Collagen-hydrogel composite

Abstract

The disclosure provides formulations and methods to produce 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

COLLAGEN-HYDROGEL COMPOSITERELATED APPLICATIONS

[0001] The present application claims the benefit of priority of U.S. provisional application no. 63 / 485,240 filed February 15, 2023, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

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

[0003] The disclosure also relates generally compositions and methods to restore lost soft tissue volume while promoting soft tissue regeneration and applications of dermal fillers, reconstructive and cellular therapies.BACKGROUND

[0004] Hydrogels are popular scaffold matrices for tissue restoration in the field of soft tissue repair because of their biomimetic features close to the tissue microenvironment and their ability to be tailored for different adipose tissue needs such as volume and porosity. Young, D.A., et al., biomaterialia, 2011. 7(3): p. 1040-1049; and Varma, D.M., et al., Acta Biomaterialia, 2014. 10(12): p. 4996-5004.

[0005] Among various hydrogel products, hyaluronic acid (HA) has been widely used as fillers and foams for soft tissue augmentation because of its biocompatibility as an endogenous polysaccharide, its moderate biodegradability, and similar physical properties to soft tissues like adipose tissue, fibrous tissue, and nerves. See, Tezel, A. et al., Journal of Cosmetic and Laser Therapy, 2008. 10(1): p. 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): p.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 a robust therapeutic systemformulated by hyaluronic acid (HA) chemically bonded to polycaprolactone (PCL) nanofibers, which gives the material higher porosity and cell permeability while maintaining storage moduli to match the 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.

[0007] Although collagen, as a bio-active and bio- stimulatory component, has been extensively used as fillers and wound dressing, there still exist drawbacks, such as its volume retention and the resultant inflammatory responses. What is needed, therefore, is a system for new approaches to soft tissue engineering with one or more desirable characteristics such as improved mechanical properties, biocompatibility, enhanced injectability, feasibility for terminal sterilization and reduced costs.SUMMARY OF THE DISCLOSURE

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

[0009] In various aspects, 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 composite provides a natural, autoclavable, one-step crosslinking of both fibers and the hydrogel phase and does not require a PEG-based crosslinking agent.

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

[0012] In various other aspects, the nanofiber-hydrogel composite provides a number of advantageous biostimulatory or biocompatible features. For instance, the nanofiber-hydrogel composite exhibits monocyte recruitment and / or polarization, or enables cellular infiltration.

[0013] In various embodiments, the nanofiber-hydrogel composite provides biostimulatory effects selected from tissue remodeling, host cell infiltration, cell adhesion, cellmigration, angiogenic responses, adipogenic responses, macrophage polarization to pro-healing phenotypes, and regenerative responses.

[0014] In various aspects, the nanofiber-hydrogel composite exhibits preferred biocompatibility selected from one or more of the properties including cell adhesion, cell migration, durable soft tissue remodeling, angiogenesis, stimulatory effects by retaining the nanofiber-hydrogel composite conditioned infiltrating macrophages towards a pro-regenerative M2 phenotype, neo-vasculature formation, adipose tissue formation, vasculature formation, and accelerated tissue remodeling.

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

[0016] In various embodiments, the hydrogel material is present in the complex 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, a nanofiber-hydrogel composite comprises a hydrogel material comprising a hyaluronic acid, a nanofiber material comprising a collagen, and a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; and wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

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

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

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

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

[0023] Additionally disclosed are kits comprising the nanofiber hydrogel composite material.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 A depicts a schematic for the preparation of divinyl sulfone (DVS) crosslinked hyaluronic acid (HA) hydrogel and nanofiber-hydrogel composite (NHC), the particularization of the materials, and the injection subcutaneously to the back of Sprague Dawley (SD) rats. FIG. IB is an illustration depicting HA (double line) and collagen fibers (bold solid line) crosslinked using DVS. FIG. 1C depicts non-limiting examples of DVS crosslinking to alternative hydroxyl groups on HA.

[0025] FIG. 2 depicts a chart showing % Swelling of implanted gels at Day 1 of animal study (example #6) as measured by MRI.

[0026] FIG. 3 depicts a chart showing % Swelling of implanted gels at Day 7 of animal study (example #6) as measured by MRI with commercial comparator (Voluma XC).

[0027] FIG. 4 depicts a chart showing the swelling potential of Hyaluronic acid gels (without fibers), made in example #2, showing the inverse correlation between initial HA concentration and the swelling ratio post-gelation.

[0028] FIG. 5 depicts a chart showing the final hyaluronic acid concentration of Hyaluronic acid gels (without fibers), made in example #2, showing the effect of initial HA concentration (during gelation) on the final HA concentration post-gelation.

[0029] FIG. 6 depicts a chart showing the final hyaluronic acid concentration of Hyaluronic acid gels (without fibers), made in example #2, showing the effect of DVS concentration (during gelation) on the final HA concentration post-gelation at a fixed initial HA concentration of 30mg / mL.

[0030] FIG. 7 depicts a chart showing the final hyaluronic acid concentration of Hyaluronic acid gels (without fibers), made in example #2, showing the effect of the DVS concentration (during gelation) on the final HA concentration post-gelation. The overall correlation is weaker than when HA Concentration is fixed (such as in figure 6) or other correlations (such as figure 5).

[0031] FIG. 8 depicts a chart showing the swelling potential of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the inverse correlation between initial collagen concentration (pre-gelation) and the swelling ratio for post-gelation gel.The fibers, which are covalently linked to the hydrogel phase, reduce the swelling potential of the resulting composite in a dose-response manner. This correlation is also evidence of interfacial bonding between the fiber phase and hydrogel phase, as the fiber presence is affecting the swelling behavior of the hydrogel phase.

[0032] FIG. 9 depicts a chart showing the swelling potential of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the correlation between initial HA concentration (pre-gelation) and the swelling ratio for post-gelation gel. The correlation is much weaker than for the groups without fibers (figure 4), showing that the collagen fiber concentration has a large effect on the swelling behavior of the gels during manufacturing and is an important variable to achieve a targeted final HA concentration and thus swelling profile in vivo.

[0033] FIG. 10 depicts a chart showing the swelling potential of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the correlation between DVS crosslinking concentration (pre-gelation) and the swelling ratio for post-gelation gel. The DVS to-HA subunit ratio is inversely correlated with swelling ratio.

[0034] FIG. 11 depicts a chart showing the final gel (in finished syringe) concentration of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the correlation between collagen fiber concentration (pre-gelation) and the HA concentration in the post-gelation gel. The increasing collagen content is associated with a lower swelling ratio during manufacturing, and thus a higher final HA content (less dissolution during post-gelation manufacturing). The correlation is stronger than the correlation for the initial HA concentration (fig 12).

[0035] FIG. 12 depicts a chart showing the final gel (in finished syringe) concentration of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the correlation between the initial HA concentration (pre-gelation) and the HA concentration in the post-gelation gel. The increasing HA content is weakly associated with a higher final HA content.

[0036] FIG. 13 depicts a chart showing the final gel (in finished syringe) concentration of Hyaluronic acid composite gels (with collagen fibers), made in example #2, showing the correlation between the initial DVS crosslinker relative concentration (pre-gelation) and the HAconcentration in the post-gelation gel. The increasing DVS content is associated with a higher final HA content.

[0037] FIG. 14 depicts (A) the chart depicts the relative volumization (measured with MRI) profile of the M1-M4 formulations made in Example #4 and tested in Example #6 through 63 days. The values are normalized to the measured values immediately after injection. The Voluma doubled in volume, while the 4 composite gels are maintaining volume without initial swelling.

[0038] FIG. 15 depicts representative photographic images of the 4 implanted gels from groups during explantation after 7 days in rat subcutaneous implantation model. All 4 test group implants (M1-M4) and commercial comparator (Voluma XC) were well tolerated. 1= Ml, 2 = M2, 3=M3, 4=M4, 5=Voluma XC. All images at same scale, Scalebar = 1cm.

[0039] FIG. 16 depicts representative photographic images of the 4 implanted gels from groups during explantation after 30 days in rat subcutaneous implantation model. All 4 test group implants (M1-M4) and commercial comparator (Voluma XC) were well tolerated. Tl= Ml, T2 = M2, T3=M3, T4=M4, Voluma =Voluma XC. All images at same scale. Scalebar = 1cm.

[0040] FIG. 17 depicts histology images of Group Ml from Example #6 at day 7. Masson’s tri chrome staining (top) and H&E Staining (middle, bottom). The implanted gel is maintaing its integrity and shape and is clearly distinguishable from the surrounding tissue and is inducing cellular infiltration from the periphery inwards. Scalebar = 2.5mm (top left, middle left), 250 microns (top right), 100 microns (middle right, bottom).

[0041] FIG. 18 depicts histology images of Group M2 at day 7 from example #6. Masson’s tri chrome staining (top) and H&E Staining (bottom). Cellular infiltration is clearly visible. Scalebar = 2.5mm (left column), 250 microns (right column)

[0042] FIG. 19 depicts histology images of Group M3 at day 7. Areas of cellular infiltration are not as frequent as in groups Ml and M2, but are preseent in areas on gel periphery. Masson’s trichrome staining (top) and H&E Staining (bottom) Scalebar = 2.5mm (left column), 250 microns (right column)

[0043] FIG. 20 depicts histology images of Group M4 at day 7. Areas of cellular infiltration are infrequent compared to groups Ml and M2, but are preseent in areas on gelperiphery. Masson’s trichrome staining (top) and H&E Staining (bottom) Scalebar = 2.5mm (left column), 250 microns (right column)

[0044] FIG. 21 depicts histology images of control group (Voluma XC) at day 7. No apparent cellular infiltration into the gel. Masson’s trichrome staining (top) and H&E Staining (bottom) Scalebar = 2.5mm (left column), 250 microns (right column)

[0045] FIG. 22 depicts histology images (Masson’s Trichrome staining) of Group Ml at day 30. The implant has been infiltrated by cells, together with collagen deposition and numerous functional blood vessels. Scalebars are 2.5mm (left), 250microns (right).

[0046] FIG. 23 depicts histology images (Masson’s Trichrome staining) of Group Ml at day 30. The implant has been infiltrated by cells, together with collagen deposition and numerous functional blood vessels. Scalebar is lOOmicrons.

[0047] FIG. 24 depicts histology images (H&E staining) of Group Ml at day 30. The implant has been infiltrated by cells, together with collagen deposition and numerous functional blood vessels. Scalebars are 500 microns (left) and lOOmicrons (right).

[0048] FIG. 25 depicts histology images of Group M2 at day 30. Top row: Masson’s Trichrome staining; bottom row: H&E staining. The implant has been infiltrated by cells, together with collagen deposition and numerous functional blood vessels. Scalebars are 2.5mm (left column) and 250microns (right column).

[0049] FIG. 26 depicts histology images of Group M3 at day 30. Top row: Masson’s Trichrome staining; bottom row: H&E staining. The implant has been infiltrated by cells, together with numerous functional blood vessels. The density of cells and blood vessels is lower than groups Ml and M2, with substantial regions of the implant not yet infiltrated by cells. The implanted gels maintained the original bolus shape well (little spreading). Scalebars are 2.5mm (left column) and 250microns (right column)

[0050] FIG. 27 depicts histology images of Group M4 at day 30. Top row: Masson’s Trichrome staining; bottom row: H&E staining. The implant has been infiltrated by cells only at the periphery by day 30. Blood vessels have not formed within the implanted material by this timepoint. The density of cells inside the implant is lower than groups Ml and M2, and M3, with most regions of the implant not yet infiltrated by cells. The implanted gels maintained theoriginal bolus shape well (little spreading). Scalebars are 2.5mm (left column) and 250microns (right column)

[0051] FIG. 28 depicts histology images of Control group (Voluma XC) at day 30. Top row: Masson’s Tri chrome staining; bottom row: H&E staining. The implant is intact but does not have apparent cellular infiltration and does not have blood vessels within the implanted gel. The gel is surrounded by a thin collagenous band. Scalebars are 2.5mm (left column), 250microns (right column).

[0052] FIG. 29 depicts Photographic images of the 4 implanted gels from Ml group during explantation after 63 days in rat subcutaneous implantation model. All 4 implants were well tolerated and integrated into the surrounding tissue with similar appearance to the native tissue. Scalebar = 1cm.

[0053] FIG. 30 depicts an image of H&E Histology of Group Ml at Day 63 in rat subcutaneous implantation model. The gel maintains volumization while undergoing remodeling, with cellular infiltration and angiogenesis. Active adipogenesis is underway at the gel periphery (marked by solid border area), generating new fat tissue. Scale bar = 2.5mm.

[0054] FIG. 31 depicts a magnified image of section of Fig. 30 of H&E histology slide of Ml group at POD 63, showing the regions of active adipogenesis and resulting new adipose tissue, together with examples of blood vessels. Scale bar = 100 microns. Symbols: * = Newly Generated Adipose Tissue; + = Active Adipose Tissue Generation; # ^Intact Gel and Fibers;A= Blood vessel.

[0055] FIG. 32 depicts an image of H&E Histology of Group M2 at Day 63 in rat subcutaneous implantation model. The gel maintains volumization while undergoing remodeling, with cellular infiltration and angiogenesis. Active adipogenesis is underway at the gel periphery, generating new fat tissue. Scale bar = 2.5mm.

[0056] FIG. 33 depicts an image of H&E Histology of Group M3 at Day 63 in rat subcutaneous implantation model. The gel maintains volumization while undergoing remodeling, with cellular infiltration and angiogenesis. The gel maintains its shape and thickness over time. The Cellular infiltration rate is decreased relative to groups Ml and M2 at this timepoint, with partial, progressive cellular remodeling on left half of implant image, but little infiltration onright half of implant image. Scale bar = 2.5mm.

[0057] FIG. 34 depicts a higher magnification image of same histology slide as Fig. 33, of M3 group at POD 63. Note the progressive cellular infiltration and angiogenesis. Scale bar = 250 microns.

[0058] FIG. 35 (A) depicts an image of H&E Histology of Group M4 at Day 63 in rat subcutaneous implantation model. The gel maintains volumization while undergoing remodeling, with cellular infiltration and angiogenesis. The gel maintains its shape and thickness over time. The Cellular infiltration rate is decreased relative to groups Ml and M2 at this timepoint, with partial, progressive cellular remodeling on right lobe of implant image, but little infiltration on left lobe in implant image. Scale bar = 2.5mm. (B) Micrographs of group M4 histology slide at day 63 (H&E Staining). Scale bar = 250 micrometers. The extent of cellular infiltration is far lower for group M4 than group Ml, yet cells can still be seen progressively growing into the implant site, with natural spindly morphology. The bulk gel is maintaining integrity while awaiting the cellular infiltration. The Gel bolus maintained bolus shape well. In the areas with more extensive ingrowth, blood vessels are formed within the bulk of the gel (black arrows) and smaller white globular structures are visible. Scale bars are 100 microns (top) and 250 microns (bottom).

[0059] FIG. 36 depicts a graph of a representative trace of the injection force testing (on Instron universal test system) for the group Ml gels produced in example #4 and tested in animal study of example #6, injected at a volumetric rate of 0.25cc / min through a 27gauge needle.

[0060] FIG. 37 depicts a graph of a representative trace of the injection force testing (on Instron universal test system) for the group M2 gels produced in example #4 and tested in animal study of example #6, injected at a volumetric rate of 0.25cc / min through a 27gauge needle.

[0061] FIG. 38 depicts a graph of a representative trace of the injection force testing (on Instron universal test system) for the group M3 gels produced in example #4 and tested in animal study of example #6, injected at a volumetric rate of 0.25cc / min through a 27gauge needle.

[0062] FIG. 39 depicts a graph of a representative trace of the injection force testing (on Instron universal test system) for the group M4 gels produced in example #4 and tested in animal study of example #6, injected at a volumetric rate of 0.25cc / min through a 27gauge needle.

[0063] FIG. 40 depicts a graph of representative traces of the storage modulus (G’) for the test groups produced in example #4 and tested in animal study of example #6. Rheology testing per example #3 on oscillatory rheometer (AR2000ex, TA Instruments) at 25°C with a parallel plate geometry with a 25mm diameter top plate, gap of 900microns, and frequency of 1Hz. Groups Ml (open circle), M2 (filled circle), M3 (open square), M4 (filled square).

[0064] FIG. 41 depicts a graph of representative traces of the loss modulus (G”) for the test groups produced in example #4 and tested in animal study of example #6. Rheology testing per example #3 on oscillatory rheometer (AR2000ex, TA Instruments) at 25°C with a parallel plate geometry with a 25mm diameter top plate, gap of 900microns, and frequency of 1Hz. Groups Ml (open circle), M2 (filled circle), M3 (open square), M4 (filled square).

[0065] FIG. 42 depicts a graph of representative traces of the tan-delta values (tan-8) for the tests groups produced in example #4 and tested in animal study of example #6. Rheology testing per example #3 on oscillatory rheometer (AR2000ex, TA Instruments) at 25°C with a parallel plate geometry with a 25mm diameter top plate, gap of 900microns, and frequency of 1Hz. Groups Ml (open circle), M2 (filled circle), M3 (open square), M4 (filled square).

[0066] FIG. 43 depicts an SEM image of porcine collagen with a mean diameter of 0.57microns + / - 0.32 microns. Spun into fibers by dissolving to 7% in HFIP and spinning at 0.3ml / hour at 1 IkV voltage. Scale Bar = 30 micrometers.

[0067] FIG. 44 depicts an SEM image of Porcine gelatin (Sigma) spun producing ribbon morphology. Scale Bar = 80 micrometers.

[0068] FIG. 45 depicts an SEM image of fibers spun from recombinant collagen (Vecollan / Evonik). The image shows cylindrical or ribbon morphologies. Scale Bar = 100 micrometers.

[0069] FIG. 46 depicts line plots of volume data measured via MRI demonstrating volumization maintenance over time after implantation in a rat subcutaneous model. The top graph shows the mean measured volume in cubic centimeters for each group through 385 days with error bars denoting the standard deviation.

[0070] FIG. 47 (A) depicts the relative volume as a percentage of each individual implant’s day 7 volume. This more closely demonstrates the volume profile a patient wouldexperience, accounting for the significant swelling expected for the Voluma XC® group. FIG. 47 (B) depicts the relative volume for just the Ml and Voluma XC® groups for more clearly highlighting the improvement from the disclosed invention..

[0071] FIG. 48 depicts a micrograph of Ml group histology slide at 20 weeks. There is extensive cellular ingrowth into the gel, adipose tissue growing around the implanted gel, and substantial areas of adipose tissue growing in the pockets in the panniculus carnosus layer. Scale bar is 2.5mm.

[0072] FIG. 49 depicts a micrograph of Ml histology slide at 20 weeks (H&E staining). A large (> 1mm x 1mm) section of vascularized adipose tissue has developed within the implanted gel. Scale bar is 2.5mm (A), 250 microns (B).

[0073] FIG. 50 depicts a micrograph of M2 histology slide at 26 weeks (H&E staining) with extensive cellular ingrowth, adipose tissue formation, and numerous adipocytes present within the gel in addition to the surrounding tissue. Scale bar is 2.5mm (A), 500 microns (B).

[0074] FIG. 51 depicts a micrograph of Voluma XC® histology slide at 20 weeks (H&E staining). Scale bar is 1mm.

[0075] FIG. 52 depicts a micrograph of Ml histology slide at 40 weeks (H&E staining). Images are from a different slice of same tissue, with only a small amount of residual gel at this location. The gel is partially replaced with vascularized adipose tissue. Scale bar is 2.5mm (A), 500 microns (B).

[0076] FIG. 53 depicts a micrograph of M2 histology slide at 40 weeks (H&E staining). Top image shows a tissue slice with intact gel bleb, but also extensive adipose tissue surrounding it and in panniculus carnosus layer. The bottom image is from a different slice of same tissue, with adipose cells present within and around the gel. Scale bar is 2.5mm (A), 500 microns (B).

[0077] FIG. 54 (A) depicts a micrograph of M3 histology slide at 40 weeks (H&E staining). The interior of the gel has some cellular ingrowth across the full thickness, but is still predominantly acellular at this location. There is some adipose tissue on the left of the gel. Scale bar is 1mm. FIG. 54 (B) depicts a micrograph of M4 histology slide at 40 weeks (H&E staining). The interior of the gel is still predominantly acellular at this location at this timepoint, though there is cells on the periphery of the gel and adipose tissue to the right of the gel. Scale bar is2.5mm.

[0078] FIG. 55 depicts a photograph of gels Ml, M2, M3, and M4 during explantation at the 15month timepoint. The gels maintained integrity and shape over this timeframe.

[0079] FIG. 56 depicts a micrograph of Voluma XC® histology slide at 26 weeks (Masson’s trichrome staining). The nerve pocket (B) in the panniculus carnosus layer (C) neighboring the implanted gel (A) lacks adipocytes. Scale bar is 500 microns.

[0080] FIG. 57 depicts a Micrographs of Ml (A) at 26 weeks (Masson’s trichrome staining). Scale bars is 500 microns. Fig. 57 (a) shows tissue sections traverse to the panniculus carnosus muscle fibers. Fig. 57 (b) shows tissue sections longitudinal to the panniculus carnosus muscle fibers. Circle A shows a nerve pocket with adipocytes. Circle B shows M2. Circle C shows a panniculus carnosus layer.

[0081] FIG. 58 depicts a Micrograph of M2 at 20 weeks (H&E Staining). Scale bars is 500 microns.DETAILED DESCRIPTION OF THE DISCLOSURE

[0082] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular 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 various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.Incorporation by reference

[0083] All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.Definitions

[0084] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0085] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0086] The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0087] As used herein, "about" or "approximately" can mean within an acceptable error range, plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.

[0088] As used herein the specification, "subject" or "subjects" or "individuals" may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild animals, as well as birds and aquatic animals.

[0089] As used herein, a "scaffold complex" or "composite" includes any association of two components, e.g., a covalent association: a polymeric fiber and a hydrogel material. The scaffold complex contains the polymeric fiber and hydrogel material in a "functional network", meaning that the interactions between components results in a chemical, biochemical, biophysical, physical, or physiological benefit. In addition, a functional network may include additional components, including cells, biological materials (e.g., polypeptides, nucleic acids, lipids, carbohydrates), therapeutic compounds, synthetic molecules, and the like. In certain embodiments, the scaffold complex promotes tissue growth and cell infiltration when implanted into a target tissue present in a human subject.

[0090] 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, blends thereof) held together by covalent or non- covalent crosslinks that can absorb a substantial amount of water (e.g., 50%, 60% 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or greater than 99% per unit of non-water molecule) to form an elastic gel. The polymeric matrix may be formed of any suitable synthetic or naturally occurring polymer material. As used herein, the term "gel" refers to a solid three-dimensional network that spans the volume of a liquid medium and ensnares it through surface tension effects.

[0091] As used herein, the term "Nanofiber” describes the individual unit of a fibrous material with a high specific surface area, made of individual units with a high aspect ratio (length substantially greater than width) with at least one feature that is typically in the nanometer scale (with diameters in the range of about 1-1000 nanometers). The term nanofiber can also include, but not limited to, fibers with diameters of thousands of nanometers (singledigits of microns), since such materials are produced with similar technologies and have similar traits. As a non-limiting example, “nanofibers” produced via electrospinning generally have diameters in the range of 50nm to 10 microns. The nanofibers may be produced by methods such as electrospinning, melt spinning, drawing, phase separation, or self-assembly. Nanofibers are typically comprised of polymeric synthetic or natural materials.

[0092] As used herein, the term "Microfiber” describes the individual unit of a fibrous material with a high specific surface area, made of individual units with a high aspect ratio (length substantially greater than width) with at least one feature that is typically in the micrometer scale (with diameters in the range of about 1 -100 microns).

[0093] The term "crosslinked" herein refers to a composition containing intramolecular and / or intermolecular crosslinks, whether arising through covalent or noncovalent bonding, and may be direct or include a cross-linker. "Noncovalent" bonding includes both hydrogen bonding and electrostatic (ionic) bonding.

[0094] As used herein, the term "storage modulus" is used to define the measurement of the elastic components of the dynamic modulus which illustrates how the material responds to deformation or stress. In an embodiment, "deformation" means the change in shape or size of an object due to applied force.

[0095] As used herein, the term "shear modulus" also known as the modulus of rigidity, denoted by (G’), is defined as the ratio of shear stress to the shear strain. In an embodiment,"shear stress" means the component of stress coplanar with a material cross section. In an embodiment, "shear strain" means the force perpendicular to the material cross section.

[0096] As used herein, the term "loss modulus" also known as the modulus of viscosity, denoted by (G”). The loss modulus is the inelastic part of the dynamic (or complex) modulus. Dynamic modulus is the ratio of stress to strain.

[0097] Tan Delta (Tan-5; 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 “liquidlike” material.

[0098] As used herein, the term "POD" refers to post-operative date.

[0099] 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.

[0100] 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 of skill in the art. The materials, methods, and examples are illustrative only and not intended to be limiting.Fiber Dimensions

[0101] Fibers can have one or more of various morphologies. In certain embodiments, the fibers can be cylindrical or ribboned. In various embodiments, the fibers comprise a mixed morphology. In various embodiments, the polymeric fibers comprise a cylindrical morphology. In various embodiments, the polymeric fibers comprise a ribbon morphology. In various embodiments, the polymeric fibers comprise both a cylindrical morphology and a ribbon morphology.

[0102] Cylindrical fibers need not necessarily have perfectly round cross section. Cylindrical fibers include, but are not limited to, fibers comprising similar cross-section width and height.

[0103] In various embodiments, ribbon-shaped fibers comprise a cross-section width greater than a cross-section height. In various embodiments, ribbon-shaped fibers comprise a cross-section width about 10% to about 90% greater than cross-section height. In variousembodiments, ribbon-shaped fibers comprise a cross-section width about 1% to about 10% greater than cross-section height. In various embodiments, ribbon-shaped fibers comprise a cross-section width about 10% to about 50% greater than cross-section height. In various embodiments, ribbon-shaped fibers comprise a cross-section width about 25% to about 75% greater than cross-section height.

[0104] In various embodiments, the polymeric fibers have a mean diameter of less than about 100 pm, less than about 50 pm, less than about 40 pm, less than about 30 pm, less than about 20 pm, less than about 10 pm, less than about 900 pm, less than about 800 pm, less than about 700 pm, less than about 600 pm, less than about 500 pm, less than about 400 pm, less than about 300 pm, less than about 200 pm, less than about 100 pm, less than about 50 pm, less than about 10 pm, less than about 5 pm, or less than about 1 pm.

[0105] In various embodiments, the polymeric fibers have a mean diameter of less than about 10 pm, less than about 5 pm, less than about 4 pm, less than about 3 pm, less than about 2 pm, less than about 1 pm, 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.

[0106] In various embodiments, the polymeric fibers have a mean diameter in a range of about 100 pm to 1000 pm, about 10 pm to 100 pm, about 10 pm to 50 pm. In certain embodiments, the fibers have a mean diameter in a range of about 1 pm to 1,000 pm, about 1 pm to 500 pm, or about 1 nm to 100 pm.

[0107] In various embodiments, the polymeric fibers have a mean diameter in a range of about 10 nm to 5 pm, about 100 nm to 5 pm. In certain embodiments, the fibers have a mean diameter in a range of about 1 nm to 1,000 nm, about 1 nm to 500 nm, or about 1 nm to 100 nm.

[0108] In various embodiments, polymeric fibers, have a mean diameter of from about 10 nm to about 10,000 nm, such as about 100 nm to about 8000 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.

[0109] In various embodiments, the polymeric fibers (e.g., microfibers) have a meanlength greater than about 1 gm, greater than about 5 gm, greater than about 10 gm, greater than about 20 gm, greater than about 30 gm, greater than about 40 gm, greater than about 50 gm, greater than about 60 gm, greater than about 70 gm, greater than about 80 gm, greater than about 90 gm, greater than about 100 gm, greater than about 200 gm, greater than about 300 gm, greater than about 400 gm, greater than about 500 gm, greater than about 600 gm, greater than about 700 gm, greater than about 800 gm, greater than about 900 gm, or of about 1 mm. In certain embodiments, the fibers (e.g., microfibers) have a length in the range of about 1 to 1,000 gm, of about 10 to 500 gm, or of about 100 to 500 gm.

[0110] Diameter and length of the polymeric fibers may be determined using optical microscopy (including fluorescence microscopy) or electron microscopy.

[0111] In various embodiments, the polymeric fibers may have an aspect ratio in a range of at least about 10 to about at least 10,000. It will be appreciated that, because of the very small diameter of the fibers, the fibers have a high surface area per unit of mass. This high surface area to mass ratio permits fiber-forming solutions or liquids to be transformed from liquid or solvated fiber-forming materials to solid fibers in fractions of a second.Ratio of fiber to hydrogel

[0112] Ratios of polymeric fiber to hydrogel material can be determined by any means known in the art. The ratio may be reported on a component-mass basis. For example, in various embodiments, the ratio of polymeric fiber to hydrogel material ranges from about 1 : 100 to about 100: 1. In various embodiments, ratio of polymeric fiber 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-mass basis.

[0113] The ratio of polymeric fiber to hydrogel material is also provided as a concentration basis, e.g., a given weight of polymeric fiber per volume of hydrogel material. For example, the concentration is from about 10 mg / mL to about 40 mg / mL.Collagen Fiber Composition

[0114] Any suitable from of collagen may be used. In various embodiments, the collagen is a human-type collagen. Collagen may be fibrillar or non-fibrillar. In various embodiments, collagen is a fibrillar collagen. In various embodiments, the fibrillar collagen is aType I, Type II, Type III, Type V, or Type XI collagen. In various embodiments, collagen is a non-fibrillar collagen. In various embodiments, the non-fibrillar collagen is a FACIT (Fibril Associated Collagens with Interrupted Triple Helices) (Type IX, XII, XIV, XIX, or XXI) collagen. In various embodiments, the non-fibrillar collagen is a MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Type XIII, or XVII) collagen. In various embodiments, the non-fibrillar collagen is a Short chain (Type VIII, or X). In various embodiments, the non-fibrillar collagen is a multiplexin (Multiple Triple Helix domains with Interruptions) (Type XV, or XVIII) collagen. In various embodiments, the non-fibrillar collagen is a microfibril forming (Type VI) collagen. In various embodiments, the non-fibrillar collagen is a anchoring fibrils (Type VII) collagen. In various embodiments, the collagen is a synthetic collagen.

[0115] In various embodiments, the collagen is a Type I collagen.

[0116] In various embodiments, the collagen is a Type II collagen.

[0117] In various embodiments, the collagen is a Type III collagen.

[0118] In various embodiments, the collagen is a Type IV collagen.

[0119] In various embodiments, collagen nanofibers comprise type I bovine collagen nanofiber fragments. In various embodiments, collagen nanofibers comprise porcine, ovine, chicken, fish, or recombinant forms of collagen.

[0120] In various embodiments, collagen nanofibers comprise bovine type I collagen. In various embodiments, collagen nanofibers comprise bovine type I atelocollagen.

[0121] In various embodiments, collagen nanofibers comprise porcine type I. In various embodiments, collagen nanofibers comprise porcine type I atelocollagen.

[0122] In various embodiments, collagen nanofibers comprise non-animal derived recombinant sources of collagen. In various embodiments, the collagen is a recombinant human type 3 collagen by Bloomage. In various embodiments, the collagen is Demulcent SFA by Hand Biotech. In various embodiments, the collagen is Vecollan by Evonik. Vecollan can be made by a fermentation-based process. The recombinant collagen may comprise a single type of alpha chain or multiple types of alpha chain, with or without hydroxyproline residues. In various embodiments, the recombinant collagen may comprise a single type of alpha chain. In variousembodiments, the recombinant collagen may comprise multiple types of alpha chain. In various embodiments, the recombinant collagen may comprise hydroxyproline residues. In various embodiments, the recombinant collagen may not comprise hydroxyproline residues.

[0123] In various embodiments, collagen nanofibers comprise gelatin. In various embodiments, gelatin is a denatured collagen. Gelatin can be sourced from various sources, including but not limited to, bovine, porcine, fish, chicken sources.

[0124] In various embodiments the collagen form comprise forms with and without (gelatin) triple-helix secondary structure.

[0125] In various embodiments the collagen comprises forms with and without telopeptides. In various embodiments the collagen form comprises telopeptides. In various embodiments the collagen form does not comprise telopeptides. In various embodiments the collagen is an atelocollagen. Atelocollagen is a low-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 by pepsin treatment from type I collagen of calf dermis.

[0126] Once spun, the resulting fibers from alternate sources can be processed as previously described for bovine collagen and used to form fiber-hydrogel composites. Other forms of collagen or gelatin, including other types of collagen like type III or recombinant “collagen-like” peptides from bacterial sequences can be applied.

[0127] Depending upon the raw material source, the resulting fibers may have less secondary structure as-spun than the collagen previously described (gelatin for instance is not triple-helical), or have differences in crosslinking moiety concentrations due to variances in peptide sequences. Such fibers may require more intensive crosslinking prior to contact with water. In addition to or in replacement of the EDC / NHS crosslinking previously described, the fibers can be crosslinked with DVS or BDDE, crosslinked with an aldehyde-based crosslinker such as vapor phase glutaraldehyde treatment, treated Maillard reactions such as with D-ribose and heat, dehydrothermal treatment, genipin and transglutaminases, irradiation (UV, gamma, or e-beam), plasma treatment, or other methods known in the field (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] .

[0128] Once rendered water insoluble, the fibers can be mechanically processed to a dispersed form, and covalently gelled into a composite using the methodology described in this filing.

[0129] In various embodiments, the collagen nanofibers are electrospun, melt spun, blow spun and / or cryomilled. In one particular embodiment, the collagen nanofibers are electrospun.Crosslinking of Collagen to Hyaluronic Acid

[0130] As provided herein, one form of interaction of the composite containing polymeric fiber and hydrogel includes a crosslinking agent, such as DVS (divinyl sulfone), generally present in an amount effective to introduce bonding between polymer fiber and hydrogel material, e.g., to induce cross-linking between collagen fiber and hyaluronic acid. In one particular embodiment, interfacial bonding is generated between the polymeric fiber and hydrogel material to form a composite network. For instance, the nanofibers are introduced to the HA network while crosslinking with DVS to generate interfacial bonding between the HA network and the nanofibers.Composite Production

[0131] The method of producing the collagen hydrogel composite may further include a step of swelling the collagen hydrogel composite. In certain embodiments, the step of swelling is performed by incubating the collagen hydrogel composite in an aqueous buffer solution (e.g., PBS). In certain embodiments, the buffer solution may be used in greater than 10 times, greater than 50 times, greater than 100 times, greater than 200 times, greater than 300 times, greater than 400 times, greater than 500 times, or greater than 1000 times, of volume of the collagen hydrogel composite. In certain embodiments, the swelling is performed (continuously or discontinuously) for more than an 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. Preferably, the swelling is performed (continuously or discontinuously) for 24 to 72 hours at room temperature.

[0132] The method of producing the collagen hydrogel composite may further include processing the collagen hydrogel composite to particularize into to microbeads. In certainembodiments, the microbeads have a mean diameter in a range of about 1 gm to about 1000 gm, about 10 gm to about 1000 gm, about 20 gm to about 1000 gm, about 30 gm to about 1000 gm, about 40 gm to about 1000 gm, about 50 gm to about 1000 gm, about 50 gm to about 500 gm, about 50 gm to about 400 gm, or about 100 gm to about 500 gm. As described above, the collagen hydrogel composite is formed into particulate formulations (e.g., microbead or microgel), enabling use of higher concentrations of each component and enhanced stability. In certain embodiments, a system of particulation may be employed wherein the pre-formed collagen hydrogel composite is physically modulated, such as by being pushed through one, two, three, or more than three mesh screens, creating a population of nonspherical beads that are relatively similar to one another in shape and size. In certain embodiments, the processing preferably may include mechanical milling or mechanical screening by applying shear using a mesh. This two-screen system allows for tight 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, the collagen hydrogel composite is particularized by applying mechanical shear through meshes with defined sizes in the range of 50 to 400 pm.

[0133] In certain embodiments, the collagen hydrogel composite produced herein may be in a biphasic gel or monophasic gel. For example, the collagen hydrogel composite is a biphasic gel with similar bead sizes made by forcing the gel through screens. Alternatively, the fiberhydrogel composite is a monophasic gel with a continuous distribution of gel bead sizes made by homogenizing the gel or mechanically disrupting the gel during the crosslinking reaction.

[0134] Methods described herein may include the step of autoclaving. In various embodiments, an autoclave temperature is about 100°C to about 150°C. In various embodiments, an autoclave temperature is about 110°C to about 140°C. In various embodiments, an autoclave temperature is about 110°C to about 130°C. In various embodiments, an autoclave temperature is about 115°C to about 125°C. In various embodiments, an autoclave temperature is about 118°C. In various embodiments, an autoclave temperature is about 121 °C. In various embodiments, an autoclave temperature is about 138°C. In various embodiments, an autoclave time is about 1 minute to about 1 hour. In various embodiments, an autoclave time is about 5 minutes to about 45 minutes. In various embodiments, an autoclave time is about 5 minutes to about 30 minutes. In various embodiments, an autoclave time is about 5 minutes, about 10 minutes, about 20 minutes, or about 30 minutes.

[0135] The method of producing the collagen hydrogel composite may further include a step of sterilizing the collagen hydrogel composite. Any sterilizing method or process in the art may be used without limitation. For example, sterilizing the collagen hydrogel composite may include sterilizing via autoclave at 121 °C for about 5 to 30 minutes. In various embodiments, the method of producing the collagen hydrogel composite comprises the step of autoclaving. In various embodiments, the method of producing the collagen hydrogel composite comprises the step of autoclaving at 121 °C for about 5 to 30 minutes. In various embodiments, the method of producing the collagen hydrogel composite comprises the step of autoclaving at 121 °C for about 5 to 30 minutes.

[0136] In various embodiments, the collagen hydrogel composite is part of a packaged composite. In various embodiments, the packaged composite is sterilized. In various embodiments, a method of producing the packaged composite comprises a step of sterilization. For example, sterilizing the packaged composite may include sterilizing via autoclave at 121 °C for about 5 to 30 minutes. In various embodiments, the method of producing the packaged composite comprises the step of autoclaving. In various embodiments, the method of producing the packaged composite comprises the step of autoclaving at 121 °C for about 5 to 30 minutes. In various embodiments, the method of producing the packaged composite comprises the step of autoclaving at 121 °C for about 5 to 30 minutes.

[0137] The method of producing the collagen hydrogel composite may further include fabricating the collagen hydrogel composite in a sheet or an injectable fluid.

[0138] In certain embodiments, the method may further comprise incubating an infiltrated macrophage in the collagen hydrogel composite. In certain embodiments, the collagen hydrogel composite including the collagen nanofiber may condition a population of a M2 phenotype infiltrated macrophage.Medical Applications

[0139] In various aspects, the collagen hydrogel composite may be applied to medical applications, including but not limited to, dermal aesthetic, reconstruction, fat grafting, and wound care indications.

[0140] In various aspects, the collagen hydrogel composite exhibits minimal swelling,minimal tissue irritation, durable volumization, and / or robust new tissue generation that is appropriate for the target tissue without fibrosis or foreign body reactions.

[0141] In various aspects, the collagen hydrogel composite, when implanted, injected, or placed on to a target tissue or wound in vivo should fully degrade or resorb over a timeframe of 6-24 months.

[0142] Provided are methods of using the collagen-hydrogel composite to treat a subject.

[0143] In one aspect, the disclosure provides a method of forming adipose tissue formation in a subject. The method includes administering the collagen-hydrogel composite as described herein to the subject. In the subject of prolonged in vivo retention of collagen-hydrogel composite or enhanced host cell infiltration is induced by the collagen-hydrogel composite. 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.

[0144] In certain embodiments, the subject exhibits neo-vasculature formation facilitated by M2 macrophage polarization. In certain embodiments, the subject exhibits an increase in a- SMA+ cells around 2.5-fold at the site of administration. In certain embodiments, the subject exhibits localization of endothelial cells and CD 163+ M2 macrophages at the site of administration. In certain embodiments, the subject displays an increase of around 1.5-fold of CD68+ pan- macrophages at the site of administration by post-operative day 7. In certain embodiments, the subject displays an increase of around 2-fold of CD68+ pan-macrophages at the site of administration by post-operative day 14.

[0145] In an aspect, the disclosure also provides a method of delivering a cell or tissue in a subject. The method includes encapsulating one or more cells or tissues in the collagen- hydrogel composite as described herein to form a suspension; and applying the suspension to a target site in the subject.

[0146] In another aspect, the disclosure also provides a method of delivering adipose tissue in a subject. The method includes encapsulating one or more adipose tissues in the collagen-hydrogel composite as 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 suitably include adipose- derived stem cells, adipocytes, or combinations thereof.

[0147] In an aspect, the disclosure also provides a method of delivering a pharmaceutical agent in a subject. The method includes combining a pharmaceutical agent and the collagenhydrogel composite as described herein to form a mixture; and applying the mixture to an intended site of delivery.

[0148] In an aspect, the disclosure also provides a method of delivering a cell or tissue in a subject. The method includes encapsulating one or more cells or tissues in the collagenhydrogel composite as described herein to form a flowable; and applying the flowable to a target site in the subject.

[0149] In an aspect, the disclosure also provides a method of delivering a cell or tissue in a subject. The method includes encapsulating one or more cells or tissues in the collagenhydrogel composite as described herein to form a mat; and applying the mat to a target site in the subject.

[0150] In an aspect, the disclosure also provides a method of delivering a cell or tissue in a subject. The method includes encapsulating one or more cells or tissues in the collagenhydrogel composite as described herein to form an injectable; and applying the injectable to a target site in the subject.

[0151] As discussed above, the collagen-hydrogel composite as described herein can be used advantageously in numerous tissue repair situations, as well as in other applications, such as providing coatings on catheters and other surgical devices and implants. The collagen-hydrogel composite can also be used to deliver active agents described herein, such as antibiotics, growth factors, and immunosuppressive agents. For example, the disclosure provides a method for healing a soft tissue defect comprising applying the collagen-hydrogel composite to a soft tissue defect.

[0152] It will be appreciated that advantageous properties of the collagen-hydrogel composite described herein include the ability to: I) provide easy characterization and quality control; 2) integrate with existing tissue matrices; 3) directly incorporate into newly formed matrices; 4) directly include cells and bioactive factors; 5) maintain biocompatibility; 6) control bioresorption; 7) cast easily into complicated anatomical shapes due to greater structural rigidity owing to the nanostructures; 8) exhibit the mechanical properties of native tissues such as articular cartilage; 9) crosslink fibers and hydrogel phase in one step; and 10) retain mechanicalproperties after terminal sterilization by autoclave.

[0153] In another aspect, the collagen-hydrogel composite can be used to repair cartilage tissue.

[0154] Current biologically-based surgical procedures for cartilage repair include autologous chondrocyte implantation, drilling, abrasion chondroplasty, microfracture, and mosaic arthroplasty. All these procedures treat only focal articular cartilage injuries, and not cartilage denuded joint surfaces such as seen in severe osteoarthritis and rheumatoid arthritis. Also, they use either cartilage tissue plugs or expanded chondrocytes harvested from the patient to fill cartilage defects. These tissues or chondrocytes are expected to fill the defect by synthesizing entirely de novo material, such as newly synthesized hyaline cartilage, that has integrated with existing cartilage matrices and has the biomechanical properties of normal cartilage. However, such procedures all promote the formation of a reparative tissue (fibrocartilage) rather than true hyaline cartilage with further mechanical damage to fibrocartilage thought to predispose the joint to osteoarthritis. Furthermore, the availability of endogenous cartilage as a repair material is quite limited with its acquisition presenting its own risks and morbidity to the patient. As evident from the foregoing discussion, the resulting collagen hydrogel compositions disclosed herein present practical materials for promising new therapies in patients suffering from cartilage degenerative diseases.

[0155] As described herein, the collagen-hydrogel composite can be prepared having widely varying properties that are suitable for any number of synthetic tissue implantation or augmentation, as well as other clinical applications. As already described, the collagen-hydrogel composite can be used to repair cartilage defects produced as a result of either injury or disease. Defects due to injury that can be so repaired can be sports- or accident-related, and may involve only the superficial cartilage layer, or may include the underlying subchondral bone. Defects due to disease which can be repaired using the compositions described herein include those resulting from osteoarthritis and rheumatoid arthritis. Whether from injury or disease, such defects may be in either mature or growth plate cartilage. Formulations for hydrogels for synthetic growth plate cartilage may require the inclusion of unsubstituted scaffold material to allow for controlled bioresorption of the biomaterial during growth.

[0156] Another field where the collagen-hydrogel composite described herein can beuseful is the repair, reconstruction or augmentation of cartilaginous as well as soft tissues of the head and neck. The availability of biomaterials for soft tissue augmentation and head and neck reconstruction has remained a fundamental challenge in the field of plastic and reconstructive surgery. Significant research and investment has been undertaken for the development of a material with appropriate biological compatibility and life span. The outcomes of this research have not been promising. When placed in immunocompetent animals the structural integrity of the collagen-hydrogel composite has been shown to fail as the framework is absorbed. Furthermore, though conventional synthetic materials offer excellent lifespan, they presented certain unavoidable pitfalls. For example, silicones have been fraught with concerns of safety and long- term immune related effects. Synthetic polymers PTFE (Gore-Tex) and silastic offer less tissue reactivity but do not offer tissue integration and can represent long term risks of foreign body infections and extrusion. The collagen-hydrogel composite will be useful to prepare a synthetic soft- tissue scaffold material for the augmentation or repair of soft-tissue defects of the head and neck. In particular, the collagen hydrogel compositions, which are noninflammatory, non- immunogenic, and which can be prepared having the appropriate degree of viscoelasticity (see description herein), could be used as an effective implantable scaffold material.

[0157] In addition, the collagen-hydrogel composite can be used, for example, as a novel, biocompatible and biocompliant materials to prepare cartilage implants which are frequently used in reconstructive procedures of the head and neck to repair cartilaginous or bony defects secondary to trauma or congenital abnormalities. Applications specific to the ear include otoplasty and auricular reconstruction, which are often undertaken to repair cartilaginous defects due to trauma, neoplasm (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, tip, shield and spreader grafts are frequently used in cosmetic rhinoplasty. Nasal reconstruction following trauma, neoplasm, autoimmune diseases such as Wegener’s granulomatosis, or congenital defects require cartilage for repair. Septal perforations are difficult to manage and often fail treatment.

[0158] Cartilage grafts would be ideal for these applications, as autologous or donor cartilage is often unavailable. Applications specific to the throat include laryngotrachealT1reconstruction, which in children usually requires harvesting costal cartilage, which is not without morbidity. Auricular and septal cartilage is often inadequate for this application. Synthetic cartilaginous materials prepared from hydrogels disclosed herein can be synthesized to suit each of the foregoing applications, based on tuning parameters of hydrogel synthesis such as reagent concentration, substitution and cross-linking rates. Laryngotracheal reconstruction is usually performed for airway narrowing due to subglottic or tracheal stenosis. The etiology may be traumatic (i.e., intubation trauma, or tracheotomy) or idiopathic. Other possibilities include chin and cheek augmentation, and use in ectropion repair of the lower eyelid, in addition to numerous craniofacial applications. It should be noted that these applications may not need cartilage with the exacting mechanical properties of articular cartilage. Inclusion of a cell population or bioactive agents may also be desirable.

[0159] The collagen-hydrogel composite described herein can be used for repair and narrowing of the nasal cavity, normally following overly aggressive surgical resection, to prevent the chronic pooling of fluid in the nasal passages that leads to infection and encrustation. Another promising application is in laryngotracheal reconstruction in both children and adults, as a result of laryngotracheal injury due for example to intubation during a surgical procedure such as cardiovascular surgery. The collagen-hydrogel composite as herein described also can be used to provide cricoid ring replacements to protect the carotid artery following neck resection for cancer - the collagen-hydrogel composite can be placed between the carotid artery and the skin as a protective barrier for the carotid artery against loss of the skin barrier. As a protective coating during neuronal repopulation of a resected nerve-often fibrous tissue forms faster than the neuronal repopulation preventing its eventual formation. Placement of the nerve ends within the collagen-hydrogel composite pre-cast tube could exclude fibrous tissue formation from the site of repopulation.

[0160] The collagen-hydrogel composite can also be used for repair of soft tissue defects of any internal or external organs. For example, the collagen-hydrogel composite can be used to for chin and cheek augmentation, and use in ectropion repair of the lower eyelid, in addition to numerous craniofacial applications. For cosmetic and reconstructive purposes in sites other than the head and neck, for example use as breast implants for breast augmentation, for body sculpting, or as a wound sealant, for example to fill the void left after removal of lymph nodes (i.e. due to cancer) in the breast or neck, to seal the lymphatics and abate uncontrolled fluiddrainage into the resection site that may lead to infection and other complications.

[0161] In addition to the above uses, the collagen-hydrogel composite described herein can be used in other tissue engineering applications to produce synthetic orthopedic tissues, including, but not limited to, bone, tendon, ligament, meniscus and intervertebral disc, using similar strategies and methodologies as described above for the synthesis of artificial forms of cartilage. The collagen hydrogel composite also can be used to make synthetic non-orthopedic tissues including but not limited to vocal cord, vitreous, heart valves, liver, pancreas and kidney, using similar strategies and methodologies as described above for the synthesis of artificial forms of cartilage.

[0162] Another field where the collagen-hydrogel composite 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 abdominal or gastrointestinal organs. There already are a number of products at various stages of clinical and FDA approval, which generally are termed "hydrogels," that are designed or intended to be useful in the treatment and prevention of scarring and / or stricture formation. The collagen-hydrogel composite is superior to other known hydrogels in that the ones disclosed here can include a nanostructure which can provide support, shape, and strength to hydrogel materials. The collagen-hydrogel composite disclosed herein can be used in similar applications as the already known hydrogels are used or intended to be used, including the following: for treatment of strictures or scarring of the gastrointestinal tract. The treatment involves injection of the collagen-hydrogel composite at the site of an anticipated stricture to prevent scarring, or at a site of existing stricture after therapy to enlarge the narrowed GI tract to prevent the stricture from reoccurring.

[0163] The collagen-hydrogel composite as described herein can also be used for the treatment of esophageal strictures. Esophageal strictures are a common complication of gastroesophageal reflux disease (GERD). GERD is caused by acid, bile and other injurious gastric contents refluxing into the esophagus and injuring the esophageal lining cells. Approximately 7-23% of GERD patients develop an esophageal stricture, or fibrous scarring of the esophagus. Esophageal scarring also can be caused by ablative therapies used to treat Barrett's esophagus. The major complication of such ablative therapies is that the ablative injury extends too deeply into the esophageal wall and results in an esophageal scar or stricture.Esophageal strictures prevent normal swallowing and are a major cause of patient morbidity. The materials described herein may be used to treat or prevent esophageal strictures resulting from GERD, Barrett's esophagus, and esophageal ablative therapies.

[0164] The collagen-hydrogel composite may also be used for treatment of Crohn's disease. Crohn's disease causes strictures or scars that block off or narrow the lumen of the bowel, preventing normal bowel function. For example, the collagen-hydrogel composite may be useful to treat or prevent such strictures.

[0165] The collagen-hydrogel composite can also be used in methods for treating primary sclerosing cholangitis (PSC). PSC is a rare disease of the bile ducts of the liver. The bile ducts form a branching network within the liver and exit the liver via two main branches that are combined into the common bile duct which drains the liver and gallbladder of bile into the duodenum. The bile ducts are very narrow in diameter, measuring only up to 2 mm normally at their largest most distal portions, and yet they must normally drain liters of bile every day from the liver into the duodenum. Any blockage of these ducts can result in a serious condition known as jaundice, which allows many toxins and especially hemoglobin breakdown products to accumulate in the body. PSC is a scarring or structuring disease of the bile ducts within the liver and in the extrahepatic bile ducts described above that connect the liver to the small intestine. The bile duct strictures of PSC may be treated or prevented with the present collagen hydrogel compositions.

[0166] The collagen-hydrogel composite can also be used to treat chronic pancreatitis. Chronic pancreatitis is a chronic inflammatory disease of the pancreas that may be complicated by scars or strictures of the pancreatic ducts. These strictures block the drainage of pancreatic juice, which normally must exit the pancreas through a system of ducts or drainage conduits into the small intestine. The pancreatic juice contains many digestive enzymes and other elements important to normal digestion and nutrient absorption. Blockage or narrowing of the pancreatic ducts by chronic pancreatitis can result in severe complications in which the pancreas autodigests and forms life-threatening abdominal infections and or abscesses. The pancreatic strictures of chronic pancreatitis may be treated or prevented with the present hydrogels.

[0167] The collagen-hydrogel composite may also be used for treatment of gallstone- induced bile duct and pancreatic duct strictures. Gallstones are a very common disorder, aprincipal complication of which is the formation of bile duct and pancreatic duct strictures, which may be treated or prevented with the hydrogels, for treatment of ischemic bowel disease. The intestines are prone to the formation of scars or strictures when their blood supply is compromised. Compromised blood flow is called ischemia, and can be caused by many pathologies, including cardiovascular disease, atherosclerosis, hypotension, hypovolemia, renal or hepatic disease- induced hypoalbuminemia, vasculitis, drug-induced disease, and many others. The end stage result of all of these etiologies can result in intestinal strictures that block off the bowel and prevent its normal function. The present collagen hydrogel composites may be used to treat or prevent ischemic bowel strictures.

[0168] The collagen-hydrogel composite may also be used for treatment of radiation- induced intestinal strictures. Radiation therapy for cancer is associated with numerous morbidities, important among which is intestinal stricture formation. The collagen-hydrogel composite may be used to treat or prevent radiation-induced intestinal strictures.

[0169] In addition to making synthetic tissues or repairing native tissues, the collagen- hydrogel composite disclosed here also can be used to provide a coating for non-biological structures or devices to be used in surgery or otherwise for in vivo implantation, such as surgical instruments, or ceramic or metal prostheses. Such a coating would provide a barrier between the non-biologic device material and living tissue. The role of collagen-hydrogel composite as a barrier for non- biologic devices includes, but is not limited to: I) prevention of absorption of macromolecules and / or cells on the surfaces of non-biologic devices, which can lead to protein fouling or thrombosis at the device surface; 2) presentation of a non-toxic, non-inflammatory, non- immunogenic, biologically compatible surface for devices made from otherwise non- biologically compatible materials; 3) compatibility with device function such as diffusion of glucose for a glucose sensor, transmission of mechanical force for a pressure sensor, or endothelization of a vascular graft or stent; 4) enhancement of device function, such as providing a charge barrier to an existing size barrier in a MEMS based artificial nephron; 5) incorporation into non-biologic devices of a viable cell population entrapped within an aqueous, physiologically compatible environment; and 6) inclusion of drugs or bioactive factors such as growth factors, anti-viral agents, antibiotics, or adhesion molecules designed to encourage vascularization, epithelization or endothelization of the device.

[0170] Based on the foregoing, the collagen-hydrogel composite may be used to provide a non-allergenic coating for a variety of implantable devices including an implantable glucose sensor for management of diabetes. In addition, the collagen-hydrogel composite may be used to provide: a charge barrier for the development of MEMS-based artificial nephrons; an aqueous, physiologically compatible environment in which embedded kidney cells such as podocytes can be incorporated into a MEMS-based artificial nephron design; and a coating for implantable MEMS devices designed for a variety of purposes including, but not limited to, drug delivery, mechanical sensing, and as a bio-detection system.

[0171] The disclosed collagen-hydrogel composite, and particularly including HA, also may be covalently attached to silicon-based devices, e g. through first covalent attachment of the primary amine of tyramine to the silicon surface to provide a hydroxyphenyl coated surface chemistry.

[0172] This may use the same chemistry used to bind DNA that has been modified with a free amine to silicon surfaces. The HA-based collagen-hydrogel composite then is covalently coupled to the hydroxyphenyl coated surface by the same peroxidase driven chemistry used in its preferred cross-linking mode described above.

[0173] The collagen-hydrogel composite also can be used for coating non-biologic cardiovascular devices such as catheters, stents and vascular grafts. These would include devices made from materials conventionally not used because of their biological incompatibility, but which have superior design characteristics to those devices currently in use. Bioactive factors could be incorporated into the hydrogels to promote endothelization or epithelization of the hydrogel, and thus of the implanted device.

[0174] Composites maintaining 85% of its initial volume through more than a year and generated vascularized adipose tissue, making it an excellent candidate for dermal fdling applications and body sculpting.

[0175] Composites exhibiting more gel spreading for more a diffuse effect, may be wanted for superficial placement in the body. The lower crosslinking and collagen concentrations and resulting larger pore size allows for more rapid cellular movement and diffusion for therapeutic indications for cell or drug delivery or increasing cell homing and interactions within the implanted gel.

[0176] Composites exhibiting slower cellular infiltration, and maintained the original shape, resisting spreading. These formulation groups are good candidates for use where implant traits like slow degradation, geometric stability are desired. Examples include implantation of the device into high tissue tension regions such as supraperiosteal placement in the body or weightbearing locations, or for indications where a more permanent plug is desired, such as for fistula repair, hernia repair, coating permanent implants, etc.

[0177] The ideal soft tissue filler would initially immediately fill the defect site with 100% of the target volume, with similar mechanical properties to the surrounding soft tissue, without migration or flow of the filler, and without inducing pain or irritation. The filler should not induce swelling, and then should maintain nearly 100% of its volume indefinitely, while paradoxically degrading away completely without causing scarring or adverse events (such as nodules or granulomas). The present formulations disclosed herein come much closer to achieving this “ideal” profile than any current products such as the Voluma XC® control included in this study.HA DVS Crosslinking Formulation Development

[0178] One advantage of the present disclosure is to limit post-procedural swelling.

[0179] In various embodiments, the methods of manufacturing the collagen hydrogel composites has the advantage of obtaining gels that have good mechanical properties and a hyaluronic acid concentration of about 7-12mg / mL to limit post-procedural swelling.

[0180] By varying the HA concentration and DVS to HA ratio, various embodiments vary how much the HA gel swells during the manufacturing processes (swelling ratio), and also vary the HA concentration of the final device.

[0181] In general, the higher the HA concentration during gelation, the more tightly crosslinked the resulting gel becomes, and thus, the lower the swelling ratio. But the higher the initial HA concentration during gelation, the greater the swelling ratio needs to be during purification in order to lower the HA concentration to the target range. At 21mg / mL gelation HA concentration, the gel swells 4.88fold, dropping the final device HA concentration to 4.3mg / mL, lower than the target. With these experiments, gelation HA concentrations of 27 or 30mg / mL were able to produce final gels after purification that had the targeted 7-12mg / mL window (withresulting concentrations of 7.8mg / mL to 10.7mg / mL in the experiment).

[0182] At a fixed initial gelation HA concentration of 30mg / mL, DVS ratios of 3.22, 4.5, and 5.0 created gels that fell within the targeted final HA concentration range (FIG. 6). The increasing DVS concentration had a modest decrease in the swelling ratio and thus increased final gel HA concentration (increasing crosslinking concentration leads to increased crosslinked density and lessened swelling). Within this relatively wide range of DVS ratios (3.22-5X), the HA gelation concentration is a sensitive variable for final gel properties. The chart in FIG. 7 shows the poorer correlation between DVS ratio and final HA concentration when using a wider range of HA gelation concentrations (21-30mg / mL).

[0183] Based upon the HA-experiments, various embodiments of the disclosure comprise a hyaluronic acid concentration ranging from about 27 to about 30mg / mL at the time of gelation with a DVS-to-HA-subunit ratio of 3.22 to 5X (or a DVS to HA hydroxyl molar ratio of 0.8: 1 to 1.25: 1)

[0184] Collagen content had an unexpectedly large effect upon the gel swelling ratio, requiring a further evaluation of gelation conditions to achieve targeted final gel properties. Meanwhile, the HA gelation concentration had a much smaller direct effect on the swelling ratio, in the presence of the collagen fibers. Without being bound by theory, the fibers may restrict the ability of the hydrogel to swell as much during manufacturing, since the fibers are a second more solid phase that is covalently bonded into the hyaluronic acid gel to form a composite. HA needs to be set at a lower concentration during gelation in order to achieve the target final concentration, since the collagen content restricted the amount that the gels swell during purification.

[0185] In one embodiment, a nanofiber-hydrogel composite comprises a hydrogel material comprising a hyaluronic acid, a nanofiber material comprising a collagen, and a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; and wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL.

[0186] In one embodiment, a nanofiber-hydrogel composite comprises a hydrogel material comprising a hyaluronic acid, a nanofiber material comprising a collagen, and acrosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL; and wherein the divinyl sulfone is present in an amount ranging from about 3 : 1 to about 5 : 1 prior to crosslinking.

[0187] In one embodiment, a nanofiber-hydrogel composite comprises a hydrogel material comprising a hyaluronic acid, a nanofiber material comprising a collagen, and a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the amount of crosslinking (HA subunit linked via DVS to another subunit of HA or Collagen) ranges from about 0.1% to about 30%; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; and wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL

[0188] In one embodiment, a nanofiber-hydrogel composite comprises a product by the process comprising a step of reacting a hyaluronic acid material, a collagen fiber, and divinyl sulfone whereby the hyaluronic acid is covalently crosslinked to the collagen fiber by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL; and wherein the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; and wherein the divinyl sulfone is present in an amount of a ratio ranging from about 0.5: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.

[0189] In one embodiment, the disclosure provides a method for manufacturing a nanofiber-hydrogel composite comprising a step of reacting a hyaluronic acid material, a collagen fiber, and divinyl sulfone; whereby the hyaluronic acid is covalently crosslinked to the collagen fiber by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL; and wherein the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; and wherein the divinyl sulfone is present in an amount of a ratio ranging from about 0.5: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Composite swelling

[0190] The composites of the present disclosure may swell during manufacture (in vitro)'or following administration to a subject such as by injection, implantation or application on (in vivo). For clarity, "Swelling Ratio" refers to swelling during manufacturing, while "Swelling Percentage" (Swelling %) refers to swelling of the final device in vivo. Swelling Ratio is a multiple of increase in volume from an initial volume. Swelling Ratio may be expressed as a rational number over the integer of one. Swelling % is a percentage increase in volume of the implant in the body or physiological-equivalent condition.Swelling ratio - During manufacture

[0191] In various aspects, the composite swells certain amounts during the manufacturing process.

[0192] In various embodiments, the composite comprises a swelling ratio ranging from about 0. 1 to about 4.0 during manufacture. In various embodiments, composite comprises a swelling ratio ranging from about 1.5 to about 4.0 during manufacture. In various embodiments, composite comprises a swelling ratio ranging from about 1.0 to about 3.0 during manufacture.

[0193] In various embodiments, the composite comprises a swelling ratio ranging from about 2.0 to about 3.0 during manufacture. In various embodiments, the composite comprises a swelling ratio ranging from about 1.0 to about 2.0 during manufacture. In various embodiments, the composite comprises a swelling ratio ranging from about 0.1 to about 1.0 during manufacture.

[0194] In various embodiments, composite comprises a swelling ratio ranging from about 2.0 to about 3.0 during manufacture. In various embodiments, composite comprises a swelling ratio ranging from about 2.2 to about 3.0 during manufacture. In various embodiments, composite comprises a swelling ratio ranging from about 2.3 to about 2.8 during manufacture.

[0195] In various embodiments, the composite comprises a swelling ratio of about 2.3 during manufacture.

[0196] In various embodiments, the composite comprises a swelling ratio of about 2.4 during manufacture.

[0197] In various embodiments, the composite comprises a swelling ratio of about 2.6 during manufacture.

[0198] In various embodiments, the composite comprises a swelling ratio of about 2.8during manufacture.Post-procedural swelling (in vivo)

[0199] In various aspects, the composite swells certain amounts when placed in vivo.

[0200] In various embodiments the composite comprises a swelling % of at most 100% in vivo.

[0201] In various embodiments the composite comprises a swelling % of at most 75% in vivo. In various embodiments the composite comprises a swelling % of at most 50% in vivo. In various embodiments the composite comprises a swelling % of at most 40% in vivo. In various embodiments the composite comprises a swelling % of at most 30% in vivo. In various embodiments the composite comprises a swelling % of at most 20% in vivo. In various embodiments the composite comprises a swelling % of at most 15% in vivo. In various embodiments the composite comprises a swelling % of at most 12% in vivo. In various embodiments the composite comprises a swelling % of at most 10% in vivo.

[0202] In various embodiments the composite comprises a swelling % of at most 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in vivo.

[0203] In various embodiments the composite comprises a swelling % ranging from about 0% to about 100% in vivo. In various embodiments the composite comprises a swelling % ranging from about 1% to about 100% in vivo. In various embodiments the composite comprises a swelling % ranging from about 1% to about 50% in vivo. In various embodiments the composite comprises a swelling % ranging from about 1% to about 20% in vivo. In various embodiments the composite comprises a swelling % ranging from about 1% to about 10% in vivo. In various embodiments the composite comprises a swelling % ranging from about 10% to about 20% in vivo. In various embodiments the composite comprises a swelling % ranging from about 10% to about 15% in vivo.

[0204] In various embodiments the composite comprises a swelling % of about 50%, 40%, 30%, 20%, or 10% in vivo.

[0205] In various embodiments the composite comprises a swelling % of about 10%, 11%, 12%, 13%, 14%, or 15% in vivo.

[0206] In various embodiments the composite comprises a swelling % of about 20% invivo.

[0207] In various embodiments the composite comprises a swelling % of about 12% in vivo.

[0208] In one embodiment, swelling is reduced by 86% compared to Voluma; 14% vs. 100%.

[0209] In one embodiment, swelling is reduced by 80% compared to Voluma. 20% vs. 100%.

[0210] In one embodiment, swelling is reduced by 88% compared to Voluma. 12% vs. 100%.

[0211] In one embodiment, swelling is reduced by 84% compared to Voluma. 16% vs. 100%.

[0212] In various embodiments, the composite comprises a swelling ratio ranging from about 0.1 to about 3.0 when placed in vivo.

[0213] 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.

[0214] 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 ranging from about 12% to about 20% in or on the body (in vivo).

[0215] Exemplary data is provided in Figure 14.Hyaluronic acid

[0216] As used herein the specification, “hyaluronic acid” or “HA” is typically referred to as a nonsulfated glycosaminoglycan. HA is a polymer of disaccharide monomers. Disaccharide monomers of hyaluronic acid may be referred to as “hyaluronic acid subunits” or “HA subunits.” HA subunits comprise D-glucuronic acid and N-acetylglucosamine. HA subunits are linked to one another by alternating beta-1,4 and beta-1,3 glycosidic linkages.

[0217] The IUPAC name of hyaluronic acid is (l^-4)-(2-Acetamido-2-deoxy-D-gluco)- (1— >3)-D-glucuronoglycan. A chemical structure drawing of hyaluronic acid is shown inFigure 1. Exemplary representations of divinyl-sulfone-crosslinked hyaluronic acid are shown in Figure 2.

[0218] In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about IMDa to about 2MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about IMDa to about 1 5MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 1.5MDa to about 2MDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 500kDa to about IMDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about 500kDa to about 500kDa. In various embodiments, the hyaluronic acid comprises a molecular weight ranging from about l OOkDa to about 500kDa.

[0219] In various embodiments, the hyaluronic acid comprises a molecular weight of about lOOkDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 200kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 300kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 400kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 500kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 600kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 700kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 800kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 900kDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about IMDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1. IMDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.2MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.3MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.4MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.5MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1 ,6MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.7MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1.8MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 1 9MDa. In various embodiments, the hyaluronic acid comprises a molecular weight of about 2MDa.Concentration of hyaluronic acidHyaluronic acid concentrations at gelation

[0220] In various embodiments, the gelling conditions for the composite comprises 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.

[0221] In various embodiments, the gelling conditions for the composite comprises hyaluronic acid present in an amount of about 23 mg / mL, or about 25 mg / mL.Concentration of hyaluronic acid in composite (finished device / post-gelling)

[0222] In various embodiments, the composite 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.

[0223] 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 10.5mL, about 1 ImL, about 11.5mL, about 12mL, or about 12.5mL.Concentration of CollagenCollagen fiber concentrations at gelation

[0224] In various embodiments, the gelling conditions for the composite comprises 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 about 45 mg / mL to about 65 mg / mL.

[0225] In various embodiments, the gelling conditions for the composite comprises collagen fibers present in an amount of about 45 mg / mL, or about 65 mg / mL.Concentration of Collagen fiber in composite

[0226] 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.

[0227] 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.Concentration of DVS

[0228] In various embodiments, the gelling conditions for the composite comprises 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.

[0229] In various embodiments, the gelling conditions for the composite comprises 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.

[0230] In various embodiments, the gelling conditions for the composite comprises divinyl sulfone present in an amount of about 230mM of di vinyl sulfone, about 240mM of divinyl sulfone, about 260mM of divinyl sulfone, or about 270mM of divinyl sulfone.Pores

[0231] The scaffold complex is not generally a uniform solid material. Instead, scaffold complexes contain a plurality of pores present on or within a surface of the scaffold complex. The presence, size, distribution, frequency and other parameters of the pores can be modulated during the creation of the scaffold complex. Mean Pore Size can be from below about 1 micron to up to 100 microns, including 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60 70, 80, 90 100, or 200 microns. Mean pore size may be narrowly tailored, e.g., such that at least 40%, such as 50%, 60%>, 70%, 80%, 90%, 95% or greater than 95 > of the pores are in a desired size or within a desired size range.

[0232] In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 100 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 75 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 50 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 25 microns. In various embodiments, the scaffoldcomplex comprises a mean pore size ranging from about 1 micron to about 10 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 5 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 1 micron to about 2 microns.

[0233] In various embodiments, the scaffold complex comprises a mean pore size ranging from about 10 micron to about 100 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 25 micron to about 100 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 50 micron to about 100 microns. In various embodiments, the scaffold complex comprises a mean pore size ranging from about 75 micron to about 100 microns.

[0234] In various embodiments, the scaffold complex comprises a mean pore size of about 1 micron. In various embodiments, the scaffold complex comprises a mean pore size of about 2 micron2. In various embodiments, the scaffold complex comprises a mean pore size of about 3 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 4 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 5 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 6 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 7 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 8 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 9 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 10 microns.

[0235] In various embodiments, the scaffold complex comprises a mean pore size of about 15 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 20 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 25 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 30 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 40 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 50 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 60 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 70 microns. In various embodiments, the scaffold complex comprises a mean pore size ofabout 75 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 80 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 90 microns. In various embodiments, the scaffold complex comprises a mean pore size of about 100 microns.Pore size uniformity

[0236] In various embodiments, pore size is substantially uniform. In various embodiments, pore size are within a narrow size distribution.

[0237] In various embodiments, pores comprise substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 100 microns of each other.

[0238] In various embodiments, pores comprise substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 10 microns of each other.

[0239] In various embodiments, pores comprise substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 1 micron of each other.

[0240] In various embodiments, pores comprise a substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 100 microns of the mean pore size.

[0241] In various embodiments, pores comprise substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 10 microns of the mean pore size.

[0242] In various embodiments, pores comprise substantially uniform size distribution wherein at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the pores are within a range of about 1 micron of the mean pore size.

[0243] These pores may also be provided in sufficient densities that two or more pores are in contact with each other such that their volumes overlap to form tunnels between them. These tunnels can be mostly free of the composite material forming the scaffold of pores. These tunnels may be mostly circular in shape, or may be more irregular in shape. The shortestdistances between these tunnels’ edges can be from below about 1 micron to up to 100 microns, including 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60 70, 80, 90 or 100 microns.Reducing Toxicity

[0244] In various aspects, the present disclosure describes a method of reducing toxicity of a divinyl sulfone crosslinked material comprising pendant vinyl groups, said method comprising the step of reacting the pendant vinyl sulfone group with a quenching agent whereby the pendant vinyl sulfone group is converted to a less toxic material. This present disclosure also reduces the toxicity of any unreacted, free-floating DVS that may remain in the gel even after processing. Without being bound by theory, the compositions of the present converted to a less toxic compositions because the materials are converted to groups that lacks or has limited reactivity to crosslink or alkylate proteins and biological constituents in vivo. In various embodiments, a “less toxic material” is a material that lacks or that has limited reactivity to crosslink or alkylate proteins and biological constituents in vivo. In various embodiments, “reducing toxicity” means limiting reactivity of a material to crosslink or alkylate proteins and biological constituents in vivo.

[0245] Quenching Agent

[0246] In various embodiments, quenching agents comprise 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, quenching agents comprise molecules that comprise thiols (also known as sulfhydryl groups), amines, hydroxyls, or halogens. In various embodiments, the halogen is fluorine, chlorine, bromine, or iodine.

[0247] In various embodiments, the quenching agents comprise one or more amine groups. In various embodiments, the quenching agents comprise one or more thiol groups. In various embodiments, the quenching agents comprise one or more hydroxyl groups. In various embodiments, the quenching agents comprise any combination of amine, thiol or hydroxyl groups.

[0248] In various embodiments, the quenching agent comprise two or more nucleophiles. The two or more nucleophiles may be the same or different. In various embodiments, quenching agent comprises an amine and a thiol.

[0249] In various embodiments, quenching agent comprises two or more amines.

[0250] In various embodiments, quenching agent comprises two or more thiols.

[0251] In various embodiments, quenching agent comprises two or more hydroxyls.

[0252] In various embodiments, quenching agent comprises an amine and a thiol.

[0253] In various embodiments, quenching agent comprises an amine and a hydroxyl.

[0254] In various embodiments, quenching agent comprises a thiol and a hydroxyl.

[0255] 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.

[0256] In various embodiments, the quenching agent comprises more than one nucleophile.

[0257] In various embodiments, the quenching agent comprises two or more nucleophiles, wherein at least one nucleophile comprises an amine.

[0258] In various embodiments, the quenching agent comprises two or more nucleophiles, wherein at least one nucleophile comprises an amine, wherein the amine remains available for subsequent reactions following the quenching reaction. .

[0259] In various embodiments, the amine in a primary amine following the quenching reaction.

[0260] In various embodiments, the quenching reaction forms a quenched pendant group, wherein the quenched pendant group comprises an amine, thiol or 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 an 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,

[0261] In various embodiments, amino acids include, but are not limited to, cysteine (with reactive thiol group), lysine (with primary amines), arginine, histidine, glycine, glutamate or aspartate (anionic), leucine (increasing hydrophobicity), and asparagine (amide), methionine, serine (hydroxyl) and threonine (hydroxyl).

[0262] In various embodiments, amino acids include, but are not limited to, essential amino acids, non-essential amino acids, and amino acid derivatives.

[0263] A “non-essential” amino acid residue, as used herein, is an amino acid residue present in a wild-type sequence of a polypeptide that can be altered without abolishing or substantially altering essential biological or biochemical activity (e.g., receptor binding or activation) of the polypeptide.

[0264] An “essential” amino acid residue, as used herein, is an amino acid residue present in a wild-type sequence of a polypeptide that, when altered, results in abolishing or a substantial reduction in the polypeptide's essential biological or biochemical activity (e.g., receptor binding or activation).

[0265] In various embodiments, amino acids include any isomer of the amino acid. In various embodiments, amino acids or amino acid analogs are 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 chiral centers that are 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.

[0266] In various embodiments, amino acids include, but are not limited to, amino acids comprising unblocked nucleophilic side chains. In various embodiments, amino acids include, but are not limited to, amino acids comprising an amino group of a P-amino acid is substituted with a protecting group. Amino protecting groups include but not limited to, tert-butoxycarbonyl (BOC group), 9-fluorenylmethyloxy carbonyl (FMOC), and toluenesulfonyl (tosyl, Ts). In various embodiments, amino acids comprise an amino group substituted with a protecting group selected from the group consisting of tert-butoxy carbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), and toluenesulfonyl (tosyl, Ts). In various embodiments, the carboxylic acid functional group of a P-amino acid is protected. In various embodiments, the carboxylic acid is protected as an ester derivative. In various embodiments, thenucleophilic 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.

[0267] In various embodiments, the amino acid comprises a capping group. The term “capping group” refers to the chemical moiety occurring at either the carboxy or amino terminus of a polypeptide chain. A capping group of a carboxy terminus includes an unmodified carboxylic acid (i.e., -COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus can be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary and secondary amines, including but not limited to, pegylated secondary amines. A capping group of an amino terminus includes an unmodified amine (i.e., -NH2) or an amine with a substituent. For example, the amino terminus can be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including Ci-Ce carbonyls, C7- C30 carbonyls, and pegylated carbamates. Polypeptides can also be used as quenching agents.

[0268] In various embodiments, the quenching agent comprises a polypeptide. In various embodiments, the polypeptide comprises a bioactive sequences.

[0269] In various embodiments, the bioactive sequence comprises cell recognition or cell attachment activity. In various embodiments, the bioactive sequence comprises a sequence present in a cell-attachment domain of a biological polymer. In various embodiments, the bioactive sequence comprises a sequence present in a cell-attachment domain of a biological polymer comprising 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).

[0270] In various embodiments, the quenching agent comprises an amine (also referred to as an amine quenching agent). In various embodiments, a quenching agent includes, but in not limited to, an amine quenching agent.

[0271] In various embodiments, the quenching agent comprises a polyamine. In various embodiments, polyamines include, but are not limited to, ethylenediamine (EDA), polyethylene amines, putrescine, spermine, thermospermine, and spermidine.

[0272] In various embodiments, the quenching agent comprises a thiol. In various embodiments, thiols include, but are not limited to, propane-1, 3 -dithiol and dimercaprol.

[0273] In various embodiments, the quenching agent comprises an aminothiol. An aminothiol, as referred to herein, is a molecule containing both an amine and a thiol functional groups. In various embodiments, include, but are not limited to, cysteamine, and ((aminopropyl)amino)ethanethiol, 3-(methylamino)-2-((methylamino)methyl)propane-l-thiol or 3-amino-2-(arninomethyl)propane-l-thiol.

[0274] An advantage of using amino acids, amines, polyamines or their derivatives, as quenching agents is the resulting ability to encourage cellular attachment and migration. Without being bound by theory, incorporation of amino acids such as lysine or amine-containing molecules such as EDA may result in amines being present on the polymer backbone, thereby encouraging cellular attachment and migration. Cellular attachment and migration may be desirable for cell permeable gels.

[0275] Quenching Reaction pH

[0276] Without being bound by theory, functional groups with higher reactivity allow for faster reaction kinetics. For example, lower reaction times is one example of lower reaction kinetics factor that may allow for a broader range of pH conditions to be used. In various embodiments, quenching agents comprise highly reactive functional groups. Without being bound by theory, a quenching agent is more reactive if it comprises a functional group that deprotonates more readily, or at a lower pH.

[0277] In various embodiments, the quenching agent is any suitable agent able to react as a nucleophile with a vinyl group during a Michael Addition reaction. In various embodiments, the quenching agent is any suitable agent able to react as a nucleophile with a vinyl sulfone group during a Michael Addition reaction. In various embodiments, the quenching agent comprises a functional group that deprotonates. In various embodiments, the quenching agent comprises a functional group that deprotonates readily. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 14. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 13. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 12. In various embodiments, the quenching agent comprises afunctional group that deprotonates at a pH of less than 11 . In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 10. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 9. In various embodiments, the quenching agent comprises a functional group that deprotonates at a pH of less than 8.

[0278] An advantage of using a quenching agent comprising a more reactive or nucleophilic functional group species (compared to the groups being crosslinked, such as the hydroxyls of hyaluronic acid) such as a thiol or a primary amine, the quenching can alternatively be performed at lower pH, halting the further modification of HA with DVS and limiting the degradation of constituents due to their hydrolysis at high pH. Instead, the pH can be dropped to 10 or below before addition of quenching agent, as well as being reacted for a significantly longer time if needed for reducing the pendant concentration further. Thiols can react with the vinyl sulfone groups at neutral pH, so if using a quenching agent like cysteine, the pH can be dropped to near 7 and reacted for a long time to complete the pendant group quenching. (See: Yu Y. et al., Biomacromolecules 13.3 (2012): 937-942.)

[0279] In various embodiments, quenching agents comprise amines and / or thiols.Without being bound to theory, amines and thiols maximize reaction rates, thereby minimizing quenching time at high pH. A quenching pH that is above the pKa value for the quenching group (such as the ionized amine side chain and / or peptide amine group of an amino acid), but below the pH where substantial HA hydrolysis occurs, can allow for longer quenching times for more complete quenching while minimizing degradation of the HA backbone. For example, since the pKa of lysine’s 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), the amine group may be deprotonated and suitable for reaction with vinyl sulfone at a pH above 10.4 yet below the pH were substantial HA degradation occurs (pH > 11). A high pH, such as 11 or above, and especially 13, is associated with HA degradation through 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, he pKa of cysteine’s thiol side chain in water is 8.6, providing a suitable range of quenching pH below the onset pH of accelerated HA hydrolysis (G. Roos, et al., "Understanding the pKa ofredox cysteines: The key role of hydrogen bonding." Antioxidants & redox signaling 18.1 (2013): 94-127).

[0280] In various embodiments, the quenching reaction step is performed at a pH relative to the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at a pH equal to or above the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at a pH about equal to the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at a pH above the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at about a 1.0 pH unit (relative to pKa) above the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at about 2.0 pH units above the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed at about 3.0 pH units above the pKa of the quenching agent. In various embodiments, the quenching reaction step is performed 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 performed 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 performed at a pH ranging from about 0.5 to about 1.0 pH units above the pKa of the quenching agent.

[0281] In one non-limiting example, lysine has a side-chain amine with a pKa of about 10.8. In various embodiments, a quenching step using lysine as a quenching agent is performed 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, a quenching step using cysteine as a quenching agent is performed at a pH ranging from about 9 to about 10.

[0282] In various embodiments, the quenching reaction step is performed at a pH lower than about 12. In various embodiments, the quenching reaction step is performed at a pH lower than about 11. In various embodiments, the quenching reaction step is performed at a pH lower than about 10.0. In various embodiments, the quenching reaction step is performed at a pH lower than about 9.0. In various embodiments, the quenching reaction step is performed at a pH lower than about 8.0.

[0283] In various embodiments the quenching reaction is performed at a pH ranging from about 8.0 to about 12.0. In various embodiments the quenching reaction is performed at a pHranging from about 9.0 to about 11 .0. In various embodiments the quenching reaction is performed at a pH ranging from about 8.0 to about 9.0. In various embodiments the quenching reaction is performed at a pH ranging from about 9.0 to about 10.0. In various embodiments the quenching reaction is performed at a pH ranging from about 10.0 to about 11.0.

[0284] In one embodiment the quenching reaction is performed at a pH ranging from about 8.6 to about 12.0. In one embodiment the quenching reaction is performed at a pH ranging from about 10.4 to about 11.0. In one embodiment the quenching reaction is performed at a pH ranging from about 10.4 to about 11.4. In one embodiment the quenching reaction is performed at a pH ranging from about 8.6 to about 10.

[0285] In one embodiment the quenching reaction is performed at a pH of less than 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.

[0286] 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.

[0287] 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 for 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 for the crosslinking reaction.

[0288] In various embodiments, the quenching reaction comprises a quenching agent and a DVS at 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 reactioncomprises a quenching agent and a DVS at 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 a DVS at 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 a DVS at 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 a DVS at a molar ratio ranging from about 1 : 1 to about 1 :80 (quenching agent: DVS concentration used in the crosslinking reaction).

[0289] In various embodiments, the quenching reaction comprises a quenching agent and a DVS at 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 a DVS at 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 a DVS at 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 a DVS at 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 a DVS at 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 a DVS at 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 a DVS at 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 a DVS at 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 a DVS at a molar ratio ranging from about 1 : 1 to about 1 :2 (quenching agent: DVS).

[0290] In various embodiments, the quenching reaction comprises a quenching agent and a DVS at a molar ratio of about 1: 1, (quenching agent: DVS). In various embodiments, the quenching reaction comprises a quenching agent and a DVS at a molar ratio of about 1 : 10, (quenching agent: DVS). In various embodiments, the quenching reaction comprises a quenching agent and a DVS at a molar ratio of about 1 : 100, (quenching agent: DVS).

[0291] Concentration of unquenched pendant groups

[0292] In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 10 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 5 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 2.5 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 2 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 1.5 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 1.2 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups of at most about 1.0 micromoles / mL.

[0293] In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups ranging from about 0.1 micromoles / mL to about 10 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups ranging from about 0.1 micromoles / mL to about 1 micromole / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups ranging from about 1 micromole / mL to about 10 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups ranging from about 1 micromole / mL to about 5 micromoles / mL. In various aspects, the crosslinked polymer comprises a concentration of unquenched pendant groups ranging from about 1 micromole / mL to about 2 micromoles / mL.

[0294] Quenching Reaction Conditions

[0295] In various embodiments, a quenching agent (a nucleophile such as lysine, glycine, cysteine, or equivalent) is added to the reactant mixture at the end of gel crosslinking reaction, but before the acid neutralization. In various embodiments, the quenching agent is allowed time to diffuse and react with residual vinyl sulfone groups, prior to acid-neutralizing the reaction. In various embodiments, the quenching reaction time ranges from at least about 1 minute to about 1 hour.

[0296] 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 quenchingreaction time ranges from at least about 5 minute to about 15 minutes at room temperature or above.

[0297] In various embodiments, the quenching reaction takes place at a temperature below about the boiling point of an aqueous solution or mixture. In various embodiments, the quenching reaction takes place at a temperature below about the boiling point of water. In various embodiments, the quenching reaction takes place at a temperature below about 100°C.

[0298] In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 90°C . In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 80°C . In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 70°C . In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 60°C . In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 50°C. In various embodiments, the quenching reaction takes place at a temperature ranging from about 25°C to about 40°C. In various embodiments, the quenching reaction takes place at a temperature ranging from about 25 °C to about 30°C.

[0299] In various embodiments, the quenching reaction takes place at about room temperature or above. In various embodiments, the quenching reaction takes place at about room temperature.

[0300] In various embodiments, the quenching reaction takes place at room temperature or above, especially 5-15minutes, though up to several hours when at or under 25°C.

[0301] In various embodiments, the quenching reaction takes place at about 0°C. In various embodiments, the quenching reaction takes place below about 0°C.

[0302] Reduction in Residual Pendant Vinyl Sulfone Groups

[0303] In various embodiments, the composition comprises a diminished number of pendant vinyl sulfone groups.

[0304] In various aspects, quenching of pendant vinyl sulfone groups results in a reduction of the number of residual unreacted pendant vinyl sulfone groups.

[0305] In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 0.1 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 0.5 pg / mL, 1.0 pg / mL, 1.5 pg / mL, 2.0 pg / mL.

[0306] In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 1.7 pg / mL.

[0307] In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 1.4 pg / mL In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 1.0 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration of at most about 0.5 pg / mL.

[0308] In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.1 pg / mL to about 2.0 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.1 pg / mL to about 1.5 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.1 pg / mL to about 1.0 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.5 pg / mL to about 1.0 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.5 pg / mL to about 1.5 pg / mL. In various embodiments, the crosslinked polymer comprises residual sulfone groups at a concentration ranging from about 0.5 pg / mL to about 1.4 pg / mL.

[0309] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by a range of about 10% to about 99.9%.

[0310] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by a range of about 10% to about 80%, a range of about 10% to about 70%, a range of about 10% to about 60%, a range of about 10% to about 50%, a range of about 10% to about 40%, a range of about 10% to about 30%, or a range of about 10% to about 20%.[0031 1] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 10%.

[0312] In various embodiments, the methods of the present disclosure result in a reduction of residual vinyl sulfone groups by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0313] 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%.Additional Benefits of compositions and methods:

[0314] Besides quenching vinyl sulfone groups to improve the sugar polymer hydrogel’s safety profile, the chemical quenching also provides an opportunity to further tune the gel properties through choice of quenching agent. This allows for conjugation of chemical groups to the hydrogel network after primary gel formation.Tuning Gel Properties

[0315] Quenching agents can be used to tune the overall gel properties, such as hydrophobicity / hydrophilicity, electronegativity, or other functions. For example, cysteine can increase the mucoadhesive properties of the gel, with cysteine-quenched gels being substantially tackier, qualitatively, when handling after production. For example, this tacky trait could be used to mitigate implant migration in vivo or for mucosal targeting of drug delivery. Additional sulfur groups could also result in a more immunostimulatory gel, which could be useful for immunomodulation products. Methionine could be used as a quenching agent in a similar fashion. These agents, however, may not be preferentially chosen when adhesion is undesired,such as some surgical mesh use cases in which adhesion formations between two normally separate tissues may be undesirable.

[0316] With this invention, one can tune the electronegativity of the crosslinked sugar polymer backbone. For example, on my choose choice of groups with different electronegativity in their side chains, such as positively charged (such as histidine, lysine, arginine, polylysine, etc.), negatively charged (such as aspartate or glutamate), or uncharged (such as glycine, alanine, serine, threonine, asparagine, glutamine, etc.) to be used as crosslinking agents.Hydrophilicity / Hydrophobicity

[0317] With this invention, one can tune the hydrophilicity / hydrophobicity of the gel backbone to a degree with a choice of the hydrophilicity / hydrophobicity of the quenching agent. For example, one may quench the residual vinyl sulfone pendants with polar or ionic agents such as lysine or glutamate to increase a hydrogel’s hydrophilicity, or quench with non-polar agents such as alanine or leucine (or other small molecules such as an aliphatic chain with an amine or thiol group) in order to increase a hydrogel’s hydrophobicity.Adding Functionality

[0318] The quenching step can be used to provide additional functionality to the gel. The quenching step can provide additional functionality to the gel, by appending a functional moiety or functional agent to the gel.

[0319] 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 biologically active sequence. In various embodiments, the bioactive sequence comprises cell recognition or cell attachment activity. In various embodiments, the bioactive sequence comprises a sequence present in a cell attachment domain of a biological polymer. In various embodiments, the bioactive sequence comprises a sequence present in a cell attachment domain of a biological polymer comprising 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).

[0320] In a non-limiting example, the quenching step can provide additional functionality to the gel, such as by appending a biologically active peptide sequence such as RGD or IKVAV,

[0321] In various embodiments, the quenching step can provide additional functionality by adding a functional group that allows for a further step of subsequent modification. For example, the quenching agent can comprise at least 1 group that can be used to react with vinyl sulfone, and at least 1 group that can be used thereafter to conjugate a second agent.

[0322] This two-step conjugation may be necessary if the intended cargo is incompatible with the potentially harsh alkaline conditions present at the quenching step. The target cargo can then be added after the reaction mixture’s pH has been lowered to a compatible level. Alternatively, the target cargo can then be added after sterilization, or even at the point-of-care if the cargo is unsuitable for autoclave sterilization and / or long-term storage conditions.Toxicity of Waste Streams

[0323] Chemical quenching has the benefit of rendering unreactive one or more of the reactive groups of residual unbound crosslinking agents, uncrosslinked groups, which decreases the toxicity of the manufacturing waste streams.Durability of Hydrogels

[0324] The current trend of on-market sugar polymer hydrogels such as HA dermal fillers has been toward longer-lasting effects by making the gels more resistant to degradation after their implantation. This typically is accomplished by making denser gels with high HA concentrations and high crosslinking degrees. Furthermore, DVS crosslinking has been mostly superseded by BDDE crosslinking in the current dermal filler market.

[0325] Employing the use of amino acids as nucleophilic quenching agents can be used to obtain benefits in addition to the reduction of possible pendant VS crosslinker toxicities. Amino acid conjugation was thought to lead to cell-mediated hydrogel degradation and a shorter gel duration and thus a shorter clinical effect as the hydrogel backbone could increase and encourage cellular motility and material interaction. Cellular interaction is a major cause of hyaluronic acid degradation (particularly enzymatic cleavage of hyaluronic acid, such as through HYAL-2).

[0326] Also, it is unintuitive that such a small modification degree (only pendant vinyl sulfone groups are available for quencher modification) had such a large effect on resulting macroscopic gel properties such as gel tackiness.

[0327] In previous examples acid has been used to quench the DVS-crosslinking reaction. However, acid quench merely halts reactions between the vinyl sulfone groups and the hydrogel backbone. It does not have any direct effect upon the pendant vinyl sulfone groups.

[0328] For example, US Patent 8,481,080 describes making crosslinked HA gels with divinyl sulfone, with the DVS-crosslinking reaction followed by acidic treatment during the swelling process, to give final gel concentrations of 1.1%-1.4%, with no mention of any chemical quenching, residual vinyl sulfone concentrations, or pendant vinyl sulfone groups. In another example, US20050142152A1 uses acidic saline and PBS washes to remove impurities after DVS reaction, which is a solely acid quenching. (See: Y. Yu, et al., Biomacromolecules 13.3 (2012): 937-942.)

[0329] EMBODIMENTS i. A nanofiber-hydrogel composite comprising: a. a hydrogel material comprising a hyaluronic acid, b. a nanofiber material comprising a collagen, and c. a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL.ii. A nanofiber-hydrogel composite comprising: a. a hydrogel material comprising a hyaluronic acid, b. a nanofiber material comprising a collagen, and c. a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; and wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL. iii. A nanofiber-hydrogel composite comprising: a. a hydrogel material comprising a hyaluronic acid, b. a nanofiber material comprising a collagen, and c. a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL; and wherein the divinyl sulfone is present prior to crosslinking in an amount ranging from about 3: 1 to about 5: 1 divinyl sulfone molecules to hyaluronic acid subunit.iv. A nanofiber-hydrogel composite comprising: a. a hydrogel material comprising a hyaluronic acid, b. a nanofiber material comprising a collagen, and c. a crosslinking agent comprising divinyl sulfone; wherein the hyaluronic acid is covalently crosslinked to the collagen by the divinyl sulfone; wherein the amount of crosslinking ranges from about 0.5% to about 30%; wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 15 mg / mL; and wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL. v. A nanofiber-hydrogel composite product by the process comprising: a step of reacting a hyaluronic acid material, a collagen fiber, and divinyl sulfone; whereby the hyaluronic acid is covalently crosslinked to the collagen fiber by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, and wherein the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; and wherein the divinyl sulfone is present in an amount ranging from about 0.5: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.vi. A method for manufacturing a nanofiber-hydrogel composite comprising a step of reacting a hyaluronic acid, a collagen fiber, and divinyl sulfone; whereby the hyaluronic acid is covalently crosslinked to the collagen fiber by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, and wherein the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; and wherein the divinyl sulfone is present in an amount ranging from about 0.5: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.vii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 0.1 to about 3.0 during manufacture. viii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 1.0 to about 3.0 during manufacture. ix. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 1.5 to about 4.0 during manufacture. x. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 2.0 to about 3.0 during manufacture. xi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 2.2 to about 3.0 during manufacture. xii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 2.3 to about 2.8 during manufacture.xiii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 1.0 to about 2.0 during manufacture. xiv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio ranging from about 0.1 to about 1.0 during manufacture. xv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio of about 2.3 during manufacture. xvi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio of about 2.4 during manufacture. xvii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio of about 2.6 during manufacture. xviii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling ratio of about 2.8 during manufacture. xix. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 100% in vivo. xx. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 75% in vivo.xxi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 50% in vivo. xxii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 40% in vivo. xxiii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 30% in vivo. xxiv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 20% in vivo. xxv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 15% in vivo. xxvi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 12% in vivo. xxvii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 10% in vivo.xxviii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of at most 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in vivo. xxix. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 0% to about 100% in vivo. xxx. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 1% to about 100% in vivo. xxxi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 1% to about 50% in vivo. xxxii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 1% to about 20% in vivo. xxxiii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 1% to about 10% in vivo.xxxiv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 10% to about 20% in vivo. xxxv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % ranging from about 10% to about 15% in vivo. xxxvi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of about 50%, 40%, 30%, 20%, or 10% in vivo. xxxvii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of about 10%, 11%, 12%, 13%, 14%, or 15% in vivo. xxxviii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of about 20% in vivo. xxxix. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the composite comprises a swelling % of about 12% in vivo. xl. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount ranging from about 5 mg / mL to about 10 mg / mL.xli. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount ranging from about 9 mg / mL to about 10 mg / mL. xlii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 9 mg / mL. xliii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 9.5 mg / mL. xliv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 10 mg / mL. xlv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount ranging from about 10 mg / mL to about 40 mg / mL. xlvi. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount ranging from about 15 mg / mL to about 30 mg / mL. xlvii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount ranging from about 20 mg / mL to about 30 mg / mL.xlviii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount ranging from about 25 mg / mL to about 30 mg / mL. xlix. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount of about 15 mg / mL.1. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount of about 30 mg / mL. li. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the collagen is present in an amount of about 25 mg / mL. lii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 9 mg / mL; and wherein the collagen is present in an amount of about 15 mg / mL. liii. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 10 mg / mL; and wherein the collagen is present in an amount of about 30 mg / mL. liv. The nanofiber-hydrogel composite of any one embodiments i to iv, wherein the hyaluronic acid is present in an amount of about 9.5 mg / mL; andwherein the collagen is present in an amount of about 25 mg / mL.Iv. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 30 mg / mL.Ivi. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 25 mg / mL.Ivii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount ranging from about 23 mg / mL to about 25 mg / mL.Iviii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 23 mg / mL. lix. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 25 mg / mL. lx. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the collagen is present in an amount ranging from about 30 mg / mL to about 100 mg / mL.Ixi. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the collagen is present in an amount ranging from about 40 mg / mL to about 70 mg / mL.Ixii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the collagen is present in an amount ranging from about 45 mg / mL to about 65 mg / mL.Ixiii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the collagen is present in an amount ranging of about 45 mg / mL.Ixiv. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the collagen is present in an amount ranging of about 65 mg / mL.Ixv. A nanofiber-hydrogel composite product by the process of embodiment v wherein the divinyl sulfone is present in an amount ranging from about 1 : 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixvi. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount ranging from about 2: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixvii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount ranging from about 3: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixviii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount ranging from about 3.5: 1 to about 4: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixix. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount of about 3.5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixx. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount of about 4: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixxi. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the divinyl sulfone is present in an amount of about 4.5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixxii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 25 mg / mL; wherein the collagen is present in an amount ranging from about 45 mg / mL; and wherein the divinyl sulfone is present in an amount of about 4: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixxiii. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 25 mg / mL; wherein the collagen is present in an amount ranging from about 45 mg / mL; and wherein the di vinyl sulfone is present in an amount of about 3.5: 1 di vinyl sulfone molecules per hyaluronic acid subunit.Ixxiv. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 23 mg / mL; wherein the collagen is present in an amount ranging from about 65 mg / mL; and wherein the di vinyl sulfone is present in an amount of about 4.5: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixxv. A nanofiber-hydrogel composite product by the process of embodiment v, wherein the hyaluronic acid is present in an amount of about 23 mg / mL; wherein the collagen is present in an amount ranging from about 65 mg / mL; andwherein the divinyl sulfone is present in an amount of about 4: 1 divinyl sulfone molecules per hyaluronic acid subunit.Ixxvi. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the collagen nanofibers comprise Type I, Type II, Type III, or Type IV collagen.Ixxvii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the collagen nanofibers comprise recombinant collagen.Ixxviii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the collagen nanofibers comprise type I bovine collagen nanofiber fragments.Ixxix. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the collagen nanofibers are electrospun, melt spun, blow spun and / or cryomilled.Ixxx. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the collagen nanofibers are retained inside the nanofiber-hydrogel composite.Ixxxi. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the interfacial bonding between the collagen nanofibers and the HA enhances the composite stiffness with relatively low fiber loading density.Ixxxii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite exhibits monocyte recruitment, monocyte polarization, or both.Ixxxiii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as exhibiting higher porosity, increased cell permeability and / or maintains storage moduli.Ixxxiv. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite enables cellular infiltration.Ixxxv. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of 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.Ixxxvi. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is formulated in one or more product forms selected from mats, injectables and flowables.Ixxxvii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite accommodates pass through of a 27-gauge or smaller needle.Ixxxviii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the crosslinking agentreacts with the hydroxyl groups of the HA and hydroxy groups, thiol groups, or amine groups of the collagen nanofiber.Ixxxix. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the crosslinking agent reacts with the hydroxyl groups of the HA and hydroxy or amine groups of the collagen nanofiber. xc. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite forms one or more porous structures. xci. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the one or more porous structures induces elevated host cell infiltration. xcii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized in displaying a biostimulatory effect selected from tissue remodeling, host cell infiltration, cell adhesion, cell migration, angiogenic responses, adipogenic responses, and macrophage polarization to pro-healing phenotypes. xciii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite comprises a population of infiltrated macrophages and wherein the collagen nanofibers condition the population of infiltrated macrophages to a M2 phenotype. xciv. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as exhibiting tissue remodeling effect induced by the nanofiber- hydrogel composite without incorporation of cells or growth factors.xcv. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying a biostimulatory effect due to the low crosslinking density of the composite materials. xcvi. The nanofiber-hydrogel composite of any one of embodiments i to iv, wherein the nanofiber- hydrogel composite is suitable for terminal sterilization by autoclaving. xcvii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the composite is characterized as having increased thermal stability and / or results in shelf stability at ambient temperatures. xcviii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying biocompatibility. xcix. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying cell adhesion. c. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying cell migration. ci. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as enabling durable soft tissue remodeling.cii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as promoting angiogenesis. ciii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as prolonging a biostimulatory effect by retaining and / or conditioning infiltrating macrophages towards a pro-regenerative M2 phenotype, thereby facilitating neo-vasculature formation. civ. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as improving adipose tissue formation. cv. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying vasculature formation. cvi. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite modulates host cell infiltration and / or shape retention of the composite. cvii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the storage modulus is within the range of most soft tissues. cviii. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the storage modulus is within a range of about 150Pa to about 500Pa.cix. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiber- hydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is particularized to a set of microgels with diameters selected from the group consisting of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 pm. ex. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite mimics a host tissue structure. cxi. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite mimics a natural extracellular matrix.EXAMPLESExample 1Collagen Fiber Production:

[0330] Bovine type I collagen

[0331] Lyophilized bovine type I collagen (bovine skin-derived, from Collagen Solutions) was dissolved in l,l,l,3,3,3-Hexafluoro-2-propanol (HFIP) at room temperature overnight to give a viscous cloudy solution (5.2% w / w). The collagen solution was loaded into 5mL plastic syringes with a 23G metallic blunt needle. The electrospinning was performed on a Ucalery electrospinning unit with the following parameters: 2.2mL / h of the flow rate; two syringes in parallel; 17.4 kV of the voltage applied to the needle; 10.8kv 8kV (negative) applied to the collector; 12.5 cm of the collecting distance; 200 rpm of the rotation rate of the metallic collector. The resulting collagen nanofibers / microfibers were crosslinked in ethanol containing 50mM l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 20mM N- Hydroxysuccinimide (NHS) for 20 hours. After the crosslinking, fibers were washed with ethanol three times (30 minutes each) to remove the excessive reagents. The collagen fibers were then broken down into fragments using cryomilling (SPEX SamplePrep).

[0332] The amount of intra-crosslinking or inter-crosslinking of collagen fibers may be measured using13C-NMR spectra. (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 uncrosslinked to crosslinked collagen can be measured with the method in example #5.

[0333] Gelatin

[0334] Fibers made from porcine gelatin (Sigma) were produced by dissolving gelatin to 15%w / v in TFE , then spinning at 1.35mL / hour at a voltage of 12kV onto a grounded drum rotating at 640rpm. Fibers made from porcine gelatin are shown in Figure 44.

[0335] Porcine Collagen

[0336] Porcine type I atelocollagen fibers were produced by dissolving Porcine type I atelocollagen (Nitta) to 7% in HFIP and spinning at 0.3mL / hour at 1 IkV voltage. Figure 43 is an image showing the resulting fibers.

[0337] Non-animal -derived collagen and collagen like-peptides.

[0338] Vecollan recombinant collagen from Evonik were spun into fibers. Fibers were produced by dissolving Vecollan to 20%w / v in TFE, then spinning at E5mL / hour with 16kV voltage onto grounded drum spinning at 480 rpm. Figure 45 is an image showing the resulting fibers. The parameters can be tuned to produce cylindrical or ribbon morphologies. Both cylindrical and ribbon fibers are seen in Figure 45.

[0339] Once spun, the resulting fibers from alternate non-bovine sources can be processed as described for bovine collagen and used to form fiber-hydrogel composites. Other forms of collagen or gelatin, including other types of collagen like type III or recombinant “collagen-like” peptides from bacterial sequences can be applied.

[0340] Depending upon the raw material source, the resulting fibers may have less secondary structure as-spun than bovine collagen described (gelatin for instance is not triplehelical), or have differences in crosslinking moiety concentrations due to variances in peptide sequences. Such fibers may require more intensive crosslinking prior to contact with water. , In various embodiments, fibers may be crosslinked using EDC / NHS, DVS, BDDE, an aldehyde- based crosslinker such as vapor phase glutaraldehyde treatment, treated Maillard reactions such as with D-ribose and heat, dehydrothermal treatment, genipin and transglutaminases, irradiation (UV, gamma, or e-beam), plasma treatment, other methods known in the field (See for example 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.Once rendered water insoluble, the fibers can be mechanically processed to a dispersed form, and covalently gelled into a composite using the methodology described herein.Example 2Preparation of HA Hydrogels and NHCs Crosslinked by DVS:

[0341] Test lots of a series of gels were produced, with and without a fibrous phase, with a lot size of 3cc (during gelation stage). Stock solutions of the reagents were prepared. Sodium hyaluronate (HA, MW 1.5MDa) was dissolved in distilled water at a concentration of 30 mg / mL. Sodium hydroxide was dissolved in water to make a 10M solution. Lysine monohydrochloridewas dissolved in water at a concentration of 2M. Firstly, gels without collagen were produced with a gelation HA concentration of 21,24,27, or 30 mg / mL and a DVS-to-HA subunit ratio of 3.22, 3.5, 4, 4.5, or 5X, by varying the amount of each stock solution added, with 0.03mL of the sodium hydroxide stock added to achieve the targeted 0. IM concentration. Each group was mixed between two 5mL syringes connected with a luer-lok connector. After vigorous mixing, the mixture was incubated in sealed syringes in a 30°C water bath for 2 hours. The reaction was chemically quenched by lysine solution (molar ratio of 1 to 1 of lysine to DVS molecules), with syringe-syringe mixing followed by a 5-minute incubation at room temperature. The gels are then neutralized in 50mL syringes containing 21mL 20mM monobasic sodium phosphate solution. The hydrogel was transferred into dialysis tubing (13,000 MWCO) and dialyzed against IL 0.9% sodium chloride for 16 hours and IL DPBS for 3 hours at room temperature. The resulting gel suspension was transferred to 1.5mL microcentrifuge tubes through 18-gauge blunt needles then centrifuged at 17,000 x g for 3 minutes to separate excess water. After removing the supernatant, the gels were collected in lOmL syringes and weighed in order to calculate the swelling ratio (relative to weight of initial gelation solution, see example #5). The gels were beaded through 150-micron screens, then loaded into ImL glass syringes and autoclaved at 121°C for 30 minutes. The swelling ratio of the gels during manufacturing and the resulting final HA concentrations are plotted in Figures #4-7.

[0342] Secondly, gels were formulated with the same methodology, but additionally with gelatin fibers (porcine, purchased from Sigma) or collagen fibers (bovine, from Collagen Solutions) prepared via the method of example 1. The collagen fiber concentration in the pre-gel solution varied from 37 to 70mg / mL, the pre-gel HA concentration was set to 21,23, or 25mg / mL, and the DVS-to-HA subunit ratio was varied from 2.3 to 4.5X. Each group was mixed between two 5mL syringes connected with a luer-lok connector. After vigorous mixing, the mixture was incubated in sealed syringes in a 30°C water bath for 2 hours. The reaction was chemically quenched by lysine solution (molar ratio of 1 to 1 of lysine to DVS molecules), with syringe-syringe mixing followed by a 5-minute incubation at room temperature. The gels are then neutralized in 50mL syringes containing 2 ImL 20mM monobasic sodium phosphate solution. The composite hydrogel was transferred into dialysis tubing (13,000 MWCO) and dialyzed against IL 0.9% sodium chloride for 16 hours and IL DPBS for 3 hours at room temperature. The resulting gel suspension was transferred to 1.5mL microcentrifuge tubesthrough 18-gauge blunt needles then centrifuged at 17,000 x g for 3 minutes to separate excess water. After removing the supernatant, the gels were collected in lOmL syringes and weighed in order to calculate the swelling ratio (relative to weight of initial gelation solution, see example #5). The composite gels were beaded through 150-micron screens, then loaded into ImL glass syringes and autoclaved at 121°C for 30 minutes. The swelling ratio of the composite gels during manufacturing and the resulting final HA concentrations are plotted in Figures #8-13.Example 3Mechanical Properties of Hydrogels and NHCs:

[0343] The rheological characterizations of hydrogels and composite gels were performed as described previously. Rheometry (AR2000ex, TA Instruments) was harnessed to measure various shear mechanical properties of hydrogels and composite gels using a parallel plate geometry of 25mm with 0.9 mm gap at 25°C. A linear viscoelastic region was measured for the gels by a strain sweep with an increasing shear strain amplitude at a set frequency (1Hz). The three main properties were storage modulus (G’), loss modulus (G”) and tan delta (G’7G’). Representative traces are depicted in figures #40-42. The injection forces of bulk gels and microgels of HA and NHC were measured by extruding ImL sample through a 27-gauge needle in 1 mL BD syringe using Instron load frame (34SC-05, Norwood, MA). Representative traces are in figures # 36-39.Example 4Preparation of DVS crosslinked collagen-HA composite groups for Animal Study

[0344] Sodium hyaluronate (HA, MW 1.5MDa) was dissolved in distilled water at a concentration of 30 mg / mL. Sodium hydroxide was dissolved in water to make a 10M solution. Lysine monohydrochloride was dissolved in water at a concentration of 2M.Table 1HA waterGroup fiber(g) NaOH(mL) DVS(mL) lysine(mL) solution(mL) (mL)Ml 5.00 1.00 0.270 0.060 0.159 0.792M2 5.00 1.00 0.270 0.060 0.139 0.692M3 4.98 1.52 0.422 0.065 0.178 0.887M4 4.60 1.40 0.390 0.060 0.146 0.727

[0345] According to Table 1, for each group, the HA stock solution volume specified in above table was blended with the specified amount of water, collagen fibers and sodium hydroxide solution in two lOmL syringes connected with a luer-lok connector for syringesyringe mixing. The specified DVS volume is then added to each group and mixed via-syringe- syringe mixing. . After vigorous mixing, the mixture was incubated in sealed syringes in a 30°C water bath for 2 hours. The reaction was chemically quenched by lysine solution (molar ratio of 1 to 1 of lysine to DVS molecules), with syringe-syringe mixing followed by a 5 minute incubation at room temperature. The gels are then neutralized in 60mL syringes containing 42mL 20mM monobasic sodium phosphate solution. The resulting hydrogel suspension was transferred into dialysis tubing (13,000 MWCO) and dialyzed against IL DPBS 3 times (8 to 16 hours each time, room temperature) then particularized with 150pm stainless steel wire cloth discs in 25mm filter holders. The resulting gel suspension was centrifuged at 4,000 x g for 10 minutes to separate excess water. After removing the supernatant, the gel composites were weighed in order to calculate the swelling ratio (relative to weight of initial gelation solution). The swelling ratio was then verified by directly measuring the hyaluronic acid content and the collagen content ofthe final gel . The gel composite was loaded into ImL glass syringes then autoclaved at 121°c for30 minutes.Example 5Measurement of component concentrations in gel composite

[0346] Carbazole assay for hyaluronic acid content in gel composite

[0347] The concentration of hyaluronic acid in the final gel sample can be calculated through a carbazole-based assay. Briefly, 3.00 mL 0.025M sodium tetraborate in sulfuric acid was added into a 7-mL glass tube containing 0.030-0.040 g gel composite sample and heated in a boiling water bath for 15 min before being cooled down at room temperature for 15 minutes. Then, 0.80 mL 0.125% carbazole in absolute ethanol (w / w) was added into each tube and heated in boiling water bath for 15 min. After being cooled down at room temperature for 15 minutes, 100 pL of 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 gel was calculated from a standards curve (generated via identical testing of a known-concentration hyaluronic acid solution).

[0348] Sirus Red adsorption assay for collagen fiber content in gel composite

[0349] The concentration of collagen in the final gel sample can be calculated through a colorimetric assay. Briefly, 10.00 ml Img / 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 gel composite and vortex at maximum speed for 1 minute. The sample was covered with aluminum foil and shaken on an orbital shaker at lOOrpm for 5 hours at room temperature. 1 mL solution was transferred into a microcentrifuge tube and spin down at 10,000 x g for 3 minutes. 0.05ml of supernatant was diluted into a microcentrifuge tube containing ImL of water. 0.2ml diluted solution was put into a 96-well plate to read at 530 nm. A series of samples of known amounts of collagen (0.2-2mg) were used to make a standard curve that was used to calculate the concentration of the test sample.

[0350] Free (uncrosslinked) collagen in collagen fiber

[0351] 1 .OOmL water was added to 1 5mL microcentrifuge tubes containing 0.020-0.025 g collagen fiber. The tubes were agitated on a vortex mixer at maximum speed for 1 minute, followed by incubation on an orbital shaker (lOOrpm) at room temperature for 4 hours. Fiber was spun down at 17,000 x g for 3 minutes and 0. ImL supernatant was mixed with 0.8mL BCA working solution (Thermo Scientific), then incubated at 37°C for 30 minutes. When the samples were cooled down to room temperature. 0.2mL was transferred to a 96-well plate and the absorbance was read at 562nm on an Epoch microplate spectrophotometer (Bio Tek instruments). Bovine serum albumin (Thermo Scientific) was used as standards. The free collagen content in collagen fiber was determined as 3.1 ± 0.05%.

[0352] Free (uncrosslinked) HA in Matrix-C gel composite

[0353] 1.OOrnL water was added to the centrifuge tubes containing 0.500g Matrix-C samples. The tubes were agitated on a vortex mixer at maximum speed for 1 minute, followed by incubation on an orbital shaker (lOOrpm) at room temperature for 2 hours. Samples were spun down at 17,000 x g for 3 minutes. 0.050mL was added to 0.500mL 0.005% Stains-All in 55% DMSO, followed by mixing on a vortex mixer for 1 minute. O.lOOmL sample was transferred to a 96-well plate and read at 655nm. Sodium Hyaluronate (LifeCore) was used as a reference material. Free HA content in Ml and M2 were ranging from 5.6-5.9%.Example 6In Vivo Animal Study

[0354] Four treatment groups were prepared in Example #4 from the same batch of raw materials and are manufactured to be as similar as possible aside from the experimental variables (pre-gelation concentrations of the HA, Collagen fiber, and crosslinker (DVS) concentration). Collagen fibers were produced from Collagen Solutions Type I bovine collagen as per example #1. The Hyaluronic Acid was purchased from LifeCore Biomedical Corporation. The DVS was purchased from TCI (Tokyo Chemical Industry). At the end of gelation, the residual sulfone groups are chemically quenched with a lysine addition (Sigma).Test Groups

[0355] Four formulations of collagen-fiber-hyaluronic acid composite gels (formulationsdesignated Ml , M2, M3, M4) were injected subcutaneously in a rat model for assessing biocompatibility and efficacy (in maintaining volume). The groups were compared with a commercial control, Voluma XC. The gels are biocompatible and effective in maintaining volume and encouraging cellular ingrowth through 69 weeks.

[0356] The four test groups have varying levels of collagen fiber loading and crosslinking to assess the effects of those variables on the biologic response. Each group used 1 ,6MDa Hyaluronic acid.

[0357] Group Ml : Gel produced with the gelation conditions of 25mg / mL HA, 45mg / mL Collagen, and a [DVS] / [HA Repeat Unit] ratio of 4.0: 1 (264mM of DVS). The resulting gel has final gel concentrations of approximately 9.7mg / mL of HA and 17.5mg / mL of collagen in DPBS with a stiffness (G’) of 628 Pa and an injection force of 6. IN.

[0358] Group M2: Gel produced with the gelation conditions of 25mg / mL HA, 45mg / mL Collagen, and a [DVS] / [HA Repeat Unit] ratio of 3.5: 1 (231mM of DVS). The resulting gel has final gel concentrations of approximately 8.7mg / mL of HA and 15.7mg / mL of collagen in DPBS with a stiffness (G’) of 534 Pa and an injection force of 7.8N.

[0359] Group M3 : Gel produced with the gelation conditions of 23mg / mL HA, 65mg / mL Collagen, and a [DVS] / [HA Repeat Unit] ratio of 4.5: 1 (273 mM of DVS). The resulting gel has final gel concentrations of approximately 10.2mg / mL of HA and 27.5mg / mL of collagen in DPBS with a stiffness (G’) of 610 Pa and an injection force of 4.9N.

[0360] Group M4: Gel produced with the gelation conditions of 23mg / mL HA, 65mg / mL Collagen, and a [DVS] / [HA Repeat Unit] ratio of 4.0: 1 (242mM of DVS). The resulting gel has final gel concentrations of approximately 9.5mg / mL of HA and 25.1mg / mL of collagen in DPBS with a stiffness (G’) of 548 Pa and an injection force of 5.2N.

[0361] Control Group C: Voluma XC, a commercial market-leading soft tissue filler comprised of crosslinked hyaluronic acid.

[0362] Test SystemSpecies: RatBreed: Sprague DawleySource: Single USDA licensed supplierSex: FemaleAge: Young AdultNumber of Animals: 27(4 rats per test group timepoint and 6 timepoints, with 3 control rats)Identification Method: Ear tag or marking.

[0363] Injection procedure

[0364] The rats are anesthetized with isoflurane and their backs are shaved with clippers. The back is then disinfected (Iodine) immediately before injection.

[0365] At each injection site, 200ul injected as a single, round bolus subcutaneously in dorsal region of rat with 27G x 1 / 2" needle. Each injection bolus has a margin of at least 5mm from each other bolus.

[0366] Site Location Map: the injections will be at the back of the rat per the implant scheme below:

[0367] Implant Scheme:Test GroupsCranialCaudalT1 = Test Group 1, T2 = Test Group 2, T3 = Test Group 3, T4 = Test Group 4Control Group:CranialCaudalC = Control group (Voluma XC)

[0368] Sample size

[0369] Test groups: 4 replicates per group per timepoint, each replicate coming from a separate animal. (4 test group rats per timepoint, for a total of 16 test sites combined per timepoint).

[0370] Control: 4 replicates per timepoint, in the same animal.

[0371] Live Assessment Timepoints

[0372] The Animals will be scored for erythema and edema immediately after injection, and 4 days after injection. They will be graded on the scale from ISO 10993-10 table 3.

[0373] MRI Timepoints

[0374] The rats from TBD group 2 will be imaged at every timepoint. Additionally, at each harvest timepoint, the rats-to-be-harvested may be imaged prior to tissue harvesting. The timepoints correspond to 0 days (establishing baseline immediately after injection) 1 day post injection (to assess swelling), and at designated tissue harvest timepoints.

[0375] MRI Timepoints: 0 days, 1 day, 1 week, 4 weeks, 8 weeks, 26 weeks, TBD 1, TBD 2

[0376] Tissue Harvest Timepoints

[0377] Timepoints for this study were 0 day, 1 day, 1 Week, 4 weeks, 8 weeks, 20 weeks, 26 weeks, 40 weeks, and 69 weeks. The last timepoints are assigned based upon the evaluation at the 26-week timepoint. Unless otherwise noted, the Iweek timepoint will be tested exactly 7 days after the procedure and the 4-week timepoint will be tested within 2 days of the planned timepoint. The 8 week and beyond test groups will be tested within 7 days of the planned timepoint. 1 rat of the control group will be harvested at the 26-week timepoint and at each of the TBD timepoints.

[0378] Euthanasia. After any MRI imaging, the designated rats will be humanely euthanized. The dorsal skin is dissected open such that all four injection sites can be visually identified. The test and control sites are excised as full tissue blocks and fixed in formalin overnight, then processed for histological and immunofluorescent assessments.

[0379] Acceptance Criteria

[0380] Swelling: Less than 200% initial volume (which is the POD 0 measurement, expected to be approximately 200pL) and less than commercial control (Voluma XC) at first measurement (1 day post-injection, POD 1).

[0381] Edema: Mean scores less than or equal to the mean scores for Voluma XC® per rubric of Table 3 of ISO- 10993- 10 at POD 4

[0382] Irritation: Erythema mean scores less than or equal to the mean scores for Voluma XC® per Table 3 of ISO 10993-10 (See attachments) at POD 4

[0383] Degradation: Degradation will be considered to be complete when all viewed sites score 4 on the ISO 10993-6 degradation scale (as interpreted by NAMSA and LifeSprout for Lumina, see attachment 2 below). First timepoint with all 4’s (when analyzing at least 3 sites) will be considered time of complete degradation.

[0384] Volumization: The mean implant volume will be measured for each group at each MRI timepoint. Each test group will be considered to have acceptable volumization properties if the mean measured volume is greater than 50% of original (PODO) volume at each timepoint through 26 weeks.

[0385] Cellular Infiltration Rate: Cell infiltration rate is calculated by the percentage area covered by the infiltrated cells inside the injected materials. The acceptance criteria for cellular infiltration rate is at least 50% increase of the average cell infiltration rate compared with control (Voluma XC) at 26 week timepoint.

[0386] Cellular Density: Cell density is counted based on the immunofluorescence images stained with DAPI (staining individual cell nucleus). The acceptance criteria of cellular density is at least 50% higher mean cell density inside the injected implant compared with

[0387] Angiogenesis.: The measurements of angiogenesis will be based on the immunofluorescence images where tissue slides are stained with RECA-1 (endothelial cells) anda-SMA (pericytes). The criteria for endothelial cells will be the distance infiltrating from the periphery to the middle, and that for pericyte will be the densities per unit area (# / mmA2).

[0388] Cell type, adipogenesis, macrophage polarization: Adipogenesis and macrophage polarization measurements will be based on immunofluorescence imaging. Perilipin- 1 is used to stain the adipocytes forming inside the materials, while CD68, CD38 and CD163 are used to stain the macrophage phenotypes inside the materials. For both adipogenesis and macrophage polarization, cell density will be a method to quantitatively compare the test groups with the Voluma control.Table 2. Grading system for intracutaneous (intradermal) reactions

[0389] Results:

[0390] Live assessments:

[0391] All 4 treatment groups were well tolerated by the animals, without inducing infection, necrosis, or gross inflammation in the live animals. The implants remained as persistent bumps protruding from the skin at the same location as originally injected. At POD4 (post-operative day 4), the animals were inspected and scored for erythema and oedema per the grading system from ISO 10993-10. All 4 groups met the acceptance criteria described in the protocol. The erythema score were all mild, and all below the score of Voluma XC. The Oedema scores were also all below that of Voluma. This indicates that the formulations are low irritation and low swelling, making them excellent candidates for dermal applications.

[0392] During explantation, the implantation sites were photographed intact. The injected boluses of material had remained in place, and maintained a bolus shape. The color of all implant sites (all 4 groups, all replicates, at both day 7 and 30 and 63) was white to yellow, remarkably similar to native fat tissue. Blood vessels can be discerned either on or near the boluses. The implanted samples are distinct from the surrounding tissues and are the only discernable feature protruding from the skin (no surrounding fat pads, etc.).Table 3. Mean Erythema and Oedema scoring of prototypes and commercial control at postoperative day 4, scored per rubric in Table 2. All treatment groups met their acceptance criteria, with substantially lower erythema and oedema results than Voluma XC, indicating excellent biocompatibility.Table 3.

[0393] Oedema / Swelling

[0394] Acceptance Criteria: Mean scores less than or equal to the mean scores for Voluma XC® per rubric of Table 3 of ISO- 10993- 10 at POD 4, with scores ranging from 0 for no oedema and 4 for severe oedema. Result: PASS. The 4 formulation groups all had a lower Oedema score than the Voluma XC® control. The values of the Oedema scores are in line with expectations for a gel that maintains volumization and thus maintains a visible bleb at the injection site.

[0395] Explantation:

[0396] During explantation, the implantation sites were photographed intact. The injected boluses of material had remained in place, and maintained a bolus shape. The color of all implant sites (all 4 groups, all replicates, at days 7, 30, and 63) was white to yellow, remarkably similar to native fat tissue. Blood vessels can be discerned either on or near the boluses. The boluses are distinct from pre-existing tissue structures.

[0397] Erythema / Irritation

[0398] Acceptance criterion: Mean erythema mean scores less than or equal to the mean scores for Voluma XC® per Table 3 of ISO 10993-10 at POD 4, with scores ranging from 0 for no erythema and 4 for severe erythema.

[0399] Result: PASS. The 4 formulation groups all had a lower Erythema score than the Voluma XC® control. Groups Ml and M2 exhibited no erythema score at all, while groups M3 and M4 exhibited a very mild score, lower than that of Voluma XC. Table 4A.Table 4A

[0400] MRI volume measurement

[0401] The 4 groups showed little to no post-procedural swelling at days 1 and 7, maintaining their volume as intended.Table 4B shows the quantification of the swelling via MRI at day 1 for the test formulations Ml - M4 of example #6 one day after injection. The swelling % is the percentage of additional volume measured at day 1 over day 0. All 4 formulations had 20% swelling or less, while the Voluma XC® had 100% swelling (doubled in volume).Table 4B.

[0402] The mean swelling percentage for the treatment groups was 12-20%. This is in contrast to Voluma, which doubled in size due to swelling by day 1 (100% swelling), which persisted through day 7 (and then slowly decreased through day 63). This shows a variant of the Matrix-C gel can be made without undesired swelling. The 4 groups and Voluma had broadly similar volumization profdes through day 63, with no-to-slight declines from the day 1 values. The Voluma group had the most absolute volume due to the extensive initial swelling. Normalizing to the day 1 volumes, the groups all maintained 75% or more of the original volume through 63 days. Group Ml had the best volume maintenance, retaining 97% of its day 1 volume at day 63, virtually indistinguishable from Voluma’s 94% volume maintenance.

[0403] Swelling:

[0404] Acceptance Criteria: Less than 200% initial volume (which is the POD 0 measurement, expected to be approximately 200pL) and less than commercial control (Voluma XC) at first measurement (1 day post-injection, POD 1).

[0405] Result: PASS. All 4 formulation groups passed this criterion. The mean relative volumes are in the table below (p-values are 2-sided t-tests relative to Voluma XC). All 4 groups maintained volumization (>100% volume) yet still had less swelling than the commercial control, Voluma XC. These formulation groups also show substantially lower swelling than the hyaluronic acid-collagen fiber gel of disclosure D17311, which increased its volume by approximately 80% by day 7. Table 4C.

[0406] Table 4C.

[0407] At the 7 day timepoint, the 4 formulation groups have returned to baseline volumes near the injection volume, while the Voluma XC® group is still swollen to double its volume. Table 4D.

[0408] Table 4D.00409] Volumization:

[0410] Acceptance criterion: The mean implant volume will be measured for each group at each MRI timepoint. Each test group will be considered to have acceptable volumization properties if the mean measured volume is greater than 50% of original (PODO) volume at each timepoint through 26 weeks.

[0411] Result: PASS. All 4 treatment groups had a mean relative volume of greater than 50% (relative to time 0) at the 26 week timepoint. The results were statistically insignificant relative to the Voluma XC® control, which also passed this criterion. Table 4E.

[0412] When assessing relative volumes once accounting for initial swelling (volumes relative to the 7-day timepoint), the four formulations all had equivalent or superior volume retention compared with Voluma XC, all with higher mean relative volumes than Voluma XC. Table 4F.

[0413] Table 4F.

[0414] Groups Ml, M2, and M3 still maintained over 50% relative volume at the 55week timepoint, with mean relative volumes greater than the Voluma XC® control. Only the M4 formulation had a relative volume below that of Voluma XC® and below 50%. Table 4G.

[0415] Table 4G.

[0416] When assessing relative volumes at 55 weeks once accounting for initial swelling (volumes relative to the 7-day timepoint), groups Ml, M2, and M3 still maintained >50% volume, and were much higher than Voluma XC. The Ml group maintained over twice as much volume as Voluma XC® (72% to 33%). Table 4H.

[0417] Table 4H.

[0418] Histological Assessment:

[0419] Degradation

[0420] Acceptance Criterion: Degradation will be considered to be complete when all viewed sites score 4 on the ISO 10993-6 degradation scale (as interpreted by NAMSA and LifeSprout for Lumina, see attachment 2 below). The First timepoint with all 4’s (when analyzing at least 3 sites) will be considered time of complete degradation.

[0421] Prototypes will be assessed at terminal timepoint. The absorption scores for the 9.5month histology slides are below Table 41:

[0422] Table 41.

[0423] Day 7 histology

[0424] At the day 7 timepoint, the gels can be easily discerned from the surrounding tissue using standard histology practices and stains, such as H&E and Masson’s trichrome stains. In Masson’s trichrome stained slides, the HA gel component is stained as a light blue, and the fibers are stained as cells can be seen migrating into implant site from periphery in all 4 test groups, with cells interdigitating into the implant, not just as a surrounding tissue layer. The cells The Ml and M2 groups had faster cellular ingrowth, with cells visible up to 200 microns into the implant by day 7. The infiltrating cells have a natural, spindled shape. There are numerous blood vessels in the surrounding space, but none can be seen yet within the implanted gels. The Voluma XC® control, however, did not have infiltrating cells into the gel, but rather had flat bands of cells encapsulating the outer layer of the gels.

[0425] The gel was clearly discernable in the subcutaneous space, as a light blue gel with dark blue-to-purple fibers. The gel samples remained as coherent boluses at the injection site, and groups M3 and M4 were most resistant to any spreading, with a high gel thickness at all timepoints. The gel groups integrated well with the surrounding tissue, without necrosis or encapsulation. In groups Ml and M2, bands of cellular ingrowth were apparent at the POD7 timepoint, and were extensive in the POD 30 and 63 timepoints, with a plurality of blood vessels formed within the material together with light collagen deposition. Cellular ingrowth and angiogenesis was also seen in groups M3 and M4, but was less extensive, with large sections still acellular at the day 63 timepoint. At the 63day timepoint, active adipogenesis appears to beoccurring in the Ml group in particular, with vascularized adipose tissue at the periphery of the gel bolus and globular, adipocyte-like structures forming within the neighboring gel. The Voluma group appeared to be biocompatible, without necrosis or inflammation, with a thin collagenous band around the periphery of the gel bolus, but without cellular migration into the gel, nor blood vessel growth, or adipose tissue generation.

[0426] Through day 63, the matrix-C prototypes are excellent candidates for dermal fdler applications, and compare favorably to a commercial leader, Juvederm® Voluma XC®. In particular, Group Ml had a better irritation and swelling profiles than Voluma, equally maintained the injection volume through 63 days, yet additionally induces extensive cellular integration, and angiogenesis without fibrosis or encapsulation. At the day 63 timepoint, the histology slides indicate that active adipogenesis is underway in the Ml gel, which is generating new, healthy-looking fat in replacement of the gel. This indicates that Ml can be injected safely, with no swelling and predictable response, and maintaining the volumization even as it is turned into vascularized, healthy fat.

[0427] 20 & 26 Week Timepoints

[0428] At the 20 week timepoint, the adipose pockets in the panni cuius carnosus layer have grown extensively in the Ml group. A large vascularized adipose tissue greater than 1mm in length and width can be seen within the center of the Ml gel in one example.

[0429] The groups M3 and M4 gels exhibited cellular ingrowth rates slower than that of groups Ml and M2.

[0430] The Voluma XC® group still does not show cellular ingrowth into the gel. There is some small areas of adipose tissue in surrounding tissue, but none within the gel or panniculus carnosus near the gel. The gel has been encapsulated in a thin band of cells and collagen.

[0431] Figure 48 shows a micrograph of Ml group histology slide at 20 weeks. There is extensive cellular ingrowth into the gel, adipose tissue growing around the implanted gel, and substantial areas of adipose tissue growing in the pockets in the panniculus carnosus layer. Scale bar is 2.5mm.

[0432] Figure 49 shows a micrograph of Ml histology slide at 20 weeks (H&E staining). A large (> 1mm x 1mm) section of vascularized adipose tissue has developed within theimplanted gel. Scale bar is 2.5mm (top), 250 microns (bottom)

[0433] Figure 50 shows a micrograph of M2 histology slide at 26 weeks (H&E staining) with extensive cellular ingrowth, adipose tissue formation, and numerous adipocytes present within the gel in addition to the surrounding tissue. Scale bar is 2.5mm (top), 500 microns (bottom)

[0434] Figure 51 shows a micrograph of Voluma XC® histology slide at 20 weeks (H&E staining) Scale bar is 2.5mm (top), 250 microns (bottom). There is still virtually no cellular ingrowth into the gel at 20 weeks. Scale bar is 5mm.

[0435] 40 Week Histology

[0436] At 40 weeks, the same trend continues. Depending upon the slice location within the implant, the gel has been partially replaced by adipose tissue. The groups Ml 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, especially the concentration of collagen fibers and crosslinking agent, DVS.

[0437] Figure 52 shows a micrograph of Ml histology slide at 40 weeks (H&E staining). Top image shows a tissue slice with intact gel bleb, but also extensive adipose tissue surrounding it and in panni cuius carnosus layer. The middle and bottom images are from a different slice of same tissue, with only a small amount of residual gel at this location. The gel is partially replaced with vascularized adipose tissue. Scale bar is 2.5mm, 500 microns

[0438] Figure 53 shows a micrograph of M2 histology slide at 40 weeks (H&E staining). Top image shows a tissue slice with intact gel bleb, but also extensive adipose tissue surrounding it and in panniculus carnosus layer. The bottom image is from a different slice of same tissue, with adipose cells present within and around the gel. Scale bar is 2.5mm, 500 microns.

[0439] Figure 54 (A) shows a micrograph of M3 histology slide at 40 weeks (H&E staining). The interior of the gel has some cellular ingrowth across the full thickness, but is still predominantly acellular at this location. There is some adipose tissue on the left of the gel. Scale bar is 1mm.

[0440] Figure 54 (B) shows a micrograph of M4 histology slide at 40 weeks (H&E staining). The interior of the gel is still predominantly acellular at this location at this timepoint,though there is cells on the periphery of the gel and adipose tissue to the right of the gel. Scale bar is 2.5mm.

[0441] 69 Week Histological Assessment

[0442] The animal study has been terminated with the final timepoint group harvested at the 69-week timepoint (16months) due to the age and size of the animals (too large to be imaged alive in the MRI apparatus). The implant sites were harvested en bloc with the surrounding skin tissues and immediately imaged ex vivo in the MRI machine to get final volumization data, before being prepared for histological assessment. The gels are still visibly present at the implant site and are well tolerated, with no signs of localized irritation or fibrosis. Histological assessments have yet to be completed on these tissues.

[0443] Figure 55 shows a photograph of gels Ml, M2, M3, and M4 during explantation at the 15month timepoint. The gels maintained integrity and shape over this timeframe.

[0444] Differential adipogenesis in or near panniculus carnosus layer in matrix-c prototypes compared to Voluma XC.

[0445] In the histological slides, there is enhanced adipogenesis seen in the panniculus carnosus layer of the rat skin compared to that seen in the Voluma XC® group. This is visible in either enlarged regions of vascularized adipose tissue immediately above or below the muscle layer when near the implanted gel, or in a greatly increased number of adipocytes in the pockets within the panniculus carnosus layer that typically surround nerves. This adipocyte generation in the pockets of the panniculus carnosus neighboring the implanted gel site is also seen in sections cut longitudinally to the panniculus carnosus muscle layer. This shows that the adipocyte structures are not a sectioning artifact. Without being bound by theory, the adipogenesis may be directly the result of gel that flowed into the pocket during injection, or indirectly caused by the biological signals from the cells directly interacting with the nearby gel. Potential precursors for the adipocytes generated include pericytes from the vasculature and mesenchymal stem cells or fibro-adipogenic progenitors from the muscle. This muscle layer is not a major feature of human skin, unlike in the rat model, but this indicates that the Matrix-C test articles may be more capable of inducing targeted adipogenesis than Voluma XC.

[0446] Figure 56 shows a micrograph of Voluma XC® histology slide at 26 weeks(Masson’s trichrome staining). The nerve pocket in the panniculus carnosus layer neighboring the implanted gel lacks adipocytes. Scale bar is 500 microns.

[0447] Figure 57 shows a micrograph of Ml at 26 weeks, with tissue sections both traverse to and longitudinal to the panniculus carnosus muscle fibers (Masson’s trichrome staining).

[0448] Figure 58 shows a micrograph of M2 group at 20 weeks (H&E Staining) All scale bars are 500microns.

[0449] All 4 prototype gel groups were well-tolerated, with minimal-to-no acute adverse response, with an erythema and oedema profile superior to that of Voluma XC. No adverse events were noted for any of the rats with the M1-M4 test groups over the entirety of the long term study. The test gels maintained volume with little-to-no swelling in the initial days after injection (days 1 and 7), compared to Voluma XC, which doubled in volume. From Day 7 onwards, the gel groups maintained volume, with less volume loss than the Voluma XC® control through 385 days, with a flatter slope in both plots of absolute volume per time and relative volume per time. While still maintaining volumization, the gels also showed marked cellular infiltration, with cells growing into the implanted gel with natural dendritic cell morphology by day 7. The cells grew progressively into the gel over the course of the study, replacing the implanted material with vascularized, cellular tissue complete with diffuse collagen. Potential preadipocytes can be seen on the periphery of early timepoints, and extensive pools of vascularized adipose tissue can be seen intermingled with the remaining gel in the Ml and M2 test groups by 9.5months. Adipocytes are only seen on the periphery of the gels in groups M3 and M4, and only in the surrounding tissue for the Voluma XC® control group. This volumization maintenance together with cellular ingrowth demonstrates the optimal balance between gel degradation rate and the replacement by cells and new tissue. The extent of cellular infiltration varied greatly from group to group, demonstrating the effectiveness and importance of the specific formulation choices for each test group.

[0450] The Ml group maintained 85% of its initial volume through more than a year and generated vascularized adipose tissue, making it an excellent candidate for dermal filling applications and body sculpting.

[0451] The M2 group exhibited more gel spreading for more a diffuse effect, as might bewanted for superficial placement in the body. The lower crosslinking and collagen concentrations and resulting larger pore size allows for more rapid cellular movement and diffusion for therapeutic indications for cell or drug delivery or increasing cell homing and interactions within the implanted gel.

[0452] The M3 and M4 groups had much slower cellular infiltration, and maintained the original shape, resisting spreading. These formulation groups are good candidates for use where implant traits like slow degradation, geometric stability are desired. Examples include implantation of the device into high tissue tension regions such as supraperiosteal placement in the body or weight-bearing locations, or for indications where a more permanent plug is desired, such as for fistula repair, hernia repair, coating permanent implants, etc.

[0453] An ideal soft tissue filler would initially immediately fill the defect site with 100% of the target volume, with similar mechanical properties to the surrounding soft tissue, without migration or flow of the filler, and without inducing pain or irritation. The filler should not induce swelling, and then should maintain nearly 100% of its volume indefinitely, while paradoxically degrading away completely without causing scarring or adverse events (such as nodules or granulomas). The test formulations come much closer to achieving this “ideal” profile than any current products such as the Voluma XC® control included in this study.

[0454] As additional evidence, the test gels induce a large increase in adipocytes in the panniculus carnosus, the striated cutaneous muscle layer in many lower mammals such as rats. The panniculus carnosus includes periodic pockets of nerves surrounded by collagen, blood vessels, and occasionally a small number of adipocytes. The pockets in the panniculus carnosus layer of the Voluma XC® control are Without being bound by theory, the adipogenesis may be directly the result of gel that flowed into the pocket during injection (gel can be seen in some pockets at early timepoints), or indirectly caused by the biological signals from the nearby gel, either from cells directly interacting with the nearby gel or gel degradation products.

Claims

CLAIMSWhat is claimed is:

1. A method for manufacturing a nanofiber-hydrogel composite comprising a step of reacting a hyaluronic acid, a collagen fiber, and divinyl sulfone; whereby the hyaluronic acid is covalently crosslinked to the collagen fiber by the divinyl sulfone; wherein the hyaluronic acid is present in an amount ranging from about 20 mg / mL to about 100 mg / mL, and wherein the collagen is present in an amount ranging from about 30 mg / mL to about 200 mg / mL; and wherein the divinyl sulfone is present in an amount ranging from about 0.5: 1 to about 5: 1 divinyl sulfone molecules per hyaluronic acid subunit.

2. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 0.1 to about 3.0 during manufacture.

3. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 1.0 to about 3.0 during manufacture.

4. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 1.5 to about 4.0 during manufacture.

5. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 2.0 to about 3.0 during manufacture.

6. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 2.2 to about 3.0 during manufacture.

7. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 2.3 to about 2.8 during manufacture.

8. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 1.0 to about 2.0 during manufacture.

9. The method of claim 1, wherein the composite comprises a swelling ratio ranging from about 0.1 to about 1.0 during manufacture.

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 comprises a swelling % of at most 100% in vivo.

15. The method of claim 1, wherein the composite comprises a swelling % of at most 75% in vivo.

16. The method of claim 1, wherein the composite comprises a swelling % of at most 50% in vivo.

17. The method of claim 1, wherein the composite comprises a swelling % of at most 40% in vivo.

18. The method of claim 1, wherein the composite comprises a swelling % of at most 30% in vivo.

19. The method of claim 1, wherein the composite comprises a swelling % of at most 20% in vivo.

20. The method of claim 1, wherein the composite comprises a swelling % of at most 15% in vivo.

21. The method of claim 1, wherein the composite comprises a swelling % of at most 12% in vivo.

22. The method of claim 1, wherein the composite comprises a swelling % of at most 10% in vivo.

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

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

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

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

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

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

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

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

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

32. The method of claim 1, wherein the composite comprises a swelling % of about 10%, 11%, 12%, 13%, 14%, or15% in vivo.

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

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

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

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

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

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

40. 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. The method of claim 1, wherein the composite comprises collagen present in an amount ranging from about15 mg / mL to about 30 mg / mL.

42. The method of claim 1, wherein the composite comprises collagen present in an amount ranging from about20 mg / mL to about 30 mg / mL.

43. The method of claim 1 wherein the composite comprises collagen present in an amount ranging from about25 mg / mL to about 30 mg / mL.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

64. 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. 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. 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. The method of claim 1, wherein the hyaluronic acid is present in an amount of about 25 mg / mL; wherein the collagen is present in an amount ranging from about 45 mg / mL; and wherein the di vinyl sulfone is present in an amount of about 4: 1 divinyl sulfone molecules per hyaluronic acid subunit.

68. The method of claim 1, wherein the hyaluronic acid is present in an amount of about 25 mg / mL; wherein the collagen is present in an amount ranging from about 45 mg / mL; and wherein the di vinyl sulfone is present in an amount of about 3.5: 1 divinyl sulfone molecules per hyaluronic acid subunit.

69. The method of claim 1, wherein the hyaluronic acid is present in an amount of about 23 mg / mL; wherein the collagen is present in an amount ranging from about 65 mg / mL; and wherein the di vinyl sulfone is present in an amount of about 4.5: 1 di vinyl sulfone molecules per hyaluronic acid subunit.

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

71. 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 inside the nanofiber- hydrogel composite.

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

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

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

79. The method of claim 1, wherein the nanofiber-hydrogel composite enables cellular infiltration.

80. 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 mats, injectables and flowables.

82. The method of claim 1, wherein the nanofiber-hydrogel composite accommodates pass through of a 27-gauge or smaller needle.

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

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

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 induces elevated host cell infiltration.

87. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized in displaying a biostimulatory effect selected from tissue remodeling, host cell infiltration, cell adhesion, cell migration, angiogenic responses, adipogenic responses, and macrophage polarization to pro-healing phenotypes.

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

89. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as exhibiting tissue remodeling effect induced by the nanofiber-hydrogel composite without incorporation of cells or growth factors.

90. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as displaying a biostimulatory effect due to the low crosslinking density of the composite materials.

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

92. The method of claim 1, wherein the composite is characterized as having increased thermal stability and / or results in shelf stability at ambient temperatures.

93. The nanofiber-hydrogel composite of any one of embodiments i to iv, or the nanofiberhydrogel composite product by the process of embodiment v, wherein the nanofiber-hydrogel composite is characterized as displaying biocompatibility.

94. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as displaying cell adhesion.

95. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as displaying cell migration.

96. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as enabling durable soft tissue remodeling.

97. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as promoting angiogenesis.

98. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as prolonging a biostimulatory effect by retaining and / or conditioning infdtrating macrophages towards a pro-regenerative M2 phenotype, thereby facilitating neo-vasculature formation.

99. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as improving adipose tissue formation.

100. The method of claim 1, wherein the nanofiber-hydrogel composite is characterized as displaying 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. The method of claim 1, wherein the storage modulus is within the range of most soft tissues.

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

104. The method of claim 1, wherein the nanofiber-hydrogel composite is particularized to a set of microgels with diameters selected from the group consisting of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 pm.

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

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

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

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

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

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

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

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

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

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

115. The method of claim 15, wherein the hydrogel comprises residual sulfone groups at a concentration of at most about 2.0 pg / mL hydrogel.

116. The composition of claim 15, wherein the hydrogel comprises residual sulfone groups at a concentration of at most about 1.7 pg / mL hydrogel.

117. The composition of claim 15, wherein the hydrogel comprises residual sulfone groups at a concentration of at most about 1.4 pg / mL hydrogel.

118. The composition of claim 15, wherein the hydrogel comprises residual sulfone groups at a concentration of at most about 1.0 pg / mL hydrogel.

119. The composition of claim 15, wherein the hydrogel comprises residual sulfone groups at a concentration of at most about 0.5 pg / mL hydrogel.

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

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