Bioengineered scaffolds and methods of making and using the same

Abstract

The present disclosure provides tissue scaffolds and hydrogels as well as methods of making and using the same. Also provided are tissue scaffolds with improved biological properties and methods of utilizing said tissue scaffolds for the treatment of wounds.

Description

Attorney Docket No. FES-003WOBIOENGINEERED SCAFFOLDS AND METHODS OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 735,491, filed December 18. 2024. the content of which is herein incorporated by reference in its entirety'.BACKGROUND

[0002] The care of acute and chronic wounds has a significant social, medical, and economic impact on the global economy. This burden is exacerbated by the adverse effects of infections, prolonged recovery, and escalated treatment costs of associated complications. Wounds can be treated with a variety of approaches, which include surgery, negative pressure wound therapy, wound dressings, and hyperbaric oxygen therapy. However, each of these strategies has an array of limitations. The existing dry' wound dressings lack functionality' in promoting wound healing and exacerbating pain by adhering to the wound, while wet wound dressings, such as hydrogels, have poor mechanical strength and are susceptible to degradation.

[0003] Thus, there is a need to improve upon current hydrogel products to enhance their structural and biological properties.FIELD

[0004] The present disclosure relates to hydrogels and methods of utilizing the same. The disclosure also relates to methods of engineering hydrogels with improved biological properties.SUMMARY

[0005] In one aspect, the disclosure provides a method of bonding a hydrogel to a substrate, the method comprising: contacting at least a portion of Ute hydrogel with a substrate; and irradiating the portion of the hydrogel and the substrate in an amount effective to bond the portion of the hydrogel to the substrate, wherein the hydrogel comprises collagen. In embodiments, the method comprises irradiating with a wavelength of about I pm to about 10 nm. In embodiments, the irradiating comprises a kinetic energy of about 10 keV to about 250 MeV. In embodiments, the irradiating comprises X-ray, gamma irradiation, or electron -beam.Attorney Docket No. FES-003WOIn embodiments, the irradiating comprises electron-beam. In embodiments, the electron-beam produces a radiation of about 5 kGy to about 100 kGy. In embodiments, the electron-beam produces a radiation of about 30 kGy. In embodiments, the electron-beam operates under an inert atmosphere. In embodiments, the inert atmosphere is argon, nitrogen, or vacuum. In embodiments, the bonding is determined by a peelability assay. In embodiments, peelability is reduced by at least about 1 -point, 2-points, or 3-points, as determined by peelability assay. In embodiment, the irradiating comprises a kinetic energy of about 1 MeV, 5 MeV. 10 MeV. 15 MeV. 20 MeV, 25 MeV, 30 MeV, 35 MeV, 40 MeV, 45 MeV or up to about 50 MeV In embodiments, the irradiating comprises akinetic energy of about 10 MeV.

[0006] In embodiments, the substrate is selected from any one of silicone, polyesters, polyamides, thermoplastic polyurethane (TPU), polyethylene terephthalate (PET) and combinations thereof. In embodiments, the substrate comprises silicone. In embodiments, the substrate comprises polyester. In embodiments, the substrate comprises polyamide. In embodiments, the substrate comprises TPU. In embodiments, the substrate comprises PET. In embodiments, the PET has a density of at least about 1.33 g / cm3. In embodiments, die silicone substrate has a density of at least about 1.05 g / cm3In embodiments, the TPU substrate has a density of at least about 1.1 g / cnr’. In embodiments, the substrate comprises a film comprising a polymer. In embodiments, the substrate comprises a water content of about 90% w / v to about 99,9% w / v. In embodiments, the substrate further comprises one or more polymers. In embodiments, the polymer is a low weight percent polymer. In embodiments, the polymer comprises hydroxyl or carboxylic acid groups. In embodiments, the irradiating further sterilizes the portion of the hydrogel and the substrate as determined by ISO 11137 In embodiments, the collagen is type I collagen. In embodiments, the collagen is atelocollagen or telocollagen.

[0007] In another aspect, the disclosure provides a tissue scaffold material comprising a bonded hydrogel and substrate made utilizing the method described herein In embodiments, the scaffold further comprises cells In embodiments, the scaffold further comprises an antimicrobial agent.

[0008] In another aspect, the disclosure provides a tissue scaffold material that comprises: a) a microsphere comprising a polymer; and b) one or more of a cell and an anti-microbial In embodiments, the tissue scaffold material further comprises a hydrogel comprising a polymer. In embodiments, the microsphere is embedded in the hydrogel. In embodiments, the microspheres have a different density than the hydrogel. In embodiments, the tissue scaffold material comprises a cell. In embodiments, the tissue scaffold material comprises an anti¬ microbial. In embodiments, the tissue scaffold material comprises an anti-microbial and a cell.9Attorney Docket No. FES-003WOIn embodiments, the tissue scaffold material comprises cells, and wherein the cells are human cells. In embodiments, the tissue scaffold material comprises cells, and wherein the cells are non-human. In embodiments, the human cells are regenerative cells. In embodiments, the human cells are non-regenerative cells In embodiments, the regenerative cells are embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow- derived stem or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSC). skeletal muscle-derived cells, multipotent adult progenitor cells (MAPC). skin tissue derived cells, bone tissue derived cells, cardiac stem cells (CSC), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, or aortic endothelial cells, bone marrow-derived stem cells, endothelial or vascular stem or progenitor cells, endothelial progenitor cells (EPC), and combinations thereof. In embodiments, the non-regenerative cells comprise cardiomyocytes, neurons, lens cells, or combinations thereof. In embodiments, the tissue scaffold material comprises cells, and wherein the cells are derived from one or more of a tissue and organ. In embodiments, the cells are derived from skin tissue.

[0009] In embodiments, the tissue scaffold material comprises at least 1,000. 10.000, 100,000, 1,000,000, 10,000,000, or 100,000,000 cells. In embodiments, the tissue scaffold material comprises from about 1,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 10,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 1,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 100,000 cells / mg tissue, about 1,000 cells / mg tissue to about 10,000 cells / mg tissue, about 10,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 100,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 1,000,000 cells / mg tissue to about 100,000,000 cells / mg tissue, or about 10,000.000 cells / mg tissue to about 100.000,000 cells / mg tissue, including any ranges therebetween. In embodiments, the human ceils are autologous to a subject in need. In embodiments, the human cells are allogeneic to a subject m need. In embodiments, the non- human cells are pig, murine, bovine, sheep, or canine.

[0010] In embodiments, the polymer is selected from the group consisting of: collagen, atelocollagen, telocollagen, gelatin, elastin, hyaluronate, cellulose, fibrinogen, poly(lactic-co-gly colic acid) (PLGA), polyfglycolic acid) (PGA), poly(lactic acid) (PLA), poly(caprolactone), poly(butylene succinate), poly(trimethylene carbonate), poly(p-dioxanone), and poly(butylene terephthalate); a polyester amide, a polyurethane, poly [(carboxy phenoxy) propane-sebacic acid]. poly[bis(hydroxyethyl) terephthalate-ethyl orthophosphorylate / terephthaloyl chloride], a po]y(ortho ester), a poly(alky) cyanoacrylate), polylethylene glycol), a microbial polyester,Attorney Docket No. FES-003WOpoly(P-hydroxy alkanoate), and a tyrosine derived polycarbonate. In embodiments, the polymer is collagen. In embodiments, the polymer is atelocollagen. In embodiments, the polymer is telocollagen. In embodiments, the microspheres are comprised of 1 % w / v, 1 % w / w, or 1 % v / v of the polymer. In embodiments, the tissue scaffold material comprises an anti -microbial, and wherein the anti-microbial is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, antiseptics, broad-spectrum antimicrobials, broad-spectrum biocidal materials and any combination thereof. In embodiments, the tissue scaffold material comprises an anti -microbial, and wherein the antimicrobial prevents or treats an infection selected from the group consisting of bacteria, fungi, viruses, parasites, prions and any combination thereof. In embodiments, the tissue scaffold material comprises an anti-microbial, and wherein the anti -microbial is a metal selected from any one of silver, copper, zinc, gold, titanium, nickel, tin, platinum, or a combination thereof. In embodiments, the metal is silver, copper, or a combination thereof. In embodiments, the tissue scaffold material comprises an anti-microbial, and wherein the anti-microbial acts through a mechanism comprising inhibition of cell wall synthesis, disruption of cell membrane function, inhibition of protein synthesis, inhibition of nucleic acid synthesis, inhibition of metabolic pathways, or any combination thereof. In embodiments, the tissue scaffold material comprises an anti-microbial, and wherein the tissue scaffold material has at least about 2-fold higher microbicidal activity than a reference scaffold without the anti-microbial that is otherwise identical.

[0011] In another aspect, the disclosure provides a method of wound healing comprising administering the tissue scaffold material described herein to a subject in need thereof.BRIEF DESCRIPTION OF FIGURES[00121 FIGs. 1A-1C show representative images seven days post-implantation, wherein cells infiltrate microsphere scaffolds (MSS) (FIG. 1C) but do not infiltrate 1% bulk alone (FIG.1A) and poorly infiltrate 0.3% bulk (FIG. IB).

[0013] FIGs. 2A-2C show representative images fourteen days post-implantation, wherein cells show excellent infiltration of MSS scaffolds (FIG. 2C) but do not infiltrate beyond outer portion of 1 % bulk (FIG. 2A) and show only modest infiltration of 0.3% bulk (FIG. 2B).

[0014] FIGs. 3A-3D show representative images at seven days post-implantation, wherein cells show more complete infiltration of MSS scaffolds with 1% microspheres in 0.3% bulk (FIG. 3C). and 0.6% microspheres in 0.3% bulk (FIG. 3D), with less infiltration of 0.4% microspheres in 0.6% bulk (FIG. 3A) and 0.4% microspheres in 0,2% bulk (FIG. 3B).Altomev Docket No. FES-003WO

[0015] FIGs. 4A-4B show representative images at seven (FIG. 4A) and fourteen days post¬ implantation (FIG. 4B), cellular infiltration of 1% microspheres in 0.3% bulk (blue staining, 4',6-diamidino-2-phenylindole (DAPI)) includes endothelial precursor CD31 + cells (red staining)

[0016] FIGs. 5A-5D show representative images at seven days post-implantation. FIGs. 5A-5C show MSS, 0.3% bulk, 1% bulk and INTEGRA scaffolds in mouse. FIG. 5D shows the relative sizes of scaffolds after implantation.

[0017] FIGs. 6A-6C show representative images seven days post-implantation, where cells infiltrate the MSS scaffold all the way to the center of the scaffold (FIG. 6A) but do not infiltrate 1% bulk except where scaffold is split (FIG. 6B) and poorly infiltrate 3% bulk (FIG.6C).

[0018] FIG. 7 shows a representative image seven days post-implantation. DAPI nuclear staining (blue) demonstrating cell invasion to the center of the MSS and CD31+ endothelial precursors (red).

[0019] FIGs. 8A-8E show representative images fourteen days post-implantation. FIGs. 8A- 8D show the MSS. 0.3% bulk, 1% bulk and INTEGRA scaffolds in mouse. FIG. 8E shows relative sizes of scaffolds after implantation.

[0020] FIGs. 9A-9D show representative images fourteen days post-implantation. FIG. 9A show's significant cellular invasion in MSS scaffold. FIG. 9B shows 1% collagen with minimal invasion (except along fissures). FIG. 9C show's 0.3% collagen scaffold with sparse invasion. FIG. 9D shows INTEGRA at 14 days also with less robust appearing invasion.

[0021] FIG. 10 shows the cell count per unit scaffold area that indicates significantly more cells invaded the MSS scaffold at 7 and 14 days (approximately 7 cells and 10 cells per area, respectively) relative to 1% hydrogel (approximately 3 and 5 cells per unit area) and 0.3% hydrogel (approximately 3 and 7 cells per unit area).

[0022] FIGs. 11A-11D show representative images twenty eight days post-implantation.FIGs. 11A-11C shows MSS, 0.3% bulk, 1% bulk and INTEGRA scaffolds in mouse, FIG.11D shows relative sizes of scaffolds after implantation. Note, 0.3% hydrogel is significantly shrunken.

[0023] FIGs. 12A-12D show' representative images twenty eight days post-implantation. FIG.12A show's excellent cellular invasion in MSS scaffold. FIG. 12B show’s 1% collagen maintains minimal invasion (except along fissures). FIG. 12C shows 0.3% collagen scaffold shows uniform moderate invasion. FIG. 12D shows INTEGRA also demonstrated reasonable invasion.Attorney Docket No. FES-003WO

[0024] FIG. 13 shows a representative image of scanning electron microscopy of the microspheres.

[0025] FIGs. 14A-14F show representative images of the irradiated microsphere containing hydrogels post-peeling for each of the three substrates evaluated, including polyethylene terephthalate (PET) (FIG. 14A), thermoplastic polyurethane (TPU) (FIG. 14C), and silicone (FIG. 14E) in comparison to non-irradiated controls, including PET (FIG. 14B), thermoplastic polyurethane (TPU) (FIG. 14D), and silicone (FIG. 14F).

[0026] FIG. 15 shows a comparison of the relative score of peelability for each substrate tested among the irradiated versus non-irradiated tissue scaffold samples.

[0027] FIGs. 16A-16D show the antimicrobial activity7of MSS loaded with copper ion or silver ion. FIG. 16A represents MSS loaded with silver ion against Staphylococcus aureus. FIG. 16B represents MSS loaded with silver ion against Klebsiella pneumoniae. FIG. 16C represents MSS loaded with copper ion against Staphylococcus aureus. FIG. 16D represents MSS loaded with copper ion against Klebsiella pneumoniae.DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] Disclosed herein are compositions comprising tissue scaffold materials containing hydrogels, microbeads, or a combination thereof, for wound healing and tissue regeneration. Also provided are tissue scaffolds comprising hydrogels and / or microbeads. Provided are also hydrogels and / or microbeads comprising cells and / or antimicrobial agents. The disclosed scaffolds, hydrogels, and compositions comprising the same can be employed m a variety of indications. In aspects, the tissue scaffold materials are utilized to promote wound healing. Additionally provided herein are methods for attaching tissue scaffold materials (e.g., hydrogels and / or microbeads) to substrates.[0029| In patients requiring surgical treatment of traumatic injury, successful and definitive closure of the wound is often jeopardized by microbial contamination or even colonization. The risk of infection is increased due to potential exposure of critical structures like tendons, ligaments, and bone, these structures can quickly deteriorate without adequate coverage. Traditional treatments and wound care devices may be limited by the need for specific handling or storage conditions, which can be challenging to maintain in dynamic or resource-constrained environments.

[0030] Without wishing to be bound by theory, the compositions disclosed herein (e.g., antimicrobial MSS) could be an antimicrobial dermal template tailored for wound healing in a potentially contaminated environment. In some embodiments, the compositions could preventAttorney Docket No. FES-003WOMSS bacterial colonization and / or achieve wound healing in a potentially contaminated environment.Definitions

[0031] The terms '‘tissue scaffold"’, “tissue scaffold material’", “dermal substitute”, “dermal substitute material” and “material” are used interchangeably herein to refer to a cell growth support structure made of biocompatible polymer. These materials are capable of regenerating damaged tissues by providing a biocompatible template that promotes cellular invasion and tissue regeneration. In embodiments, the tissue scaffold materials comprise hydrogels, microbeads, or a combination thereof. In embodiments, the hydrogels and / or microgels comprise collagen,

[0032] As used herein, the terms "about" and “approximately” are used interchangeably and refer to plus or minus 10% of the referenced number, unless otherwise stated or otherwise evident by the context, and except where such a range would exceed 100% of a possible value, or fall below 0% of a possible value, such as less than 0% content of an ingredient, or more than 100% of the total contents of a composition. For example, reference to about 10% microparticle by weight of the tissue scaffold material means that the microparticle can be present in any amount ranging from 9% to 11 % by weight of the tissue scaffold material. Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used m the specification and claims are contemplated to be able to be modified in all instances by the term "about".

[0033] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an animal, including mammals such as non-primates (e.g, cows, pigs, horses, cats, dogs, rats etc.) and pnmates (e.g., monkey and human).Tissue Scaffold Materials

[0034] Provided are tissue scaffold materials (e.g., hydrogels and / or microbeads) and various compositions comprising tissue scaffold materials (e.g., hydrogels and / or microbeads). In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise a three- dimensional structure or framework comprising biomaterials that can support cell adherence and proliferation and formation of functional tissue. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) may resemble an extracellular matrix. Thus, the tissue scaffold materials (e.g., hydrogels and / or microbeads) provided herein may serve as a template for tissue repair and regeneration.Attorney Docket No. FES-003WO

[0035] Tissue scaffold materials (e.g., hydrogels and / or microbeads) of the disclosure can be formed with one or more materials. In embodiments, a tissue scaffold material comprises one or more of microspheres and / or hydrogels. The disclosed tissue scaffold are configured to facilitate cellular invasion and / or vascularization to optimize wound healing and tissue regeneration. Specifically, disclosed configurations utilize different densities to promote cellular invasion of any of the cells of the disclosure.

[0036] In embodiments, a tissue scaffold material (e.g, hydrogels and / or microbeads) comprises a hydrogel support. In embodiments, a hydrogel support comprises microspheres. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) are configured to have varying densities between materials forming a scaffold. For example, microspheres can have a greater density7than a hydrogel or vice versa. In embodiments, polymers are utilized to achieve varying densities. In embodiments, microspheres and hydrogels comprise the same polymer. In embodiments, microspheres and hydrogels comprise different polymers. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise additional materials. In embodiments, the additional materials comprise one or more bioactive factors.

[0037] In embodiments, bioactive factors are incorporated into tissue scaffold materials (e.g., into hydrogels and / or microbeads) to help to elicit biological responses from cells and tissues. In embodiments, the factors are naturally occurring biomolecules or synthetic compounds. In embodiments, bioactive factors comprise growth factors, extracellular matrix components, cell-adhesive peptides, anti-inflammatory agents, antimicrobial agents, oxygen carriers, mineralization factors, antioxidants, anti-fibrotic agents, hemostatic agents, neurotrophic factors, and angiogenic factors. In embodiments, growth factors comprise transforming growth factor-beta, platelet-derived growth factor, and fibroblast growth factor. In embodiments, extracellular membrane components comprise collagen, fibronectin and laminin. In embodiments, proteins comprise bone morphogenetic proteins, osteopontin, and decorin. In embodiments, bioactive factors may comprise hyaluronic acid and glycosaminoglycans. Tn embodiments, antimicrobial agents compnse antibiotics, antifungals, antiparasitics, antivirals, broad-spectrum antimicrobials, broad-spectrum biocidals, and antiseptics. In embodiments, antimicrobial agents comprise broad-spectrum antimicrobials or biocidals In embodiments, the broad-spectrum antimicrobials or biocidals comprise metal ions. In embodiments, the broad-spectrum antimicrobials or biocidals comprise silver or copper ions. In embodiments, the broad-spectrum antimicrobials or biocidals comprise halogens, alcohols, quaternary ammonium compounds, hydrogen peroxide, aldehydes, phenolic compounds, biguanides,Attorney Docket No. FES-003WOozone, peracetic acid, metallic ions, or essential oils. In embodiments, the broad-spectrum antimicrobials or biocidals comprise iodine, chlorine, bromine, ethanol, isopropanol, benzalkonium chloride, alkyl dimethyl benzyl ammonium chloride, dodecylbenzenesulfonic acid (DBSA). formaldehyde, glutaraldehyde, phenol, cresols, hexachlorophene, chlorhexidine, silver, copper, tea tree oil, and thyme oil.

[0038] In embodiments, anti-inflammatory agents comprise corticosteroids and non-steroidal anti-inflammatory drugs. In embodiments, bioactive factors may comprise vascular endothelial growth factor, nerve growth factor and brain-derived neurotrophic factor. In embodiments, bioactive factors may also comprise minerals, ions and exosome and growth-factor loaded nanoparticles.

[0039] In embodiments, provided herein are tissue scaffold materials comprising a microsphere and / or a hydrogel. In embodiments, the microsphere and / or hydrogel comprise a polymer (e.g., collagen). In embodiments, the microsphere and / or hydrogel comprise a polymer (e.g., collagen) and one or more of an antimicrobial and a cell. In embodiments, the microspheres have a different density than the hydrogel. In embodiments, the density of the microsphere is greater than the density of the hydrogel. In embodiments, the density of the microsphere is less than the density of the hydrogel

[0040] In embodiments, the tissue scaffold materials (e.g., into hydrogels and / or microbeads) comprise at least one antimicrobial. In embodiments, the tissue scaffold materials (e.g,, into hydrogels and / or microbeads) comprise at least 1. at least 2, at least 3. or at least 4 antimicrobials. In embodiments, the tissue scaffold materials (e.g., into hydrogels and / or microbeads) comprises an antimicrobial, and the antimicrobial prevents or treats an infection selected from the group consisting of bacteria, fungi, viruses, parasites, prions and any combination thereof.[00411 In embodiments, the tissue scaffold materials (e.g., into hydrogels and / or microbeads) comprise an antimicrobial, and the antimicrobial is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, antiseptics, and any combination thereof. In embodiments, the antibacterial agents comprise antibiotics. In embodiments, the antibiotics comprise penicillins, cephalosporins, carbapenems, monobactams, beta-lactams, glycopeptides, aminoglycosides, macrolides, tetracyclines, chloramphenicol, hncosamides, oxazolidinones, fluoroquinolones, rifamycins, nitrofurans, sulfonamides, trimethoprim, polymyxins, daptomycin, or a combination thereof. In embodiments, the antibiotics comprise penicillin G, penicillin V, amoxicillin, ampicillin, cioxacillin, piperacillin, cephalexin, cefazohn, cefuroxime, cefoxitin, ceftriaxone, ceftazidime,Attorney Docket No. FES-003WOcefepime, ceftaroline, meropenem, imipenem, ertapenem, donpenem, aztreonam, vancomycin, teicoplanin, gentamicin, amikacin, erythromycin, azithromycin, clarithromycin, gentamicin, amikacin, tobramycin, streptomycin, doxycycline, minocycline, clindamycin, linezolid, tedizolid, ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, rifampin, rifabutin, nitrofurantoin, sulfamethoxazole, trimethoprim, teicoplanin, polymyxin B, colistin, linezolid, tedizolid. nitrofurantoin, chloramphenicol, fusidic acid, rifampin, daptomycin, fosfomycin, quinupristin. dalfopristin, mupirocin, bacitracin, neomycin or any combination thereof. In embodiments, the antibacterial agent comprises broad-spectrum antibiotics. In embodiments, the broad-spectrum antibiotics comprise amoxicillin, ceftriaxone, azithromycin, tetracycline, doxycycline, levofloxacin and meropenem. In embodiments, the tissue scaffold materials (e.g., into hydrogels and / or microbeads) comprise one or more, two or more, three or more, or four or more antibiotics. In embodiments, the antibiotic comprises an antibiotic that is slowly released over time.

[0042] In embodiments, the antimicrobial comprises an antiseptic. In embodiments, the antiseptic comprises alcohol-based antiseptics, halogen-based antiseptics, hydrogen peroxide, phenolic compounds, biguanides, quarternary ammonium compounds, silver-based antiseptics, oxidizing agents, acids, ointments, essential oils, sulfur-based antiseptics, or any combination thereof. In embodiments, the antiseptic comprises ethyl alcohol, isopropyl alcohol, iodine, iodophors, chlorhexidine, phenol, chloroxylenol, benzalkonium chloride, cetylpyridinium chloride, silver sulfadiazine, colloidal silver, potassium permanganate, chlorine dioxide, acetic acid, citric acid, tea tree oil. lavender oil, thyme oil, eucalyptus oil, povidone-iodine or any combination thereof.

[0043] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise an anti-microbial, wherein the antimicrobial is a metal selected from any one of silver, copper, zmc, gold, iron, titanium, manganese, bismuth, mercury, vanadium, nickel, tin, platinum, or a combination thereof In embodiments, the metal is silver, copper, or a combination thereof. In embodiments, the metals comprise nanoparticles, salts, ions, complexes, chelates, or any combination thereof. In embodiments, the silver comprises silver nanoparticles, silver salts, silver complexes or chelates, or silver-containing polymers. In embodiments, the copper comprises copper nanoparticles, copper salts, copper complexes or chelates, or copper-containing polymers.

[0044] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise an antimicrobial, and wherein the antimicrobial acts through a mechanism comprising inhibition of cell wall synthesis, disruption of cell membrane function, inhibitionAttorney Docket No. FES-003WOof protein synthesis, inhibition of nucleic acid synthesis, inhibition of metabolic pathways, or any combination thereof.

[0045] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) are coated with a solution comprising the antimicrobial.

[0046] In embodiments, the antimicrobial is present in the solution at a concentration ranging from about 0.001 mM to about 500 mM. In some embodiment, the antimicrobial is present in the solution at a concentration of about 0.001 mM, about 0.01 mM, about 0.1 mM, about 1 mM, about 5 mM, about 1 mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM (including all values and ranges therein). In embodiments, the antimicrobial is present in the solution at a concentration ranging from 50 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 0.001 mM to about 150 mM, from about 50 mM to about 150 mM, from about 100 mM to about 150 mM, from about 0.001 mM to about 100 mM, from about 50 mM to about 100 mM, or from about 0.001 mM to about 50 mM (including any ranges or values therein).

[0047] In embodiments, the antimicrobial is present at a concentration comprising about 0.1% w / v to about 2% w / v (e g., 0.1, 0.2, 0 3, 0.4, 05, 0.6, 0.7, 0 8, 0.9, 1, 1.1, 1 2, 1.3, 1.4, 1 5, 1.6, 1.7, 1.8, or 1.9% w / v, including all values and ranges therein). In embodiments, the antimicrobial is present a concentration comprising about 0,5% w / v to about 2% w / v, about 1% to about 2% w / v, about 1.5% to about 2% w / v, about 0.1% w / v to about 1.5% w / v, about 0.5% w / v to about 1.5% w / v, about 1% w / v to about 1.5% w / v. about 0.1% w / v to about 1% w / v, about 0.5% w / v to about 1% w / v, or about 0.1% w / v to about 0.5% w / v, including any ranges or values therein.

[0048] In embodiments, the tissue scaffold matenals (e.g.. hydrogels and / or microbeads) comprise at least one cell type. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise one or more, two or more, three or more or four or more cell types. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise ceils selected from the group consisting of human cells, insect cells, bacterial cells, yeast cells, plant cells, and animal cells. In embodiments, the cells are human. In embodiments, the cells are non-human. In embodiments, the human cells are regenerative cells. In embodiments, the human cells are non-regenerative cells. In embodiments, the regenerative cells comprise embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSC), skeletal muscle-derived cells, or multipotentAttorney Docket No. FES-003WOadult progenitor cells (MAPC), skin tissue derived cells, bone tissue derived cells, cardiac stem cells (CSC), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, or aortic endothelial cells, bone marrow-derived stem cells, endothelial or vascular stem or progenitor cells, endothelial progenitor cells (EPC), and combinations thereof. In embodiments the non-regenerative cells comprise cardiomyocytes, neurons, lens cells, or combinations thereof. In embodiments the human cells are autologous to a subject in need. In embodiments, the human cells are allogenic to a subject in need. In embodiments, the non-human cells are pig, murine, bovine, sheep, or canine.

[0049] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise cells, wherein the cells are derived from one or more of a tissue and organ. In embodiments, the cells are derived from epithelial, connective, muscle, or nervous tissue In embodiments, the cells are derived from skin tissue, an amnion tissue, an artery' tissue, a cartilage tissue, a connective tissue, a chorion tissue, a colon tissue, a non-calcified dental tissue, a dermal tissue, a duodenal tissue, an endothelial tissue, an epithelial tissue, a fascial tissue, a gastrointestinal tissue, a gingival tissue, a growth plate tissue, an intervertebral disc tissue, an intestinal mucosal tissue, an intestinal serosal tissue, a ligament tissue, a liver tissue, a lung tissue, a mammary tissue, a membranous tissue, a meniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, a parenchymal organ tissue, a pericardial tissue, a periosteal tissue, a peritoneal tissue, a placental tissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendon tissue, a testes tissue, an umbilical cord tissue, a urological tissue, a vascular tissue, a vein tissue, other non-calcified tissues, and a combination thereof. In embodiments, the cells are derived from skin tissue. In embodiments, skin cells comprise pluripotent stem cells, MSCs, skin progenitors and stroma obtained from skin tissue.

[0050] In embodiments, the tissue scaffold matenals (e.g.. hydrogels and / or microbeads) comprise cells associated with wound healing. In embodiments, the wound healing cells comprise keratinocytes, fibroblasts, endothelial cells, macrophages, platelets, mast cells, neutrophils, myofibroblasts, or a combination thereof. In embodiments, the wound healing cells comprise keratinocytes. In embodiments, the wound healing cells comprise fibroblasts. In embodiments, the wound healing cells comprise endothelial cells. In embodiments, the wound healing cells comprise macrophages. In embodiments, the wound healing cells comprise platelets. In embodiments, the wound healing cells comprise mast cells. In embodiments, the wound healing cells comprise neutrophils. In embodiments, tire wound healing cells comprise myofibroblasts.Attorney Docket No. FES-003WO

[0051] In embodiments, the tissue scaffold materials (e.g., hydrogels and or microbeads) comprise cells that are derived from a tissue sample obtained from a subject. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise all cell types derived from the tissue sample. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise one or more cell types derived from the tissue sample. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise one or more of keratinocy tes. fibroblasts, endothelial cells, macrophages, platelets, mast cells, neutrophils, T cells, B cells, or myofibroblasts derived from the tissue sample.

[0052] In embodiments, the cells comprise one or more of mesenchymal stem cells (MSCs), fibroblasts, chondrocytes, neural stem cells (NSCs), endothelial cells, hepatocytes, epithelial cells, adipose-derived stem cells (ADSCs), and immune cells. In embodiments, the cells comprise MSCs. fibroblasts, chondrocytes, NSC's, endothelial cells, hepatocytes, epithelial cells, ADSCs and immune cells. In embodiments, the cells comprise MSCs. In embodiments, the MSCs comprise osteoblasts, chondrocytes, and adipocytes. In embodiments, the cells comprise fibroblasts. In embodiments, the cells comprise dermal fibroblasts. In embodiments, the cells comprise chondrocytes. In embodiments, the cells comprise NSCs. In embodiments, the cells comprise endothelial cells. In embodiments, the cells comprise hepatocytes. In embodiments, the cells comprise epithelial cells. In embodiments, the cells comprise ADSCs. In embodiments, the cells comprise immune cells. In embodiments, the cells comprise fibroblast and endothelial cells. In embodiments, the cells comprise fibroblasts. In embodiments, the cells comprise endothelial cells. In embodiments, the cell type depends on the specific tissue or therapeutic goal. In embodiments, the tissue scaffolds materials (e.g., hydrogels and / or microbeads) described herein are combined with cell types comprising fibroblast and endothelial cells. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) described herein are combined with fibroblasts and endothelial cells, in embodiments, the tissue scaffold materials (e g., hydrogels and / or microbeads) described herein are combined with fibroblasts. In embodiments, the tissue scaffold materials (e.g, hydrogels and / or microbeads) described herein are combined with endothelial cells.

[0053] In embodiments, the tissue scaffold materials (e.g.. hydrogels and / or microbeads) comprise at least 1,000, 10,000. 100,000, 1,000,000, 10,000,000, or 100,000,000 cells. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise from about 1,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 10,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 1,000.000 cells / mg tissue, about 1,000 cells / mg tissue to about 100,000 cells / mg tissue, about 1.000 cells / mg tissueAttorney Docket No. FES-003WOto about 10,000 cells / mg tissue, about 10,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 100,000 cells / mg tissue to about 100.000,000 cells / mg tissue, about 1,000,000 cells / mg tissue to about 100,000,000 cells / mg tissue, or about 10,000,000 cells / mg tissue to about 100,000,000 cells / mg tissue, including any values or ranges therebetween.

[0054] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise about 103to about 108cells (e.g., 103, 104, 105, 106, 10'. or about 108cells, including all values and ranges therein). In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprises about IO4cells to about 108cells, about 103cells to about 108cells, about 10° cells to about I08cells, about 107cells to about 108cells, about 103cells to about 107ceils, about 104cells to about 107cells, about 105cells to about 107cells, about IO6cells to about IO7cells, about 10Jcells to about 106cells, about 104cells to about IO6cells, about I05cells to about 106cells, about 103cells to about I05cells, about 104cells to about 103cells or about 103cells to about 104cells, including all values and ranges therebetween.

[0055] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise about IO3to about 108cells / mg tissue (e.g., I03, 104. IO3, 106. 107, or about IO8cells / mg tissue, including all values and ranges therein). In embodiments, the tissue scaffold materials (e g., hydrogels and / or microbeads) comprise about 104to about 108cells / mg tissue, about 103to about 108cells / mg tissue, about 106to about 108cells / mg tissue, about 107to about 108cells / mg tissue, about 103to about 10' cells / mg tissue, about IO4to about IO7cells / mg tissue, about 103to about 107cells / mg tissue, about 106to about 107cells / mg tissue, about 103to about 106cells / mg tissue, about 104to about IO6cells / mg tissue, about 103to about 106cells / mg tissue, about 103to about 10scells / mg tissue, about 104to about 103cells / mg tissue or about IO3to about 104cells / mg tissue, including all values and ranges therein. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise from about 1,000 cells / mg tissue to about 10,000,000 cells / mg tissue.

[0056] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise any antimicrobial described herein and / or any cell described hereinPolymers

[0057] In embodiments, the microspheres and / or hydrogels of the tissue scaffold materials disclosed herein comprise polymers. A “polymer” is a macromolecule composed of repeating subunits. Exemplary polymer materials for tissue engineering include natural polymers, including, but not limited to, collagen, atelocollagen. telocollagen, gelatin, elastin, hyaluronate, cellulose, alginate; fibrinogen; and synthetic polymers, including polyesters such asAttorney Docket No. FES-003WOpoly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poiy(lactic acid) (PLA), poly(caprolactone), polylbutylene succinate), poly(trimethylene carbonate), poly(p-dioxanone), and poly (butylene terephthalate); polyester amides, such as HYBRANE SI 200 (DSM, The Netherlands); polyurethanes, such as DEGRAPOL (Abmedica, Italy); polyanhydrides, such as poly [(carboxy phenoxy) propane-sebacic acid]; polyphosphoesters, such as poly|bis(hydroxyelliyl) terephthalate-ethyl orthophosphorylate / terephthaloyl chloride]; poly(ortho esters); poly(alkyl cyanoacrylates); polyethers, such as polytethylene glycol); microbial polyesters, such as poly (P-hydroxyalkanoate); and poly(amino acids), such as tyrosine derived polycarbonate (for review, see Marin et al., Int. J. Nanomed. 8:3071-3091 (2013)). In embodiments, the polymer is selected from the group consisting of collagen, hyaluronic acid. poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), and poly(lactic acid) (PLA). In embodiments, the polymers are collagen and / or collagen-based biomatenals comprising collagen ty pes I, II, III, IV, and V. In embodiments, the poly mers comprise type 1 collagen. In embodiments, the collagen and / or collagen-based biomaterials are derived from a source comprising a microorganism, fungi, human, bovine, porcine, or ovine. In embodiments, the collagen and / or collagen-based biomatenals are type 1 collagen derived from a source comprising a microorganism, fungi, human, bovine, porcine, or ovine In an example, human and bovine collagens can be used in human subjects, wherein the human or bovine collagen comprise type I collagen. In embodiments, the collagen and / or collagen-based biomatenals comprise recombinant collagen. In embodiments, the collagen and / or collagen- based biomaterials comprise recombinant collagen derived from a microorganism or fungi. In embodiments, the polymer is atelocollagen or telocollagen.Microspheres[0058| •‘Microspheres” are small particles, made of a polymer. The terra “‘microspheres’' as used herein encompasses small particles that can be spherical or non-spherical; accordingly, any reference to “microspheres” in this application can be used interchangeably with the term “‘microstructures"’, as the microspheres disclosed herein include both spherical and non-spherical small particles Although microspheres can encompass any diameter from 1 pm - 1 mm, microspheres as disclosed herein typically have a diameter of between 5-500 pm in diameter, between 50-250 pm in diameter, between 50-150 pm in diameter, or between 100-200 pm in diameter, for example. In embodiments, microspheres in tissue scaffold material are fairly uniform in size and shape. In embodiments, all the microspheres in a given scaffold can be roughly spherical and have a diameter of about 50-150 pm (e.g., 50, 55, 60, 65. 70, 75, 80,Attorney Docket No. FES-003WO85, 90, 95, 100, 105, 110, 115. 120, 125, 130, 135, 140, 145 or 150 m, including any values or ranges therebetween) or about 100-200 pm (e.g., 100. 105, 110, 115. 120, 125, 130. 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 195, or 200 gm, including ranges or values therebetween). In embodiments, microspheres in a given tissue scaffold material can differ in shape. In embodiments, the microspheres have a shape comprising flattened, curved, oblong, irregularly shaped, or spherical. In embodiments, microspheres in a given tissue scaffold material differ in size. In embodiments, microspheres having different sizes range from about 5-500 pm (e.g., 5, 50. 100, 1 0. 200, 250, 300. 350, 400, 450, or 500 pm, including any ranges or values therebetween), or about 1-1000 pm (1, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 pm, including any values or ranges there between) in diameter.

[0059] In embodiments, the microspheres are made of about 0.1% w / v to about 25% w / v of a polymer (e.g.. 0.1, 0.5, I, 2. 3, 4, 5, 6, 7, 8, 9. 10. 11. 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 % w / v, including any values or ranges therebetween). In embodiments, the microspheres are made of an amount of polymer ranging from about 0.1% w / v to about 25% w / v, 5% w / v to about 25% w / v, 10% w / v to about 25% w / v, 15% w / v to about 25% w / v, 20% vv / v to about 25% w / v, 0.1% w / v to about 20% w / v, 5% w / v to about 20% w / v, 10% w / v to about 20% w / v, 15% w / v to about 25% w / v, 0.1 % w / v to about 15% w / v. 5% w / v to about 15% w / v, 10% w / v to about 25% w / v, 0.1% w / v to about 10% w / v, 5% w / v to about 10% w / v, or 0 1% w / v to about 5% w / v, including any values or ranges therebetween. In embodiments, the microspheres are made from about 0.1 % to about 2 % vv / v of a polymer. In embodiments, the microspheres are made of about 0.1% w / v to about 10% w / v of a polymer (e.g.. 0.1, 0.5, 1. 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v, including any values or ranges therebetween). In embodiments, the microspheres are made of an amount of polymer ranging from about 0.1% w / v to about 10% w / v, about 2% w / v to about 10% w / v, about 4% w / v to about 10% w / v, about 6% w / v to about 10% w / v, about 8% w / v to about 10% w / v. 0.1% w / v to about 5% w / v, about 2% w / v to about 5% w / v, about 4% w / v to about 5% w / v, or about 0 1 % w / v to about 1 % w / v, including any values or ranges therebetween. In embodiments, the microspheres are made from about 0.1 % to about 2 % w / v of a polymer. In embodiments, the microspheres are made from about 0.2% to 2.0%, 0.4% to 1.2%, 0.4% to 0.8%, or 0.2%. 0.3%, 0.4%. 0.5%, 0.6%. 0.7%, 0.8%, 0.9%, 1.0%, 1.1 %, 1.2 %, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%. 1.8 %. 1.9%, or 2 % w / v of a polymer, including any ranges or values therebetween. In embodiments, the microspheres are comprised of 1% w / v, 1% w / w, or 1% v / v of the polymer.

[0060] In embodiments, the polymer is selected from any one of collagen, atelocollagen, telocollagen, gelatin, elastin, hyaluronate, cellulose, alginate, fibrinogen, poly(lactic-co-gly colicAttorney Docket No. FES-003WOacid) (PLGA). poly(glycolic acid) (PGA), polyllactic acid) (PLA), poly (caprolactone), poly(butylene succinate), poly (trimethylene carbonate), poly(p-dioxanone). and polyibutylene terephthalate); a polyester amide, a polyurethane, poly[(carboxyphenoxy) propane-sebacic acid], poly [bi s(hydroxy ethyl) terephthalate-ethyl orthophosphorylate / terephthaloyl chloride], a poly(ortho ester), a poly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester, poly (p-hydrox alkanoate), a tyrosine derived polycarbonate, atelocoilagen, and telocollagen. In embodiments, the polymer is collagen and / or a collagen-based biomaterial. In embodiments, the polymer is atelocoilagen or telocollagen. In embodiments, the polymer comprises a collagen and / or collagen-based biomaterial derived from a source comprising a microorganism, fungi, human, bovine, porcine, or ovine. In embodiments, the collagen and / or collagen-based biomaterials comprise recombinant collagen. In embodiments, the collagen and / or collagen-based biomaterials comprise recombinant collagen derived from a microorganism or fungi.

[0061] The microspheres can further include bioactive factors in addition to the polymer. A “bioactive factor’ can be a small organic molecule, a nucleic acid, or a polypeptide that can stimulate or promote one or more cellular invasion, cellular growth, angiogenesis, vascularization, nen e regeneration, or cellular differentiation In embodiments, the bioactive factor is a growth factor contained within the microsphere or mixed with the polymer matrix of the microsphere prior to preparing the tissue scaffold material. In embodiments, the bioactive factor is a growth factor selected from the group consisting of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), neurotrophin-3 (NT -3), brain derived growth factor (BDNF), acidic and basic fibroblast growth factor (FGF), pigment epithelium-derived factor (PEDF), glial derived growth factor (GDNF), angiopoietin. and erythropoietin (EPO). In embodiments, the bioactive factor is a nucleic acid comprising an antisense siRNA molecule. In embodiments, the microspheres do not include other bioactive factors.Hydrogels

[0062] In embodiments, tissue scaffold materials (e.g., into hydrogels and / or microbeads) provided herein are hydrogels Hydrogels can refer to a broad class of polymeric materials which are swollen extensively in w ater, but which do not dissolve in water. Hydrogels can be formed by' polymerizing a hydrophilic monomer in an aqueous solution under conditions where the polymer becomes crosslinked so that a three dimensional polymer netw ork is formed whichAttorney Docket No. FES-003WOis sufficient to gel the solution. Hydrogels are described in more detail in Hoffman. D. S., " Polymers in Medicine and Surgery." Plenum Press, New York, pp 33-44 (1974).

[0063] In embodiments, the hydrogels provided herein comprise one or more polymers. Tn embodiments the polymer is any polymer disclosed herein. In embodiments, the hydrogel contains 0.1% to 0.6%, 0.2 to 0.4%, or 0.3% w / v of a polymer selected from the group consisting of collagen, atelocollagen, telocollagen gelatin, elastin, hyaluronate, cellulose, fibrinogen. poly(lactic-co-gly colic acid) (PLGA), poly(gly colic acid) (PGA). poly(lactic acid) (PLA), poly(caprolactone). poly (butylene succinate). poly(trimethylene carbonate), poly(p-dioxanone), and polylbutylene terephthalate); a polyester amide, a polyurethane, poly [(carboxyphenoxy) propane-sebacic acid], poly [bis(hydroxy ethyl) terephthalate-ethyl orthophosph oryl te / terephthaloyl chloride], a polytortho ester), a poly(alkyl cyanoacrylate), poly(ethylene glycol), a microbial polyester, poly(P-hydroxy alkanoate), and a tyrosine derived polycarbonate. In embodiments, the hydrogel comprises collagen. In embodiments, the hydrogel comprises atelocollagen or telocollagen. In embodiments, die hydrogel comprises collagen in an amount of 0.1 % to 0,6%. 0.2 to 0.4%, or 0.3% w / v. In embodiments, the hydrogel comprises atelocollagen or telocollagen in an amount of 0.1% to 0.6%, 0.2 to 0.4%, or 0.3% w / v.Substrates

[0064] In embodiments, the tissue scaffold materials (e.g.. hydrogels and / or microbeads) disclosed herein can be bound to one or more substrates. In embodiments, the hydrogels are bound to one or more, two or more, three or more, four or more, five or more or six or more substrates. In embodiments, the hydrogels are bound to a substrate. In embodiments, the substrate comprises silicone, polyethylene, polyesters, polyamides, polyurethane, thermoplastic polyurethane (TPU), polyethylene terephthalate (PET) or combinations thereof. In embodiments, Hie substrate is selected from a group consisting of silicone, polyethylene, polyesters, polyamides, polyurethane, and combinations thereof In embodiments, the substrate is of a category’ selected from the group consisting of: silicone, polyesters, polyamides, thermoplastic polyurethane (TPU), polyethylene terephthalate (PET) and combinations thereof.

[0065] In embodiments, the substrate comprises silicone. In embodiments, the substrate comprises polyester. In embodiments, the substrate comprises poly amide. In embodiments, the substrate comprises polyurethane (PU). In embodiments, the substrate comprises polyamide. In embodiments, the substrate comprises TPU. In embodiments, the substrate comprises PET.Attorney Docket No. FES-003WOIn embodiments, the substrate comprises high density polyethylene. In embodiments, the substrate comprises a low-density polyethylene and / or gelatin.

[0066] In embodiments, the substrate has a density of at least 0.1 g / cm3. In embodiments, the substrate has a density from about 0.1 g / cm3to about 5 g / cm3(e.g., 0.1, 05, 1, 1.5, 2, 25, 3, 3.5, 4, 4.5, or 5 g / cm3, including all values and ranges therein). In embodiments, the substrate has a density comprising at least about 0.88 g / cm3. In embodiments, the substrate has a density consisting of about 0.88 g / cm3. In embodiments, the PET substrate has a density of at least about 1.33 g / cm3. In embodiments, the silicone substrate has a density of at least about 1.05 g / cm3. In embodiments, the TPU substrate has a density of at least about 1.1 g / cm3.

[0067] In embodiments, the substrate is in a form of a film that can be peeled off. In embodiments, the substrate is in a polymeric film. In embodiments, the substrate comprises a similar thickness to the hydrogel. In embodiments, the substrate comprises a different thickness to the hydrogel. In embodiments, the substrate comprises a thickness of about 25 pm to 254 pm (e.g., 25, 50, 75, 100, 125, 150, 175, 200, 225, or 254 pm, including all values and ranges therein). In embodiments, the substrate comprises a thickness of about 50 pm to about 254 pm, about 75 pm to about 254 pm. about 100 pm to about 254 pm, about 125 pm to about 254 pm, about 150 pm to about 254 pm, about 175 pm to about 254 pm, about 200 pm to about 254 pm, about 225 pm to about 254 pm, about 25 pm to about 225 pm, about 50 pm to about 225 pm, about 75 pm to about 225 pm, about 100 pm to about 225 pm, about 125 pm to about 225 pm, about 150 pm to about 225 pm. about 175 pm to about 225 pm, about 200 pm to about 225 pm, about 25 pm to about 200 pm, about 50 pm to about 200 pm, about 75 pm to about 200 pm, about 100 pm to about 200 pm, about 125 pm to about 200 pm, about 150 pm to about 200 pm, about 175 pm to about 200 pm, about 25 pm to about 175 pm, about 50 pm to about 175 pm, about 75 pm to about 175 pm, about 100 pm to about 175 pm, about 125 pm to about 175 pm. about 150 pm to about 175 pm. about 25 pm to about 150 pm, about 50 pm to about 150 pm, about 75 pm to about 150 pm, about 100 pm to about 150 pm, about 125 pm to about 150 pm. about 25 pm to about 125 pm, about 50 pm to about 125 pm, about 75 pm to about 125 pm, about 100 pm to about 125 pm, about 25 pm to about 100 pm, about 50 pm to about 100 pm. about 75 pm to about 100 pm. about 25 pm to about 75 pm, about 50 pm to about 75 pm, or about 25 pm to about 50 pm, including all values and rangers therein.

[0068] In embodiments, the substrate comprises a thickness of about 10 pm to about 35 pm, about 15 pm to about 35 pm. about 20 pm to about 35 pm, about 25 pm to about 35 pm, about 30 pm to about 35 pm. about 10 pm to about 30 pm, about 15 pm to about 30 pm, about 20Attorney Docket No. FES-003WOpm to about 30 m, about 25 pm to about 30 pm. about 10 pm to about 25 pm, about 15 pm to about 25 pm, about 20 pm to about 25 pm, about 10 pm to about 20 pun, about 15 pm to about 20 pun, or about 10 pun to about 15 pim, including any ranges or values therebetw een. In embodiments, the substrate comprises a thickness of about 23.4 im. In embodiments, the substrate comprises a thickness of about 25.4 piM.

[0069] In embodiments, the substrate comprises a thickness of about 40 pim to about 60 pun, about 45 pim to about 60 pun, about 50 pim to about 60 pim, about 55 ptm to about 60 pm, about 40 pm to about 55 pm, about 45 pm to about 55 pm, about 50 pm to about 55 pm, about 40 pm to about 50 pm, about 45 pm to about 50 pm, or about 40 pm to about 45 pm, including any ranges or values therebetween. In embodiments, the substrate comprises a thickness of about 50.8 pm.

[0070] In embodiments, the substrate comprises a thickness of about 120 pm to about 140 pm, about 125 pm to about 140 pm, about 130 pm to about 140 pm, about 135 pm to about 140 pm, about 120 pm to about 135 pm, about 125 pm to about 135, about 130 pm to about 135 pm, about 120 pm to about 130 pm, about 125 pm to about 130 pm, or about 120 pm to about 130 pm, including any values or ranges therebetween. In embodiments, the substrate comprises a thickness of about 127 pm.

[0071] In embodiments, the substrate comprises a thickness of about 65 pm to about 85 pm, about 65 pm to about 85 pm, about 70 pm to about 85 pm, about 75 pm to about 85 pm, 80 pm to about 85 pm, about 65 pm to about 80 pm. about 70 pm to about 80 pm, about 75 pm to about 80 pm, about 65 pm to about 75 pm, about 70 pm to about 75 pm, or about 65 pm to about 70 pm, including any values or ranges therebetween. In embodiments, the substrate comprises a thickness of about 75 pm. In embodiments, the substrate is PET.

[0072] In embodiments, the substrate comprises a thickness of about 47 pm to about 67 pm, about 52 pm to about 67 pm, about 57 pm to about 67 pm, about 62 pm to about 67 pm, about 47 pm to about 62 pm, about 52 pm to about 62 pm, about 57 pin to about 62 pm, about 47 pm to about 57 pm, about 52 pm to about 57 pm, or about 47 pm to about 52 pm, including any values or ranges therebetween. In embodiments, the substrate comprises a thickness of about 57 pm. In embodiments, the substrate is silicone.

[0073] In embodiments, the substrate comprises a thickness of about 15 pm to about 35 pm, about 20 pm to about 35 pm, about 25 pm to about 35 pm, about 30 pm to about 35 pm, about 15 pm to about 30 pm, about 20 pm to about 30 pm, about 25 pm to about 30 pm, about 15 pm to about 25 pm, about 20 pm to about 25 pm, or about 15 pm to about 20 pm, including any values or ranges therebetween. In embodiments, the substrate comprises a thickness ofAttorney Docket No. FES-003WOabout 25 pm. In embodiments, the substrate comprises a thickness of about 25.4 pm. In embodiments, the substrate is TPU.

[0074] In embodiments, the substrate comprises a high water content. In embodiments, the substrate comprises a water content of at least about 50% w / v. In embodiments, the substrate comprises a water content of at least about 60% w / v, at least about 70% w / v, at least about 80% w / v, al least about 90% w / v, at least about 95% w / v or at least about 99% w / v. In embodiments, the substrate comprises a water content of at least about 90% w / v. In embodiments, the substrate comprises a water content of at least about 99% w / v. In embodiments, the substrate comprises a water content from about 60% w / v to about 99.99% w / v (e.g., 60, 65, 70, 75, 80, 85, 90, 95, or 99.99% w / v, including all values and ranges therebetween). In embodiments, the substrate comprises a water content of about 90% w / v to about 999% w / v (e.g.. 90. 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% w / v. including any values or ranges therebetween).

[0075] In embodiments, the substrate further comprises one or more polymers. In embodiments, the substrate comprises one or more, two or more, three or more, four or more, five or more or six or more polymers. In embodiments, the polymer is a low weight percent polymer. In embodiments, the substrate comprises about 0.1% to about 25% polymer by weight (e.g., 0.1. 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18. 19, 20, 21, 22, 23, 24, or 25%, including any values or ranges therebetween. In embodiments, the substrate comprises about 0.1% to about 25%, about 5% to about 25%, about 10% to about 25%, about 15% to about 25%. about 20% to about 25%, about 0.1% to about 20%. about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 0.1% to about 15%, about 5% to about 15%, about 10% to about 15%, about 0.1% to about 10%, about 5% to about 10% or about 0.1% to about 5% polymer by weight, including any values or ranges therein.

[0076] In embodiments, a variety' of surfaces can be bound to hydrogels. In embodiments, the surface may be a poly meric surface. In embodiments, the polymeric surface can comprise chemical moieties in the structure of the polymer that will interact or bond with the hydroxyl groups or carboxylic acid groups of the hydrogel or hydrogel components. In embodiments, chemical moieties can compose hydroxyl, ether, ester, carboxylic acid, amine, amide, or silyl moieties. In embodiments, other chemical moieties can be used to form covalent or other chemical bonds with hydroxyl or carboxylic acid groups. In embodiments, the polymer comprises hydroxyl or carboxylic acid groups.

[0077] In embodiments, the surface may be a soft tissue. In embodiments, soft tissue can comprise muscle, tendons, ligaments, synovial tissue, fascia, which surrounds the musculoskeletal components, and other structures such as nerves, blood vessels and fat. InAttorney Docket No. FES-003WOembodiments, the soft tissue comprises cartilage, meniscus or other soft tissue that is located at a joint site such as a hip, knee, spine, finger, elbow or shoulder joint.Methods of making tissue scaffold materials

[0078] Also disclosed herein are methods of making a tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, to make the tissue scaffold materials (e.g., hydrogels and / or microbeads), suitable polymers are incorporated into compositions for production. Exemplary polymers include but are not limited to natural polymers, such as collagen, atelocollagen, telocollagen, gelatin, elastin, hyaluronate, and cellulose; fibrinogen; and synthetic polymers, including polyesters such as poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(caprolaclone), polylbutylene succinate), poly(tnmethylene carbonate), poly(p- dioxanone), and poly(butylene terephthalate); polyester amides, such as HYBRANE S1200 (DSM, The Netherlands); polyurethanes, such as DEGRAPOL (Abmedica, Italy); poly anhydrides, such as poly[(carboxyphenoxy) propane- sebacic acid]; polyphosphoesters, such as poly [bis (hydroxy ethyl) terephthalate-etliyl orthophosphorylate / terephthaloyl chloride]; poly(ortho esters); poly(alkyl cyanoacrylates); polyethers, such as polylethylene glycol); microbial polyesters, such as poly(P -hydroxy alkanoate); and poly(amino acids), such as tyrosine derived polycarbonate (for review, see Mann et al,, Int J. Nanomed, 8:3071-3091 (2013)). In embodiments, the polymer is selected from the group consisting of collagen, hyaluronic acid, poly(lactic-co-gly colic acid) (PLGA), poly(gly colic acid) (PGA), and poly(lactic acid) (PLA). In embodiments, the polymers are collagen and / or collagen-based biomaterials consisting of collagen types I, II, III, IV, and V. In embodiments, the collagen and / or collagen-based biomaterials comprise human and bovine collagens. In embodiments, the collagen and / or collagen-based biomaterial derived from a source comprising a microorganism, fungi, human, bovine, porcine, or ovine In embodiments, the collagen and / or collagen-based biomaterials comprise recombinant collagen. Tn embodiments, the collagen and / or collagen-based biomatenals comprise recombinant collagen derived from a microorganism or fungi.

[0079] In embodiments, the polymer is atelocollagen or telocollagen.

[0080] In embodiments, the collagen is derived from various sources comprising human or bovine tissue. In embodiments, the collagen is autologous to a subject for whom the tissue scaffold material (e.g., hydrogels and / or microbeads) is to be administered, and is extracted from the skin of the subject. In embodiments, after a biological sample (e.g., skin, placenta, tendon, or cultured cells) is procured, collagen is extracted from the sample by knownAttorney Docket No. FES-003WOtechniques to form a stock solution. See, for example. Epstein, J. Biol. Chem. 249:3225-3231 (1974). in embodiments, stock solutions of collagen comprise collagen in a suitable solution, comprising, for example, 0 1 % acetic acid, or Earle's or Hank's salts, L-glutamine, HEPES, and sodium bicarbonate An example of a suitable medium is a Medium 199 (MI99)-based medium. In embodiments, collagen is kept at a stock concentration higher than the final concentration, comprising concentrations of 0.2%-1.6% collagen, preferably 0.3-0.5% collagen for the hydrogel, and 0.6-2.0% collagen for the microspheres.

[0081] In embodiments, collagen is neutralized before use. In embodiments, collagen is neutralized by mixing a stock solution of collagen with sodium hydroxide to reach a pH of 7.2- 7.6, preferably pH 7.4. In embodiments, this mixture is overlayed with oil, such as mineral oil, preferably at least 5 volumes of oil per volume of collagen with NaOH, and stored with refrigeration until use.

[0082] In embodiments, to make microspheres, a polymer (e.g., collagen) composition with oil overlay is mixed at high speed to form an oil-in water emulsion. In embodiments, the polymer composition further contains at least one type of bioactive factor as disclosed hereinabove. In embodiments, the emulsion is then subject to repeated washings with increasing concentrations of ethanol comprising a first wash with about 50% ethanol, a second wash with about 80% ethanol, and a third through fifth wash with about 100% ethanol. In embodiments, the first wash comprises mixing (such as by stirring at 800-1500 rpm for 20- 40 minutes) with at least 5 volumes of ethanol per volume of collagen solution, centnfuging the mixture at about 2500-3500 rpm for about 5-10 minutes, and removing the oil and alcohol layers. In embodiments, subsequent w ashes comprise mixing with at least 5 volumes of ethanol per volume of collagen solution, centrifuging the mixture at about 2500-3500 rpm for 5-10 minutes, and removing the alcohol layer. In embodiments, after the alcohol washes, the collagen is washed three to five times with at least 5 volumes of cold saline, such as phosphate buffered saline (PBS), in embodiments, after removal of the final saline wash, the collagen microsphere composition formed by the washes is ready for use.

[0083] Another exemplary method of making microspheres is described below. In embodiments, to make microspheres, a polymer (e.g., collagen) composition with oil overlay is mixed at high speed to form an oil-in water emulsion. In embodiments, the polymer composition further contains at least one type of bioactive factor as disclosed hereinabove. In embodiments, the emulsion is added to a basic solution to stabilize microspheres, which are then washed with methanol to remove excess base. In embodiments, microspheres are furtherAttorney Docket No. FES-003WOstabilized through crosslinking with diisocyanates, and are washed with methanol, water, and buffer to remove unreacted reagents.

[0084] In embodiments, a polymer used for the hydrogel is the same or different from the polymer used to make the microspheres. In embodiments, the polymer for the hydrogel is the same as the polymer for the microspheres. In embodiments, the polymer for the hydrogel is different from the polymer for the microspheres. In embodiments, the polymer used for both the microspheres and the hydrogel is collagen. In embodiments, the microspheres and hydrogel have the same or different polymer, the density of the polymer (w7v) in the microspheres will differ from the density of polymer (w / v) in the hydrogel "bulk". In embodiments, collagen hydrogel "bulk" scaffolds are made with a collagen stock solution mixed with sodium hydroxide to reach a pH of 7, 2-7,6, preferably pH 7.4.

[0085] In embodiments, to make the tissue scaffold materials, a first composition, containing microspheres, is added to a mold or shaping platform. In embodiments, a second composition that forms the hydrogel, comprising a polymer material, is added to the first composition. In embodiments, the compositions are mixed by a method comprising stirring or pipetting to achieve uniform mixing. In embodiments, the mixture is then cross-linked. In embodiments, following cross-linking, the tissue scaffold material is used immediately or stored for future use.

[0086] In embodiments, an antimicrobial is added to the tissue scaffold materials (e.g., hydrogels and / or microbeads) via several methods. In embodiments, the antimicrobial is added to the tissue scaffold material (e.g., hydrogels and / or microbeads) prior to, during or after the formation of the hydrogel. In embodiments, the methods comprise, in situ synthesis during the gelation process, post-synthesis incorporation, addition to a precursor solution prior to gelation, physical absorption or immersion into a solution comprising the agent, or layer by layer deposition. In embodiments, the in-situ synthesis comprises reducing antimicrobials m the presence of the tissue scaffold material (e.g, hydrogels and / or microbeads) components during gelation using a reducing agent or UV light. In embodiments, post-synthesis incorporation comprises infusion or injection of a tissue scaffold material (e.g.. hydrogels and / or microbeads) with the antimicrobial. In embodiments, immersion comprises immersing the tissue scaffold material (e.g., hydrogels and / or microbeads) in a solution comprising the antimicrobial and allowing the antimicrobial to adhere to the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, the antimicrobial adheres to the inside or surface of the tissue scaffold material (e.g., hydrogels and / or microbeads).Attorney Docket No. FES-003WO

[0087] Moreover, the tissue scaffold materials (e.g., hydrogels and / or microbeads) described herein can incorporate cells through various approaches. In embodiments, the various approaches comprise simple diffusion, encapsulation during gel formation, electrostatic interactions, covalent bonding, microfluidic techniques, micropatteming, magnetic field guidance, hydrogel swelling, 3-D bioprinting, layer-by-layer assembly, spraying, aerosolization, and injection. In embodiments, the tissue scaffold material (e.g.. hydrogels and / or microbeads) are immersed into a solution comprising the cells. In embodiments, the cells are mixed with a tissue scaffold material (e.g, hydrogels and / or microbeads) precursor solution prior to gelation. In embodiments, the cells are added to the tissue scaffold material (e.g., hydrogels and / or microbeads) after gelation. In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) is immersed in a composition comprising the cells. In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) are injected or sprayed with cells post-gelation. In embodiments, the tissue scaffold material (e.g.. hydrogels and / or microbeads) are infused with cells.Methods of Bonding Tissue Scaffold Materials to Substrates

[0088] Provided herein are methods of bonding disclosed tissue scaffold materials (e.g., hydrogels and / or microbeads) to one or more substrates of the disclosure. In embodiments, bonding occurs between a hydrogel and / or microbead of a tissue scaffold material and a substrate. In embodiments, the method comprises applying the tissue scaffold material (e.g., hydrogels and / or microbeads) precursors to a substrate surface, wherein the precursors are crosslinked to form a tissue scaffold material (e.g., hydrogels and / or microbeads) coating. Examples of suitable crosslinking techniques include physical crosslinking, chemical crosslinking, and radiation crosslinking.[00891 In embodiments, physical crosslinking may be employed in the crosslinking step. Physical crosslinking uses physical, electrostatic interactions (i.e., hydrogen bonds, Van Der Waals forces, or hydrophobic interactions and the like) to form 3D hydrogel structures. Tn embodiments, physical crosslinking may be achieved by freeze-thaw solution-phase methods, which enable crosslinking through the hydrogen bonding between the hydrophilic groups of the polymers. See Peppas, et al.. Adv. Polymer Set. 153, 37 (2000). Freeze-thaw methods of crosslinking provide the advantage of enabling the formation of hydrogels without the use of potentially hazardous solvents, chemical crosslinking agents, or initiators.

[0090] In embodiments, chemical crosslinking methods may be used. Chemical crosslinking is characterized by the formation of covalent bonds between polymer chains in the hydrogelAttorney Docket No. FES-003WOstructure and provides superior mechanical stability than physical crosslinking. In embodiments chemical crosslinking agents comprise aldehydes (such as formaldehyde, acetaldehyde, or glutaraldehyde) in the presence of a solvent (such as sulfuric acid, acetic acid, or methanol), diisocyanate compounds, which can result in urethane linkages, epoxy compounds, ethylene glycol di(meth)acrylate, acrylamide compounds, or divinylbenzene) In embodiments, chemical crosslinking can be enabled by enzymes (such as a calcium-independent microbial transglutaminase), which catalyzes transamidation reactions to form N-s-(y-glutamyl)lysine crosslinks in proteins.

[0091] In embodiments, irradiation is applied to the tissue scaffold materials (e.g., hydrogels and / or microbeads) to induce chemical crosslinking. Provided herein is a method of bonding a tissue scaffold material (e.g., hydrogels and / or microbeads) to a substrate, the method comprising: contacting at least a portion of the tissue scaffold materials (e.g., hydrogels and / or microbeads) with a substrate; and irradiating the portion of the tissue scaffold materials (e.g., hydrogels and / or microbeads) and the substrate in an amount effective to bond the portion of the tissue scaffold material (e.g., hydrogels and / or microbeads) to the substrate, wherein the tissue scaffold matenal (e.g.. hydrogels and / or microbeads) comprises collagen.

[0092] When irradiation is applied to tissue scaffold materials (e.g., hydrogels and / or microbeads) it results in chemical crosslinking of polymer chains of the tissue scaffold materials (e.g., hydrogels and / or microbeads) with the substrate by way of covalent bond formation. In this instance, crosslinking comprises the irreversible linkage of tissue scaffold materials (e.g., hydrogels and / or microbeads) to substrate due to the covalent bonding enabled by said irradiation. In embodiments, irradiation induces hemolytic cleavage of C-C and C-H bonds on polymers. These generated alky l radicals are unstable and can undergo a complex series of reactions that can result in crosslinking, chain scissions, oxidation and creation of alkene bonds. These reactions can be used to attach or bond tissue scaffold materials (e.g., hydrogels and / or microbeads) to various materials. As a result, high water content tissue scaffold materials (e.g, hydrogels and / or microbeads) may exhibit strong bonding to surfaces if the adhesion force is much greater than the cohesive force of the tissue scaffold materials (e.g., hydrogels and / or microbeads). Crosslinking can also occur by chemical bonding using reagents. In embodiments, covalent crosslinks formed as a result of irradiation have increased control over the crosslinking reaction, specific location of the reaction, and exclusion of residue in comparison to chemically crosslinks.

[0093] In embodiments, the invention described herein utilizes a method of ionizing radiation energy that is converted to heat in the tissue scaffold material (e.g.. hydrogels and / orAttorney Docket No. FES-003WOmicrobeads) or tissue to enable bonding of the tissue scaffold materials (e.g., hydrogels and / or microbeads) to adjacent surfaces. In embodiments, the surfaces comprise soft tissue or collagen. In embodiments, this method is optimized by programming the ionizing radiation energy, depth of penetration and dose delivered.

[0094] In embodiments, the method comprises irradiating with a wavelength from about I pm to about 10 nm (e.g., 1 pm, 500 pm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm. 8 nm, 9 nm, or 10 nm, including any values or ranges therebetween). In embodiments, the kinetic energy of the ionizing radiation is from about 10 keV to about 250 MeV (e.g, 10 keV, 500 keV, 1 MeV, 10 MeV, 20 MeV, 30 MeV, 40 MeV, 50 MeV, 60 MeV, 70 MeV, 80 MeV, 90 MeV, 100 MeV, 110 MeV, 120 MeV, 130 MeV, 140 MeV, 150 MeV, 160 MeV, 170 MeV, 180 MeV, 190 MeV, 200 MeV, 210 MeV, 220 MeV, 230 MeV, 240 MeV, or 250 MeV, including any ranges or values therebetween). In embodiments, the ionizing energy- is from an electron beam. In embodiments, the ionizing energy is from X-ray, gamma irradiation, or electron beam.

[0095] In embodiments, crosslinking is achieved by irradiation from an electron beam source. Upon electron beam irradiation, radicals can be formed from the homolytic cleavage of C-C and C-H bonds in the polymer substrate. The generated radicals can further react w-ith the tissue scaffold materials (e g., hydrogels and / or microbeads) in a chain scission reaction to form cross-linkages. In embodiments, electron beam-induced chemical crosslinking may be formed using commercially available accelerators. In embodiments, an accelerator comprises a voltage generator, the electron gun, the accelerator tube, the scan horn, and the control system. The accelerator can create a beam of electrons approximately 2.5 centimeter m diameter and energizes it to near light speed. The beam can pass through a scan horn, where a magnet scans it back and forth at ca. 200 Hz, creating a curtain of electrons 1-2 meters wide.

[0096] In embodiments the accelerator produces an electron beam energy- (i.e. kinetic energy-) between about 1 to about 50 MeV (e.g., about 1 MeV, 5 MeV, 10 MeV, 15 MeV, 20 MeV, 25 MeV, 30 MeV, 35 MeV, 40 MeV, 45 MeV or 50 MeV, including any values or ranges therebetween). In embodiments, the electron beam energy- is between about 2 MeV to about 5 MeV. In embodiments, the electron beam energy is between about 5 MeV to about 10 MeV. In embodiments, the electron beam energy is between about 10 MeV to about 15 MeV. In embodiments, the electron beam energy is between about 15 MeV to about 20 MeV. In embodiments, the electron beam energy' is between about 25 MeV to about 30 MeV. In embodiments, the electron beam energy is between about 30 MeV to about 35 MeV. In embodiments, the electron beam energy is between about 35 MeV to about 40 MeV. In embodiments, the electron beam energy is between about 40 MeV to about 45 MeV. InAttorney Docket No. FES-003WOembodiments, the electron beam energy is between about 45 MeV to about 50 MeV. In embodiments, the electron beam energy is about 2 MeV. In embodiments, the electron beam energy is about 5 MeV. In embodiments, the electron beam energy is about 10 MeV. In embodiments, the electron beam energy is about 15 MeV Tn embodiments, the electron beam energy is about 20 MeV. In embodiments, the electron beam energy is about 25 MeV. In embodiments, the electron beam energy is about 30 MeV. In embodiments, the electron beam energy is about 35 MeV. In embodiments, the electron beam energy is about 40 MeV In embodiments, the electron beam energy is about 45 MeV. In embodiments, the electron beam energy is 10 MeV.

[0097] In embodiments, the total radiation is measured in Grays (Gy). In embodiments, the total radiation on the hydrogel is about 5 kGy to about 100 kGy. In embodiments, the total radiation is about 5 kGy to about 10 kGy. In embodiments, the total radiation is about 10 kGy’ to about 15 kGy. In embodiments, the total radiation is about 15 kGy to about 20 kGy. In embodiments, the total radiation is about 20 kGy to about 25 kGy. In embodiments, the total radiation is about 25 kGy to about 30 kGy. In embodiments, the total radiation is about 30 kGy to about 35 kGy. In embodiments, the total radiation is about 35 kGy to about 40 kGy. In embodiments, the total radiation is about 40 kGy to about 45 kGy. In embodiments, the total radiation is about 45 kGy to about 50 kGy. In embodiments, the total radiation is about 50 kGy to about 55 kGy. In embodiments, the total radiation is about 55 kGy to about 60 kGy. In embodiments, the total radiation is about 60 kGy to about 65 kGy. In embodiments, the total radiation is about 65 kGy to about 70 kGy. In embodiments, the total radiation is about 70 kGy to about 75 kGy'. In embodiments, the total radiation is about 75 kGy to about 80 kGy. In embodiments, the total radiation is about 80 kGy to about 85 kGy. In embodiments, the total radiation is about 85 kGy to about 90 kGy. In embodiments, the total radiation is about 90 kGy to about 95 kGy. In embodiments, the total radiation is about 95 kGy to about 100 kGy. in embodiments, the total radiation is about 5 kGy to about 40 kGy. In embodiments, the total radiation is about 30 kGy, In embodiments, the total radiation is 30 kGy.

[0098] In embodiments, the electron beam exposure may be carried out in an inert atmosphere. In embodiments, the inert atmosphere is argon, nitrogen, or vacuum. In embodiments, the inert atmosphere is argon. In embodiments, the inert atmosphere is nitrogen. In embodiments, the inert atmosphere is vacuum. In embodiments, the inert atmosphere is vacuum. In embodiments the atmosphere is an oxygen-scavenger atmosphere. In some embodiments, the atmosphere is air under ambient or refrigerated conditions.Attorney Docket No. FES-003WO

[0099] In embodiments, multiple crosslinking steps may be combined to facilitate the crosslinking of the tissue scaffold materials (e.g.. hydrogels and / or microbeads). A combination of crosslinking approaches may be suitable for providing additional strength or resilience to the resulting tissue scaffold materials (e.g., hydrogels and / or microbeads). In embodiments, crosslinking is achieved via physical crosslinking methods followed by chemical crosslinking methods. In embodiments, crosslinking is achieved via physical crosslinking methods followed by irradiation crosslinking methods. In embodiments, crosslinking is achieved via chemical crosslinking methods followed by physical crosslinking methods. In embodiments, crosslinking is achieved via chemical crosslinking methods followed by irradiation crosslinking methods. In embodiments, crosslinking is achieved via irradiation crosslinking methods followed by physical crosslinking methods. In embodiments, crosslinking is achieved via irradiation crosslinking methods followed by chemical crosslinking methods.

[0100] The binding strength of the tissue scaffold materials (e.g., hydrogels and / or microbeads) to the substrate can be determined via different materials characterization techniques. In embodiments, the material characterization techniques are Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), or Scanning Electron Microscopy (SEM). In embodiments, the material characterization technique is DSC. DSC is a thermoanalytical technique that measures the difference in the quantity of heat required to increase the temperature of a sample (i.e., a cross linked polymer) as a function of temperature. DSC can identify the physical transformation of polymers, such as phase changes and glass transitions, which can provide insight into the crystallization ( i.e., the reduction of flexibility) of polymer frameworks. In embodiments, the material charactenzation technique is XRD. XRD is a crystallographic characterization technique that can provide information on extent of crystallization m the crosslinked polymer. In embodiments, the material characterization technique is FT-IR. FT-IR is an infrared spectroscopic technique that can provide characteristic information the polymer composition and structure, including information on bond lengths and bond strengths. In embodiments, the material characterization technique is SEM. SEM is an electron microscopic technique that can provide information on polymer size, morphology, and three-dimensional topography.

[0101] In embodiments, the strength of the tissue scaffold material (e.g., hydrogels and / or microbeads) bonding to the substrate is determined via physical peeling (i.e., “peelability). In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) bonding is determined by a peel ability assay. Peelability assays can provide information into the physicalAttorney Docket No. FES-003WOstrength of the scaffold adhesion to the substrate under operating conditions. In an exemplary procedure, after electron beam irradiation the tissue scaffold and substrate are separated. The peeled substrate can then be graded on a scale of 1-4: (1 ) Product failure (full adhesion); (2) Residue but removable; (3) Resistance but clean peel; and (4) Clean peel and no resistance In embodiments, the substrate is a film. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) demonstrate full adhesion to the substrate. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) leave residue upon peeling from substrate. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) demonstrate a clean peel from the substrate but exhibited resistance. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) demonstrate a clean peel from the substrate with no resistance. In embodiments, the peelability is reduced by at least about 1-point, 2-pomts, or 3-points, as determined by peelability assay.

[0102] In embodiments, irradiating further sterilizes the portion of the tissue scaffold materials (e.g., hydrogels and / or microbeads) and the substrate as determined by ISO 11137. ISO 11137 refers io an International Organization for Standardization protocol for the sterilization of health care products with radiation (ISO 11137-1:2006(en) Sterilization of health care products --- Radiation --- Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices). This protocol covers radiation processes employing irradiators using the radionuclide60Co or137Cs, a beam from an electron generator or a beam from an X-ray generator.

[0103] In embodiments, a sterile tissue scaffold material (e.g., hydrogels and / or microbeads) is substantially free of viable microorganisms. In embodiments, substantially free of viable microorganisms comprises at least about 90%, at least about 95%, at least about 99% or about 100% free of viable microorganisms. In embodiments, ISO 11137 describes requirements comprising a radiation sterilization process intended to sterilize medical devices. In embodiments, the process has appropriate microbicidal activity. In embodiments, electron beam sterilization can be validated with a procedure. In embodiments, the procedure comprises assessing a minimum dose for sterility' at a determined bioburden level. Subsequently, the maximum dose of electron beam irradiation that a tissue scaffold is able to tolerate can be determined. The procedure further comprises dose map testing to assess a dose distribution within a tissue scaffold surface. Ihe dose distribution comprises a minimum and maximum internal dose. Electron beam irradiation can then be applied at a “sub-lethal’’ dose below the minimum dose for sterility at a bioburden level. These dose determinations can be combinedAttorney Docket No. FES-003WOto determine a release dose. During the sterilization process the release dose can indicate when the required internal dose for sterilization is reached.Methods of Loading Tissue Scaffold Materials with Antimicrobial

[0104] Provided herein are methods of loading disclosed tissue scaffold materials (e.g., hydrogels and / or microbeads) to one or more antimicrobials of the disclosure. In embodiments, the loading involves chelation of the antimicrobial to the collagen of the tissue scaffold material. In embodiments, the method involves conditioning (e.g., submerging) the tissue scaffold material with an conditioning buffer and an antimicrobial, to create conditions to chelation of the antimicrobial to the collagen of the tissue scaffold material.

[0105] In some embodiments, the conditioning buffer comprises piperazine-N. N'-bis(2-ethanesulfonic acid) (PIPES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-l,3-diol (bis-TRIS), 2-morpholinoethanesulfonic acid (MES), N-(2- acetamido)-2- aminoethanesulfonic acid (ACES), or any combination thereof. In embodiments, the conditioning buffer comprises PIPES. In embodiments, the conditioning buffer comprises MOPSO. In embodiments, the conditioning buffer comprises HEPES In embodiments, the conditioning buffer comprises bis-TRIS. In embodiments, the conditioning buffer comprises MES. In embodiments, the conditioning buffer comprises ACES.

[0106] In embodiments, the method comprises rinsing the tissue scaffold material with the conditioning buffer (e.g., MOPSO). In embodiments, the tissue scaffold material is rinsed with the conditioning buffer (e.g., MOPSO) one or more times. In embodiments, the tissue scaffold material is rinsed with the conditioning buffer (e.g., MOPSO) three times.

[0107] In embodiments, the method comprises submerging the tissue scaffold material in a antimicrobial solution comprising the antimicrobial (e.g., silver or copper) and the conditioning buffer (e.g., MOPSO).

[0108] In some embodiments, the antimicrobial solution comprises PIPES, MOPSO, HEPES, bis-TRIS, MES, ACES), or any combination thereof. In embodiments, the antimicrobial solution comprises PIPES. In embodiments, the antimicrobial solution comprises MOPSO. In embodiments, the antimicrobial solution comprises HEPES In embodiments, the antimicrobial solution comprises bis-TRIS. In embodiments, the antimicrobial solution comprises MES. In embodiments, the antimicrobial solution comprises ACES. In embodiments, the antimicrobial solution has a pH value ranging from about 4 to about 8, from about 4.5 to about 7.5, or from about 5 to about 7 In embodiments, the antimicrobial solution has a pH value of 6.0±2.0,Attorney Docket No. FES-003WO6.0±1.5, 6.0±1.0, 6.0±0.9, 6.0±0.8, 6.0±0.7, 6.0±0.6. 6.0+0.5, 6.0±0.4, 6.0±0.3, 6.0±0.2. or 6.0±0.1 (e.g.. 6.0).

[0109] In embodiments, the antimicrobial solution comprises the antimicrobial (e.g., silver or copper) at a concentration ranging from about 0.001 mM to about 500 mM.

[0110] In embodiments, the tissue scaffold material is submerged in the antimicrobial solution for longer than about 1 hour, longer than about 2 hours, longer than about 3 hours, longer than about 4 hours, longer than about 5 hours, longer than about 6 hours, longer than about 7 hours, longer than about 8 hours, longer than about 12 hours, longer than about 16 hours, longer than about 20 hours, or longer than about 24 hours.

[0111] In embodiments, the method comprising (e.g., upon submerging) washing the tissue scaffold material with a physiological solution. Without wishing to be bound by theory, the washing may readjust the pH value and / or osmolarity of the tissue scaffold material.

[0112] In embodiments, the physiological solution comprises a physiological buffer. In embodiments, the physiological solution comprises phosphate-buffered saline (PBS). In embodiments, the physiological solution has a pH value ranging from about 6.0 to about 9.0, from about 6.5 to about 8.5, or from about 7.0 to about 8.0. In embodiments, the antimicrobial solution has a pH value of 7.4±2 0, 7.4±1.5, 7.4±1.0, 7.4±0.9, 7.4±0.8, 7.4+0.7, 7.4±0.6, 7.4±0.5, 7.4±0.4, 7.4±0.3, 7.4±0.2, or 7.4±0.1 (e.g., 7.4).

[0113] In embodiments, the tissue scaffold material is washed with the physiological solution (e.g., PBS) for one or more times. In embodiments, the tissue scaffold material is washed with the physiological solution (e.g.. PBS) three times.Methods of Using Tissue Scaffold Materials Described Herein

[0114] Several exemplary assays and metrics can be used to assess the antimicrobial activity of the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, the assays comprise crystal violet assays of biofilms, inhibition in liquid Mueller-Hinton medium, agar diffusion assay, time-kill kinetics, quantitative suspension test, zone of inhibition method, microbial viability assays, or electrochemical methods. In embodiments, the metrics comprise zone of inhibition with disk diffusion and agar, minimum inhibitory concentration (MIC), log or concentration reduction, quantitative suspension or direct contact tests, percent inhibition, MIC50 or MIC90, and time.

[0115] In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) further comprising an antimicrobial has at least about 2-fold higher microbicidal activity than a reference scaffold without the antimicrobial that is otherwise identical. Tn embodiments, theAttorney Docket No. FES-003WOtissue scaffold materials (e.g., hydrogels and / or microbeads) comprising the antimicrobial have a least a 2-fold to 10-fold higher microbicidal activity' compared to a reference tissue scaffold material (e.g., hydrogels and / or microbeads) without the antimicrobial that is otherwise identical (e.g., 2-fold, 3-fold. 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold, including any values and ranges therein). In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising the antimicrobial have a least a 2-fold. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher microbicidal activity compared to a reference tissue scaffold material (e.g., hydrogels and / or microbeads) without the antimicrobial that is otherwise identical.

[0116] In embodiments, the tissue scaffold materials (e.g., into hydrogels and / or microbeads) disclosed herein reduce or prevent infection in a treated subject In embodiments, a subject treated with a tissue scaffold material (e.g.. hydrogels and / or microbeads) disclosed herein has about 10% to about 100% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. including any values or ranges therein). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has about a 50% to about 100% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, the increase in microbial inhibition at a wound site is about 60% to about 100%, about 70% to about 100%, about 80% to about 100%. about 90% to about 100%. about 50% to about 90%, about 60% to about 90%, about 70% to about 90%. about 80% to about 90%. about 60% to about 80%, about 70% to about 80%, or about 60% to about 70%, including any ranges and values therein.

[0117] In embodiments, a subject treated with the tissue scaffold material (e.g.. hydrogels and / or microbeads) has an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or about 100% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has at least about a 50% increase in microbial inhibition present at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and'or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g.. hydrogels and / or microbeads) has about a 50% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold materialAttorney Docket No. FES-003WO(e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has about a 75% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and'or microbeads) has about a 100% increase in microbial inhibition at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a tissue scaffold material (e.g.. hydrogels and / or microbeads) reduces or prevents infection in a treated subject.

[0118] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein reduce or prevent infection in a subject in need. Moreover, the tissue scaffold materials (e.g,, hydrogels and / or microbeads) disclosed herein may reduce incidence of infection in a treated subject as compared to an otherwise comparable subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has about 10% to about 100% reduction in incidence of infection as compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) (e.g.. 10, 15, 20, 25. 30, 35. 40, 45, 50, 55, 60, 65, 70, 75, 80. 85, 90, 95, or 100%, including any values or ranges therein). In embodiments, a subject treated wath the tissue scaffold material (e.g., hydrogels and / or microbeads) has about a 50% to about 100% reduced incidence of infection as compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In aspects, the reduction is detected from about 1 day, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 5 months, or 6 months following the initial administering of a provided tissue scaffold material (e.g., hydrogels and / or microbeads).[01191 In embodiments, tissue scaffold material (e.g., hydrogels and / or microbeads) disclosed herein may reduce or prevent infection in a treated subject. In embodiments, a subject treated with a provided tissue scaffold material (e.g., hydrogels and / or microbeads) has about 10% to about 100% reduction in microbial concentration at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. 95, or 100%, including any values or ranges therein) In embodiments, the reduction in microbial concentration present at a wound site is about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 50% to about 90%, about 60% to about 90%. about 70% to about 90%. about 80% to about 90%, about 60% to about 80%, about 70% to about 80%, or aboutAttorney Docket No. FES-003WO60% to about 70%, including any ranges and values therein. In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or about 100% reduction in microbial concentration present at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g.. hydrogels and / or microbeads) has at least about a 50% reduction in microbial concentration at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has about a 50% reduction in microbial concentration at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold material (e.g., hydrogels and / or microbeads) has about a 75% reduction in microbial concentration at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a subject treated with the tissue scaffold matenal (e.g., hydrogels and / or microbeads) has about a 100% reduction in microbial concentration at a wound site compared to a subject not treated with the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, a tissue scaffold material (e.g., hydrogels and / or microbeads) reduces or prevents infection in a treated subject.

[0120] Several exemplary assays and metrics can be used to assess cell viability and proliferation related to the tissue scaffold material (e.g., hydrogels and / or microbeads). In embodiments, the assays comprise MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) assay. MTS ((3-(4.5-Dimethylthiazol-2-Yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay, live / dead cell viability, alamar blue assay, XTT ((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)) assay, WST-1 (Water-Soluble Tetrazolium Salt-1) assay, cytotoxicity assay, flow cytometry, confocal microscopy, BrdU (Bromodeoxy uridine) incorporation assay, and / or ATP (adenosine triphosphate) bioluminescence assay.

[0121] In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) further comprising cells improves wound healing in a subject by7about 5% to about 100% in comparison to reference tissue scaffold material (e.g., hydrogels and / or microbeads) without cells that is otherwise identical (e.g., 5, 10. 15. 20, 25, 30, 35, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90. 95 or 100%, including all values and ranges therein). In embodiments, the tissueAttorney Docket No. FES-003WOscaffold materials (e.g.. hydrogels and / or microbeads) comprising cells improve wound healing in a subject by about 25% to about 100%, about 50% to about 100%. about 75% to about 100%, about 5% to about 75%, about 25% to about 75%, about 50% to about 75%, about 5% to about 50 percent, about 25% to about 50%, or about 5% to about 25%, including all values and ranges therein. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising cells have at least 50% improvement in wound healing in a subject in comparison to a reference tissue scaffold material (e g., hydrogels and / or microbeads) without cells that is otherwise identical.

[0122] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising cells improve wound healing in a subject by about 2-fold to about 10-fold in comparison to reference tissue scaffold material (e.g., hydrogels and / or microbeads) without cells that is otherwise identical (e.g.. 2, 3, 4. 5, 6, 7, 8, 9, or 10-fold, including all values and ranges therein.) In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising cells improve wound healing in a subject by about 2-fold to about 10- fold, about 4-fold to about 10-fold, about 6-fold to about 10-fold, 2-fold to about 8-fold, about 4-fold to about 8-fold, about 6-fold to about 8-fold, about 2-fold to about 6-fold, about 4-fold to about 6-fold, or about 2-fold to about 4-fold, including all ranges and values therein. In embodiments, the tissue scaffold material (e.g., hydrogels and / or microbeads) comprising cells improve wound healing in a subject by at least 2-fold in comparison to a reference tissue scaffold material (e.g., hydrogels and / or microbeads) without cells that is otherwise identical.Dressings

[0123] Provided herein are dressings comprising tissue scaffold materials (e.g., hydrogels and / or microbeads). In embodiments, the dressings comprise a tissue scaffold material (e.g., hydrogels and / or microbeads) that comprises a) a microsphere comprising a polymer: and b) one or more of a cell and an anti -microbial. In embodiments, the dressing comprising a tissue scaffold material (e.g,, hydrogels and / or microbeads) further comprises a hydrogel comprising a polymer. In embodiments, the dressing comprising a tissue scaffold matenal (e.g., hydrogels and / or microbeads) comprises microspheres embedded in the hydrogel. In embodiments, the microspheres have a different density than the hydrogel. In embodiments, the dressings comprise a tissue scaffold material (e g., hydrogels and / or microbeads) comprising a cell, an anti-microbial or both.

[0124] Moreover, provided herein are wound dressings and medical products into which the tissue scaffold material (e.g., hydrogels and / or microbeads) disclosed herein are integrated. TheAttorney Docket No. FES-003WOtissue scaffold material (e.g., hydrogels and / or microbeads) described above can be embedded into the dressing, or deposited on one side of the dressing. In embodiments, the dressing can further include one or more of silicone, gauze, or other covering, and / or an antibiotic, antiinflammatory or pain reducing agent or other ointment to facilitate healing or reduce pain.

[0125] The tissue scaffold material (e.g., hydrogels and / or microbeads) product can be further suitably packaged, such as in sterile packaging, for use in wound healing and / or tissue regeneration.Methods of Treatment

[0126] Further disclosed herein are methods to promote wound healing or tissue regeneration in a subject in need thereof, by applying the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein to a wound or tissue of the subject. The tissue scaffold materials (e.g., hydrogels and / or microbeads) can be applied, for example, to any area of the subject in which tissue regeneration is desired, such as application to an open wound or during the course of a surgical procedure. In embodiments, the disclosed tissue scaffold materials (e.g., hydrogels and / or microbeads) are applied to areas of the body with exposed bone, hardware, or necrotic tissue.

[0127] Provided are also methods of wound healing comprising administering the tissue scaffold materials (e.g., hydrogels and / or microbeads) described herein to a subject in need thereof.

[0128] The tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein can be removed or remain in place. The polymer can be biodegradable and in such cases will gradually dissolve, leaving behind a new network of cells and vasculature formed from the subject’s cells.[01291 The tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein can be in a flowable form suitable for injection into a subject, or in a sheet form, for example, a sheet with a depth of 0.5-5.0 mm, or 1-2 mm.

[0130] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein are used to treat a wound of a subject in need thereof. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising an antimicrobial are used to treat a wound of a subject in need thereof. In embodiments, the antimicrobial is a metallic agent or antibiotic.

[0131] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising cells are used to treat a wound of a subject in need thereof. In embodiments, theAttorney Docket No. FES-003WOcells are derived from skin tissue. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising cells further comprise an antimicrobial. In embodiments, tissue scaffolds comprising human cells are used to treat a wound of a subject in need thereof. Tn embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising fibroblasts are used to treat a wound of a subject in need thereof. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising endothelial cells are used to treat a wound of a subject in need thereof. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising fibroblasts and endothelial cells are used to treat a wound of a subject in need thereof. In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising human regenerative cells are used to treat a wound of a subject in need thereof.

[0132] In embodiments, tissue scaffold materials (e.g., hydrogels and / or microbeads) comprising an antimicrobial metallic agent and cells are used to treat a wound of a subject in need thereof. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) comprise a mixture of a metal agent comprising silver or copper and cells comprising fibroblasts or endothelial cells. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein comprise a mixture of silver and fibroblast. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein comprise a mixture of silver and endothelial cells. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein comprise a mixture of copper and fibroblast. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein comprise a mixture of copper and endothelial cells.

[0133] In embodiments, a subject using the tissue scaffold materials (e.g., hydrogels and / or microbeads) of the disclosure results in a subject's wound healing about 1 day to about 2 months faster than a subject not using the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein. In embodiments, the subject using the tissue scaffold materials (e.g., hydrogels and / or microbeads) experience wound healing at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least one week, at least two weeks, at least three weeks, at least 4 weeks or at least two months faster than a subject not using the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein. In embodiments, the subject using the tissue scaffold materials (e.g., hydrogels and / or microbeads) experience wound healing at least 2 days faster than a subject not using the tissue scaffold materials (e.g., hydrogels and / or microbeads) disclosed herein. In embodiments, the subject using the patchesAttorney Docket No. FES-003WOdisclosed herein experience wound healing at least 2 days faster than a subject not using the patches disclosed herein.

[0134] In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) enables a wound to heal in a time period comprising about 1 day to 2 months. In embodiments, the tissue scaffold materials (e.g., hydrogels and / or microbeads) enables a wound to heal in time period comprising about 2 days to about 2 months, about one week to about 2 months, about 2 weeks to about 2 months, about 3 weeks to about 2 months, about 4 weeks to about 2 months, about 5 weeks to about 2 months, about 6 weeks to about 2 months, about 7 weeks to about 2 months, including all values and ranges therein.

[0135] Provided are tissue scaffold materials (e.g., hydrogels and / or microbeads) that can be used to treat a variety of wounds. In embodiments, the wounds can range in severity. In embodiments, the wounds can comprise minor wounds, moderate wounds, and severe wounds. In embodiments, minor wounds may comprise scrapes. In embodiments, moderate wounds may comprise deeper cuts or surgical incisions. In embodiments, severe wounds may comprise large bums or significant trauma. In embodiments, wounds may comprise excisional wounds, incisions, lacerations, abrasions, puncture wounds, avulsions, bums, pressure ulcers, gunshot wounds, contusions, or chronic wounds.

[0136] Tn embodiments, the tissue scaffold materials (e g., hydrogels and / or microbeads) disclosed herein may heal 'ounds comprising a depth from about 0.1 mm to 10 cm. In embodiments, the 'ound may comprise a depth from about 0.1 mm to about 2 mm. about 2 mm to about 5 mm or about 5 mm to about 10 cm, including all values and ranges therein. In embodiments, the depth of the wound comprises about 0.5 mm to about 10 cm, about 1 mm to about 10 cm, about 2 mm to about 10 cm, about 3 mm to about 10 cm, about 4 mm to about 10 cm, about 5 mm to about 10 cm, about 6 mm to about 10 cm, about 7 mm to about 10 cm, about 8 mm to about 10 cm, about 9 mm to about 10 cm, about 1 cm to about 10 cm, about 5 cm to about 10 cm, or about 0.1 mm to about 5mm, about 0.5 mm to about 5 mm, about 1 mm to about 5 mm, about 2 mm to about 5 mm, about 3 mm to about 5 mm, about 4 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.5 mm to about 2 mm, or about 1 mm to about 2 mm, including any values and ranges therein. In embodiments, the tissues scaffolds disclosed herein may treat superficial wounds, partial-thickness wounds, and full-thickness wounds.

[0137] The present disclosure is further illustrated by the following non-limiting examples.EXAMPLESExample 1. Production of tissue scaffoldsAttorney Docket No. FES-003WO

[0138] Purpose: Tissue scaffolds containing microspheres and / or hydrogels were produced.

[0139] Methods: Collagen type I was extracted from rat tail samples using standard techniques. Skin was removed from rat tails using sharp dissection and discarded. Then, starting from the distal end of the tail, tendons were extracted by breaking a joint within the vertebrae and pulling upward on the distal vertebrae until the distal vertebrae with attached tendon separated from the remaining proximal tail. The vertebrae was then sharply dissected from the tendon and discarded. Next, the tendon was placed in 70% ethanol. This was repeated until all joints within the tail were broken and tendons extracted. The extracted tendons were collected, weighed and placed in a sterile 1 L container. Thereafter, 0.1% acetic acid was added to the tendons to reach a final concentration of 75 ml of acetic acid / g of tendon in order to arrive at a stock collagen solution of 15 mg / mL (1 5% w / v) type I collagen. The collagen stock was then stored at 4 °C and agitated for approximately 1 minute daily for at least 72 hours.

[0140] After 72 hours, the collagen stock was aliquoted into 50 mL conical tubes, centrifuged at 4 °C and 8800 rpm for 90 minutes, and any pellet removed and discarded. The final 15 mg / 'mL (1.5 % w / v) collagen stock was then placed in a standard lyophilizer and lyophilized for at least 72 hours. Following lyophilization, collagen stock was stored at -4° C until use. Upon use, this lyophilized collagen was resuspended in 0.1% acetic acid to a concentration of 10 mg / mL (1% w / v). This resuspended collagen was agitated daily (for approximately 1 min) for 3 days prior to use. Stock solutions of 1.5% (w / v) collagen and 0.384% (w / v) collagen were used to create microspheres and 0.3% hydrogels, respectively.

[0141] To neutralize collagen to make 1% microspheres, 2 ml of 1.5% collagen was mixed with 656 µL of 1X M199 medium (Gibco / Life Technologies, Inc.), 300 µL of 10X M199 medium, and 44 µL NaOH (or more NaOH as needed to adjust pH to 7.4). on ice. This mixture was overlayed with at least 5 times volume (e.g., 15 ml) of mineral oil, and stored at 4° C until use.

[0142] Microspheres were produced using the following two methods. Tn one method to produce microspheres, neutralized collagen with oil overlay was mixed by highspeed vortexing for about 5 minutes to create a water-in-oil emulsion. The emulsion was then poured into a flask, combined with at least 5 volumes of 50% ethanol per volume of collagen solution minus oil, and stirred with a stir bar at 1100 rpm for 30 minutes. The stirred mixture w'as then poured into a 50 ml tube, and centrifuged at 3200 rpm at 4° C for 7 minutes to form oil and ethanol layers with a thin layer of collagen between the oil and alcohol layers. The oil and alcohol layers were removed, the collagen layer was washed with 5 volumes of 80% ethanol, vortexed and centrifuged as above, alcohol layer removed, washed with 5 volumes of 100% ethanol,Attorney Docket No. FES-003WOvortexed and centrifuged, and the alcohol layer removed. The collagen was then washed for three rounds with 5 volumes of cold Phosphate buffered saline (PBS), vortexed and centrifuged, and PBS removed. During this process, collagen microspheres were formed.

[0143] In a second method to produce microspheres, a collagen composition with oil overlay was mixed at high speed to form an oil-in water emulsion. The polymer composition further contained at least one type of bioactive factor (e.g., growth factor). The emulsion was added to a basic solution to stabilize the microspheres, which were then washed with methanol to remove excess base. Subsequently, the microspheres were further stabilized through crosslinking with diisocyanates, and were washed with methanol, water, and buffer to remove unreacted reagents.

[0144] To prepare collagen “bulk" for hydrogels. 391 pL of 0.384% collagen was mixed with 50.8 pL of IX M 199 medium, 50 pL of 1 OX Ml 99 medium, and 8.6 pL NaOH (or more NaOH as needed to adjust pH to 7.4), on ice. This mixture can then be used to make scaffolds, as follows.

[0145] To make the scaffolds, molds were used with a diameter of 7 mm and a depth of 2.5 mm to create a scaffold of approximately 96 mm3. To make microsphere scaffolds, microspheres produced by the methods above were pipetted into each well to fill each well about half full. One drop of the collagen bulk was added to each well, and mixed with the microspheres by stirring, to form a hydrogel embedded with microspheres. The scaffolds were then cured at 37° C for 30 minutes. PBS was overla ed on the cured scaffolds to prevent further drying. To make “bulk” scaffolds, collagen bulk was added to the molds, w ithout microspheres, to approximately the same level as the scaffolds with microspheres. The scaffolds were cured as above and overlayed with PBS. Alternatively, scaffolds were prepared by mixing microspheres with a telocollagen solution (composed of 5 mg / mL telocollagen, 0.1 N NaOH, lx PBS. and DI water in a ratio of 0.6:0.102:0.1:0.198) at a 70:30 ratio. The mixture was then poured into molds and allowed to gel at 37°C for 16 hours.

[0146] According to Kepler’s conjecture of close-packed spheres, approximately 74% of the volume of the scaffold should be comprised of higher density' microspheres, with the remaining volume taken up by the bulk collagen hydrogel.Example 2. Microsphere containing scaffolds promote cellular infiltration.

[0147] Scaffolds were produced one day prior to implantation. Scaffolds were implanted subcutaneously in the dorsa of 8 week old wild-type C57bl / 6 mice. 3 mice were implanted with 4 total scaffolds as follows: Two 1% microspheres in 0.3% bulk scaffolds; one 1% bulkAttorney Docket No. FES-003WOscaffold as a control; one 0.3% bulk scaffold as a control. All mice were sacrificed and harvested for histological analysis after 7 or 14 days. Hematoxylin and eosin (H& E) staining was performed on tissue samples embedded in optimal cutting temperature compound (OCT) medium, to identify cellular infiltration into scaffolds.

[0148] After 7 days of implantation, the microsphere scaffolds (MSS) show substantial and uniform cellular invasion spanning the entire depth of the scaffold (FIG. 1C). Comparatively, cells sporadically and only partially invaded the 0.3% control scaffolds (FIG. IB), and failed to invade the 1 % control scaffolds, instead proliferating along the periphery' of the scaffolds (FIG.1A).

[0149] After 14 days of implantation, MSS revealed robust cellular invasion spanning the scaffold depth (FIG 2C). Comparatively, ceils sporadically invaded 0.3% (w / v) collagen scaffolds (FIG. 2B) and failed to invade 1% (w / v) collagen scaffolds altogether, instead remaining confined to the periphery' (FIG. 2A).Example 3, Different densities of microspheres relative to hydrogel density promote cellularinfiltration.

[0150] Microsphere scaffolds with different densities (w / v) of collagen m microsphere (MS) and hydrogel (H) were prepared as follows: (A) 1% collagen MS in 0.3% H; (B) 0,6% MS / 0.3% H; (C) 0.4% MS / 0.2% H; (D) 0.4% MS / 0.6% H. See, Table 1.Table 1. Densities of Microsphere ScaffoldsMicrosphere Collagen Density (w / v) Bulk Collagen Density (w / v)1% 0.3%0.6% 0.3%0.4% 0.2%0.4% 0.6%

[0151] MSS were implanted subcutaneously in the dorsa of adult mice and harvested for immunohistochemistry at 7 and 14 days after implantation. Immunohistochemical analy sis identified cellular infiltration in all MSS (FIGS. 3A-3D), with greatest infiltration seen in 1% MS / 0.3% H, and 0.6% MS 0.3% H (FIGS. 3C- 3D). In addition, CD31 expression was seen in all MSS after 7 and 14 days of implantation (FIGS 4A-4B), indicative of invading endothelial precursors and the formation of neovasculature.Example 4. MSS promotes cellular infiltration over 28 day implantation.Attorney Docket No. FES-003WO

[0152] Eighteen mice received four subcutaneous implants (A-D) per mouse as follows: (A) MSS (1% collagen microspheres in 0.3% collagen bulk), (B) 1% bulk collagen hydrogel control, (C) 0.3% collagen hydrogel control, and (D) 7 mm diameter section of INTEGRA Dermal Regeneration Template (Integra LifeSciences, Plainsboro, NJ). Mice were sacrificed at 7, 14, and 28 days post-implantation (6 mice per time point).

[0153] At 7 days after implantation (FIGS. 5A-5D). MSS, 1% collagen control, and INTEGRA scaffolds retained similar size and morphology relative to pre-implantation, while 0.3% collagen control was noticeably reduced in size (FIG. 5D). H& E staining of MSS I week after implantation reveals invasion of cells all the way to the center of the scaffold (FIG. 6A). By comparison, there is no invasion of the 1% collagen scaffolds (FIG. 6B), except along cracks where the material has split. There was also minimal invasion into the shrunken 0.3% collagen scaffold (FIG. 6C). Fluorescent staining of tlie MSS template with CD31 antibodies (to identify endothelial progenitor cells) and DAPI (to identify infiltrating cells) shows that multiple cell types, including endothelial progenitor cells, are already infiltrating the MSS scaffold at 7 days (FIG. 7), CD31+ cells were not observed within 1% and 0.3% hydrogel controls (data not shown).

[0154] After 14 days (FIGS. 8A-8E), the MSS, 1% collagen control, and INTEGRA scaffolds are still close to pre-implantation size, while 0.3% collagen control is dramatically reduced in size (FIG. 8E). The MSS scaffold shows significant cellular invasion (FIG. 9A), the 1% collagen displays minimal invasion except along fissures (FIG. 9B), and the 0.3% collagen scaffold shows sparse invasion (FIG. 9C). The INTEGRA scaffold showed less robust invasion than in the MSS scaffold (FIG. 9D); the dense structure of the INTEGRA scaffold led to shearing of the scaffold during sectioning for H& E staining.

[0155] A comparison of cell count per unit scaffold area (FIG. 10) show's that significantly more cells invaded the MSS scaffold at 7 and 14 days (approximately 7 cells and 10 cells per area, respectively) relative to 1 % hydrogel (approximately 3 and 5 cells per unit area) and 0.3% hydrogel (approximately 3 and 7 cells per unit area)

[0156] At 28 days post-implantation (FIGS. 11A-11D), the MSS. 1% collagen control, and INTEGRA scaffolds are slightly smaller than pre-implantation size, while 0.3% collagen control is smaller than at 7 or 14 days (FIG. 1 ID). The MSS scaffold at 28 days show's good cellular invasion (FIG. 12A), the 1% collagen displays essentially no invasion (FIG. 12B), and the 0.3% collagen scaffold shows invasion despite its small size (FIG. 12C). Tire INTEGRA scaffold showed some invasion as well (FIG. 12D).Attorney Docket No. FES-003WOExample 5. Scanning electron microscopy of microspheres.

[0157] Microspheres were prepared as in Example 1 and prepared for scanning electron microscopy (SEM). As seen in FIG 13, microspheres can van' in size (between 50-300 pm) and in shape (some are highly spherical, while others are irregular in morphology).Example 6. Adherence of MSS to various substrates using electron beam irradiation

[0158] Purpose: This study sought to evaluate the adherence of a MSS to various substrates using high energy electron beam irradiation.

[0159] Methods: This study sought to assess the adherence of collagen samples sandwiched between films of varying surface chemistry. The initial study comprised a total of 6 sample groups.

[0160] The scaffolds assessed comprised the following:a. MSS irradiated;b. MSS non irradiated;

[0161] The substrates / films assessed comprised the followinga. Polyethylene terephthalate (PEI);b. Thermoplastic polyurethane (TPU): andc. Silicone.

[0162] To make the scaffolds, 3x3 cm2wells were filled with collagen solution and gelled at 37 °C for 16 hours. Scaffolds were then sandwiched between films of varying surface chemistries (silicone, PET, or TPU) and packaged. For each group corresponding to each film, 8 samples were prepared, half of w'hich (n::::3) were irradiated with electron beam at 30 kGy and the other half served as non-irradiated controls.

[0163] Upon return of the electron beam irradiated samples, the films were separated from the collagen samples and scored based on their peelability. The films were first removed from the plastic encasement and silicone mold used during shipping The first film was then separated from the collagen sample and an image was taken of the resulting peel. Representative images of peeled films from the MSS groups with each of the four substrates are shown in FIGs. 14A-14E. The peeled film was then graded on a scale of 1-4: (1) Product failure (full adhesion); (2) Residue but removable; (3) Resistance but clean peel and (4) Clean peel and no resistance. The second film was then removed and an image was taken of the resulting peel. The film w as also graded on the same scale of 1-4. The lower score of the two sides was taken. Peeling scores of the irradiated samples were then compared to the non¬ irradiated samples (controls).Attorney Docket No. FES-003WO

[0164] Results of the gradient scoring are in FIG. 15. As shown, irradiation results in a lower peeling score, which suggests that peeling the film from the hydrogel (collagen) is more difficult after irradiation. In sum, the irradiation led to bonding of the collagen onto the film.Table 2. Summary of peeling scoresPET Silicone TPU Repl Rep2 Rep3 Repl Rep2 Rep3 Repl Rep2 Rep3 Collagen 4 4 — 4 4 __ 4 4 — (irradiated)Collagen 2 2.5 2 2 1.5 2.5 2 3 3(non-irradiated)

[0165] In sum, these findings demonstrated that when a collagen hydrogel was exposed to electron beam irradiation, it successfully adhered to various substrates. Given the electron beam irradiation induces hemolytic cleavage of C-C and C-H bonds on polymers, it is likely the generated alkyl radicals are unstable and undergo a complex series of reactions. These reactions can comprise crosslinking, chain scission, oxidation, and creation of alkene bonds. Likely some of these reactions may happen at the interface between the polymer and substrate and may therefore lead to covalent bonding between the two. As a result, high water-content hydrogels may exhibit strong bonding to surfaces if the adhesion force is much greater than the cohesive force of the hydrogel.Example 7, Production and adhesion of irradiated tissue scaffolds containing an antimicrobial and / or ceils

[0166] Purpose: Tissue scaffolds containing microspheres and / or hydrogels will be produced as in Example I with an antimicrobial and / or cells. This study will seek to evaluate the adherence of tissue scaffolds containing an antimicrobial and / or cells to various substrates using high energy electron beam irradiation as in Example 6.

[0167] Methods: For this study tissue scaffolds, microspheres and microsphere containing tissue scaffolds, will be generated as in Example 1. Antimicrobials and / or cells will be added to the precursor tissue scaffold solution before gelation. After gelation, the tissue scaffolds will be adhered to a substrate with electron beam irradiation at 30 kGy using the protocol described in Example 6. Substrates may include, but are not limited to, PET, TPU and silicone. Nonirradiated samples comprising the same tissue scaffold with and without the antimicrobial and / or cells will serve as controls. Adherence with collagen samples sandwiched between films of varying surface chemistry will then be assessed.Attorney Docket No. FES-003WO

[0168] To test adherence following electron beam irradiation of the samples, the films will be separated from the collagen samples and scored based on their "‘PeelabilityT The films will first be removed from their plastic encasement and silicone mold. Images will be taken of the resulting peel. The peeled film will then be graded on a scale of 1-4: (1) Product failure (full adhesion); (2) Residue but removable; (3) Resistance but clean peel; and (4) Clean and no resistance. The lower score of the two sides will be taken. Peeling scores of the irradiated samples will then compared to the non-irradiated samples.Example 8. Assessment of antimicrobial properties of the irradiated tissue scaffolds

[0169] Purpose: The study will evaluate the antimicrobial activity' of the tissue scaffolds of Example 7 that contain an antimicrobial.

[0170] Method: The irradiated tissue scaffolds containing an antimicrobial, with or without cells, generated in Example 7 will undergo antimicrobial assays. The microbial inhibition of tissue scaffolds will be compared to tissue scaffolds not comprising the antimicrobial that are otherwise identical. Antimicrobial activity will be assessed using a variety of assays including a disk diffusion assay and / or a minimum inhibitory concentration assay. In an exemplary protocol for disk diffusion, a sample of the hydrogel containing the antimicrobial agent will be placed on an agar plate inoculated with a bacterial culture. The zone of inhibition around the sample will be measured to assess the antimicrobial effect and compared to control samples lacking tire antimicrobial. Optionally, the hydrogel samples can also be placed in well punched into an inoculated agar plate. Moreover, m an exemplary protocol of a minimum inhibitory concentration assay, a series of dilutions of the antimicrobial hydrogel will be prepared and tested in liquid culture or microtiter plates.

[0171] In addition, a combination of other antimicrobial activity assays can also be used including but not limited to a minimum bactericidal concentration assay, broth dilution method, time-kill assays, in vitro cytotoxicity assays, quantitative PCR, and microbial adhesion assays.Example 9. Assessment of cell viability and proliferation using the irradiated tissue scaffolds

[0172] Purpose: This study will evaluate cell viability and proliferation of the tissue scaffolds of Example 7 that contain cells.

[0173] Methods: The irradiated tissue scaffolds containing cells, with or without an antimicrobial, generated in Example 7 will undergo cell viability assays. Cell viability and proliferation of the tissue scaffolds containing cells will be assessed in comparison to tissueAttorney Docket No. FES-003WOscaffolds lacking cells that are otherwise identical. Cell viability and proliferation will be assessed using a variety of assays including but not limited to a live / dead assay, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) assay, and WST-1 (water-soluble tetrazolium salt-1) cell proliferation assays. In an exemplaiy protocol of the live / dead assay, cells in the tissue scaffolds will be incubated in two dyes including SYTO 9 to stain live cells and propidium iodide which stains dead cells. The stained cells will then be visualized under a fluorescence microscope. In an exemplary' protocol of the MTT assay, a yellow dye is applied to the cells that becomes reduced by metabolically active cells into a purple formazan product. After the cells embedded in or cultured on the tissue scaffold will be incubated with a MTT dye solution, the formazan product will then be measured by spectrophotometer to provide a measure of metabolic activity. In an exemplary protocol for WST-1 cell proliferation assay, a WST-1 reagent with an electrolyte solution is pipetted onto the tissue scaffolds. After incubation, the cultures w ill be shaken and the supernatant w ill be transferred to a 96-w ell plate. Photometric measurements w ill then be performed.

[0174] In addition, a combination of other cell viability and proliferation assays can also be used including but limited to a MTS assay, alamar blue assay, XTT assay, cytotoxicity' assays, confocal microscopy, flow' cytometry' Bromodeoxyuridine incorporation assay, and ATP bioluminescence assay.Example 10. Loading MSS with antimicrobial agents

[0175] A MS S (4 cm x 5 cm x 0.15 cm) w as prepared following the procedures of Example 1. The MSS was first preconditioned with an appropriate buffer of MOPSO (pH = 6). The MSS was then submerged in 20 mL of buffer w ith vary ing amounts of soluble copper / silver salt at concentrations ranging from 0.001 mM to 500 mM. After soaking for >16 hours, MSS was washed with a physiological buffer of PBS (pH = 7.4) to readjust pH and osmolarity.Example 11. Assessment of antimicrobial properties of MSS loaded with antimicrobial agents

[0176] To assess the antimicrobial properties of MSS loaded with antimicrobial agents, MSS samples (loaded and control unloaded samples) were inoculated with the bacteria and cultured for 24 hours. After 24 hours bacteria w'ere recovered and quantified.

[0177] The MSS was cut into square swatches (3.8 cmx 3.8 cm) from the treated and control templates. A bacterial suspension (Staphylococcus aureus and Klebsiella pneumoniae) was prepared, and was adjusted to a known concentration (around 1-3 x 103CFU / mL). The MSSAttorney Docket No. FES-003WOsamples were each placed into a sterile jar or flask. 1.0 mL of the inoculum was added onto the surface of each MSS sample. The inoculum were added evenly across the sample surface.

[0178] For 0-hour counts (control), 100 mL of neutralizing solution was immediately added, and the mixture was shaken to elute bacteria. For 24-hour counts (test samples and controls), the sample was incubated at 37 ± 2 °C for 24 ± 1 hours, then neutralized and shaken as above. Serial dilutions were prepared from each neutralized solution, and were plated on nutrient agar and incubate (typically 37 °C for 24 hours). Colony Forming Unit (CFU) was counted for both 0-hour and 24-hour samples

[0179] The percent reduction of bacteria was calculated using the formula below. The percent reduction was then converted to log reduction.R = (B. -. A)x 100Dwhere: A= CFU from treated MSS after 24 h; and B= CFU from untreated MSS (control = not loaded w idi antimicrobial ions) after 24 h.

[0180] Results are shown in FIGs. 15A-15D. MSS samples loaded with antimicrobial agents generally demonstrated substantial antibacterial activity' against both Staphylococcus aureus and Klebsiella pneumoniae. Across all tested concentrations and antimicrobial agent ty pes, all loaded MSS samples with > 0.1 mM antimicrobial agent treatment (regardless of ion type) achieved at least a two-fold reduction in bacterial load as compared with the control samples. Some treatments even produced multiple-log reductions, indicating strong anti-microbial effects.INCORPORATION BY REFERENCE

[0181] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form pail of the common general knowledge in any country in the w orld.

Claims

Attorney Docket No. FES-003WOCLAIMS1. A method of bonding a hydrogel to a substrate, the method comprising: contacting at least a portion of the hydrogel with a substrate: and irradiating the portion of the hydrogel and the substrate in an amount effective to bond the portion of the hydrogel to the substrate, wherein the hydrogel comprises collagen.

2. Tire method of claim I. comprising irradiating with a wavelength of about 1 pm to about 10 nm.

3. The method of claim 1 or 2, wherein the irradiating comprises a kinetic energy of about 10 keV to about 250 MeV.Ute method of any one of claims 1-3, wherein the irradiating comprises X-ray, gamma irradiation, or electron-beam5. The method of any one of claims 1-4. wherein the i rradiating comprises electron-beam.The method of claim 5, wherein the electron-beam produces a radiation of about 5 kGy to about 100 kGy.

7. The method of claim 6, wherein the electron-beam produces a radiation of about 30 kGy.

8. The method of any one of claims 4-7, wherein the electron-beam operates under an inert atmosphere.$>. The method of claim 8, wherein the inert atmosphere is argon, nitrogen, or vacuum.

10. Tire method of any one of claims 1-9, wherein the bonding is determined by a peelability assay.

11. The method of claim 10, wherein peelabilily is reduced by at least about 1 -point, 2- points, or 3-points, as determined by peelability assay.Attorney Docket No. FES-003WO12. The method of any one of claims 2-11, wherein the irradiating comprises a kinetic energy of about 1 MeV, 5 MeV. 10 MeV. 15 MeV. 20 MeV. 25 MeV. 30 MeV. 35 MeV. 40 MeV, 45 MeV or up to about 50 MeV13. The method of claim 12, wherein the irradiating comprises a kinetic energy' of about 10 MeV.

14. The method of any one of claims 1-13. wherein the substrate is selected from any one of silicone, polyesters, polyamides, thermoplastic polyurethane (TPU), polyethylene terephthalate (PET) and combinations thereof.

15. The method of claim 14, wherein the substrate comprises silicone.

16. The method of claim 14, wherein the substrate comprises poly ester.

17. The method of claim 14, wherein the substrate comprises polyamide.

18. The method of claim 14, wherein the substrate comprises TPU.

19. The method of claim 14, wherein the substrate comprises PET.

20. The method of claim 19, wherein the PET has a density of at least about 1 33 g / cm’.

21. The method of claim 15, wherein the silicone substrate has a density of at least about 1.05 g / cm³.

22. The method of claim 18, wherein the TPU substrate has a density of at least about 1.1 g / cm’.

23. The method of any one of claims 1-22, wherein the substrate comprises a film comprising a polymer.

24. The method of any one of claims 1 -23, wherein the substrate comprises a water content of about 90% w / v to about 99.9% w / v.Attorney Docket No. FES-003WO25. The method of anyone of claims 1-24. wherein the substrate further comprises one or more polymers.

26. The method of claim 25, wherein the polymer is a low weight percent polymer.

27. The method of claim 25 or 26. wherein the polymer comprises hydroxyl or carboxylic acid groups28. The method of any one of claims 1-27, wherein the irradiating further sterilizes the portion of the hydrogel and the substrate as determined by ISO 1113729. The method of any one of claims 1-28, wherein the collagen is type I collagen.

30. The method of claim 29, wherein the collagen is atelocoliagen or telocollagen.

31. A tissue scaffold material comprising a bonded hydrogel and substrate made utilizing the method of any one of claims 1-30.

32. The tissue scaffold material of claim 31, wherein the scaffold further comprises cells33. The tissue scaffold material of claim 31 or 32. wherein the scaffold further comprises an antimicrobial agent.

34. A tissue scaffold material that comprises: a) a microsphere comprising a polymer; and b) one or more of a cell and an anti-microbial.

35. The tissue scaffold material of claim 34, wherein the tissue scaffold material further comprises a hydrogel comprising a polymer.

36. The tissue scaffold material of claim 35, wherein the microsphere is embedded in the hydrogel.Attorney Docket No. FES-003WO37. The tissue scaffold material of claim 35 or 36, wherein the microspheres have a different density than the hydrogel.

38. The tissue scaffold material of any one of claims 34-37, wherein the tissue scaffold material comprises a cell.

39. The tissue scaffold material of any one of claims 34-37. wherein the tissue scaffold material comprises an anti-microbial40. The tissue scaffold material of any one of claims 34-39, wherein the tissue scaffold material comprises an anti-microbial and a cell.

41. The tissue scaffold material of any one of claims 34-40, wherein the tissue scaffold material comprises cells, and wherein the cells are human cells.

42. The tissue scaffold material of any one of claims 34-40, wherein the tissue scaffold material comprises cells, and wherein the cells are non-human43. The tissue scaffold material of claim 41, wherein the human cells are regenerative cells.

44. The tissue scaffold material of claim 41, wherein the human cells are non-regenerative cells.

45. The tissue scaffold material of claim 43. wherein the regenerative cells are embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow- derived stem or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSC), skeletal muscle-derived cells, multipotent adult progenitor cells (MAPC), skin tissue derived cells, bone tissue derived cells, cardiac stem cells (CSC), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, or aortic endothelial cells, bone marrow-derived stem cells, endothelial or vascular stem or progenitor cells, endothelial progenitor cells (EPC), and combinations thereof.

46. The tissue scaffold material of claim 44, wherein the non-regenerative cells comprise cardiomyocytes, neurons, lens cells, or combinations thereof.Attorney Docket No. FES-003WO47. The tissue scaffold material of any one of claims 34-46. wherein the tissue scaffold material comprises cells, and wherein the cells are derived from one or more of a tissue and organ.

48. Tire tissue scaffold material of claim 47, wherein the cells are derived from skin tissue.

49. The tissue scaffold material of any one of claims 34-48, wherein the tissue scaffold material comprises at least 1,000, 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000 cells.

50. The tissue scaffold material of any one of claims 34-49. wherein the tissue scaffold material comprises from about 1,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 10,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 1,000,000 cells / mg tissue, about 1,000 cells / mg tissue to about 100,000 cells / mg tissue, about 1,000 cells / mg tissue to about 10,000 cells / mg tissue, about 10,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 100,000 cells / mg tissue to about 100,000,000 cells / mg tissue, about 1,000,000 cells / mg tissue to about 100,000,000 cells / mg tissue, or about 10,000,000 cells / mg tissue to about 100.000,000 cells / mg tissue, including any ranges therebetween.

51. The tissue scaffold material of any one of claims 41 and 43-50, wherein die human cells are autologous to a subject in need.

52. The tissue scaffold material of any one of claims 41 and 43-50, wherein the human cells are allogeneic to a subject in need.

53. The tissue scaffold material of any one of claims 42 and 47-50, wherein the non-human cells are pig, murine, bovine, sheep, or canine.

54. The tissue scaffold material of any one of claims 34-53, wherein the polymer is selected from the group consisting of: collagen, atelocollagen, telocollagen, gelatin, elastin, hyaluronate, cellulose, fibrinogen. poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), polyflactic acid) (PLA), poly(caprolactone), poly(butylene succinate),Attorney Docket No. FES-003WOpolyftrimethylene carbonate), poly(p-dioxanone), and poly(butylene terephthalate); a polyester amide, a polyurethane. poly [(carboxy phenoxy) propane-sebacic acid], poly[bis(hydroxyethyl) terephthalate-ethyl orthophosphorylate / terephthaloyl chloride], a poly(ortho ester), a poly(alkyl cyanoacrylate), polyethylene glycol), a microbial polyester, poly(P- hydroxyalkanoate), and a tyrosine derived polycarbonate.

55. The tissue scaffold material of claim 54. wherein the polymer is collagen.

56. The tissue scaffold material of claim 54, wherein the polymer is atelocollagen.

57. The tissue scaffold material of claim 54. wherein the polymer is telocollagen.

58. The tissue scaffold material of any one of claims 34-57, wherein the microspheres are comprised of 1 % w / v, 1 % w / w, or 1 % v / v of the polymer.

59. The tissue scaffold material of any one of claims 34-58, wherein the tissue scaffold material comprises an anti-microbial, and wherein the anti-microbial is selected from the group consisting of antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, antiseptics, broad -spectrum antimicrobials, broad-spectrum biocidal materials and any combination thereof.

60. The tissue scaffold material of any one of claims 34-59, wherein the tissue scaffold material comprises an anti-microbial, and wherein the anti-microbial prevents or treats an infection selected from the group consisting of bacteria, fungi, viruses, parasites, prions and any combination thereof.

61. The tissue scaffold material of any one of claims 34-60, wherein the tissue scaffold material comprises an anti-microbial, and wherein the anti -microbial is a metal selected from any one of silver, copper, zinc. gold, titanium, nickel, tin, platinum, or a combination thereof.

62. The tissue scaffold material of claim 61. wherein the metal is silver, copper, or a combination thereof.Attorney Docket No. FES-003WO63. The tissue scaffold material of any one of claims 34-62, wherein the tissue scaffold material comprises an anti-microbial, and wherein the anti-microbial acts through a mechanism comprising inhibition of cell wall synthesis, disruption of cell membrane function, inhibition of protein synthesis, inhibition of nucleic acid synthesis, inhibition of metabolic pathways, or any combination thereof.

64. The tissue scaffold material of any one of claims 34-63. wherein the tissue scaffold material comprises an anti-microbial, and wherein the tissue scaffold material has at least about 2-fold higher microbicidal activity than a reference scaffold without the anti-microbial that is otherwise identical.

65. A method of promoting wound healing in a subject in need thereof comprising administering the tissue scaffold material of any one of claims 34-64 to the subject.

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