Porous scaffolds of hydrogels by cryogelation

Abstract

Compositions, method and systems comprise a freeze-dried, non-hydrated scaffold that is porous and contains bioactive components that are advantageous for tissue engineering and regenerative medicine purposes.

Description

[0001] Porous Scaffolds of Hydrogels by Cryogelation

[0002]

[0001] Government Support Clause

[0003]

[0002] This invention was made with government support under DE029463 awarded by the National Institutes of Health and under W81XWH-20-1-0839 and W81XWH-22-2-0013 awarded by the US Army Medical Research and Materiel Command. The government has certain rights in the invention.

[0004]

[0001] Introduction

[0005]

[0002] Cryogels are hydrogels that are crosslinked below the freezing temperature of the solvent and subsequently lyophilized to produce dry scaffolds with an interconnected microporous structure. While hydrogels, which are water-saturated polymer networks, have long been explored as robust materials for tissue regeneration, they are challenging to scale up and can be impractical in certain clinical and austere field settings. Cryogels provide an exciting, translatable alternative to traditional hydrogels as their pore structure allows immediate cell penetration and greater molecular diffusion compared to hydrogel scaffolds.

[0006]

[0003] Summary of the Invention

[0007]

[0004] The invention provides cyrogels and related compositions, methods and systems. The cyrogels comprise a porous scaffold of bioactive component sequestering hydrogel by cryogelation, substantially as described herein.

[0008]

[0005] The mechanical properties of the cyrogels are dependent on the concentration of polymer in the network, starting macromer molecular weight, the crosslinking density, and the freezing temperature, and their stiffness can be adjusted to match the surrounding tissues. Upon exposure to fluids (including blood), dry cryogel scaffolds swell immediately due to the capillary effect, facilitating the immediate infiltration of cells and bioactive molecules present in the fluid or blood. Cell adhesion to the cryogel scaffold can be modified and controlled by the conjugation of proteins, peptides, or other biological factors to the polymers in the networks. Growth factor and other bioactive molecules can also be sequestered in the material via the inclusion of proteins and other biological factors in the polymer network. Additional bioactive components included in the network could be released at a controllable rate to encourage conduction of endogenous cells into the material or differentiation of endogenous cells. Cryogel retention of endogenous cells and other bioactive factors, as well as their scalable synthesis and potential for controlled release properties, make them attractive as materials for tissue regeneration. By selecting polymers and crosslinkers, as well as fabrication conditions, it is possible to control the rate of degradation of the cryogel in the body.

[0009]

[0006] In an aspect the invention provides freeze-dried, non-hydrated scaffolds that are porous and contain bioactive components advantageous for tissue engineering and regenerative medicine purposes.

[0010]

[0007] Using hyaluronic acid hydrogel formulations (e.g. US9,827,272), we have developed a process to transform these hydrogels into dehydrated scaffolds by cryogelation. These scaffolds provide greater ease of long-term storage and surgical insertion, while maintaining the polymeric structure required for cellular infiltration, growth, and tissue formation.

[0011]

[0008] The hydrogels are polymerized (e.g. at -20C for 48 hours) to create cyrogels. Cryogels are then placed in a freeze dryer (e.g. at -80C and O.OlmBar for 48 hours) to dehydrate scaffolds while maintaining the hyaluronic acid polymer structure. By example, a porous scaffold of hyaluronic acid remains with peptide, heparin, and MMP crosslinker intact. Other biologies such as drugs, lipid nanoparticles, or exosomes / extracellular vesicles can survive this processing and be retained in the scaffold. This invention provides a powerful device for tissue engineering research, as well as for regenerative medicine to treat patients with significant loss of tissue injuries.

[0012]

[0009] Practical applications include muscle, tendon, ligament, nerve (tissue), bone, and vascular regeneration, wherein this process allows for this solid material to be easily implanted into a patient that has suffered traumatic injury such as volumetric muscle loss.

[0013]

[0010] Other practical applications include products for in vivo tissue regeneration, wherein the process of sub-freezing polymerization and lyophilization to produce easy-to-implant, porous microstructures is well suited for the creation of hyaluronic acid scaffolds for in vivo applications. A scaffold made from a highly tunable precursor that can be modified or even loaded with drug or other component is a powerful tissue engineering research tool.

[0014] [Oil] Other practical applications also include tissue engineering, wherein this process translates tunable hyaluronic acid hydrogel formulations into clinical treatments for loss-of- tissue disease or traumatic injury. A dehydrated scaffold can be pre-packaged and cut to size, is generally easy to surgically implant, and improves product shelf-life over its hydrated counterpart. Furthermore, maintaining scaffold porosity during the dehydration process facilitates creation of a matrix that allows a patient's native cells to infiltrate and grow within the material.

[0015]

[0012] In aspects and embodiments the invention provides:

[0016]

[0013] A composition, method or system comprising a porous scaffold of hydrogel by cryogelation, substantially as described herein.

[0014] A composition, method or system comprising a porous scaffold of bioactive component sequestering hydrogel by cryogelation, substantially as described herein.

[0017]

[0015] A composition, method or system comprising a freeze-dried, non-hydrated scaffold that is porous and contains bioactive components are advantageous for tissue engineering and regenerative medicine purposes.

[0018]

[0016] A composition, method or system herein, comprising HyA polymer chains, wherein biologically active peptides or other molecules or agents maybe engineered onto the HyA polymer chains.

[0019]

[0017] A composition, method or system herein, wherein the bioactive component is a growth factor or extracellular vesicle.

[0020]

[0018] In aspects and embodiments the invention provides:

[0021]

[0019] A method of forming a porous scaffold, the method comprising:

[0022]

[0020] (a) providing a first solution comprising a first hydrogel (preferably non-peptide) polymer (a macromer) and a second hydrogel (also preferably non-peptide) polymer conjugated with one or more cell adhesion peptides;

[0023]

[0021] (b) providing a second solution comprising a crosslinker polymer;

[0024]

[0022] (c) forming a mixture of the first and second solutions;

[0025]

[0023] (d) rapidly cooling the mixture under conditions wherein the crosslinking polymer crosslinks the first hydrogel polymer around the second hydrogel polymer and ice crystals, creating a semi-interconnected porous network cryogel, or crosslinks both the first and second hydrogel polymers around ice crystals, creating an interconnected porous network cryogel; and

[0026]

[0024] (e) lyophilizing the cryogel to remove the ice crystals, leaving a dry, porous scaffold.

[0027]

[0025] A method herein wherein:

[0028]

[0026] The first and second hydrogel polymers are selected from hyaluronic acid (HyA) polymers, polylactic acid (PLA), polyglycolic acid (PGA), polyacrylic acid (PAA), poly(hydroxyethyl methacrylate) (pHEMA), poly(ethyl methacrylate) (pEMA), polypropylene glycol) methacrylate, polyvinylpyrrolidone (PVP), poly(methyl methacrylate) (PMMA), poly(glycidyl methacrylate) (pGDMA), poly(glycol methacrylate) (pGMA), polyethylene glycol (PEG), alginate, and poly(fumaric acid).

[0029]

[0027] the first and second hydrogel polymers are hyaluronic acid (HyA) polymers.

[0030]

[0028] the first and second hydrogel polymers are hyaluronic acid (HyA) polymers, that are acrylated hyaluronic acid (Ac-HyA) polymers.

[0029] the cell adhesion peptide is a peptide containing an RGD sequence, or is an integrin or syndecan engaging peptide.

[0031]

[0030] the cell adhesion peptide comprises a sequence selected from:

[0032]

[0031] the crosslinker polymer is a crosslinker peptide.

[0033]

[0032] the crosslinker polymer is a crosslinker peptide comprising a sequence selected from: wherein additional amino acids can be added to the N and / or C termini of these core sequences to improve solubility and cross linking efficiency.

[0034]

[0033] the crosslinker polymer is a thiolated crosslinker peptide.

[0035]

[0034] the crosslinker polymer is a thiolated crosslinker peptide, that is a bis-cysteine terminated matrix metalloproteinase (MMP)-cleavable crosslinker peptide.

[0036]

[0035] the first and second solutions comprise triethanolamine buffers.

[0037]

[0036] the first solution further comprises thiolated heparin

[0038]

[0037] the cooling step the mixture is frozen for at least 1, 2, 4 or 6 hours.

[0039]

[0038] the lyophilizing step the cryogel is dried for at least 4, 6, 8 or 12 hours.

[0040]

[0039] the porous scaffold comprises macro- (>100 pm pore diameter) or micro- (<100 pm pore diameter) pores

[0041]

[0040] the first solution further comprises a bioactive molecule, such as thiolated heparin, a growth factor, lipid nanoparticles or extracellular vesicles.

[0042]

[0041] the cooling (freezing) temperature is modulated to effect a predetermined pore diameter according to a nonlinear relationship between freezing temperature and pore diameter.

[0043]

[0042] the cooling is effected in a pre-cooled stainless steel-mold, which may be configured to conform the porous scaffold to a predetermined three dimensional shape and size.

[0043] A cryogel composition comprising a porous scaffold formed by a method herein.

[0044]

[0044] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0045]

[0045] Brief Description of the Drawings

[0046]

[0046] Fig. 1. Cryogel chemistry and fabrication, (left) components of the cryogel are dissolved in 0.2 M TEOA as two separate mixtures. Part 1 : Ac-HyA, peptide-HyA, and thiolated Heparin; Part 2: Labile crosslinker. Parts are rapidly mixed while minimizing bubbles and pipetted into a stainless steel mold. The material is frozen and then lyophilized to remove all ice crystals to produce a dry, porous scaffold. (Right) Cryogel structure (100 kDa, 3wt%) is shown. Top: Scanning electron microscopy (SEM) of the dry surface of the cryogel (scale bar = 100pm). Bottom: Confocal scanning laser microscopy (CSLM) showing a maximum projection of the hydrated pore structure of the cryogel -250 pm into the scaffold (scale bar = 100pm).

[0047]

[0047] Figs. 2A-2C. Hydrated cryogel pore structure. A) Representative confocal scanning laser microscope maximum projections showing pore structure ~250pm deep into hydrated scaffolds with fluorescently tagged hyaluronic acid (HyA). Scaffolds were fabricated at various temperatures keeping molecular weight and weight percent constant at 100 kDa, 3wt%. B) Representative image showing scaffold porosity of by flowing 70kDa dextran- FITC through the fluorescently tagged HyA. Shown: -15°C cryogelation temperature, 100 kDa, 3wt%. Scale bar = 100 pm. C) Cryogel scaffold pore size and scaffold porosity decrease with colder temperatures according to nonlinear relationships (N=3-4). Pore diameter R-squared = 0.99, porosity fl- squared = 0.93.

[0048]

[0048] Figs. 3A-3D. Cryogel material properties. Rheological studies on cryogel mechanical properties showed that A) storage modulus (G’) increases with increased molecular weight and weight percent (N=2-6), B) storage modulus (G’) decreases significantly at temperatures colder than -5°C and then remains consistent (N=3-6), and C) stress relaxation behavior remains relatively consistent across molecular weights and weight percents (N=2-6, mean of each group plotted). D) Swelling experiments showed that the cryogel mass swelling ratio (Qm) increases with greater weight percent and higher molecular weight.

[0049]

[0049] Figs. 4A-4B. Cryogel degradation kinetics. Cryogels were fabricated at -15°C with varying molecular weights (60, 100, 500) and weight percents (1.5, 2.5, 3). Degradation was measured by immersing samples (n=3) into 10 ng / mL solution of human recombinant activated MMP-2 enzyme at 37°C and taking sample mass every 2 days, normalizing to initial swelled mass to achieve A) degradation curves for each condition. Nonlinear fits were found using a one phase exponential decay model to calculate the degradation rate constant k. B) Surface response plot quantifying the relationship between cryogel molecular weight, weight percent, and the degradation rate constant k.

[0050]

[0050] Figs. 5A-5B. Altering material properties impacts cell behavior. Human fibroadipogenic progenitor cells were cultured in cryogels formed at -15°C for 14 days. Quantification and representative images show that varying scaffold parameters such as molecular weight and weight percent significantly impact A) cell spreading (Phalloidin) and B) cell differentiation (Uncoupling Protein 1, or UCP-1 expression). N=4-7, One way ANOVA.

[0051]

[0051] Description of Particular Embodiments of the Invention

[0052]

[0052] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0053] We have developed a system of fabricating and characterizing hyaluronic acid (HyA) based cryogels. The cryogels are composed of modular macromers, such as synthesized according to previously reported protocols.1'2Hence, in embodiments, our cryogels can share material features as our prior hydrogels (e.g. US9, 827,272) using the same HyA chemistry system, as well as introducing additional benefits as summarized below:

[0053]

[0054] Bioactive: HyA is a major component of the extracellular matrix (ECM) of almost every tissue. It is inherently biodegradable, biocompatible, and nonimmunogenic. HyA can direct various cell functions like cell migration, proliferation, and wound healing. HyA performs this signaling via interactions with cell surface receptors like CD443’4, RHAMM5’6, stabilin- 2 / HARE7, LYVE-18"10, layilin11, and various Toll-like Receptors12'13.

[0054]

[0055] Modular Chemistry: Our cryogel may be synthesized from a system of modular components, as previously described1’2’14, and the proportions of each component are independently variable to generate hydrogels with the desired biological and mechanical properties. Additional components can also be added or removed to generate cryogels with different combinations of bioactive elements. Biological properties can be changed by varying the relative ratio of HyA precursors and mechanical properties can be controlled by changing the weight percentage and crosslinker.

[0055]

[0056] Cell Supportive: The chemical composition and physical structure of the cryogel allows for cell and bioactive molecule penetration upon exposure to cell-laden fluids, supports cell adhesion and migration within the scaffold, and avoids or reduces generation of cytotoxic by products as it degrades.

[0056]

[0057] Scalable and Tunable Synthesis: Our cryogel may be synthesized via a high throughput method using a closed freeze-dryer system. Other cryogel synthesis methods require a ‘transport’ step between freezing and lyophilizing the material, which can cause ice to melt, resulting in inconsistent pore structure between batches and severely limiting the number of scaffolds that can be made at a certain time. We can use precooled custom made, electropolished stainless steel molds to ensure our scaffolds are frozen as uniformly as possible, and require no ‘transport’ step, allowing for large scale and consistent scaffold production. We can modulate freezing temperature, freezing time, and lyophilization time and pressure. Modulating freezing temperature and time enables control over the mechanical and structural properties of the scaffold by altering its pore size and structure.

[0057]

[0058] Interconnected pore structure: Because ice crystal nucleation acts as a porogen for these materials, our scaffolds have an interconnected pore structure. We have developed a novel confocal laser scanning microscopy technique to characterize the three-dimensional pore structure of the scaffold. We have shown a nonlinear relationship between hydrated scaffold pore diameter and freezing temperature. Depending on freezing temperature, the network can have macro- (>100 pm pore diameter) or micro- (<100 pm pore diameter) pores. This interconnected pore structure allows for cell and bioactive molecule movement throughout the material, and the curvature of individual pores may contribute to differentiation signaling in cells.

[0058]

[0059] High molecular weight of HyA: The modular chemistry of HyA gel synthesis is facilitated by HyA biopolymers that have been functionalized with a reactive acryl group. Our cryogel scaffolds leverage previous work regarding the creation of a novel method of acrylate HyA (Ac-HyA) that maintains a higher molecular weight relative to previously published methods ' . This high MW Ac-HyA allows for greater control over the cryogel mechanical properties and greater stability in situ.

[0059]

[0060] Prolonged retention of growth factors and extracellular vesicles: We can tune the release kinetics of growth factors and extracellular vesicles released from the cryogel system. By example, we have demonstrated that extracellular vesicles will be retained in our materials for >7 days.

[0060]

[0061] Controlled degradation kinetics: We can also control the degradation rate of the cryogel by careful selection of peptide crosslinkers that are sensitive to specific proteolytic activity, as well as modulating HyA molecular weight and cryogel scaffold pore size.

[0062] Cryogels are hydrogels formed below the crosslinking temperature of the solvent. Ice nucleation acts as a porogen, and crosslinking chemistry links HyA polymer chains via Michael- type addition to create the bulk scaffold material. Cells or other biologies may interact with biologically active peptides or other molecules or agents engineered onto the HyA polymer chains. Light can also be used to crosslink, with addition of a photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate (LAP) or riboflavin (Vitamin B2) + co-initiator (e.g., triethanolamine) as examples. Systems, embodiments, method of synthesis and use, compositions and optional inclusions into our cryogel system are discussed and exemplified herein.

[0061]

[0063] 1. HyA molecular weight and functionalization: Using established methods1, 14, we can generate HyA biopolymers decorated with acrylate groups that can be used for additional conjugation chemistry (for more detail see herein). The invention is not limited to our HyA chemistry system, as we can use other polymers in this system including but not limited to polylactic acid (PLA), polyglycolic acid (PGA), polyacrylic acid (PAA), poly(hydroxyethyl methacrylate) (pHEMA), poly(ethyl methacrylate) (pEMA), poly(propylene glycol) methacrylate, polyvinylpyrrolidone (PVP), poly(methyl methacrylate) (PMMA), poly(glycidyl methacrylate) (pGDMA), poly(glycol methacrylate) (pGMA), polyethylene glycol (PEG), poly(fumaric acid), and the like. Cryogels can have a homo or hetero polymer composition.

[0062]

[0064] 2. HyA conjugation to a bioactive peptide: Using established conjugation chemistries, we can covalently bind proteins, peptides or other bioactive agents directly to the acrylated HyA polymers. More than one HyA-bioactive conjugates can be used in the hydrogel synthesis. In proof of principle work we have often used a 15 amino acid sequence bone sialoprotein RGD peptide (bsp-RGD(15)) conjugated onto the HyA as a cell adhesion peptide (sequence: CGGNGEPRGDTYRAY); however, cryogels may include many other peptide sequences for cell adhesion in addition to or in place of bsp-RGD(15). Exemplary suitable peptide sequence are listed below:

[0065] 3. HyA network crosslinker: The crosslinker in the cryogel network is a thiolated bifunctional peptide crosslinker which links HyA chains together via acrylated groups decorating the chains. As with the rest of our modular chemistry system, this crosslinking occurs via Michael-type addition, but other crosslinking mechanisms, such as photo-initiated crosslinking can also be used (supra). Importantly, these crosslinkers are designed with a peptide sequence that can be acted upon by endogenous proteolytic enzymes like cell secreted matrix metalloproteinases (MMPs). In our initial proof of principle demonstration the formulation of cryogel contains an MMP-13 degradable crosslinker peptide (GCQPQGLAKCG) with termi acetylated and aminated; however, the cryogel can readily contain other crosslinker peptides, such as:

[0063]

[0066] Table 1. kcat / Kmvalues for a range of protease- sensitive peptides obtained from our previous studies (bolded) and literature search. Values shown are specifically for MMP-2, -9 and -13. Downward arrows indicated MMP cleavage sites.

[0064]

[0067] wherein additional amino acids can be added to the N and C termini of these core sequences, such as to improve solubility and cross linking efficiency.

[0065]

[0068] 4 Inclusion of additional bioactive molecules: Using previously established conjugation chemistry1, 14, we can covalently bind thiolated heparin directly to acrylated HyA polymers.

[0066] Heparin can non-covalently bind with growth factors and other proteins and retain these factors for extended periods of time (>20 days), which is typically very difficult for unmodified hydrogels. Alternatively, by adjusting the molecular weight of heparin these factors can be released at a predicable rate (see, e.g., Jha et al. Ref. 26, below). Our laboratory1has shown that we can pre-treat heparinized HyA with a growth factor (e.g., TGF-beta) so the growth factor may either be retained orreleased over time from the scaffold. The heparinized HyA can be pretreated with more than one growth factor prior to cryogel synthesis, or added to the first solution, supra. We may also include other bioactive molecules and constructs, such as extracellular vesicles. Extracellular vesicles have abundant surface thiols, so they can covalently bind to acrylated HyA polymers before cryogel scaffold synthesis. Extracellular vesicle-laden cryogels can retain the extracellular vesicles and release them in a controllable manner during cryogel degradation.

[0067]

[0069] 5. Freezing temperature during gelation: The pore sizes in our cryogel are dependent on the freezing temperature of the system during fabrication. Using a novel, quantitative confocal laser scanning microscopy technique, we have analyzed the three-dimensional structure of the hydrated cryogel interconnected porous network and found a quadratic relationship between freezing temperature and pore diameter. For example, we have demonstrated freezing temperatures from -30°C to -5 °C, and this range can readily be extended to -210°C (liquid nitrogen snap freezing) to 0°C.

[0068]

[0070] A Scaffold geometry and mold: We use custom made, electropolished stainless-steel molds to fabricate our cryogel scaffolds. Molds can be any three dimensional shape up to sizes of ~30cm3. Molds are pre-cooled in the freeze dryer prior to cryogel synthesis.

[0069]

[0071] Example uses of Ac-HyA cryosels:

[0070]

[0072] Skeletal muscle regeneration (hFAPs, SCs, endothelial cells)

[0071]

[0073] As a specific embodiment of the invention, we are optimized the Ac-HyA-based cryogel for skeletal muscle regeneration to treat volumetric muscle loss. This cryogel contains 100 kDa Ac-HyA, MMP-13 degradable crosslinker (GCQPQGLAKCG) to enable proteolytic degradation of the scaffold, thiolated heparin to sequester growth factors, and either bsp-RGD(15) (CGGNGEPRGDTYRAY) or bsp-RGD(14) (CGGGEPRGDTYRAY) to enable cell adhesion via integrin binding. We have determined that a freezing temperature between -20°C and -15°C with median pore diameters ~ IOum-4Opm facilitate cell infiltration into the scaffold and subsequent matrix remodeling. In addition, human fibroadipogenic progenitor cells (hFAPs) showed high pro-myogenic differentiation (uncoupling protein 1 also known as UCP-1, expression) in 100 kDa, -15°C and -20°C scaffolds, and varied differentiation and spreading with in formulations with varied modulus and HyA molecular weight. In addition to hFAPs, satellite cells and endothelial cells are useful to study in the scaffold. These scaffolds can provide enhanced myotube formation and endothelial cell proliferation within the material.

[0072]

[0074] Detailed methods (synthesis, characterization, in vitro work):

[0073]

[0075] Acrylation of HyA

[0076] HyA derivative carrying hydrazide groups (HyA-ADH) were synthesized using previously reported method.14Specifically, 30 molar excess of adipic acid dihydrazide (ADH) was added to the HyA solution (200ml, 3 mg / ml). Solution pH was adjusted to 5 using 0.1M NaOH and 0.1MHC1. EDC (3 mmol) and HOBt (3 mmol) were dissolved separately in DMSO / H2O (1 / 1 volume ratio, 6 ml) and added to the HyA solution sequentially. The solution pH was maintained at 5 for at least 2 hrs. After 2 hrs of reaction, pH of the solution was adjusted to 7.0 and exhaustively dialyzed against deionized (DI) water. Then, NaCl was added to produce a 5% (w / v) solution, and HyA-ADH was precipitated in ethanol. The precipitate was redissolved in same amount of DI water and N- Aery loxy succinimide (NAS, 700 mg) was reacted to the HyAADH solution (600mg, 200 mL DI water) to generate acrylate groups on the HyA (Ac- HyA), and subsequently dialyzed exhaustively again to remove unreacted small molecules.21The product was then lyophilized for 3 days to obtain acrylated HyA (Ac-HyA). Proton ( ' H) NMR confirmed ~ 20% conjugation of acrylate groups on the repeating units of HyA chains.

[0077] Synthesis of Thiolated Heparin (heparin-SH)22

[0074]

[0078] Heparin (50mg) was dissolved DI water at a concentration of 5 mg / mL. Then, heparin was reacted with an excess amount of cysteamine in the presence of EDC and HOBt at pH 6.8 for overnight at room temperature. After that, TCEP (0.25mM) was added in the reaction mixture and reacted for 2hr to obtain free thiol on the heparin after the reduction of the disulfide groups on heparin. Then, final solution was dialyzed against DI water containing 2M NaCl, followed by dialysis against DI water. Then heparin-SH was lyophilized for 3 days. Percentage of conjugation of thiol group on the final product (heparin-SH) was determined by colorimetric Ellman assay.

[0075]

[0079] Synthesis of HyA bioconjugates

[0076]

[0080] Prior to reaction of CGGNGEPRGDTYRAY (bsp-RGD(15)) peptide and HyA, any disulfide bonds formed by bspRGD(15) was reduced by TCEP. Alkaline TCEP solution was made by dissolving 3 mg of NaOH and 30 mg of TCEP in 5 mL 3 mg / mL NaOH solution. Then, bspRGD(15) (50 mg) was dissolved in the TCEP solution and allowed to reduce for 1 hour at 4°C. An Ac-HyA solution (125mg, 45mL DI water) was then heated to 30°C, after which the bspRGD(15) / TCEP solution were added and allowed to react overnight, stirring in a closed system to minimize evaporation. The Ac-HyA-RGD was then dialyzed through a 10 kDa MWCO membrane against DI water. After dialysis, the solution was filtered through 0.45|im Steriflip tube (50mL, BD Bioscience) and lyophilized through the sterile filter cap. The dry product was stored at -20°C. 100% conjugation of bspRGD to Ac-HyA was confirmed by comparing the absorbance (274nm) of tyrosine residue on bspRGD(15) via1H NMR. The same protocol is used for conjugating bspRGD(14) (CGGGEPRGDTYRAY) or other peptides listed above to Ac-HyA.

[0077]

[0081] Cryogel scaffold fabrication

[0078]

[0082] To create cryogels, Ac-HyA, Ac-HyA-RGD, and SH-heparin were first dissolved in 0.2M triethanolamine buffer. In a separate tube, a bis-cysteine terminated matrix metalloproteinase (MMP)-cleavable crosslinker was also dissolved in 0.2M triethanolamine buffer. Combining these two solutions initiates gelation via Michael-type addition, so they were kept separate until just before scaffold fabrication. Stainless steel molds were pre-cooled in a freeze dryer set to the freezing temperature for at least 1 hour prior to fabrication. For fabrication, Part 1 and Part 2 were combined, rapidly mixed, briefly spun down to remove bubbles, and pipetted into the pre-cooled stainless-steel molds. Materials were then frozen for 6 hours and lyophilized for at least 12 hours. Gelation occurs on the order of 10-20 minutes whereas freezing begins almost immediately upon pipetting into the stainless-steel molds. For this reason, Michael-type addition crosslinking occurs around the already formed ice crystals, creating the interconnected porous network.

[0079]

[0083] Cell seeding

[0080]

[0084] Confluent cells were trypsinized and collected. The cell pellet was re-dispersed in media into a cell slurry with a density of 3 106cells / mL. The cell slurry was dispensed into a well plate and dry cryogel scaffolds were immersed into the slurry, resulting in immediate hydration of scaffolds simultaneous with cell infiltration into the scaffold. To allow cells to attach, scaffolds were incubated in the cell slurry for 45 minutes at 37 °C before adding cell culture media. Cells were cultured for 7 days and the medium was refreshed every day. After 4 and 7 days, fibrotic (aSMA) and pro-myogenic (uncoupling protein 1, UCP1) differentiation as well as cell spreading were assessed using immunohistochemistry with volumetric segmentation of z-stacks.

[0085] References

[0081] 1. Jha, A.K., Tharp, K.M., Ye, J., Santiago-Ortiz, J.L., Jackson, W.M., Stahl, A., Schaffer, D.V., Yeghiazarians, Y. & Healy, K.E. Enhanced survival and engraftment of transplanted stem cells using growth factor sequestering hydrogels. Biomaterials 47, 1- 12 (2015).

[0082] 2. Dienes, J., Browne, S., Farjun, B., Amaral Passipieri, J., Mintz, E.L., Killian, G., Healy, K.E. & Christ, G.J. Semisynthetic Hyaluronic Acid-Based Hydrogel Promotes Recovery of the Injured Tibialis Anterior Skeletal Muscle Form and Function. ACS Biomater Sci Eng 7, 1587-1599 (2021).

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Claims

CLAIMS1 . A method of forming a porous scaffold, the method comprising:(a) providing a first solution comprising a first hydrogel polymer and a second hydrogel polymer conjugated to a cell adhesion peptide;(b) providing a second solution comprising a crosslinker polymer;(c) forming a mixture of the first and second solutions;(d) rapidly cooling the mixture under conditions wherein the crosslinking polymer crosslinks the first hydrogel polymer around the second hydrogel polymer and ice crystals, creating a semi-interconnected porous network cryogel, or crosslinks both the first and second hydrogel polymers around ice crystals, creating an interconnected porous network cryogel; and(e) lyophilizing the cryogel to remove the ice crystals, leaving a dry, porous scaffold.

2. The method of claim 1, wherein the first and second hydrogel polymers are selected from hyaluronic acid (HyA) polymers, polylactic acid (PLA), polyglycolic acid (PGA), polyacrylic acid (PAA), poly(hydroxyethyl methacrylate) (pHEMA), poly(ethyl methacrylate) (pEMA), polypropylene glycol) methacrylate, polyvinylpyrrolidone (PVP), poly(methyl methacrylate) (PMMA), poly(glycidyl methacrylate) (pGDMA), poly(glycol methacrylate) (pGMA), polyethylene glycol (PEG), alginate, and poly(fumaric acid).

3. The method of claim 1, wherein the first and second hydrogel polymers are hyaluronic acid (HyA) polymers.

4. The method of claim 1, wherein the first and second hydrogel polymers are hyaluronic acid (HyA) polymers, that are acrylated hyaluronic acid (Ac-HyA) polymers.

5. The method of claim 1, wherein the cell adhesion peptide is a peptide containing an RGD sequence, or is an integrin or syndecan engaging peptide.

6. The method of claim 1, wherein the cell adhesion peptide comprises a sequence selected from:

7. The method of claim 2, wherein the cell adhesion peptide comprises a sequence selected from:

8. The method of claim 1, wherein the crosslinker polymer is a crosslinker peptide.

9. The method of claim 1, wherein the crosslinker polymer is a crosslinker peptide comprising a sequence selected from:

10. The method of claim 7, wherein the crosslinker polymer is a crosslinker peptide comprising a sequence selected from:

11. The method of claim 1 , wherein the crosslinker polymer is a thiolated crosslinker peptide.

12. The method of claim 1, wherein the crosslinker polymer is a thiolated crosslinker peptide, that is a bis-cysteine terminated matrix metalloproteinase (MMP)-cleavable crosslinker peptide.

13. The method of any of claims 1-12, wherein the first and second solutions comprise triethanolamine buffers.

14. The method of any of claims 1-12, wherein the first solution further comprises thiolated heparin.

15. The method of any of claims 1-12, wherein the cooling step the mixture is frozen for at least 1 hour.

16. The method of any of claims 1-12, wherein the lyophilizing step the cryogel is dried for at least 6 hours.

17. The method of any of claims 1-12, wherein the porous scaffold comprises macro- (>100 pm pore diameter) or micro- (<100 pm pore diameter) pores.

18. The method of any of claims 1-12, wherein the first solution further comprises a bioactive molecule, such as thiolated heparin, a growth factor, lipid nanoparticles, or extracellular vesicles.

19. The method of any of claims 1-12, wherein the cooling (freezing) temperature is modulated to effect a predetermined pore diameter according to a nonlinear relationship between freezing temperature and pore diameter.

20. The method of any of claims 1-12, wherein the cooling is effected in a pre-cooled stainless steel-mold, which may be configured to conform the porous scaffold to a predetermined three dimensional shape and size.

21. A cryogel composition comprising a porous scaffold formed by a method of any of claims 1-

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