as a nucleophile with a vinyl sulfone group during a Michael addition reaction. In various embodiments, the quenching agent comprises a deprotonating functional group. In various embodiments, the quenching agent comprises a functional group that readily deprotonates. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 14. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 13. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 12. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 11. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 10. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH less than 9. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH below 8.

[0219] The advantage of using a quenching agent containing a more reactive or nucleophilic functional group species, such as a thiol or primary amine (compared to the groups to be crosslinked, such as the hydroxyls of hyaluronic acid), is that quenching can alternatively be performed at a lower pH, halting further modification of HA by DVS and limiting component degradation due to hydrolysis at high pH. Alternatively, the pH can be lowered to 10 or below before adding the quenching agent, allowing for significantly longer reaction times if necessary to further reduce the pendant concentration. Thiols can react with vinyl sulfone groups at neutral pH, so when using a quenching agent such as cysteine, the pH can be lowered to around 7 and the reaction can be allowed to continue for a longer period to complete quenching of the pendant groups (see Yu Y. et al., Biomacromolecules 13.3 (2012): 937-942).

[0220] In various embodiments, the quenching agent comprises an amine and / or a thiol. Without being bound by theory, amines and thiols maximize reaction rates at high pH, ​​thereby minimizing quenching times. A quenching pH higher than the pKa value for the quenching group (e.g., ionizable amine side chains of amino acids and / or peptide amine groups) but lower than the pH at which substantial HA hydrolysis occurs may allow for longer quenching times for more complete quenching while minimizing degradation of the HA backbone. For example, because the pKa of the lysine side chain amino group in water is 10.4 (Isom, Daniel G., et al. "Large shifts in pKa values ​​of lysine residues buried inside a protein." Proceedings of the National Academy of Sciences 108.13 (2011): 5260-5265), at pHs above 10.4 but below the pH at which substantial HA degradation occurs (pH > 11), the amine group may be deprotonated and suitable for reaction with vinyl sulfone. High pHs above 11, especially 13, have been associated with HA degradation via hydrolytic cleavage of the HA backbone (A. Maleki, et al., "Effect of pH on the behavior of hyaluronic acid in dilute and semidilute aqueous solutions," Macromolecular Symposia. Vol. 274. No. 1. Weinheim: WILEY-VCH Verlag, 2008). Similarly, the pKa of the thiol side chain of cysteine ​​in water is 8.6, which provides a favorable quenching pH range below the onset pH of accelerated HA hydrolysis (G. Roos, et al., "Understanding the pKa of redox cysteines: The key role of hydrogen bonding." Antioxidants & redox signaling 18.1 (2013): 94-127).

[0221] In various embodiments, the quenching reaction step is carried out at a pH related to the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH equal to or greater than the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH about equal to the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH higher than the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH about 1.0 pH unit (relative to pKa) higher than the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH about 2.0 pH units higher than the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH about 3.0 pH units higher than the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH ranging from about 0 to about 1.0 pH units above the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH ranging from about 0.1 to about 1.0 pH units above the pKa of the quenching agent. In various embodiments, the quenching reaction step is carried out at a pH ranging from about 0.5 to about 1.0 pH units above the pKa of the quenching agent.

[0222] In one non-limiting example, lysine has a side chain amine with a pKa of about 10.8. In various embodiments, the quenching step using lysine as the quenching agent is carried out at a pH ranging from about 12 to about 13. In another non-limiting example, cysteine ​​has a side chain thiol with a pKa of about 8.3. In various embodiments, the quenching step using cysteine ​​as the quenching agent is carried out at a pH ranging from about 9 to about 10.

[0223] In various embodiments, the quenching reaction step is carried out at a pH of less than about 12. In various embodiments, the quenching reaction step is carried out at a pH of less than about 11. In various embodiments, the quenching reaction step is carried out at a pH of less than about 10.0. In various embodiments, the quenching reaction step is carried out at a pH of less than about 9.0. In various embodiments, the quenching reaction step is carried out at a pH of less than about 8.0.

[0224] In various embodiments, the quenching reaction is carried out at a pH ranging from about 8.0 to about 12.0. In various embodiments, the quenching reaction is carried out at a pH ranging from about 9.0 to about 11.0. In various embodiments, the quenching reaction is carried out at a pH ranging from about 8.0 to about 9.0. In various embodiments, the quenching reaction is carried out at a pH ranging from about 9.0 to about 10.0. In various embodiments, the quenching reaction is carried out at a pH ranging from about 10.0 to about 11.0.

[0225] In one embodiment, the quenching reaction is carried out at a pH ranging from about 8.6 to about 12.0. In one embodiment, the quenching reaction is carried out at a pH ranging from about 10.4 to about 11.0. In one embodiment, the quenching reaction is carried out at a pH ranging from about 10.4 to about 11.4. In one embodiment, the quenching reaction is carried out at a pH ranging from about 8.6 to about 10.

[0226] In one embodiment, the quenching reaction is carried out at a pH of less than about 10. In one embodiment, the quenching reaction is carried out at a pH of about 13. In one embodiment, the quenching reaction is carried out at a pH of about 12. In one embodiment, the quenching reaction is carried out at a pH of about 11. In one embodiment, the quenching reaction is carried out at a pH of about 10. In one embodiment, the quenching reaction is carried out at a pH of about 9.

[0227] In one embodiment, the quenching reaction is performed at a pH of about 10. In one embodiment, the quenching reaction is performed at a pH of about 13. In one embodiment, the quenching reaction is performed at a pH of about 12. In one embodiment, the quenching reaction is performed at a pH of about 11. In one embodiment, the quenching reaction is performed at a pH of about 10. In one embodiment, the quenching reaction is performed at a pH of about 9. In one embodiment, the quenching reaction is performed at a pH of about 10. In one embodiment, the quenching reaction is performed at a pH of about 8.

[0228] In various embodiments, the quenching agent is present in the quenching step at a concentration ranging from about 0.01 to about 100 times the concentration of DVS initially used in the crosslinking reaction. In various embodiments, the quenching agent is present in the quenching step at a concentration ranging from about 0.01 to about 1 times the concentration of DVS used in the crosslinking reaction.

[0229] In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 100:1 to about 1:100 (quenching agent:DVS concentration used in the crosslinking reaction). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 100:1 to about 1:1 (quenching agent:DVS concentration used in the crosslinking reaction). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:100 (quenching agent:DVS concentration used in the crosslinking reaction). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:90 (quenching agent:DVS concentration used in the crosslinking reaction). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:80 (quenching agent:DVS concentration used in the crosslinking reaction).

[0230] In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:70 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:60 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:50 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:40 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:30 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:20 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:10 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:5 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio ranging from about 1:1 to about 1:2 (quenching agent:DVS).

[0231] In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio of about 1:1 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio of about 1:10 (quenching agent:DVS). In various embodiments, the quenching reaction comprises a quenching agent and DVS in a molar ratio of about 1:100 (quenching agent:DVS).

[0232] Concentration of non-quenched pendant groups In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 10 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 5 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 2.5 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 2 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 1.5 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 1.2 micromoles / mL. In various embodiments, the crosslinked polymer comprises a concentration of non-quenching pendant groups of up to about 1.0 micromoles / mL.

[0233] In various embodiments, the crosslinked polymer comprises a non-quenching pendant group at a concentration ranging from about 0.1 micromoles / mL to about 10 micromoles / mL. In various embodiments, the crosslinked polymer comprises a non-quenching pendant group at a concentration ranging from about 0.1 micromoles / mL to about 1 micromoles / mL. In various embodiments, the crosslinked polymer comprises a non-quenching pendant group at a concentration ranging from about 1 micromoles / mL to about 10 micromoles / mL. In various embodiments, the crosslinked polymer comprises a non-quenching pendant group at a concentration ranging from about 1 micromoles / mL to about 5 micromoles / mL. In various embodiments, the crosslinked polymer comprises a non-quenching pendant group at a concentration ranging from about 1 micromoles / mL to about 2 micromoles / mL.

[0234] Quenching reaction conditions In various embodiments, a quenching agent (a nucleophile, e.g., lysine, glycine, cysteine, or equivalent) is added to the reaction mixture at the end of the gel cross-linking reaction but before acid neutralization. In various embodiments, the quenching agent is allowed time to diffuse and react with remaining vinyl sulfone groups before acid neutralizing the reaction. In various embodiments, the quenching reaction time ranges from at least about 1 minute to about 1 hour.

[0235] In various embodiments, the quenching reaction time ranges from at least about 1 minute to about 1 hour at room temperature or above. In various embodiments, the quenching reaction time ranges from at least about 5 minutes to about 15 minutes at room temperature or above.

[0236] In various embodiments, the quenching reaction is carried out at a temperature below about the boiling point of the aqueous solution or mixture. In various embodiments, the quenching reaction is carried out at a temperature below about the boiling point of water. In various embodiments, the quenching reaction is carried out at a temperature below about 100°C.

[0237] In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 90°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 80°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 70°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 60°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 50°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 40°C. In various embodiments, the quenching reaction is carried out at a temperature ranging from about 25°C to about 30°C.

[0238] In various embodiments, the quenching reaction is carried out at about room temperature or above. In various embodiments, the quenching reaction is carried out at about room temperature.

[0239] In various embodiments, the quenching reaction is carried out at room temperature or above, particularly for 5-15 minutes, or up to several hours at temperatures below 25°C.

[0240] In various embodiments, the quenching reaction is carried out at about 0° C. In various embodiments, the quenching reaction is carried out below about 0° C.

[0241] Reduction of residual pendant vinyl sulfone groups In various embodiments, the composition comprises a reduced number of pendant vinyl sulfone groups.

[0242] In various aspects, quenching of the pendant vinyl sulfone groups results in a reduction in the number of remaining unreacted pendant vinyl sulfone groups.

[0243] In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration of up to about 0.1 μg / mL, hi various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration of up to about 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, or 2.0 μg / mL.

[0244] In various embodiments, the crosslinked polymer contains residual sulfonic groups at a concentration of up to about 1.7 μg / mL.

[0245] In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration of up to about 1.4 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration of up to about 1.0 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration of up to about 0.5 μg / mL.

[0246] In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.1 μg / mL to about 2.0 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.1 μg / mL to about 1.5 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.1 μg / mL to about 1.0 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.5 μg / mL to about 1.0 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.5 μg / mL to about 1.5 μg / mL. In various embodiments, the crosslinked polymer comprises residual sulfonic groups at a concentration ranging from about 0.5 μg / mL to about 1.4 μg / mL.

[0247] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups ranging from about 10% to about 99.9%.

[0248] In various embodiments, the methods of the present disclosure result in a reduction in residual vinyl sulfone groups in the range of about 10% to about 80%, in the range of about 10% to about 70%, in the range of about 10% to about 60%, in the range of about 10% to about 50%, in the range of about 10% to about 40%, in the range of about 10% to about 30%, or in the range of about 10% to about 20%.

[0249] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 10%.

[0250] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0251] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 20%. In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 18%. In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 50%. In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 60%. In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 67%.

[0252] Additional advantages of the compositions and methods: In addition to quenching vinyl sulfone groups to improve the safety profile of glycopolymer hydrogels, chemical quenching also offers the opportunity to further tailor gel properties through the choice of quenching agent, allowing for the conjugation of chemical groups into the hydrogel network after primary gel formation.

[0253] Adjustment of gel properties Quenching agents can be used to tailor overall gel properties, such as hydrophobicity / hydrophilicity, electronegativity, or other features. For example, cysteine ​​can enhance the mucoadhesive properties of the gel, making cysteine-quenched gels qualitatively substantially more viscous when handled after production. This viscous nature can be used, for example, to reduce in vivo implant migration or for mucosal targeting of drug delivery. Additional sulfur groups can also result in more immunostimulatory gels, which may be useful for immunomodulatory products. Methionine can be used as a quenching agent in a similar manner. However, these agents may not be a preferred choice when adhesion is undesirable, such as in the use of some surgical meshes, where adhesion formation between two normally separated tissues may be undesirable.

[0254] The present invention can be used to tailor the electronegativity of the cross-linked glycopolymer backbone, for example, by selecting groups with varying electronegativity in their side chains to be used as cross-linkers, such as positively charged (e.g., histidine, lysine, arginine, polylysine, etc.), negatively charged (e.g., aspartic acid or glutamic acid), or uncharged (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, etc.).

[0255] Hydrophilic / Hydrophobic Using the present invention, the hydrophilicity / hydrophobicity of the gel backbone can be tuned to some extent by selecting the hydrophilicity / hydrophobicity of the quenching agent. For example, the remaining vinyl sulfone pendants can be quenched with polar or ionic agents such as lysine or glutamic acid to increase the hydrophilicity of the hydrogel, or with non-polar agents such as alanine or leucine (or other small molecules such as aliphatic chains with amine or thiol groups) to increase the hydrophobicity of the hydrogel.

[0256] Adding functionality The quenching step can be used to provide additional functionality to the gel. The quenching step can provide additional functionality to the gel by adding functional moieties or functional agents to the gel.

[0257] In various embodiments, the functional agent comprises a biologically active agent. In various embodiments, the biologically active agent comprises a biologically active peptide. In various embodiments, the biologically active peptide is a biologically active sequence. In various embodiments, the bioactive sequence comprises cell recognition or cell adhesion activity. In various embodiments, the bioactive sequence comprises a sequence present in a cell adhesion domain of a biological polymer. In various embodiments, the bioactive sequence comprises a sequence present in a cell adhesion domain of a biological polymer including fibronectin, vitronectin, laminin, or collagen. In various embodiments, the bioactive sequence comprises Arg-Gly-Asp (RGD). In various embodiments, the bioactive sequence comprises Arg-Gly-Asp-Ser (RGDS), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Thr (RGDT), and Ile-Lys-Val-Ala-Val (IKVAV).

[0258] In a non-limiting example, the quenching step can provide additional functionality to the gel by adding biologically active peptide sequences such as RGD or IKVAV.

[0259] In various embodiments, the quenching step can provide additional functionality by adding functional groups that allow for further steps of subsequent modification. For example, the quenching agent can include at least one group that can be used to react with vinyl sulfone and at least one group that can subsequently be used to conjugate a second agent.

[0260] This two-step conjugation may be necessary if the intended cargo is incompatible with the potentially harsh alkaline conditions present in the quenching step. After lowering the pH of the reaction mixture to a compatible level, the target cargo can be added. Alternatively, the target cargo can be added after sterilization or at the point of care if the cargo is not suitable for autoclaving and / or long-term storage conditions.

[0261] Toxicity of waste streams Chemical quenching has the advantage that one or more of the reactive groups of the remaining unbound crosslinker, the non-crosslinking group, becomes unreactive, which reduces the toxicity of the manufacturing waste stream.

[0262] Hydrogel durability The current trend in sugar polymer hydrogels on the market, such as HA dermal fillers, is toward longer-lasting effects by making the gels more resistant to degradation after implantation. This is typically achieved by creating denser gels with higher HA concentrations and a higher degree of crosslinking. Furthermore, DVS crosslinking has largely been replaced by BDDE crosslinking in the current dermal filler market.

[0263] The use of amino acids as nucleophilic quenching agents can be used to advantage in addition to potentially reducing the toxicity of the pendant VS crosslinker. Because the hydrogel scaffold may increase and promote cell motility and material interaction, amino acid conjugation is thought to result in cell-mediated hydrogel degradation and shorter gel duration, and therefore shorter clinical efficacy. Cell interaction is the primary cause of hyaluronan degradation (especially enzymatic cleavage of hyaluronan, such as via HYAL-2).

[0264] It is also counterintuitive that such a small degree of modification (only the pendant vinyl sulfone groups are available for quencher modification) had such a large impact on the resulting macroscopic gel properties, such as gel stickiness.

[0265] In previous examples, acid has been used to quench the DVS crosslinking reaction. However, acid quenching merely terminates the reaction between the vinyl sulfone groups and the hydrogel backbone; it has no direct effect on the pendant vinyl sulfone groups.

[0266] For example, U.S. Patent No. 8,481,080 describes making divinyl sulfone-crosslinked HA gels using an acidic treatment followed by a DVS crosslinking reaction during the swelling process to obtain a final gel concentration of 1.1% to 1.4%, without mentioning chemical quenching, residual vinyl sulfone concentration, or pendant vinyl sulfone groups. In another example, U.S. Patent No. 20050142152A1 uses acidic saline and PBS washes to remove impurities after the DVS reaction, which is simply acid quenching (see Y. Yu, et al., Biomacromolecules 13.3 (2012): 937-942).

[0267] Embodiment i. a. a hydrogel material comprising hyaluronic acid; b. a nanofiber material comprising collagen, and c. Divinyl sulfone-containing crosslinkers A nanofiber-hydrogel composite comprising: Hyaluronic acid is covalently bonded to collagen by divinyl sulfone, A nanofiber-hydrogel composite, wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL.

[0268] ii. a. a hydrogel material comprising hyaluronic acid; b. a nanofiber material comprising collagen, and c. Divinyl sulfone-containing crosslinkers A nanofiber-hydrogel composite comprising: Hyaluronic acid is covalently bonded to collagen by divinyl sulfone, hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; The nanofiber-hydrogel composite, wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0269] iii. a. a hydrogel material comprising hyaluronic acid; b. a nanofiber material comprising collagen, and c. Divinyl sulfone-containing crosslinkers A nanofiber-hydrogel composite comprising: Hyaluronic acid is covalently bonded to collagen by divinyl sulfone, hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL; A nanofiber-hydrogel composite, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules to hyaluronic acid subunits ranging from about 3:1 to about 5:1 prior to crosslinking.

[0270] iv. a. a hydrogel material comprising hyaluronic acid; b. a nanofiber material comprising collagen, and c. Divinyl sulfone-containing crosslinkers A nanofiber-hydrogel composite comprising: Hyaluronic acid is covalently bonded to collagen by divinyl sulfone, the amount of crosslinking is in the range of about 0.5% to about 30%; hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; The nanofiber-hydrogel composite, wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0271] v. reacting a hyaluronic acid material, collagen fibers, and divinyl sulfone; A nanofiber-hydrogel composite product by a method comprising the steps of: covalently bonding hyaluronic acid to collagen fibers by divinyl sulfone; hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL; collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; A nanofiber-hydrogel composite product, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 0.5:1 to about 5:1.

[0272] vi. reacting hyaluronic acid, collagen fibers, and divinyl sulfone; Thereby, a method for producing a nanofiber-hydrogel composite, comprising the steps of: covalently bonding hyaluronic acid to collagen fibers by divinyl sulfone; hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL; collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; The method wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 0.5:1 to about 5:1.

[0273] vii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 3.0.

[0274] viii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 1.0 to about 3.0.

[0275] ix. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 1.5 to about 4.0.

[0276] x. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 2.0 to about 3.0.

[0277] xi. A nanofiber-hydrogel composite described in any one of embodiments i-iv, wherein the composite comprises a swelling ratio in the range of about 2.2 to about 3.0 during manufacture.

[0278] xii. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 2.3 to about 2.8.

[0279] xiii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio in the range of about 1.0 to about 2.0 during fabrication.

[0280] xiv. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 1.0.

[0281] xv. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio of about 2.3 during fabrication.

[0282] xvi. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio of about 2.4 during fabrication.

[0283] xvii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio of about 2.6 during fabrication.

[0284] xviii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling ratio of about 2.8 during fabrication.

[0285] xix. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 100%.

[0286] xx. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 75%.

[0287] xxi. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 50%.

[0288] xxii. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 40%.

[0289] xxiii. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 30%.

[0290] xxiv. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 20%.

[0291] xxv. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 15%.

[0292] xxvi. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 12%.

[0293] xxvii. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 10%.

[0294] xxviii. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0295] xxix. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo ranging from about 0% to about 100%.

[0296] xxx. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 100%.

[0297] xxxi. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 50%.

[0298] xxxii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 20%.

[0299] xxxiii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 10%.

[0300] xxxiv. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 10% to about 20%.

[0301] xxxv. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a % swelling in vivo ranging from about 10% to about 15%.

[0302] xxxvi. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of about 50%, 40%, 30%, 20%, or 10%.

[0303] xxxvii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of about 10%, 11%, 12%, 13%, 14%, or 15%.

[0304] xxxviii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of about 20%.

[0305] xxxix. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the composite comprises a swelling percentage in vivo of about 12%.

[0306] xl. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 10 mg / mL.

[0307] xli. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the hyaluronic acid is present in an amount ranging from about 9 mg / mL to about 10 mg / mL.

[0308] xlii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the hyaluronic acid is present in an amount of about 9 mg / mL.

[0309] xliii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the hyaluronic acid is present in an amount of about 9.5 mg / mL.

[0310] xliv. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the hyaluronic acid is present in an amount of about 10 mg / mL.

[0311] xlv. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0312] xlvi. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount ranging from about 15 mg / mL to about 30 mg / mL.

[0313] xlvii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount ranging from about 20 mg / mL to about 30 mg / mL.

[0314] xlviii. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount ranging from about 25 mg / mL to about 30 mg / mL.

[0315] xlix. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount of about 15 mg / mL.

[0316] l. A nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the collagen is present in an amount of about 30 mg / mL.

[0317] li. A nanofiber-hydrogel composite described in any one of embodiments i-iv, wherein the collagen is present in an amount of about 25 mg / mL.

[0318] lii. hyaluronic acid is present in an amount of about 9 mg / mL; The collagen is present in an amount of about 15 mg / mL. A nanofiber-hydrogel composite according to any one of embodiments i-iv.

[0319] liii. hyaluronic acid is present in an amount of about 10 mg / mL; The collagen is present in an amount of about 30 mg / mL. A nanofiber-hydrogel composite according to any one of embodiments i-iv.

[0320] liv. hyaluronic acid is present in an amount of about 9.5 mg / mL; The collagen is present in an amount of about 25 mg / mL. A nanofiber-hydrogel composite according to any one of embodiments i-iv.

[0321] lv. A nanofiber-hydrogel composite product according to embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 30 mg / mL.

[0322] lvi. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 25 mg / mL.

[0323] lvii. A nanofiber-hydrogel composite product according to embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 23 mg / mL to about 25 mg / mL.

[0324] lviii. The nanofiber-hydrogel composite product according to embodiment v, wherein the hyaluronic acid is present in an amount of about 23 mg / mL.

[0325] lix. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein hyaluronic acid is present in an amount of about 25 mg / mL.

[0326] lx. A nanofiber-hydrogel composite product according to embodiment v, wherein the collagen is present in an amount ranging from about 30 mg / mL to about 100 mg / mL.

[0327] lxi. A nanofiber-hydrogel composite product according to embodiment v, wherein the collagen is present in an amount ranging from about 40 mg / mL to about 70 mg / mL.

[0328] lxii. A nanofiber-hydrogel composite product according to embodiment v, wherein the collagen is present in an amount ranging from about 45 mg / mL to about 65 mg / mL.

[0329] lxiii. The nanofiber-hydrogel composite product according to embodiment v, wherein the collagen is present in an amount of about 45 mg / mL.

[0330] lxiv. The nanofiber-hydrogel composite product according to embodiment v, wherein the collagen is present in an amount of about 65 mg / mL.

[0331] lxv. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 1:1 to about 5:1.

[0332] lxvi. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 2:1 to about 5:1.

[0333] lxvii. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 3:1 to about 5:1.

[0334] lxviii. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 3.5:1 to about 4:1.

[0335] lxix. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of about 3.5:1 divinyl sulfone molecules per hyaluronic acid subunit.

[0336] lxx. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit.

[0337] lxxi. A nanofiber-hydrogel composite product according to the method of embodiment v, wherein the divinyl sulfone is present in an amount of about 4.5:1 divinyl sulfone molecules per hyaluronic acid subunit.

[0338] lxxii. hyaluronic acid is present in an amount of about 25 mg / mL; collagen is present in an amount of about 45 mg / mL; The divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit. A nanofiber-hydrogel composite product according to embodiment v.

[0339] lxxiii. hyaluronic acid is present in an amount of about 25 mg / mL; collagen is present in an amount of about 45 mg / mL; The divinyl sulfone is present in an amount of about 3.5:1 divinyl sulfone molecule per hyaluronic acid subunit. A nanofiber-hydrogel composite product according to embodiment v.

[0340] lxxiv. hyaluronic acid is present in an amount of about 23 mg / mL; collagen is present in an amount of about 65 mg / mL; The divinyl sulfone is present in an amount of about 4.5:1 divinyl sulfone molecule per hyaluronic acid subunit. A nanofiber-hydrogel composite product according to embodiment v.

[0341] lxxv. hyaluronic acid is present in an amount of about 23 mg / mL; collagen is present in an amount of about 65 mg / mL; The divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit. A nanofiber-hydrogel composite product according to embodiment v.

[0342] lxxvi. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the collagen nanofibers comprise type I, type II, type III, or type IV collagen.

[0343] lxxvii. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the collagen nanofibers comprise recombinant collagen.

[0344] lxxviii. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the collagen nanofibers comprise type I bovine collagen nanofiber fragments.

[0345] lxxix. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the collagen nanofibers are electrospun, melt spun, blow spun and / or cryomilled.

[0346] lxxx. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein collagen nanofibers are retained within the nanofiber-hydrogel composite.

[0347] lxxxi. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the interfacial bonding between the collagen nanofibers and HA enhances the stiffness of the composite with a relatively low fiber loading density.

[0348] lxxxii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite exhibits monocyte recruitment, monocyte polarization, or both.

[0349] lxxxiii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite is characterized by exhibiting higher porosity, increased cell permeability, and / or maintaining storage modulus.

[0350] lxxxiv. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the nanofiber-hydrogel composite allows cell infiltration.

[0351] lxxxv. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the pH of the nanofiber-hydrogel composite is about 7.0, 7.1, 7.2, 7.3, or 7.4 in an isotonic solution.

[0352] lxxxvi. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite is formulated in one or more product forms selected from a mat, an injectable solution, and a flowable solution.

[0353] lxxxvii. A nanofiber-hydrogel composite described in any one of embodiments i-iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the nanofiber-hydrogel composite is suitable for passage through a needle of 27 gauge or less.

[0354] lxxxviii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the crosslinking agent reacts with the hydroxyl groups of the HA and the hydroxyl groups, thiol groups, or amine groups of the collagen nanofibers.

[0355] lxxxix. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the crosslinking agent reacts with the hydroxyl groups of the HA and the hydroxyl groups or amine groups of the collagen nanofibers.

[0356] xc. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the nanofiber-hydrogel composite forms one or more porous structures.

[0357] xci. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein one or more porous structures induce increased host cell infiltration.

[0358] xcii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits a biostimulatory effect selected from tissue remodeling, host cell infiltration, cell adhesion, cell migration, angiogenic response, adipogenic response, and macrophage polarization toward a pro-healing phenotype.

[0359] xciii. A nanofiber-hydrogel composite according to any one of embodiments i-iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, wherein the nanofiber-hydrogel composite comprises a population of infiltrating macrophages and the collagen nanofibers condition the population of infiltrating macrophages to an M2 phenotype.

[0360] xciv. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits a tissue remodeling effect induced by the nanofiber-hydrogel composite without incorporating cells or growth factors.

[0361] xcv. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits a biostimulatory effect due to the low crosslinking density of the composite material.

[0362] xcvi. The nanofiber-hydrogel composite of any one of embodiments i-iv, wherein the nanofiber-hydrogel composite is suitable for terminal sterilization by autoclaving.

[0363] xcvii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the composite is characterized by increased thermal stability and / or provides shelf stability at ambient temperatures.

[0364] xcviii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits biocompatibility.

[0365] xcix. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits cell adhesion.

[0366] c. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits cell migration.

[0367] ci. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite enables durable soft tissue remodeling.

[0368] cii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite promotes angiogenesis.

[0369] ciii. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product according to the method of embodiment v, characterized in that the nanofiber-hydrogel composite prolongs the biostimulatory effect by retaining and / or conditioning infiltrating macrophages towards a pro-regenerative M2 phenotype, thereby promoting neovasculature formation.

[0370] civ. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite improves adipose tissue formation.

[0371] cv. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, characterized in that the nanofiber-hydrogel composite exhibits vasculature formation.

[0372] cvi. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite modulates host cell infiltration and / or shape retention of the composite.

[0373] cvii. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, having a storage modulus within the range of most soft tissues.

[0374] cviii. A nanofiber-hydrogel composite according to any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method according to embodiment v, having a storage modulus in the range of about 150 Pa to about 500 Pa.

[0375] cix. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product by the method described in embodiment v, wherein the nanofiber-hydrogel composite is micronized into a series of microgels having diameters selected from the group consisting of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μm.

[0376] cx. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite mimics host tissue structure.

[0377] cxi. A nanofiber-hydrogel composite described in any one of embodiments i to iv, or a nanofiber-hydrogel composite product produced by the method described in embodiment v, wherein the nanofiber-hydrogel composite mimics the natural extracellular matrix. [Example]

[0378] Example 1 Collagen fiber formation: Bovine type I collagen Freeze-dried bovine type I collagen (derived from bovine skin, Collagen Solutions) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) overnight at room temperature to obtain a viscous, cloudy solution (5.2% w / w). The collagen solution was loaded into a 5 mL plastic syringe with a 23 G blunt metal needle. Electrospinning was performed on a UCalery electrospinning unit using the following parameters: flow rate of 2.2 mL / h, two syringes in parallel, 17.4 kV applied to the needle, 10.8 kV applied to the collector (negative), 8 kV applied to the collector (negative), 12.5 cm collection distance, and 200 rpm metal collector rotation speed. The resulting collagen nanofibers / microfibers were cross-linked for 20 hours in ethanol containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mM N-hydroxysuccinimide (NHS). After cross-linking, the fibers were washed with ethanol three times (30 min each) to remove excess reagents. The collagen fibers were then disaggregated into fragments using cryomilling (SPEX SamplePrep).

[0379] The amount of intra-crosslinking or inter-crosslinking of collagen fibers is 13The cross-linking ratio can be measured using C-NMR spectroscopy (see A.L. Gatta, et al. "Hyaluronan Hydrogels: Rheology and Stability in Relation to the Type / Level of Biopolymer Chemical Modification," Polymers 2022, 14, 2402). The ratio of non-cross-linked to cross-linked collagen can be measured using the method in Example 5.

[0380] gelatin Fibers made from porcine gelatin (Sigma) were produced by dissolving the gelatin in TFE to 15% w / v and then spinning at 1.35 mL / hr at a voltage of 12 kV on a grounded drum rotating at 640 rpm. Fibers made from porcine gelatin are shown in Figure 44.

[0381] Pig collagen Porcine type I atelocollagen fibers were produced by dissolving porcine type I atelocollagen (Nitta) in HFIP at 7% and spinning at 0.3 mL / hr at 11 kV. Figure 43 is an image showing the resulting fibers.

[0382] Non-animal derived collagen and collagen-like peptides. Vecollan recombinant collagen from Evonik was spun into fibers. Fibers were produced by dissolving Vecollan in TFE at 20% w / v and then spinning at 1.5 mL / hr using a voltage of 16 kV on a grounded drum rotating at 480 rpm. Figure 45 is an image showing the resulting fibers. Parameters can be adjusted to produce either cylindrical or ribbon morphologies. Both cylindrical and ribbon fibers are seen in Figure 45.

[0383] Once spun, fibers obtained from alternative non-bovine sources can be processed as described for bovine collagen and used to form fiber-hydrogel composites. Other forms of collagen or gelatin are applicable, e.g., other types of collagen such as type III, or recombinant "collagen-like" peptides from bacterial sequences.

[0384] Depending on the raw material source, the resulting fibers may have less secondary structure when spun than the described bovine collagen (e.g., gelatin is not triple helical) or may have different crosslinking moiety concentrations due to variations in peptide sequence. Such fibers may require more intensive crosslinking before contact with water. In various embodiments, the fibers may be crosslinked using EDC / NHS, DVS, BDDE, aldehyde-based crosslinkers, e.g., vapor-phase glutaraldehyde treatment, Maillard reactions such as those using D-ribose and heat, dehydrothermal treatment, genipin and transglutaminase, irradiation (UV, gamma, or e-beam), plasma treatment, other methods known in the art (see, e.g., Ehrmann A. Non-Toxic Crosslinking of Electrospun Gelatin Nanofibers for Tissue Engineering and Biomedicine—A Review. Polymers (Basel). 2021 Jun 15;13(12):1973), or any combination thereof.

[0385] Once rendered water insoluble, the fibers can be mechanically processed into a dispersed form and covalently gelled into a composite using the methods described herein.

[0386] Example 2 Preparation of HA hydrogels and NHCs crosslinked by DVS: A series of test gel lots with and without a fibrous phase (during the gelation stage) were generated, each with a lot size of 3 cc. Reagent stock solutions were prepared. Sodium hyaluronate (HA, MW 1.5 MDa) was dissolved in distilled water at a concentration of 30 mg / mL. Sodium hydroxide was dissolved in water to create a 10 M solution. Lysine monohydrochloride was dissolved in water at a concentration of 2 M. First, gels without collagen were generated by adding 0.03 mL of sodium hydroxide stock to achieve a target concentration of 0.1 M, using gelling HA concentrations of 21, 24, 27, or 30 mg / mL and DVS to HA subunit ratios of 3.22, 3.5, 4, 4.5, or 5x by varying the amount of each stock solution added. Each group was mixed between two 5 mL syringes connected with a Luer-Lok connector. After vigorously mixing, the mixtures were incubated in sealed syringes in a 30°C water bath for 2 hours. The reaction was chemically quenched with a lysine solution (1:1 molar ratio of lysine to DVS molecules) by syringe-to-syringe mixing followed by incubation at room temperature for 5 minutes. The gel was then neutralized in a 50 mL syringe containing 21 mL of 20 mM monobasic sodium phosphate solution. The hydrogel was transferred to a dialysis tube (13,000 MWCO) and dialyzed against 1 L of 0.9% sodium chloride for 16 hours and against 1 L of DPBS for 3 hours at room temperature. The resulting gel suspension was transferred to a 1.5 mL microcentrifuge tube through an 18-gauge blunt needle and then centrifuged at 17,000 × g for 3 minutes to separate excess water. After removing the supernatant, the gel was collected in a 10 mL syringe and weighed to calculate the swelling ratio (relative to the weight of the initial gelling solution; see Example 5). The gel was beaded through a 150 micron screen and then loaded into a 1 mL glass syringe and autoclaved for 30 min at 121 °C. The swelling ratio of the gel during preparation and the resulting final HA concentration are shown in Figures 4-7.

[0387] Second, gels were formulated using the same method but with gelatin fibers (porcine, purchased from Sigma) or collagen fibers (bovine, from Collagen Solutions) prepared by the method in Example 1. The collagen fiber concentration in the pregel solution varied from 37 to 70 mg / mL, the pregel HA concentration was set at 21, 23, or 25 mg / mL, and the ratio of DVS to HA subunits varied from 2.3 to 4.5x. Each group was mixed between two 5 mL syringes connected with a Luer-Lok connector. After vigorously mixing, the mixture was incubated in a sealed syringe in a 30°C water bath for 2 hours. The reaction was chemically quenched with a lysine solution (1:1 molar ratio of lysine to DVS molecules) by syringe-to-syringe mixing followed by incubation at room temperature for 5 minutes. The gel was then neutralized in a 50 mL syringe containing 21 mL of 20 mM monobasic sodium phosphate solution. The composite hydrogel was transferred to a dialysis tube (13,000 MWCO) and dialyzed against 1 L of 0.9% sodium chloride for 16 hours and against 1 L of DPBS for 3 hours at room temperature. The resulting gel suspension was transferred to a 1.5 mL microcentrifuge tube through an 18-gauge blunt needle and then centrifuged at 17,000 × g for 3 minutes to separate excess water. After removing the supernatant, the gel was collected in a 10 mL syringe and weighed to calculate the swelling ratio (relative to the weight of the initial gelling solution; see Example 5). The composite gel was beaded through a 150-micron screen, then loaded into a 1 mL glass syringe and autoclaved at 121 °C for 30 minutes. The swelling ratio of the composite gel during preparation and the resulting final HA concentration are shown in Figures 8–13.

[0388] Example 3 Mechanical properties of hydrogels and NHCs: Rheological characterization of hydrogels and composite gels was performed as previously described. Various shear mechanical properties of hydrogels and composite gels were measured using a rheometer (AR2000ex, TA Instruments) at 25 °C using a 25 mm parallel-plate geometry with a 0.9 mm gap. The linear viscoelastic region of the gels was measured by strain sweeps with increasing shear strain amplitude at a set frequency (1 Hz). The three main properties were storage modulus (G'), loss modulus (G''), and tan delta (G'' / G'). Representative traces are shown in Figures 40-42. The injection force of HA and NHC bulk gels and microgels was measured by extruding 1 mL of sample in a 1 mL BD syringe through a 27-gauge needle using an Instron load frame (34SC-05, Norwood, MA). Representative traces are shown in Figures 36-39.

[0389] Example 4 Preparation of DVS-crosslinked collagen-HA composites for animal studies Sodium hyaluronate (HA, MW 1.5 MDa) was dissolved in distilled water at a concentration of 30 mg / mL. Sodium hydroxide was dissolved in water to make a 10 M solution. Lysine monohydrochloride was dissolved in water at a concentration of 2 M.

[0390] [Table 1]

[0391] For each group, the volume of HA stock solution specified in the table above was mixed with the specified amounts of water, collagen fibers, and sodium hydroxide solution in two 10 mL syringes connected with a Luer-Lok connector for syringe-to-syringe mixing. The specified volume of DVS was then added to each group and mixed by syringe-to-syringe mixing. After vigorous mixing, the mixture was incubated in a sealed syringe in a 30°C water bath for 2 hours. The reaction was chemically quenched with a lysine solution (1:1 molar ratio of lysine to DVS molecules) by syringe-to-syringe mixing followed by incubation at room temperature for 5 minutes. The gel was then neutralized in a 60 mL syringe containing 42 mL of 20 mM monobasic sodium phosphate solution. The resulting hydrogel suspension was transferred to a dialysis tube (13,000 MWCO) and dialyzed three times against 1 L of DPBS (8–16 h each at room temperature), then microparticulated using 150 μm stainless steel wire cloth discs in a 25 mm filter holder. The resulting gel suspension was centrifuged at 4,000 × g for 10 min to separate excess water. After removing the supernatant, the gel composite was weighed and the swelling ratio was calculated (relative to the weight of the initial gelling solution). The swelling ratio was then verified by directly measuring the hyaluronic acid and collagen contents of the final gel. The gel composite was loaded into a 1 mL glass syringe and then autoclaved at 121 °C for 30 min.

[0392] Example 5 Determination of component concentrations in gel composites Carbazole assay for hyaluronic acid content in gel composites The concentration of hyaluronic acid in the final gel samples can be calculated through a carbazole-based assay. Briefly, 3.00 mL of 0.025 M sodium tetraborate in sulfuric acid was added to 7 mL glass tubes containing 0.030–0.040 g of gel composite sample, heated in a boiling water bath for 15 minutes, and then cooled at room temperature for 15 minutes. Next, 0.80 mL of 0.125% carbazole (w / w) in absolute ethanol was added to each tube, and heated in a boiling water bath for 15 minutes. After cooling at room temperature for 15 minutes, 100 μL of the final solution was transferred to a 96-well plate and read at 530 nm on a microplate reader (Epoch, BioTek). The hyaluronic acid content of the gels was calculated from a standard curve (generated by identical testing of hyaluronic acid solutions of known concentrations).

[0393] Sirius Red Adsorption Assay for Collagen Fiber Content in Gel Composites The concentration of collagen in the final gel sample can be calculated through a colorimetric assay. Briefly, 10.00 ml of a 1 mg / ml Sirius Red F3B solution in 1:10 (v:v) acetic acid was added to a 15 mL centrifuge tube containing 0.080–0.100 g of gel composite and vortexed at maximum speed for 1 minute. The sample was covered with aluminum foil and shaken at room temperature on an orbital shaker at 100 rpm for 5 hours. 1 mL of the solution was transferred to a microcentrifuge tube and spun down at 10,000 × g for 3 minutes. 0.05 mL of the supernatant was diluted into a microcentrifuge tube containing 1 mL of water. 0.2 mL of the diluted solution was placed in a 96-well plate and read at 530 nm. A series of samples with known amounts of collagen (0.2–2 mg) was used to generate a standard curve, which was used to calculate the concentration of the test samples.

[0394] Free (non-crosslinked) collagen in collagen fibers 1.00 mL of water was added to a 1.5 mL microcentrifuge tube containing 0.020–0.025 g of collagen fibers. The tube was vortexed at maximum speed for 1 minute and then incubated at room temperature for 4 hours on an orbital shaker (100 rpm). The fibers were spun down at 17,000 × g for 3 minutes, and 0.1 mL of the supernatant was mixed with 0.8 mL of BCA working solution (Thermo Scientific) and then incubated at 37°C for 30 minutes. After the sample cooled to room temperature, 0.2 mL was transferred to a 96-well plate, and absorbance was read at 562 nm on an Epoch microplate spectrophotometer (BioTek Instruments). Bovine serum albumin (Thermo Scientific) was used as a standard. The free collagen content in the collagen fibers was determined to be 3.1 ± 0.05%.

[0395] Free (non-crosslinked) HA in matrix-C gel composite 1.00 mL of water was added to a centrifuge tube containing 0.500 g of Matrix-C sample. The tube was vortexed at maximum speed for 1 minute and then incubated on an orbital shaker (100 rpm) at room temperature for 2 hours. The sample was spun down at 17,000 × g for 3 minutes. 0.050 mL was added to 0.500 mL of 0.005% Stains-All in 55% DMSO, followed by vortex mixing for 1 minute. 0.100 mL of sample was transferred to a 96-well plate and read at 655 nm. Sodium hyaluronate (LifeCore) was used as a standard. The free HA content in M1 and M2 ranged from 5.6 to 5.9%.

[0396] Example 6 in vivo animal studies Four treatment groups were prepared from the same batch of raw materials in Example 4 and were manufactured to be as similar as possible, except for experimental variables (pre-gelation concentrations of HA, collagen fibers, and cross-linker (DVS) concentration). Collagen fibers were generated from type I bovine collagen from Collagen Solutions according to Example 1. Hyaluronic acid was purchased from LifeCore Biomedical Corporation. DVS was purchased from TCI (Tokyo Chemical Industry Co., Ltd.). At the end of gelation, remaining sulfonic acid groups were chemically quenched by the addition of lysine (Sigma).

[0397] Test group Four formulations of collagen fiber-hyaluronic acid composite gel (designated M1, M2, M3, and M4) were injected subcutaneously into a rat model to evaluate their biocompatibility and efficacy (in maintaining volume). These groups were compared with a commercial control, Voluma XC. The gels were biocompatible and effective in maintaining volume and promoting cell ingrowth for up to 69 weeks.

[0398] The four test groups had different levels of collagen fiber loading and crosslinking to assess the effect of these variables on biological response. Each group used 1.6 MDa of hyaluronic acid.

[0399] Group M1: Gels produced using gelation conditions of 25 mg / mL HA, 45 mg / mL collagen, and a [DVS] / [HA repeat unit] ratio of 4.0:1 (264 mM DVS). The resulting gels had a final gel concentration of approximately 9.7 mg / mL HA and 17.5 mg / mL collagen in DPBS, and had a stiffness (G') of 628 Pa and an injection force of 6.1 N.

[0400] Group M2: Gels produced using gelation conditions of 25 mg / mL HA, 45 mg / mL collagen, and a [DVS] / [HA repeat unit] ratio of 3.5:1 (231 mM DVS). The resulting gels had a final gel concentration of approximately 8.7 mg / mL HA and 15.7 mg / mL collagen in DPBS, and had a stiffness (G') of 534 Pa and an injection force of 7.8 N.

[0401] Group M3: Gels produced using gelation conditions of 23 mg / mL HA, 65 mg / mL collagen, and a [DVS] / [HA repeat unit] ratio of 4.5:1 (273 mM DVS). The resulting gels had a final gel concentration of approximately 10.2 mg / mL HA and 27.5 mg / mL collagen in DPBS, and had a stiffness (G') of 610 Pa and an injection force of 4.9 N.

[0402] Group M4: Gels produced using gelation conditions of 23 mg / mL HA, 65 mg / mL collagen, and a 4.0:1 [DVS] / [HA repeat unit] ratio (242 mM DVS). The resulting gels had a final gel concentration of approximately 9.5 mg / mL HA and 25.1 mg / mL collagen in DPBS, and had a stiffness (G') of 548 Pa and an injection force of 5.2 N.

[0403] Control Group C: Voluma XC, a commercially available, market-leading soft tissue filler composed of cross-linked hyaluronic acid.

[0404] Test System Species: Rat Variety: Sprague Dawley Source: Single USDA-approved supplier Gender: Female Age: Young adult Number of animals: 27 (4 rats per test group time point, and 6 time points, with 3 control rats) Identification method: Ear tag or marking.

[0405] Injection procedure Rats are anesthetized with isoflurane and their backs are shaved with clippers, then disinfected (iodine) immediately before injection.

[0406] At each injection site, 200 μl was injected subcutaneously as a single round bolus into the dorsal region of the rat using a 27G×½″ needle. Each injection bolus was separated from each other by at least 5 mm.

[0407] Map of site location: Injections follow the following implantation scheme in the dorsum of the rat:

[0408] Embedding scheme: Test group Head side

[0409] JPEG2026507614000003.jpg20133

[0410] caudal T1 = Study group 1, T2 = Study group 2, T3 = Study group 3, T4 = Study group 4

[0411] Control group: Head side

[0412] JPEG2026507614000004.jpg20137

[0413] caudal C = control group (Voluma XC)

[0414] Sample Size Test Groups: 4 replicates per group per time point (each replicate coming from a different animal). (4 test group rats per time point, 16 total test sites per time point).

[0415] Control: 4 replicates per time point in the same animal.

[0416] Time of vital evaluation Animals are scored for erythema and edema immediately after injection and 4 days after injection. Animals are graded on a scale from ISO 10993-10 Table 3.

[0417] Time of MRI Rats from TBD Group 2 will be imaged at all time points. Additionally, at each collection time point, rats to be harvested may be imaged prior to tissue harvest. Time points correspond to day 0 (to establish a baseline immediately after injection), day 1 post-injection (to assess swelling), and the designated tissue harvest time point.

[0418] MRI time points: 0 days, 1 day, 1 week, 4 weeks, 8 weeks, 26 weeks, TBD1, TBD2

[0419] Time of tissue collection The time points for this study were 0 days, 1 day, 1 week, 4 weeks, 8 weeks, 20 weeks, 26 weeks, 40 weeks, and 69 weeks. The final time point was assigned based on the assessment at 26 weeks. Unless otherwise noted, the 1 week time point was tested exactly 7 days after treatment, and the 4 week time point was tested within 2 days of the scheduled time point. The 8 week and higher test groups were tested within 7 days of the scheduled time point. One rat from the control group was harvested at 26 weeks and at each of the TBD time points.

[0420] Euthanasia. Following any MRI imaging, designated rats are humanely euthanized. The dorsal skin is incised to allow visual identification of all four injection sites. Test and control sites are excised as intact tissue blocks, fixed overnight in formalin, and then processed for histological and immunofluorescence evaluation.

[0421] Acceptance Criteria Swelling: At the first measurement (1 day after injection, POD1), less than 200% of the initial volume (expected to be approximately 200 μL, POD0 measurement) and less than the commercial control (Voluma XC).

[0422] Edema: Mean score less than or equal to the mean score for Voluma XC® as described in Table 3 of ISO-10993-10 on POD4

[0423] Irritation: Erythema mean score less than or equal to the mean score for Voluma XC® according to Table 3 of ISO 10993-10 (see appendix) on POD4

[0424] Degradation: Degradation is considered complete when all examined sites score 4 on the ISO 10993-6 degradation scale (as interpreted by NAMSA and LifeSprout for Lumina; see Appendix 2 below). The first time point with all 4s (when at least three sites are analyzed) is considered the time of complete degradation.

[0425] Volumization: The mean implant volume is measured for each group at each MRI time point. Each study group is considered to have acceptable volumization characteristics if the mean measured volume is greater than 50% of the original (POD0) volume at each time point up to 26 weeks.

[0426] Cellular infiltration rate: Cellular infiltration rate is calculated by the percentage of the area covered by infiltrating cells within the injected material. The acceptance criterion for cellular infiltration rate is an increase of at least 50% in the mean cellular infiltration rate compared to the control (Voluma XC) at 26 weeks.

[0427] Cell density: Cell density is counted based on immunofluorescence images stained with DAPI (which stains individual cell nuclei). The acceptance criterion for cell density is an average cell density at least 50% higher inside the injected implant compared to the control.

[0428] Angiogenesis: Measurement of angiogenesis is based on immunofluorescence images where tissue slides are stained with RECA-1 (endothelial cells) and α-SMA (pericytes). The measure for endothelial cells is the distance invaded from the periphery to the center, and the measure for pericytes is the density per unit area (# / mm^2).

[0429] Cell Types, Adipogenesis, Macrophage Polarization: Measurement of adipogenesis and macrophage polarization is based on immunofluorescence imaging. Perilipin-1 is used to stain adipocytes forming within the material, while CD68, CD38, and CD163 are used to stain macrophage phenotypes within the material. For both adipogenesis and macrophage polarization, cell density is a way to quantitatively compare test groups to the Voluma control.

[0430] [Table 2]

[0431] result: Biometric evaluation: All four treatment groups were well tolerated by the animals, without inducing infection, necrosis, or significant inflammation in live animals. The implants remained as permanent bumps protruding from the skin at the same location as the initial injection. On POD4 (postoperative day 4), the animals were examined and scored for erythema and edema according to the grading system from ISO 10993-10. All four groups met the acceptance criteria described in the protocol. All erythema scores were mild and all were below the scores of Voluma XC. All edema scores were also below the edema score of Voluma. This indicates that the formulations are mild and low-swelling, making them excellent candidates for dermal application.

[0432] Implantation sites were photographed in situ during explantation. The bolus of injected material remained in place and maintained its bolus shape. All implantation sites (all four groups, all replicates, at days 7, 30, and 63) were white to yellow in color, remarkably similar to native adipose tissue. Blood vessels were discernible either on or near the bolus. The implanted specimens, unlike the surrounding tissue, were the only discernible feature protruding from the skin (there was no surrounding fat pad, etc.).

[0433] [Table 3]

[0434] edema / swelling Acceptance Criteria: Mean score less than or equal to the mean score for Voluma XC® according to the instructions in Table 3 of ISO-10993-10 on POD4, with scores ranging from 0 for no edema to 4 for severe edema. Result: Pass. All four formulation groups had lower edema scores than the Voluma XC® control. The edema score values ​​are consistent with expectations for a gel that maintains volumization and therefore a visible bleb at the injection site.

[0435] Explant: During explantation, the implantation sites were photographed in situ. The bolus of injected material remained in place and maintained its bolus shape. The color of all implantation sites (all four groups, all replicates, at days 7, 30, and 63) was white to yellow, remarkably similar to native adipose tissue. Blood vessels could be identified either on or near the bolus. The bolus was distinct from existing tissue structures.

[0436] Erythema / irritation Acceptance criteria: A mean erythema mean score less than or equal to the mean score for Voluma XC® according to Table 3 of ISO 10993-10 on POD4, with scores ranging from 0 for no erythema to 4 for severe erythema.

[0437] Results: Pass. All four formulation groups had lower erythema scores than the Voluma XC® control. Groups M1 and M2 showed no erythema scores, while groups M3 and M4 showed very mild scores lower than that of Voluma XC. Table 4A.

[0438] [Table 4]

[0439] MRI volumetry The four groups showed little or no post-treatment swelling on days 1 and 7 and maintained their volume as intended.

[0440] Table 4B shows quantification of swelling by MRI on day 1 for test formulations M1-M4 of Example 6 one day after injection. % swelling is the percentage of the additional volume measured on day 1 relative to day 0. All four formulations had 20% or less swelling, while Voluma XC® had 100% swelling (doubling in volume).

[0441] [Table 5]

[0442] The average swelling percentage for the treatment groups ranged from 12 to 20%. This contrasts with Voluma, which doubled in size (100% swelling) by day 1 and continued through day 7 (then slowly decreased through day 63). This indicates that Matrix-C gel variants can be made without undesirable swelling. The four groups and Voluma had broadly similar volumization profiles through day 63, with no or only slight decreases from day 1 values. The Voluma group had the largest absolute volume due to extensive initial swelling. When normalized to day 1 volume, all groups maintained 75% or more of their original volume through day 63. Group M1 had the best volume retention, retaining 97% of its day 1 volume on day 63, nearly indistinguishable from Voluma's 94% volume retention.

[0443] Swelling: Acceptance criteria: At the first measurement (1 day after injection, POD1), less than 200% of the initial volume (expected to be approximately 200 μL, POD0 measurement) and less than the commercial control (Voluma XC).

[0444] Results: Pass. All four formulation groups passed this criterion. The mean relative volumes are in the table below (p values ​​are two-tailed t-tests versus Voluma XC). All four groups maintained volumization (>100% volume) but still had less swelling than the commercial control, Voluma XC. These formulation groups also show substantially less swelling than Disclosure D17311's hyaluronic acid-collagen fiber gel, which increased in volume by approximately 80% by day 7. Table 4C.

[0445] [Table 6]

[0446] At 7 days, the four formulation groups returned to baseline volumes near the injected volume, while the Voluma XC® group still swelled to double its volume. Table 4D.

[0447] [Table 7]

[0448] Volumizing: Acceptance Criteria: The mean implant volume is measured for each group at each MRI time point. Each study group is considered to have acceptable volumizing characteristics if the mean measured volume is greater than 50% of the original (POD0) volume at each time point up to 26 weeks.

[0449] Results: Pass. All four treatment groups had a mean relative volume greater than 50% (relative to time 0) at 26 weeks. Results were not statistically significant compared to the Voluma XC® control (which also passed this criterion). Table 4E.

[0450] [Table 8]

[0451] Once relative volume (volume relative to the 7 day time point), which accounts for initial swelling, was assessed, all four formulations had comparable or superior volume retention compared to Voluma XC, and all had higher mean relative volumes than Voluma XC. Table 4F.

[0452] [Table 9]

[0453] Groups M1, M2, and M3 still maintained greater than 50% of the relative volume at week 55, with a mean relative volume greater than the Voluma XC® control. Only the M4 formulation had a relative volume below and less than 50% of that of Voluma XC®. Table 4G.

[0454] [Table 10]

[0455] Once relative volume (volume relative to the 7-day time point) at 55 weeks was assessed to account for initial swelling, groups M1, M2, and M3 still maintained >50% of their volume, significantly higher than Voluma XC. Group M1 maintained more than twice the volume of Voluma XC® (72% vs. 33%). Table 4H.

[0456] [Table 11]

[0457] Histological evaluation: Decomposition Acceptance Criteria: Degradation is considered complete when all examined sites score 4 on the ISO 10993-6 decomposition scale (as interpreted by NAMSA and LifeSprout for Lumina; see Appendix 2 below). The first time point with all 4s (when at least three sites are analyzed) is considered the time of complete decomposition.

[0458] The prototypes are evaluated at the end of the time point. The absorption scores for the histology slides at 9.5 months are in Table 4I below:

[0459] [Table 12]

[0460] Day 7 histology At day 7, the gels could be easily distinguished from the surrounding tissue using standard histological practices and stains, such as H&E and Masson's Trichrome staining. In Masson's Trichrome-stained slides, the HA gel components stained light blue, and the fibers were stained, allowing cells to be seen migrating from the periphery to the implant site in all four test groups. The cells penetrated the implant, rather than just forming a layer of surrounding tissue. The M1 and M2 groups had more rapid cell ingrowth, with cells visible up to 200 microns into the implant by day 7. The infiltrating cells had a natural spindle shape. Numerous blood vessels were present in the surrounding space, but none were yet visible within the implanted gel. However, the Voluma XC® control did not have cells infiltrating the gel, but rather had a flat band of cells encapsulating the outer layer of the gel.

[0461] The gel was clearly identifiable in the subcutaneous space as a light blue gel with dark blue to purple fibers. The gel samples remained as a solid bolus at the injection site, with groups M3 and M4 being the most resistant to any spreading and having high gel thickness at all time points. The gel groups integrated well with the surrounding tissue without necrosis or encapsulation. In groups M1 and M2, bands of cellular ingrowth were observed at POD 7 and were extensive at POD 30 and 63, with multiple blood vessels forming within the material along with thin collagen deposition. Cell ingrowth and angiogenesis were also observed in groups M3 and M4, but were less extensive, and large areas remained acellular at day 63. Active adipogenesis appeared to be occurring, particularly in group M1, with vascularized adipose tissue surrounding the gel bolus and spherical adipocyte-like structures forming within the adjacent gel. The Voluma group appeared biocompatible, with a thin collagenous band around the gel bolus without necrosis or inflammation, but without cell migration, vascular growth, or adipose tissue formation into the gel.

[0462] By day 63, the Matrix-C prototype was an excellent candidate for dermal filler applications, comparing favorably with the commercially available, lead product, Juvederm® Voluma XC®. Notably, Group M1 had a better irritation and swelling profile than Voluma, maintaining injection volume equivalently up to day 63, while also inducing extensive cellular integration and angiogenesis without fibrosis or encapsulation. At day 63, histology slides showed that active adipogenesis was ongoing in the M1 gel, generating new, healthy-looking fat in place of the gel. This indicates that M1 can be safely injected without swelling and predictable response, and can maintain volumization even as it turns into vascularized, healthy fat.

[0463] At 20 and 26 weeks At 20 weeks, fat pockets in the cutaneous muscle layer were extensively proliferated in the M1 group, and in one example, a large vascularized adipose tissue measuring over 1 mm in length and width could be seen within the center of the M1 gel.

[0464] Groups M3 and M4 gels showed slower cell ingrowth rates than those of groups M1 and M2.

[0465] The Voluma XC® group still shows no ingrowth of cells into the gel. There are some small areas of adipose tissue in the surrounding tissue, but none within the gel or in the cutaneous muscle layer adjacent to the gel. The gel is encapsulated in a thin band of cells and collagen.

[0466] Figure 48 shows a photomicrograph of the M1 group histology slide at 20 weeks. There is extensive cellular ingrowth into the gel, adipose tissue growing around the implanted gel, and significant areas of adipose tissue growing within pockets in the cutaneous muscle layer. The scale bar is 2.5 mm.

[0467] Figure 49 shows a photomicrograph (H&E stain) of an M1 histology slide at 20 weeks. Large (>1 mm x 1 mm) areas of vascularized adipose tissue have developed within the embedded gel. Scale bars are 2.5 mm (top), 250 microns (bottom).

[0468] Figure 50 shows a photomicrograph (H&E stain) of an M2 histology slide at 26 weeks, with extensive cellular ingrowth, adipose tissue formation, and many adipocytes present within the gel in addition to the surrounding tissue. Scale bars are 2.5 mm (top), 500 microns (bottom).

[0469] Figure 51 shows photomicrographs (H&E stain) of Voluma XC® histology slides at 20 weeks. Scale bars are 2.5 mm (top) and 250 microns (bottom). At 20 weeks, there is still very little cellular ingrowth into the gel. Scale bar is 5 mm.

[0470] 40-week histology At 40 weeks, the same trend continued. Depending on the slice location within the implant, the gel was partially replaced by adipose tissue. Groups M1 and M2 had substantially more cellular ingrowth and remodeling than groups M3 and M4, demonstrating the sensitivity of the body's response to the gel formulation, particularly the concentration of collagen fibers and the crosslinker DVS.

[0471] Figure 52 shows a photomicrograph (H&E stain) of an M1 histology slide at 40 weeks. The top image shows a tissue slice with an intact gel bleb, but also extensive adipose tissue surrounding it and in the cutaneous muscle layer. The middle and bottom images are from different slices of the same tissue, and show only a small amount of residual gel in this location. The gel is partially replaced by vascularized adipose tissue. The scale bar is 2.5 mm, 500 microns.

[0472] Figure 53 shows photomicrographs (H&E stain) of M2 histology slides at 40 weeks. The top image shows a tissue slice with intact gel blebs, but also extensive adipose tissue surrounding it and in the cutaneous muscle layer. The bottom image is from a different slice of the same tissue, with adipocytes present within and around the gel. Scale bar is 2.5 mm, 500 microns.

[0473] Figure 54(A) shows a photomicrograph (H&E stain) of an M3 histology slide at 40 weeks. The interior of the gel has some cellular ingrowth throughout the full thickness, but is still primarily acellular at this location. There is some adipose tissue on the left side of the gel. The scale bar is 1 mm.

[0474] Figure 54(B) shows a photomicrograph (H&E stain) of an M4 histology slide at 40 weeks. The interior of the gel is still primarily acellular at this point, but there are cells at the periphery of the gel and adipose tissue to the right of the gel. The scale bar is 2.5 mm.

[0475] Histological evaluation at 69 weeks The animal study was terminated with a final time point group harvested at 69 weeks (16 months) due to the age and size of the animals (too large to be imaged live in an MRI machine). The implant sites were harvested en bloc with surrounding skin tissue and immediately imaged ex vivo in an MRI machine to obtain final volumetric data, which were then prepared for histological evaluation. The gel is still visibly present at the implant site and was well tolerated, with no signs of local irritation or fibrosis. Histological evaluation has not yet been completed on these tissues.

[0476] Figure 55 shows photographs of gels M1, M2, M3, and M4 during explantation at 15 months. The gels maintained their integrity and shape over this time frame.

[0477] Differential adipogenesis in or near the cutaneous muscle layer in the Matrix-c prototype compared to Voluma XC.

[0478] Histological slides showed enhanced adipogenesis in the dermomuscularis layer of rat skin compared to that seen in the Voluma XC® group. This was seen in enlarged areas of vascularized adipose tissue immediately above or below the muscle layer near the implanted gel, or in significantly increased numbers of adipocytes in pockets within the dermomuscularis layer that normally surround nerves. This adipogenesis in pockets of the dermomuscularis layer adjacent to the implanted gel site was also seen in sections cut longitudinally across the dermomuscularis layer, indicating that the adipocyte structure was not a sectioning artifact. Without being bound by theory, adipogenesis could be a direct result of gel flowing into the pocket during injection, or it could be indirectly triggered by biological signals from cells that directly interact with the nearby gel. Potential precursors of the adipocytes generated include pericytes from the vasculature and mesenchymal stem cells or fibroadipogenic progenitors from the muscle. This muscle layer is not a major feature of human skin, unlike in the rat model, indicating that the Matrix-C test article may be more capable of inducing targeted adipogenesis than Voluma XC.

[0479] Figure 56 shows a photomicrograph (Masson's Trichrome stain) of a Voluma XC® histology slide at 26 weeks. The nerve pocket in the dermomuscularis layer adjacent to the implanted gel is devoid of adipocytes. The scale bar is 500 microns.

[0480] FIG. 57 shows a photomicrograph of M1 at 26 weeks, with histological sections both transverse and longitudinal to the muscle fibers of the cutaneous muscle layer (Masson's Trichrome stain).

[0481] Figure 58 shows photomicrographs (H&E staining) of the M2 group at week 20. All scale bars are 500 microns.

[0482] All four prototype gel groups were well tolerated, with minimal to no acute adverse reactions, and erythema and edema profiles superior to those of Voluma XC. No adverse events were observed in any of the rats using the M1-M4 test groups throughout the long-term study. The test gels maintained volume on the early days after injection (days 1 and 7) with minimal to no swelling compared to Voluma XC, which doubled in volume. From day 7 onward, the gel groups maintained volume, losing less volume than the Voluma XC® control up to day 385, with flatter slopes in both absolute volume per time and relative volume per time plots. While still maintaining volumization, the gels also showed significant cellular infiltration; cells proliferated into the embedded gels by day 7, with native dendritic cell morphology. Cells gradually proliferated into the gels over the course of the study, replacing the embedded material with a fully integrated, vascularized cellular tissue, even down to diffuse collagen. Potential preadipocytes are visible in the periphery at early time points, and extensive pools of vascularized adipose tissue can be seen intermingled with the remaining gel in the M1 and M2 test groups by 9.5 months. Adipocytes are only seen at the periphery of the gel in groups M3 and M4, and only in the surrounding tissue for the Voluma XC® control group. This maintenance of volumization, coupled with cellular ingrowth, demonstrates an optimal balance between the rate of gel degradation and replacement by cells and new tissue. The degree of cellular infiltration varied significantly between groups, demonstrating the effectiveness and importance of specific formulation selection for each test group.

[0483] The M1 group maintained 85% of its initial volume for over one year and produced vascularized adipose tissue, making this group an excellent candidate for dermal filler applications and body sculpting.

[0484] The M2 group exhibited more gel spreading for a greater diffusion effect, as may be desired for superficial placement in the body. The lower cross-linking and collagen concentration, and resulting larger pore size, allow for more rapid cell migration and diffusion for therapeutic applications for cell or drug delivery, or to increase cell homing and interaction within the implanted gel.

[0485] The M3 and M4 groups had much slower cell infiltration, maintained their original shape, and resisted spreading. These formulations are excellent candidates for use when implant characteristics such as slow degradation and geometric stability are desirable. Examples include implanting devices in areas of high tissue tension or weight bearing, such as supraperiosteal placement in the body, or for applications where a more permanent plug is desired, such as fistula repair, hernia repair, or coating of permanent implants.

[0486] An ideal soft tissue filler would initially immediately fill the defect site with 100% of the target volume, have mechanical properties similar to the surrounding soft tissue, avoid filler migration or flow, and induce no pain or irritation. The filler should not induce swelling and should also maintain nearly 100% of its volume indefinitely, while paradoxically degrading completely without causing scarring or adverse events (e.g., nodules or granulomas). The test formulation came much closer to achieving this "ideal" profile than any current product, such as the Voluma XC® control included in this study.

[0487] As additional evidence, the test gel induces a significant increase in adipocytes in the striated dermomuscularis, a layer of skin and muscle, in many lower mammals, such as rats. The dermomuscularis contains periodic pockets of nerves surrounded by collagen, blood vessels, and occasionally a small number of adipocytes. Pockets in the dermomuscularis of Voluma XC® control. Without being bound by theory, adipogenesis may be a direct result of gel flowing into the pockets during injection (gel is visible in some pockets at early time points) or may be indirectly triggered by biological signals from nearby gel, from cells that directly interact with the nearby gel or gel degradation products.

Claims

1. Hyaluronic acid, collagen fibers, and divinyl sulfone are reacted to form Thereby, a method for producing a nanofiber-hydrogel composite, comprising the steps of: covalently bonding hyaluronic acid to collagen fibers by divinyl sulfone; hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL; collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; The method wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 0.5:1 to about 5:

1.

2. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 3.

0.

3. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 1.0 to about 3.

0.

4. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 1.5 to about 4.

0.

5. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 2.0 to about 3.

0.

6. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 2.2 to about 3.

0.

7. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 2.3 to about 2.

8.

8. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 1.0 to about 2.

0.

9. The method of claim 1, wherein the composite comprises a swelling ratio during manufacture ranging from about 0.1 to about 1.

0.

10. The method of claim 1 , wherein the composite comprises a swelling ratio of about 2.3 during manufacture.

11. The method of claim 1 , wherein the composite comprises a swelling ratio of about 2.4 during manufacture.

12. The method of claim 1 , wherein the composite comprises a swelling ratio of about 2.6 during manufacture.

13. The method of claim 1 , wherein the composite comprises a swelling ratio of about 2.8 during manufacture.

14. The method of claim 1 , wherein the composite has a swelling percentage in vivo of up to 100%.

15. The method of claim 1, wherein the composite has a swelling percentage in vivo of up to 75%.

16. The method of claim 1, wherein the composite has a swelling percentage of up to 50% in vivo.

17. The method of claim 1 , wherein the composite has a swelling percentage in vivo of up to 40%.

18. The method of claim 1 , wherein the composite has a swelling percentage of up to 30% in vivo.

19. The method of claim 1 , wherein the composite has a swelling percentage in vivo of up to 20%.

20. The method of claim 1, wherein the composite has a swelling percentage in vivo of up to 15%.

21. The method of claim 1, wherein the composite has a swelling percentage in vivo of up to 12%.

22. The method of claim 1 , wherein the composite has a swelling percentage in vivo of up to 10%.

23. 10. The method of claim 1, wherein the composite comprises a swelling percentage in vivo of up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

24. The method of claim 1 , wherein the composite comprises a % swelling in vivo ranging from about 0% to about 100%.

25. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 100%.

26. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 50%.

27. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 20%.

28. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 1% to about 10%.

29. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 10% to about 20%.

30. The method of claim 1, wherein the composite comprises a % swelling in vivo ranging from about 10% to about 15%.

31. The method of claim 1 , wherein the composite comprises a swelling percentage in vivo of about 50%, 40%, 30%, 20%, or 10%.

32. The method of claim 1 , wherein the composite comprises a swelling percentage in vivo of about 10%, 11%, 12%, 13%, 14%, or 15%.

33. The method of claim 1 , wherein the composite comprises a swelling percentage in vivo of about 20%.

34. The method of claim 1 , wherein the composite comprises a swelling percentage in vivo of about 12%.

35. 10. The method of claim 1, wherein the complex comprises hyaluronic acid present in an amount ranging from about 5 mg / mL to about 10 mg / mL.

36. 10. The method of claim 1, wherein the composite comprises hyaluronic acid present in an amount ranging from about 9 mg / mL to about 10 mg / mL.

37. 10. The method of claim 1, wherein the complex comprises hyaluronic acid present in an amount of about 9 mg / mL.

38. 10. The method of claim 1, wherein the complex comprises hyaluronic acid present in an amount of about 9.5 mg / mL.

39. 10. The method of claim 1, wherein the complex comprises hyaluronic acid present in an amount of about 10 mg / mL.

40. 10. The method of claim 1, wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

41. 10. The method of claim 1, wherein the composite comprises collagen present in an amount ranging from about 15 mg / mL to about 30 mg / mL.

42. 10. The method of claim 1, wherein the composite comprises collagen present in an amount ranging from about 20 mg / mL to about 30 mg / mL.

43. 10. The method of claim 1, wherein the composite comprises collagen present in an amount ranging from about 25 mg / mL to about 30 mg / mL.

44. 10. The method of claim 1, wherein the composite comprises collagen present in an amount of about 15 mg / mL.

45. 10. The method of claim 1, wherein the composite comprises collagen present in an amount of about 30 mg / mL.

46. 10. The method of claim 1, wherein the composite comprises collagen present in an amount of about 25 mg / mL.

47. the composition comprises hyaluronic acid present in an amount of about 9 mg / mL; the composite comprising collagen present in an amount of about 15 mg / mL; The method of claim 1.

48. the composite comprises hyaluronic acid present in an amount of about 10 mg / mL; the composite comprises collagen present in an amount of about 30 mg / mL; The method of claim 1.

49. the composite comprises hyaluronic acid present in an amount of about 9.5 mg / mL; the composite comprises collagen present in an amount of about 25 mg / mL; The method of claim 1.

50. 10. The method of claim 1, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 30 mg / mL.

51. 10. The method of claim 1, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 25 mg / mL.

52. 10. The method of claim 1, wherein the hyaluronic acid is present in an amount ranging from about 23 mg / mL to about 25 mg / mL.

53. 10. The method of claim 1, wherein the hyaluronic acid is present in an amount of about 23 mg / mL.

54. 10. The method of claim 1, wherein the hyaluronic acid is present in an amount of about 25 mg / mL.

55. 10. The method of claim 1, wherein the collagen is present in an amount ranging from about 30 mg / mL to about 100 mg / mL.

56. 10. The method of claim 1, wherein the collagen is present in an amount ranging from about 40 mg / mL to about 70 mg / mL.

57. 10. The method of claim 1, wherein the collagen is present in an amount ranging from about 45 mg / mL to about 65 mg / mL.

58. 10. The method of claim 1, wherein the collagen is present in an amount of about 45 mg / mL.

59. 10. The method of claim 1, wherein the collagen is present in an amount of about 65 mg / mL.

60. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 1:1 to about 5:

1.

61. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 2:1 to about 5:

1.

62. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 3:1 to about 5:

1.

63. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of divinyl sulfone molecules per hyaluronic acid subunit ranging from about 3.5:1 to about 4:

1.

64. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of about 3.5:1 divinyl sulfone molecules per hyaluronic acid subunit.

65. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit.

66. 2. The method of claim 1, wherein the divinyl sulfone is present in an amount of about 4.5:1 divinyl sulfone molecules per hyaluronic acid subunit.

67. hyaluronic acid is present in an amount of about 25 mg / mL; collagen is present in an amount of about 45 mg / mL; The divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit. The method of claim 1.

68. hyaluronic acid is present in an amount of about 25 mg / mL; collagen is present in an amount of about 45 mg / mL; The divinyl sulfone is present in an amount of about 3.5:1 divinyl sulfone molecule per hyaluronic acid subunit. The method of claim 1.

69. hyaluronic acid is present in an amount of about 23 mg / mL; collagen is present in an amount of about 65 mg / mL; The divinyl sulfone is present in an amount of about 4.5:1 divinyl sulfone molecule per hyaluronic acid subunit. The method of claim 1.

70. hyaluronic acid is present in an amount of about 23 mg / mL; collagen is present in an amount of about 65 mg / mL; The divinyl sulfone is present in an amount of about 4:1 divinyl sulfone molecules per hyaluronic acid subunit. The method of claim 1.

71. 10. The method of claim 1, wherein the collagen nanofibers comprise type I, type II, type III, or type IV collagen.

72. The method of claim 1 , wherein the collagen nanofibers comprise recombinant collagen.

73. The method of claim 1 , wherein the collagen nanofibers comprise type I bovine collagen nanofiber fragments.

74. The method of claim 1 , wherein the collagen nanofibers are electrospun, melt spun, blow spun, and / or cryomilled.

75. The method of claim 1 , wherein the collagen nanofibers are retained within the nanofiber-hydrogel composite.

76. 10. The method of claim 1, wherein the interfacial bonding between the collagen nanofibers and HA enhances the stiffness of the composite with a relatively low fiber loading density.

77. 10. The method of claim 1, wherein the nanofiber-hydrogel composite exhibits monocyte recruitment, monocyte polarization, or both.

78. 10. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized by exhibiting higher porosity, increased cell permeability, and / or maintains storage modulus.

79. The method of claim 1 , wherein the nanofiber-hydrogel composite allows for cell infiltration.

80. 10. The method of claim 1, wherein the pH of the nanofiber-hydrogel composite is about 7.0, 7.1, 7.2, 7.3, or 7.4 in an isotonic solution.

81. The method of claim 1, wherein the nanofiber-hydrogel composite is formulated in one or more product forms selected from a mat, an injectable solution, and a flowable solution.

82. 10. The method of claim 1, wherein the nanofiber-hydrogel composite is compatible for passage through a needle of 27 gauge or smaller.

83. 2. The method of claim 1, wherein the crosslinking agent reacts with the hydroxyl groups of the HA and the hydroxyl, thiol, or amine groups of the collagen nanofibers.

84. The method of claim 1, wherein the crosslinking agent reacts with the hydroxyl groups of the HA and the hydroxyl or amine groups of the collagen nanofibers.

85. The method of claim 1 , wherein the nanofiber-hydrogel composite forms one or more porous structures.

86. The method of claim 1 , wherein the one or more porous structures induce increased host cell infiltration.

87. 10. The method of claim 1, wherein the nanofiber-hydrogel composite exhibits a biostimulatory effect selected from tissue remodeling, host cell infiltration, cell adhesion, cell migration, angiogenic response, adipogenic response, and macrophage polarization toward a pro-healing phenotype.

88. 10. The method of claim 1, wherein the nanofiber-hydrogel composite comprises a population of infiltrating macrophages, and the collagen nanofibers condition the population of infiltrating macrophages to an M2 phenotype.

89. 10. The method of claim 1, wherein the nanofiber-hydrogel composite exhibits nanofiber-hydrogel composite-induced tissue remodeling effects without incorporating cells or growth factors.

90. The method of claim 1, wherein the nanofiber-hydrogel composite exhibits biostimulatory effects due to the low crosslinking density of the composite.

91. 10. The method of claim 1, wherein the nanofiber-hydrogel composite is suitable for terminal sterilization by autoclaving.

92. 10. The method of claim 1, wherein the composite is characterized by increased thermal stability and / or provides shelf stability at ambient temperatures.

93. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber-hydrogel composite product of the method of embodiment v, wherein the nanofiber-hydrogel composite is biocompatible.

94. The method of claim 1 , wherein the nanofiber-hydrogel composite exhibits cell adhesion.

95. The method of claim 1 , wherein the nanofiber-hydrogel composite exhibits cell migration.

96. 10. The method of claim 1, wherein the nanofiber-hydrogel composite enables durable soft tissue remodeling.

97. The method of claim 1 , wherein the nanofiber-hydrogel composite promotes angiogenesis.

98. 10. The method of claim 1, wherein the nanofiber-hydrogel composite prolongs the biostimulatory effect by retaining and / or conditioning infiltrating macrophages toward a pro-regenerative M2 phenotype, thereby promoting neovasculature formation.

99. The method of claim 1 , wherein the nanofiber-hydrogel composite improves adipose tissue formation.

100. The method of claim 1 , wherein the nanofiber-hydrogel composite exhibits vasculature formation.

101. The method of claim 1 , wherein the nanofiber-hydrogel composite modulates host cell infiltration and / or shape retention of the composite.

102. 10. The method of claim 1, wherein the storage modulus is within the range of most soft tissues.

103. 10. The method of claim 1, wherein the storage modulus is in the range of about 150 Pa to about 500 Pa.

104. 10. The method of claim 1, wherein the nanofiber-hydrogel composite is micronized into a series of microgels having diameters selected from the group consisting of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μm.

105. The method of claim 1 , wherein the nanofiber-hydrogel composite mimics host tissue structure.

106. The method of claim 1 , wherein the nanofiber-hydrogel composite mimics the natural extracellular matrix.

107. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups.

108. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups in the range of about 10% to about 90%.

109. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups of at least 10%.

110. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups of at least 20%.

111. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups of at least 18%.

112. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups by at least 50%.

113. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups of at least 60%.

114. 10. The method of claim 1, wherein the method results in a reduction of residual vinyl sulfone groups of at least 67%.

115. 16. The method of claim 15, wherein the hydrogel comprises residual sulfonic groups at a concentration of up to about 2.0 μg / mL of hydrogel.

116. 16. The composition of claim 15, wherein the hydrogel contains residual sulfonic acid groups at a concentration of up to about 1.7 μg / mL of hydrogel.

117. 16. The composition of claim 15, wherein the hydrogel comprises residual sulfonic groups at a concentration of up to about 1.4 μg / mL of hydrogel.

118. 16. The composition of claim 15, wherein the hydrogel contains residual sulfonic groups at a concentration of up to about 1.0 μg / mL of hydrogel.

119. 16. The composition of claim 15, wherein the hydrogel contains residual sulfonic groups at a concentration of up to about 0.5 μg / mL of hydrogel.

120. 16. The composition of claim 15, wherein the hydrogel comprises residual sulfonic groups at a concentration ranging from about 0.5 μg / mL to about 1.4 μg / mL of hydrogel.

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