WO2019148140A2 - Implantable biomaterials that enhance stem cell survival and function - Google Patents

Abstract

The present invention is directed to formulations comprising implantable or injectable biomaterials and methods for transplantation using the biomaterials to support enhanced survival of stem cells in tissues or organs after transplantation. In one embodiment, an implantable biomaterial comprises a scaffold comprising a matrix and one or more dendrimers crosslinked to the matrix. One more or peptides or peptide analogs are crosslinked to the dendrimers, or the matrix, or both. The matrix itself comprises one or more of a collagen, a hyaluronic acid, a chondrointin sulfate, or an extracellular matrix component. The biomaterials and the methods for administering them disclosed herein facilitate a slow release of the peptides or peptide analogs to prolong the stem cell survival, cell growth or both.

Description

IMPLANTABLE BIOMATERIALS THAT ENHANCE STEM CELL SURVIVAL AND

FUNCTION

PRIORITY PARAGRAPH

[0001] This application claims benefit of U.S. Provisional Patent Application No. 62/622659, filed Jan. 26, 2018, titled“DEVELOPMENT OF FORMULATIONS TO

ENHANCE STEM CELL SURVIVAL AND FUNCTION”, which application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods and compositions used in regenerative medicine and treatment of disease, especially ones that use stem cells in one or more stages. It is directed to formulations comprising injectable or implantable biomaterials and methods for transplantation using these biomaterials to support enhanced survival of stem cells in tissues or organs after transplantation. The biomaterials and methods for implanting them disclosed herein facilitate the slow release of the peptides or peptide analogs to prolong the stem cell survival, cell growth or both. Further, stem cells may also be incorporated or associated with these implantable or injectable biomaterials further increasing their utility during transplantation.

BACKGROUND OF THE INVENTION

[0003] Stem cell-based therapies hold great promise for regenerative medicine and the treatment of human disease. Capitalizing on their intrinsic ability for self-renewal and on their potential to differentiate into a variety of cell lineages, stem cells have been widely employed for therapeutic applications. However, a vast majority of these cells have been shown to die within a few weeks of transplantation. Unfortunately, the clinical translation of stem cells has been limited by acute donor-cell death. Although transplantation of stem cells showed significant improvements in myocardial repair, earlier studies have demonstrated a >98% mortality of adult stem cells (including Cardiac Progenitor Cells or CPCs) only 6-8 weeks after implantation (Lee et al. 2018, Nature Biomedical Engineering, 2(2): 104-113). Acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Robust methods to enhance stem cell survival, or cell growth, or both, are highly desirable for increasing the success of stem cell therapy in regenerative medicine and treatment of disease. In addition, the ability to target these methods to the affected areas such as injured tissue or organ sites in actual patients, animal models, and ex vivo systems would further increase the success of these methods for clinical translation.

[0004] Previous studies have attempted to use biomaterials and/or growth factors in an effort to reduce donor-cell death following in vivo delivery of therapeutic stem cell populations. However, these studies have not been able to demonstrate long-term cell survival in the ischemic microenvironment under chemically defined conditions. Although Matrigel-based solutions have been identified to promote the survival of hESC-derived cardiomyocytes in infarcted rat hearts, the clinical use of Matrigel in human patients is not feasible due to its origination from the extracellular matrix of murine Engelbreth-Holm- Swarm sarcoma cells. Therefore, there was a need to develop a clinically applicable and chemically defined biomaterial for efficient cell transplantation.

[0005] Chemically defined biomaterials offer a potential niche for the maintenance and precise control of stem cell fate with more efficiency and safety. The chemically defined injectable or implantable biomaterials disclosed herein seeks to significantly improve the in vivo survival of stem cell grafts for regenerative medical applications by enabling the slow release of pro-survival factors conjugated to delivered cells. The CollagenxDendrimerxpeptide pro- survival biomaterials disclosed herein meet these criteria.

[0006] There are several desirable properties of the Collagen scaffold in the present invention. Biocompatibility, controllable biodegradability, capability of being modified into cross-linked higher order structures such as lattices or gels, are some of them. Moreover, the protein is abundant in the animal kingdom and already plays a natural role in biological functions, such as tissue formation, cell attachment and proliferation, all of which are important properties desirable in an injectable or implantable material to enhance stem-cell growth, survival and differentiation. The presence of reactive groups on Collagen are however limited. This reduces the amounts of molecules that can be incorporated directly into Collagen. The same is true of other naturally occurring or naturally sourced extracellular matrix components.

However, the capacity of Collagen and other extracellular matrix components to carry desirable molecules in amounts that are beneficial for enhancing stem cell growth may be increased by crosslinking other molecules where reactive groups are abundant.

[0007] The present invention designed the CollagenxDendrimerxpeptide (ColxDxpep) pro-survival biomaterials to improve cell engraftment in vivo by combining the advantages of a collagen scaffold with the slow release of pro-survival growth factors. To crosslink the peptides to collagen, a conjugation scheme based on a dendrimer-intermediate conjugation was used. Further, dendrimers were chosen because the multi-terminal-free amine groups of the dendrimers increase the limited quantities of amine groups in collagen available for peptide crosslinking and also produce a crosslinked stable collagen-based material. This crosslinking method produces an injectable crosslinked collagen-based scaffold that can be functionalized by different ligands. The resulting ColxDxpep matrix promotes the engraftment of several types of therapeutic cells in vivo , resulting in functional improvements in animal models of hind-limb ischemia and myocardial infarction.

[0008] Previous studies have employed physical encapsulation, biotin-streptavidin conjugation, click chemistry, and other covalent crosslinking methods to enable the slow release of growth factors. However, these studies have largely failed to demonstrate a sustained high level cell survival in vivo at both early and late stages of delivery or have been non-compatible with FDA standards of human safety. To remedy this shortcoming, the present invention applied a technique to immobilize growth factor peptides on dendrimerized collagen to produce a stabilized and injectable cell delivery matrix for slow release of pro-survival factors.

[0009] Compared to full-length proteins, peptide analogs maintaining the same or partial biological effects may serve as more desirable therapeutic agents because of improved stability, reduced manufacturing cost, fewer side effects, and better delivery. To improve the half-life of peptide analogs of BMP2, EPO, and FGF2 in vivo as well as the retention of the injected cells, the present invention utilizes peptide analogs covalently crosslinked to a collagen matrix scaffold via dendrimers (colxDxpep,“col”=collagen;“x”=crosslinked;“D”=dendrimer; “pep”=peptide) to provide a controlled delivery system and a defined implantable biomaterial. BMP2, EPO and FGF2 peptides and their analogs are used to demonstrate the utility of the platform technology. Other growth-promoting peptides or their analogs may be used in a similar manner as desired depending upon choice of tissues and target organs targeted by stem cell therapy.

[0010] The injectable and implantable platform technologies disclosed herein improves survival and differentiation of transplanted stem cells to construct new tissues in the affected areas in the injury site. The injectable or implantable biomaterials when transplanted along with stem cells results in better cell survival and function. Embodiments of the invention disclosed herein would make stem cell implants a viable option for patients in need of tissue repair and reduce the risk of cell-rejection. With the implants and methods to use them disclosed herein, stem cells are ensured of an increased and/or continuous supply of the stimulants necessary to promote tissue regeneration adjacent to their sites of implants such as injury sites and wound sites and disease sites. This will prevent early cell death of stem cells. The slow delivery of cell growth-promoting peptides or peptide analogs and/or molecules or drugs that induce the production of these peptides in situ would trigger desirable changes such as increased vasculature around the transplanted cells leading to improve perfusion of the tissue. In addition to the role in therapy, the biomaterials and methods to use them may also serve as important tools to investigate the role of specific cell-growth promoting peptides and molecules that induce them on tissue regeneration.

[0011] The solubility of collagen is improved by cross linking with dendrimer and PEG and results in fetal tissue like micro domains [Zgheib, et ah, 2014, Ann Thorac Surg. 97: 1643- 1650, and Porrello et al., 2013,. Proc Natl Acad Sci ETSA. 110: 187] These formulations show enhanced affinity for the extracellular matrix proteins of the host tissues. Collagen I when mixed with hyaluronic acid using micro fluidic chambers result in Collagen 1/ Hyaluronic acid micro domains with reticular collagen structures.

[0012] In this invention, erythropoietin peptide analogues, fibroblast growth factor-2 (FGF2), and analogues of bone morphogenetic protein-2 (BMP2) peptides were crosslinked with dendrimer modified collagen resulting in a gel that deliver these peptides in animal tissues for more than two months. These long surviving cells could be also used to check the drug toxicity as these cells are imaged in vivo using luciferase and Infrared imaging. These cells are also engineered to interact with PET-labeled biotin on cell surface. Collagen-dendrimer biomaterial crosslinked with pro-survival peptide analogues and proteins adhere to the extracellular matrix. They slowly release the peptides and proteins in the transplanted sites significantly prolonging stem cell survival in mouse models of ischemic injury. The biomaterials can serve as some generic delivery systems to improve functional outcomes in cell-replacement therapy. This would be an added advantage.

[0013] Further, replacing cell-growth or cell-survival promoting peptides or proteins with molecules or drugs that are inducers of these peptides or proteins, expands the functionality of the biomaterials disclosed herein. For example, the present invention discloses functional replacement of BMP2 and FGF2 with small molecules Tacrolimus and Amitriptyline respectively. In addition, the biomaterials disclosed herein, for example, Collagen/ Hyaluronic acid gels, may be used to deliver recombinant human erythropoietin rhEPO, along with the stem cells. The gel provides a temporary microdomain scaffold for the implanted cells and allows slow delivery of the rhEPO and other growth factor analogues.

SUMMARY OF THE INVENTION

[0014] Disclosed herein are formulations comprising implantable biomaterials as well as methods for transplantation using these implantable biomaterials to support enhanced survival of stem cells in tissues or organs. According to one embodiment of the invention, an implantable biomaterial comprises a scaffold. The scaffold comprises a matrix and one or more dendrimers crosslinked to the matrix. Further, one more or peptides or peptide analogs are crosslinked to the dendrimers, or the matrix, or both. The matrix itself comprises one or more of a collagen, a hyaluronic acid, a chondroitin sulfate, or an extracellular matrix component. Use of the biomaterial during transplantation ensures that stem cells have an increased and/or continuous supply of the stimulants necessary to promote tissue regeneration adjacent to their sites of implants such as injury sites and wound sites and disease sites. This will prevent early cell death of stem cells.

[0015] In some embodiments, the matrix comprises a collagen and a hyaluronic acid. In some embodiments, the collagen comprises telopeptides.

[0016] In one embodiment, the one or more peptides or peptide analogs crosslinked to the matrix, or the dendrimers, or both, promote cell survival, cell growth, or both. In some embodiments, the peptide or peptide analog is selected from one or more of a bone morphogenetic protein-2 peptide (BMP2) or BMP2 analog, a erythropoietin peptide (EPO) or EPO analog, or a fibroblast growth factor-2 peptide (FGF2) or FGF2 analog, or combinations thereof. In some embodiments, the one or more peptide analogs is present at a concentration of from about 5% to about 75% of wild type levels of the corresponding peptide in an animal onto which the biomaterial is transplanted. In some embodiments, the crosslinking chemistry is selected from one or more of 1 -Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC), N- hydroxysulfosuccinimide (sulfo-NHS), Bis-N-PEG-carbamoylated lysine, and Boc-B-alanine-N- diacetic diacid.

[0017] In one embodiment, the EPO comprises a recombinant human

Erythropoietin (rhEPO) peptide. In another embodiment, the FGF2 comprises a recombinant human fibroblast growth factor-2 (rhFGF2) peptide. In another embodiment, the BMP2 comprises a recombinant human bone morphogenetic protein-2 (rhBMP2). In one embodiment, the EPO peptide analog comprises EPO peptide analog 1 (GGTYSCHFGPLTWVCKPQGG, disulfide:C6-Cl5; SEQ ID: l), EPO peptide analog 1 (GGT YSCHF GPLTW V CKPQGG; no disulfide bond; SEQ ID:2), EPO peptide analog 2 (TYSCHFGPLTWVCKPQGG, disulfide:C6- C15; SEQ ID:3), EPO peptide analog 2 (TYSCHFGPLTWVCKPQGG, no disulfide bond; SEQ ID:4), or EPO peptide analog 3 (GGQEQLERALNSS; SEQ ID:5) or combinations thereof. In some embodiments, the FGF2 analog comprises FGF2 analog 1

(YRSRKYS S W Y V ALKRK( YRSRK Y S S W Y V ALKR)- Ahx- Ahx- Ahx-RKRLDRI AR-NH2 ; SEQ ID:6). In some embodiments, the BMP2 analog comprises BMP2 peptide analog 1 (KIPKASSVPTELSAISTLYL; SEQ ID:7), or BMP2 peptide analog 2 (

CGKIPKASSVPTELSAISTLYL; SEQ ID:8), or combinations thereof

[0018] In one embodiment, the biomaterial further comprises molecules or drugs that induce cell growth-promoting or cell-survival promoting peptides. In some embodiments, these molecules comprise EPO-inducing, BMP2 -inducing, or FGF2-inducing molecules. In one embodiment, the FGF2 inducing molecule comprises Amitriptyline. In another embodiment, the BMP2 inducing molecule comprises Tacrolimus. In another embodiment, EPO inducing molecule comprises a EPO mimicking molecule PAMAM-HMB linked methyl (2-(2-([l,T- biphenyl]-4-yl)-6-chloro-5-methyl-lH-indol-3-yl)acetyl)-L-lysinate.

[0019] In one embodiment, the biomaterial further comprised stem cells. In some embodiments, the stem cells are cord blood stem cells, amniotic fluid stem cells, placental stem cells, mesenchymal stem cells, endothelial stem cells, progenitor cells, bone marrow stem cells, embryonic stem cells or non-embryonic stem cells, adult stem cells, induced pluripotent stem cells, or combinations thereof. In some embodiments, the stem cells are co-implantable with the biomaterial to a wound or injury site. In some embodiments, the stem cells are embedded adsorbed, or attached to the biomaterial. In some embodiments, the stem cells form a monolayer on the biomaterial.

[0020] In one embodiment, the biomaterial is injectable or implantable in form of a gel, or a patch, or combinations thereof.

[0021] In one embodiment, the dendrimers comprise a Poly(amidoamine) (PAMAM). In some embodiments, the PAMAMs are selected from one or more of first, second or third generation PAMAMs.

[0022] Another embodiment of the present invention comprises a method for transplantation in which a scaffold comprising a matrix and one or more dendrimers crosslinked to the matrix is prepared, wherein the matrix itself comprises one or more of a collagen, a hyaluronic acid, a chondroitin sulfate, or an extracellular matrix component. Further, one more or peptides or peptide analogs are crosslinked to the dendrimers or the matrix, or both, to yield an implantable biomaterial. Subsequently, the biomaterial is administered on a tissue or an organ site.

[0023] In one embodiment administering is carried out on in vivo on an animal. In another embodiment, administering is carried out in vitro or ex vivo , on experimental models. In some embodiments, administering is carried out to treat or investigate a disease condition.

[0024] In some embodiments, the method further comprises providing stem cells for transplantation. In some embodiments, the stem cells are cord blood stem cells, amniotic fluid stem cells, placental stem cells, mesenchymal stem cells, endothelial stem cells, progenitor cells, bone marrow stem cells, embryonic stem cells or non-embryonic stem cells, adult stem cells, induced pluripotent stem cells, or combinations thereof. In some embodiments, the stem cells are co-injectable or co-implantable with the biomaterial to the injury site or wound site. In some embodiments, the stem cells are provided at a concentration of from about 1 million cells/milliliter to about 25 million cells/milliliter.

[0025] In one embodiment, the method comprises administering is carried out in an exosome or with a poly(lactic-co-glycolic acid) (PLGA) or a poly lactic acid (PLA) encapsulation. In another embodiment, administering is intranasal, delivering the biomaterial to the brain.

[0026] In one embodiment of the method, the one or more peptides or peptide analogs promote cell survival, cell growth or both. In some embodiments the peptide or peptide analog is selected from one or more of a bone morphogenetic protein-2 peptide (BMP2) or BMP2 analog, a erythropoietin peptide (EPO) or EPO analog, or a fibroblast growth factor-2 peptide (FGF2) or FGF2 analog, or combinations thereof. In one embodiment the EPO comprises a recombinant human Erythropoietin (rhEPO) peptide. In another embodiment the FGF2 comprises a recombinant human fibroblast growth factor-2 (rhFGF2) peptide. In another embodiment the BMP2 comprises a recombinant human bone morphogenetic protein-2

(rhBMP2).

[0027] In some embodiments of the method, the EPO peptide analog comprises

EPO peptide analog 1 (GGT YSCHF GPLTW V CKPQGG, disulfide:C6-Cl5; SEQ ID: l), EPO peptide analog 1 (GGTYSCHFGPLTWVCKPQGG; no disulfide bond; SEQ ID:2), EPO peptide analog 2 (T YSCHF GPLTW V CKPQGG, disulfide:C6-Cl5; SEQ ID:3), EPO peptide analog 2 (TYSCHFGPLTWVCKPQGG, no disulfide bond; SEQ ID:4), or EPO peptide analog 3

(GGQEQLERALNSS; SEQ ID:5) or combinations thereof. In some embodiments, the FGF2 analog comprises FGF2 analog 1 ( YRSRK Y S S W Y V ALKRK( YRSRK Y S S W Y V ALKR)- Ahx- Ahx-Ahx-RKRLDRIAR-NH2; SEQ ID:6). In some embodiments, the BMP2 analog comprises BMP2 peptide analog 1 (KIPKASSVPTELSAISTLYL; SEQ ID:7), or BMP2 peptide analog 2 ( CGKIPKASSVPTELSAISTLYL; SEQ ID: 8), or combinations thereof.

[0028] In one embodiment, the method further comprises crosslinking molecules or drugs that induce cell growth-promoting or cell-survival promoting peptides to the matrix, or dendrimers, or both. In some embodiments, the molecules are EPO-inducing, BMP2-inducing, or FGF2-inducing molecules. In one embodiment the FGF2 inducing molecule comprises Amitriptyline. In one embodiment the BMP2 inducing molecule comprises Tacrolimus. In another embodiment, the EPO inducing molecule comprises a EPO mimicking molecule PAMAM-HMB linked methyl (2-(2-([l,l'-biphenyl]-4-yl)-6-chloro-5-methyl-lH-indol-3- yl)acetyl)-L-lysinate.

[0029] In one embodiment of the method, the one or more peptide analogs is present at a concentration of from about 5% to about 75% of wild type levels of the corresponding peptide in an animal onto which the biomaterial is transplanted.

[0030] In one embodiment of the method, the collagen comprises telopeptides.

[0031] In one embodiment of the method, the dendrimers comprise a

Poly(amidoamine) (PAMAM). In one embodiment the PAMAMs are selected from one or more of first, second or third generation PAMAMs.

BRIEF DESCRIPTION OF FIGURES

[0032] FIG. 1 A shows a schematic for a method for preparing an implantable biomaterial according to one embodiment.

[0033] FIG. 1B shows a quantification of amine groups on collagen before and after crosslinking dendrimers using TNBSA assay according to one embodiment. [0034] FIG. 1C shows detection of free dendrimers after crosslinking by PAGE according to one embodiment.

[0035] FIG. 1D shows a click reaction scheme showing the fluorescent labeling of acetylene-labeled peptides with an azide probe according to one embodiment.

[0036] FIG. 1E shows quantification of peptides crosslinked to collagen by click chemistry according to an embodiment.

[0037] FIG. 1F shows detection of collagen and free peptides after click chemistry according to one embodiment.

[0038] FIG. 1G shows scanning electron microscope (SEM) images of crosslinked collagens according to one embodiment.

[0039] FIG. 2A shows slow release of BMP2 peptides in vitro from a biomaterial prepared according to one embodiment.

[0040] FIG. 2B shows slow release EPO peptides in vitro from a biomaterial prepared according to one embodiment.

[0041] FIG. 2C shows slow release of FGF2 peptides in vitro from a biomaterial prepared according to one embodiment.

[0042] FIG. 2D shows peptide release from a biomaterial in vivo in SCID mice according to one embodiment.

[0043] FIG. 3 A shows representative bioluminescence (BLI) during evaluation of cell survival in SCID mice after implanting a biomaterial according to an embodiment.

[0044] FIG. 3B shows quantification of results in FIG. 3 A.

[0045] FIG. 3C shows representative Doppler images during evaluation of limb perfusion in immunocompetent mice after implanting a biomaterial according to an embodiment.

[0046] FIG. 3D shows quantification of results in FIG. 3C. [0047] FIG. 4A shows representative Doppler images during an evaluation of limb perfusion in immunocompetent mice after implanting a biomaterial according to one embodiment.

[0048] FIG. 4B shows quantification of results in FIG. 4A.

[0049] FIG. 5A shows representative bioluminescence images showing promotion of long-term cell survival in vivo in SCID mice after implanting a biomaterial according to one embodiment.

[0050] FIG. 5B shows quantification of results in FIG. 5 A.

[0051] FIG. 5C shows representative bioluminescence images of showing promotion of long-term cell survival in vivo in immunocompetent mice after intra-myocardial injection of a biomaterial according to one embodiment.

[0052] FIG. 5D shows quantification of results in FIG. 5C.

[0053] FIG. 6A shows evaluation of graft function after implanting cardiac progenitor cells with a biomaterial in a SCID model of myocardial infarction according to one embodiment. Representative GFP signals overlaid over bright-field images from hearts harvested from mice 30 days post injection is shown.

[0054] FIG. 6B shows evaluation of graft function after implanting cardiac progenitor cells with a biomaterial in a SCID model of myocardial infarction according to one embodiment. Immunofluorescence staining of heart tissues for phosphorylated ERK1/2 and ART after implanting biomaterials is shown.

[0055] FIG. 6C shows evaluation of graft function after implanting cardiac progenitor cells with a biomaterial in a SCID model of myocardial infarction according to one embodiment. Representative M-mode echocardiographic data of infarcted hearts after implanting biomaterials is shown.

[0056] FIG. 6D shows evaluation of graft function after implanting cardiac progenitor cells with a biomaterial in a SCID model of myocardial infarction according to one embodiment. Comparison of fractional shortening after implanting biomaterials is shown.

[0057] FIG. 6E shows evaluation of graft function after implanting cardiac progenitor cells with a biomaterial in a SCID model of myocardial infarction according to one embodiment. Comparison of left ventricular end diastolic dimension and end systolic dimension after implanting biomaterials is shown. [0058] FIG. 7 A shows evaluation of the effects of CPC delivery on post-infarct ventricular function according to one embodiment. Representative M-mode echocardiographic data of infarcted hearts in immunocompetent animals after implanting biomaterials is shown.

[0059] FIG. 7B shows evaluation of the effects of CPC delivery on post-infarct ventricular function according to one embodiment. Comparison of fractional shortening after implanting biomaterials is shown.

[0060] FIG. 7C shows evaluation of the effects of CPC delivery on post-infarct ventricular function according to one embodiment. Representative short-axis MRI images of hearts in immunocompromised mice after implanting biomaterials is shown.

[0061] FIG. 7D shows evaluation of the effects of CPC delivery on post-infarct ventricular function according to one embodiment. Quantitative MRI assessments of left ventricular ejection fraction of infarcted mice after implanting biomaterials is shown.

[0062] FIG. 8 A shows evaluation of LV remodeling in immunodeficient mice by

MRI after implanting biomaterials according to one embodiment. Quantitative MRI assessments of left end diastolic volume and end systolic volume of infarcted mice are shown.

[0063] FIG. 8B shows evaluation of LV remodeling in immunodeficient mice by MRI after implanting biomaterials according to one embodiment. Quantification of the amount of scar and viable tissue by histology after implanting biomaterials is shown.

[0064] FIG. 8C shows evaluation of LV remodeling in immunodeficient mice by MRI after implanting biomaterials according to one embodiment. Representative hematoxylin and eosin and Masson’s tri chrome staining of left ventricular tissue of mice after implanting biomaterials is shown. Blue on the Masson’s trichome tissue signifies scar tissue.

[0065] FIG. 9 shows of screening of pro-survival factor cocktail according to one embodiment.

[0066] FIG. 10 shows in vitro up-regulation of pro-survival pathways by EPO, FGF, and BMP2 peptide analogs according to one embodiment.

[0067] FIG. 11 shows characterization of the collagen-linked factors by size exclusion chromatography according to one embodiment.

[0068] FIG. 12 shows CD spectra for secondary structure analysis of collagen according to one embodiment. [0069] FIG. 13 shows characterization of the collagen-linked factors by Raman spectroscopy at amide-I and C-H vibration regions according to one embodiment.

[0070] FIG. 14 shows turbidity assay to detect fiber formation according to one embodiment.

[0071] FIG. 15 shows Dynamic light scattering (DLS) analysis according to one embodiment.

[0072] FIG. 16 shows microgel structure by AFM analysis according to one embodiment.

[0073] FIG. 17 shows a possible mechanism of peptide release according to one embodiment.

[0074] FIG. 18 shows peptide sequences according to one embodiment.

[0075] FIG. 19 shows characterization of the peptides released from colxDxpep analyzed by MALDI-TOF and electrophoresis according to one embodiment.

[0076] FIG. 20 shows a BMMNC survival model for in vivo evaluation of colxDxpep factor analogs according to one embodiment.

[0077] FIG. 21 shows preparation of the collagen-based slow release delivery system with D-Luciferin as a model compound according to one embodiment.

[0078] FIG. 22 shows slow release of luciferin-labeled collagen without diffusion according to one embodiment.

[0079] FIG. 23 shows peptide release from the collagen dendrimer gel around the injected cells according to one embodiment.

[0080] FIG. 24 shows binding of ColxDxpep to extracellular matrix components (ECM) by enzyme-linked immunosorbent assay (ELISA) method according to one embodiment.

[0081] FIG. 25 shows RNA-seq analysis of CPCs treated with ColxDxpep according to one embodiment.

[0082] FIG. 26 shows effect of injecting crosslinked collagen gel using different needle gauges on the release of the linked molecules according to one embodiment.

[0083] TABLE 1 shows a list of pro-survival factors tested in an initial screen according to one embodiment. [0084] TABLE 2A shows a list of biomaterials and crosslinking chemistry strategies used for each of the biomaterial for EPO and EPO analogs and small molecules according to some embodiments.

[0085] TABLE 2B shows a list of biomaterials and crosslinking chemistry strategies used for each of the biomaterial for FGF2 and FGF2 analogs and small molecules according to some embodiments.

[0086] TABLE 2C shows a list of biomaterials and crosslinking chemistry strategies used for each of the biomaterial for BMP2 and BMP2 analogs and small molecules according to some embodiments.

DETAILED DESCRIPTION

[0087] According to one embodiment, an implantable biomaterial comprises a scaffold. The scaffold comprises a matrix and one or more dendrimers crosslinked to the matrix. Further, one more or peptides or peptide analogs are crosslinked to the dendrimers, or the matrix, or both. The matrix itself comprises one or more of a collagen, a hyaluronic acid, a chondroitin sulfate, or an extracellular matrix component. Use of the biomaterial ensures that stem cells supplied with the biomaterial, or stem cells or other types of cells within the vicinity of the biomaterial after it has been administered, have an increased and/or continuous supply of the stimulants necessary to promote tissue regeneration adjacent to their sites of implants such as injury sites and wound sites and disease sites. This will prevent early cell death of stem cells or other types of cells within the vicinity of the biomaterial after it has been administered.

“Vicinity” means a reasonable proximity to the biomaterial where a biological effect may be directly attributable to the presence of the biomaterial or its administration either as disclosed herein or any modified formulation that is routine in the art.

[0088] The biomaterial may be used to treat a disease condition in a subject by administering it to a desired tissue or organ site. Another embodiment of the present invention comprises a method for transplantation in which a scaffold comprising a matrix and one or more dendrimers crosslinked to the matrix is prepared, wherein the matrix itself comprises one or more of a collagen, a hyaluronic acid, a chondroitin sulfate, or an extracellular matrix

component. Further, one more or peptides or peptide analogs are crosslinked to the dendrimers or the matrix, or both, to yield an implantable biomaterial. Subsequently, the biomaterial is administered on a tissue or an organ site. Administering may be done to treat or mitigate a disease condition in a subject to investigate, or interrogate, or influence, or impact conditions of tissues and organs in subjects or experimental systems in vivo , or in vitro , or ex vivo.

Administering may be carried out with an effective amount of the biomaterial.

[0089] The term“administering” means to administer a biomaterial to a patient or living being or an ex vivo organ or tissue model whereby the biomaterial influences or impacts the tissue or the organ to which it is targeted in a desirable or predictable manner; when used for a therapeutic purpose, the impact or influence would be in a positive. The biomaterials described herein can be administered either alone or in combination (concurrently or serially) with other pharmaceuticals. For example, the therapeutic agents can be administered in combination with other vaccines, antibiotics, antiviral agents, anti-cancer or anti-neoplastic agents, or in combination with other treatment modalities such as herbal therapy, acupuncture, naturopathy, etc. [0090] A subject treated by the presently disclosed biomaterial compositions, or methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).

[0091] The term“treatment” or“treating” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly,“treatment” can refer to therapeutic treatment or prophylactic or preventative measures. In some embodiments, the treatment is for therapeutic treatment. In some

embodiments, the treatment is for prophylactic or preventative treatment. Those in need of treatment can include those already with a disorder or a disease as well as those in which the disorder is to be prevented. In some embodiments, the treatment is for experimental treatment. In some embodiments the treatment may be carried out in lab on chip systems or ex vivo tissue or organ model systems.

[0092] The term“effective amount” as used herein generally refers to a sufficient amount of the biomaterial that is added to decrease, prevent or inhibit a disease. The amount will vary for each biomaterial and upon known factors related to the item or use to which the biomaterial is applied. Further, administering may involve using desirable doses of the biomaterial. Effective amounts and doses that may be measured against number of cells/body weight unit of the subject or system in which the biomaterial is administered to bring desirable changes with respect to any of the features monitored routinely in the art. Effective amounts and doses may also be measured against any other reliable parameter of the subject or system on which the biomaterial is used to exert an effect.

[0093] In some embodiments, the method involves administering a single biomaterial. In some embodiments, the method involves administering a combination of one or more biomaterials. In the methods disclosed herein, a first biomaterial may be administered with a second biomaterial concomitantly or subsequently. In some embodiments, there might be a lag period of few hours to days between administration of the first and the second biomaterial. In some embodiments, a single biomaterial may carry one growth-promoting peptide or peptide analog or molecules/drugs that induce them, and/or stem cells. In some others, it may carry two or more growth-promoting peptide or peptide analog or molecules/drugs that induce them and/or stem cells. In some embodiments, the biomaterial may have only one type of matrix component. In others, it may have more than one kind of matrix component. In some embodiments, the biomaterial may carry only one type of dendrimer. In others, it may carry more than one type of dendrimer.

[0094] In some embodiments, the dendrimers comprise a Poly(amidoamine)

(PAMAM). In some embodiments, the PAMAMs are selected from one or more of first, second or third generation PAMAMs. Some embodiments may use non-modified dendrimers that are cationic or anionic. Some embodiments may use modified dendrimers.

[0095] In some embodiments, the dose of biomaterial for administering may be varied by adjusting it relative to the body weight of the subject or volume of the experimental system or any other parameter against which a dose is generally defined. In some embodiments, the biomaterial may be administered for different durations as desired measured in hours, days, weeks, months and years and intervals thereof. In some embodiments, the biomaterials may be administered at a desired frequency within an hour, a day, a week, a month or a year.

Administration may be once, or a more than one time, and/or over one or more periods such as one to several days, weeks or months.

[0096] Administration of the biomaterials prepared by the methods disclosed herein may include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral or non-parenteral routes of administration, for example, injection or infusion. The parenteral routes may be intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. The non-parenteral routes may be oral, topical, epidermal or mucosal, intranasal, vaginal, rectal, sublingual or topical. [0097] In some embodiments, a subject in need thereof is preconditioned prior to or simultaneously with the administration of the biomaterial. The subject may be

preconditioned according to any method known in the art. Preconditioning may involve modulating the levels of one or more other factors in the subject or the system in which the biomaterial is used. Modulation may be mediated by delivering synthetic or natural molecules to the subject or system such as RNA, DNA, proteins, peptides, hormones, or drugs. Modulation may also be mediated by delivering natural or engineered cells. In some embodiments, persistence of the effects due to the presence biomaterial may be measured by following the amount of time the biomaterial exerts an effect that is attributable to it directly or indirectly.

[0098] In some embodiments, the peptide or peptide analogs crosslinked to the dendrimers, or matrix, or both are growth factors. A growth factor may any substance that may stimulate growth, survival or differentiation of living cells. In some embodiments, they may be selected for treating or investigating a particular disease or condition. Selection may be based on screening for desirable effects based on existing knowledge or employing specialized screens using cell-, tissue- or organ-cultures or experimental animal models. The effects evaluated may be changes in number or activity of enzymes, or enzyme-linked assay molecules, promoter or gene expression, cellular components such as RNA, or protein, or peptides or metabolites, post- transcriptional changes in RNA, post-translational changes in proteins, epigenetic changes in DNA, cells, cell cycle, cell division, cell adhesion, cell differentiation, cell signaling, or cell signaling molecules. The effects may be upregulation, or downregulation, or stabilization, or sustenance of desirable levels of concentrations or activity of any of one or more of the aforementioned factors or processes. [0099] In some embodiments, the matrix comprises a collagen and a hyaluronic acid. In some embodiments, the collagen comprises telopeptides. In some embodiments, the collagen may not comprise telopetides. A collagen, a hyluronic acid or another extracellular matrix component from any animal may be used to create a matrix. The number of reactive amine or carboxyl groups on any one or more of the matrix components may be increased by the crosslinking one or more dendrimers to the amine or carboxyl groups present on one or more of the components of the matrix. One of the functions of the dendrimers is to increase the number of active acidic or carboxyl groups on one or more of the matrix components for attaching growth factors, or growth-factor inducing molecules, or any other desired factor or molecule, in numbers beyond what is originally present on these components. Acidic or basic amino acids on one or more of the components of the matrix may serve as the reactive groups for attaching intermediate molecules.

[00100] In some embodiments, a dendrimer is crosslinked first to one or more of the matrix components before crosslinking growth factors, or growth-factor inducing molecules, or any other desired factor or molecule that enhances stem cell growth and survival. In some embodiments, the crosslinking of a dendrimer and the desired factor or factor-inducing drug or molecule are carried out simultaneously. In some embodiments it is carried out in overlapping steps. In some embodiments, the desired factor or factor-inducing drug or molecule is first crosslinked to a dendrimer following which the complex is crosslinked to one or more of the matrix components. Chemical modifications such as blocking of undesirable reactive sites, or activation of desirable reactive sites may be carried out on the dendrimers, or factors, or factor- inducing drug or molecules, or one or more components of the matrix to facilitate a desired crosslinking or level of crosslinking. The same crosslinkers, or chemical modifications may be used for crosslinking dendrimers (or other intermediate molecules) to one or more of the matrix components, and crosslinking the peptides, or peptide analogs or molecules/drugs that induce them to the dendrimers (or other intermediate molecules). In some embodiments, different crosslinkers, or chemical modifications may be used for crosslinking dendrimers (or other intermediate molecules) to one or more of the matrix components, and crosslinking the peptides, or peptide analogs or molecules/drugs that induce them to the dendrimers.

[00101] Crosslinking may be monitored by changes to the scaffold or its matrix components. This may include one or more of a chemical, a physical, or a biological method, or combinations thereof. Enzyme-linked assays, immunologic assays, antibody-based assays, ELISAs, amine reactivity, carboxylic acid reactivity, electrophoretic methods, dialysis methods, click chemistry methods, fluorescence-based methods, spectroscopic methods (such as circular dichroism and Raman spectroscopy), spectrophotometric methods, calorimetric methods (such as differential scanning calorimetry), colorimetric methods, light-scattering methods, microscopy methods (such as Atomic Force and Scanning electron microscopy), selective extraction methods, chromatographic methods (such as size exclusion, ion exchange, and affinity-based methods), centrifugation-based methods, diffusion-based methods, imaging techniques (such as magnetic resonance, Doppler imaging), and mass spectroscopic methods are non-limiting examples that may be used. Cell-death assays, cell-survival assays, cell-differentiation assays, monitoring changes in perfusion, monitoring changes in physiological consequences, bioavailability assays, gene expression analysis methods, DNA and RNA sequencing methods, metabolite analysis methods, metabolomics, transcriptome analysis, proteome analysis, staining methods, labeling methods, and detection methods are some other non-limiting examples that may be used. [00102] In some embodiments, in vivo , or in vitro release kinetics of peptides may last from one day to a few months. Functional improvements in animal models, ex vivo models, or lap-on-chip models maybe used to evaluate the release kinetics and their benefits. Any of the assays mentioned before may be used alone or in combination for this evaluation. Vascularizaton and microdomain formation in tissues or organs, changes in tissue or organ morphology, and survival of subjects or models, are some other parameters that may be used to evaluate the release kinetics and their benefits.

[00103] In some embodiments, the one or more peptides or peptide analogs crosslinked to the matrix, or the dendrimers, or both, are present at a concentration of from about 5% to about 75%, from about 1% to about 50%, or from 1% to about 300% of the wild type levels of the corresponding peptide in an animal onto which the biomaterial is transplanted. In some embodiments, the concentration of the peptides, peptide analogs and/or drugs or molecules that induce them may be calibrated to deliver desirable amounts of these molecules in a subject or a system it is administered. In some embodiments, the information gained from one or more of the assays mentioned above to evaluate the crosslinking, release kinetics, and benefits may be used to arrive at preparing biomaterials with desired molecules and desired kinetics of release.

[00104] In some embodiments, the peptides comprise natural amino acids. In some embodiments, they comprise amino acid analogues and/or modified amino acids. Although the present invention uses recombinant peptides and their analogs of a certain sequence, it may be realized that modifications to the length, sequence and composition of these peptides and peptide analogs may be carried out in some embodiments while preparing and/or administering these biomaterials to achieve desired results depending on their application. [00105] Makkar et al. have reported no adverse effects or tumor formations following CPC transplantation (Makkar et al, Lancet. 20l2;379(98l9):895-904). The biomaterials and methods to use them disclosed herein improve viable heart mass and reduction in the scar volume without tumor formation. Survival of injected CPCs in the heart would help researchers and clinicians to use this technology for the regeneration of heart. The biomaterials and methods disclosed herein may be modified by alternatives, variations or improvements by those skilled in the art for the regeneration of other organs, each of which is also intended to be encompassed by the disclosed embodiments.

[00106] It is to be understood that even though numerous characteristics and advantages of various embodiments of the present disclosure have been set forth in the foregoing materials, together with details of the structure and function of various embodiments of the invention, this detailed description is illustrative only, and changes may be made in detail, especially in matters of structure and arrangements of parts within the principles of the embodiments of the present invention to the full extent indicated by the broad general meaning of the terms disclosed. Several of the above disclosed features and functions, or alternatives thereof, may be combined into many other embodiments. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be

subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

EXAMPLES

EXAMPLE 1 [00107] Identification of pro-survival components for use in biomaterials:

Treatment of ischemic injury was selected as a model to evaluate the biomaterial disclosed herein. To identify the peptide, or peptide analogs to be used as pro-survival components in the biomaterials, a selection of growth factors (TABLE 1) were evaluated. For the initial screening, bone marrow mononuclear cells (BMMNCs) were used, given their prevalence in clinical trials and their potential applications to human patients. BMMNCs were harvested from transgenic L2G mice, which constitutively express the firefly luciferase (FLuc) and green fluorescence protein (GFP) reporter genes driven by the b-actin promoter (FIG. 9A-B). BMMNCs were co- injected with individual pro-survival factors at separate sites under the dorsum of adult FVB donor mice, and in vivo cell survival was monitored by bioluminescence imaging (BLI) (FIG. 9C). BMMNCs co-injected with bone morphogenetic protein-2 peptide analog (BMP2), erythropoietin peptide analog (EPO), and fibroblast growth factor-2 peptide analog (FGF2) were observed to survive longer than cells delivered alone or with other molecules, although all cells were observed to die by day 17 post-injection due to the short half-lives of the BMP2, EPO, and FGF2 factors. In vitro lactate dehydrogenase (LDH) assays confirmed decreased cytotoxicity in BMMNCs cultured under hypoxic conditions when incubated with BMP2, EPO, and FGF2 (FIG. 9D). Western blot of BMMNCs demonstrated activation of BMP2, EPO, and FGF2 recombinant protein activated AKT (Protein kinase B, also known as AKT) and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) pro-survival signaling pathways (FIG. 9E).

[00108] Dose dependent activation of AKT and ERK was further detected when cells were treated with the peptides (FIG. 10A) as well as up-regulation of anti-apoptotic and pro-survival proteins Hsp70 and Bcl-xL (B-cell lymphoma-extra large) and down-regulation of cleaved Caspase 3, indicating reduced amount of apoptosis (FIG. 10B).

[00109] A combination of BMP2, EPO, and FGF2 individually crosslinked to dendrimized collagen (e.g., Col^xDxBMP2, ColxDxEPO, and Col^xDxFGF2) were used in many of our assays described in the other examples disclosed herein to evaluate and demonstrate the efficiency of our biomaterial implant’s Slow Release Delivery System (SRDS).

EXAMPLE 2

[00110] Design of an implantable Collagen-Dendrimer-Peptide biomaterial (ColxDxpep): In order to increase the amine functionality on collagen (Col) matrix, collagen was crosslinked with first generation of polyamidoamine (PAMAM) dendrimers, which are rich in amine groups (FIG. 1 A). Dendrimer (D) crosslinking was achieved by coupling the amine groups on dendrimers to the carboxyl groups of collagen’s -12 % acidic amino acids (e.g., aspartic acid and glutamic acid) through the standard peptide coupling method utilizing l-Ethyl- 3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide (EDC/sulfo-NHS) to obtain a CoDD scaffold. Pro-survival peptides BMP2, FGF2, and EPO were separately crosslinked to the dendrimers on collagen using the same crosslinkers.

[00111] The primary amine content of the collagen was determined via a colorimetric assay using trinitrobenzene sulfonic acid (TNBSA). The amine content was normalized to the collagen concentration, which was determined by hydroxyproline assay, as described later. A significant increase (~5-fold) of amine groups was observed after conjugation of dendrimers (FIG. 1B). Collagen samples were further analyzed by Tris-Borate-EDTA- polyacrylamide gel electrophoresis (TBE-PAGE), an electrophoresis technique optimized to detect free dendrimers. Prior to dialysis, only traces of free dendrimers were detected, indicating a large extent of crosslinking (FIG. 1C). After dialysis, no free dendrimer was found to migrate into the gel, confirming that the increased amine functionality was due to immobilized dendrimers.

[00112] To quantify the amount of pro-survival peptides crosslinked to the collagen in our ColxDxpep biomaterial, the A-terminus of the peptides was modified with hexynoic acid (hexynoyl group contains a carbon-carbon triple bond), which can be

quantitatively measured by an azide fluorescent probe using a click chemistry approach (FIG.

1D). Crosslinking occurred between 7-19 nmol peptide/collagen by weight, resulting in an average crosslinking efficiency of 33±12% SD (range between 26% - 56%) for all three peptides (FIG. 1E). Peptide crosslinking was further confirmed by Sodium Dodecyl Sulfate- Poly Acrylamide Gel Electrophoresis (SDS-PAGE) (FIG. 1F). Most of the free peptides were removed after dialysis as evidenced by the presence of low molecular weight peptides (5-10% by densitometry) in the dialyzed samples. Self-crosslinking of collagen was identified as a very high-molecular weight species in the wells of SDS-PAGE (-20% by densitometry). Therefore, it is concluded that the signals detected after the click reaction were from peptides that were covalently immobilized to collagen. Further Size-Exclusion Chromatography (SEC) (FIG. 11) indicated that the collagen-dendrimer complexes (ColxD) and collagen-dendrimer-peptide complexes (ColxDxpep) elute at high molecular weight region similar to collagen. The low molecular weight species (non-crosslinked peptides) were present < 5%.

[00113] The secondary structures of collagen in ColxDxpep preparations both with and without linked peptides were examined by circular dichroism (CD) (FIG. 12). Positive peak at 221 nm was consistent with the triple helix structure of collagen (FIG. 12A). Temperature dependent CD spectra of collagen with linked peptides demonstrated the same trend as control (e.g., collagen only) (FIG. 12B). Differential scanning calorimetry (DSC) further demonstrated the thermal stability of collagen samples was unaltered when modified with dendrimer and peptides (FIG. 12C). ColxDxpep was then analyzed by Raman spectroscopy. Investigation of the region, characteristic of the amide I band (1620-1720 cm ¹) showed that the triple helix conformation was preserved upon grafting of dendrimers and linking to peptides even though a decrease in the intensity of the peaks was observed without any peak shift (FIG. 13 A). The region characteristic to C-H vibrations, demonstrated a strong peak around 2995 cm ¹ for the dendrimer-functionalized collagen, which is attributed to NH₃ ⁺ group in the material (FIG. 13B). Another well-known property of collagen I, namely, fibril formation from acid soluble collagen I monomers, was observed in vitro by raising the pH and temperature of the collagen solution. As quantified by turbidity assay, unmodified collagen exhibited a typical turbidity curve of increased optical density as collagen fibrils formed (FIG. 14). Interestingly, no increase in turbidity was detected for peptide-linked collagen, indicating that crosslinking stabilized collagen preparations remained in a non-fibrillar form. Dynamic light scattering (DLS) relaxation curves (FIG. 15) showed the absence of a plateau around the time points approaching 1 second, which indicated the presence of large aggregates in the peptide-linked collagen. A plateau below 1000 microseconds showed no relaxation, indicating the absence of any individual small particles in solution. The morphology of collagen before and after crosslinking with BMP2, EPO, and FGF2 were also examined by scanning electron microscope (SEM).

Einmodified collagen showed a fibrous structure, whereas peptide crosslinked collagens appeared as aggregates (FIG. 1G). Further, atomic force microscopy (AFM) (FIG. 16) showed non- fibrillar structural features that were ~l nm in height, which excludes the possibility of fiber bundles, indicating the presence of non-fibrillar aggregates.

EXAMPLE 3

[00114] Gradual release of peptide factors from ColxDxpep biomaterial: To test whether covalent conjugation results in a slower release of the peptide analogs, the release kinetics of crosslinked versus unlinked peptides were compared in a cell free system by using a one-sided slab at the bottom of a micro centrifuge tube with phosphate buffered saline (PBS) layer on top. Peptides were labeled with an azide fluorescent probe using click chemistry for tracking and quantification. Unlinked peptides were rapidly released within the first two to three days when physically mixed with collagen, which is consistent with the short peptide half-lives reported previously in the literature (FIG. 2). By comparison, crosslinked peptides demonstrated prolonged release lasting for periods longer than 15 days. A mixture of CoUD and free peptides (EPO, or BMP2, or FGF2) was included as an additional control to examine the effect of dendrimer crosslinking on free peptide release. Interestingly, dendrimer crosslinking resulted in different release profiles for different peptides, possibly due to the differential alterations in collagen structure and charges (FIG. 2A-C). The slowest release profile, however, was observed only when peptides were covalently crosslinked to the CoUD scaffold (FIG. 2D).

[00115] Peptides covalently crosslinked are released gradually as collagen degrades and autolysis occurs. The presence of the dendrimer moieties promotes hydrolysis of the amide bonds, fostering peptide release (FIG. 17). Of the two cleavable sites: dendrimer- peptide and dendrimer-collagen, the dendrimer-peptide link has greater exposure to solvent, is more accessible for chemical transformations, and is thus more prone to autolysis. After autolytic cleavage, the release kinetics depends on the binding affinity of the peptide to dendrimers in ColxD scaffold, which varies according to the number of hydrogen donor groups in each peptide. The quicker release of peptides from“ColxD + peptide” compared to“Col + peptide” is likely due to the low binding affinity of peptides to the highly positively charged dendrimers present in ColxD (FIG. 2A-C). Of the three peptides in our cocktail, BMP2 and EPO have the lower number of hydrogen bond donor groups in their sequences (FIG. 18), resulting in a lower binding affinity and a faster release (FIG. 2A-C). The Raman spectroscopic results also indicate a strong peak around 2995 cm ¹, which is attributed to the NH₃ ⁺ group in ColxDxBMP2 and

ColxDxEPO, indicating higher charge density (FIG. 13B). Mass Spectrometry (MALDI) was then used to determine the molecular weight of released peptides (FIG. 19 A), whose molecular weight was comparable to free peptides as shown by stained SDS-PAGE and fluorescence imaging (FIG. 19B-C).

[00116] In vivo release kinetics of peptides labeled with an azide fluorescent probe was then assessed by injecting collagen crosslinked peptides into the hind limbs of severe combined immune-deficient (SCID) mice and explanting the gastrocnemius muscle at defined time points after delivery. Because the released peptides are constantly removed (either by consumption or via clearance), the results represent the quantity of the ColxDxPep that is present/remaining in the tissue. For non-conjugated biomaterial preparations, free peptides were observed to fall to low levels as early as 10 days after injection (FIG. 2A-C). In contrast, crosslinked biomaterial preparations yielded detectable levels of free peptide through day 60 post-injection (FIG. 2D). Subcutaneous injection of L2G BMMNCs into the dorsum of SCID mice confirmed that the slow release of growth factors by ColxDxpep prolonged cell survival in vivo as compared to BMMNCs delivered with unlinked collagen or peptides (FIG. 20A-C). EXAMPLE 4

[00117] Functional improvements in murine models of hind limb ischemia and myocardial infarction: To test the bioavailability and the diffusion of collagen-based slow release delivery system in vivo , firefly D-luciferin (Luc) was linked to the biomaterial

(ColxDxLuc) (FIG. 21). Animals were injected with ColxDxLuc in the left gastrocnemius muscle, and Col + Luc or PBS + Luc alone was injected in the right gastrocnemius muscle as control. BLI was performed at various time points until only background signals were measured. These results confirmed that the crosslinked molecules were delivered slowly (FIG. 22).

Movement or diffusion was also restricted due to the high molecular weight (FIG. 19) of the biomaterial slow release delivery system and the binding of the collagen element to factors in the extracellular matrix (ECM) (FIG. 23), including collagen in the ECM, the fibronectin heparin complex, and glycosaminoglycans (e.g., heparin and heparin sulfate). These interactions helped retain the injected complex for at least 14 days (FIG. 22). To study the binding of ColxDxpep with extracellular matrix, different concentrations of ColxDxpep was titrated against chondroitin sulfate, heparin sulfate, hyaluronic acid, collagen-IV, collagen-I and fibronectin. These results (FIG. 24) indicate that the ColxDxpep interacts and binds to ECM components.

[00118] The pro-survival effects of the ColxDxpep biomaterial’s slow release delivery system on BMMNCs were evaluated in a murine model of hind limb ischemia.

Preparations of PBS, unlinked peptides, or ColxDxpep with l x lO⁶ BMMNCs were delivered into the right hind limb of SCID mice and immunocompetent mice following unilateral hind limb ischemia. Cell survival was monitored by BLI at days 2, 7, and 14 after transplantation. BLI signal from cells injected with PBS alone or unlinked peptides diminished by day 14, indicating significant cell death as previously reported. By comparison, cells delivered with ColxDxpep were found to engraft robustly for the duration of the experiment (FIG. 3 A-B). To assess the physiological consequences of the implanted cells and/or materials, limb perfusion was monitored by laser Doppler imaging (FIG. 3C-D; FIG. 4). Mice injected with PBS had only low levels of revascularization due to spontaneous recovery. Mice that received ColxDxpep alone or BMMNCs with unlinked peptides demonstrated a mildly improved recovery. In contrast, the injection of cells with the ColxDxpep with cells resulted in marked improvement of perfusion, reaching statistical significance on day 2 post-transplantation as compared to other groups for SCID recipients and day 14 for immunocompetent animals (FIG. 3C-D; FIG. 4).

[00119] ColxDxpep biomaterial slow release delivery system’s was evaluated for application to other stem cell populations more relevant to treatment of myocardial infarction (MI). Previous studies have demonstrated the potential of cardiac progenitor cells (CPCs) for cardiac regeneration but suffered from poor survival in the ischemic heart. To evaluate the biomaterial (pro-survival matrix) disclosed herein, CPCs derived from the hearts of L2G mice were transplanted these cells into the myocardial border zone of SCID mice and

immunocompetent mice undergoing ligation of the left anterior descending artery. To ensure that any effects in CPC survival were due to ColxDxpep slow release, a number of control preparations were used including: 1) PBS + cells, 2) collagen + cells, 3) free peptides + cells, and 4) collagen + uncrosslinked peptides + cells.

[00120] CPCs delivered without pro-survival matrix demonstrated poor survival, with 80% cell loss by day 4 post-injection, and over 90% loss by day 10, matching previously published findings. In contrast, BLI signal from cells mixed with ColxDxpep persisted at extremely robust levels for up to 8 weeks following delivery in SCID mice (FIG. 5A-B). Cells mixed with PBS, collagen alone, free peptides alone, or unlinked collagen and peptides failed to have the same effects, indicating that the slow release of peptides is required for prolonged survival. Although cell survival was also prolonged in immunocompetent mice treated with ColxDxpep, the improvement was not as robust as seen in the SCID mice (FIG. 5C-D).

[00121] Cell engraftment in the border zone of the infarcted myocardium was confirmed by fluorescence dissecting scope of explanted hearts from a subset of animals at day 30 post-cell injection. GFP expression was detected at robust levels only in the ColxDxpep + cells group, but not in other control groups, validating the pro-survival effects of the slow release peptide delivery system (FIG. 6A). Activation of the AKT and ERK survival signaling pathways in heart tissues by ColxDxpep was revealed by immunofluorescence staining several weeks after cell delivery (FIG. 6B). Taken together, these results suggest that collagen crosslinked pro- survival peptides promote long-term survival of CPCs after transplantation into the mouse heart by activating the AKT and ERK pathways.

[00122] Next cardiac function was assessed in treatment groups by

echocardiogram and small animal magnetic resonance imaging (MRI) through 8 weeks post- surgery. At day 2 post infarction, echocardiogram demonstrated a significant decrease in fractional shortening for all animals compared with that seen at baseline, consistent with successful induction of myocardial infarction (MI). At week 2, no significant change was found in any group, likely due to the residual effects of myocardial stunning from acute injury.

However, at weeks 4 and 8, SCID animals treated with ColxDxpep + cells were found to have a statistically improved left ventricular function as measured by echocardiography (FIG. 6C-E). Although this improvement in cardiac function was observed to persist in immunodeficient mice up to 8 weeks post-MI, it was only maintained in immunocompetent mice up to 6 weeks post- transplantation (FIG. 7A-B). These findings are consistent with the BLI results above, which demonstrate improved cell survival in SCID mice as compared to immunocompetent mice (FIG. 5). MRI confirmed improvement in ejection fraction for SCID animals at week 4 and 8 (FIG. 7C- D), as well as left ventricular remodeling at weeks 2, 4, and 8 following MI (FIG. 8A).

Importantly, histological analysis further corroborated our in vivo imaging results, demonstrating that mice treated ColxDxpep had less infarct and more viable tissue than other treatment groups (FIG. 8B-C).

EXAMPLE 5

[00123] RNA sequencing confirms ColxDxpep activates pro-survival pathways: To confirm that ColxDxpep activated pro-survival pathways leading to improvement in cell survival and tissue function, we performed RNAseq of CPCs treated with ColxDxpep, unlinked collagen+peptide, and peptide-only treated samples following a 96h in vitro incubation. Gene expression analysis suggests that the ColxDxpep replicate samples were significantly different from either the unlinked collagen + peptide or the peptide treated samples, suggesting a synergistic effect on the recipient cell’s gene expression from the crosslinking. Pathway analysis of the differential expression data suggests that, compared to collagen+peptide and peptide-only treated samples, treatment with the ColxDxpep led to increased expression of genes involved in MAPK and PI3K-AKT pro-survival signaling pathways as well as reduced pro-apoptotic signaling from extra-cellular stimuli (FIG. 25). In particular, expression of multiple genes along MAPK cascade including HRAS, RAF1, MAP2K1/MAP2K2, and MAPK1 were found to be increased, along with PI3K-AKT signaling genes including PI3KCA, PI3KCG, and PI3KR1, which also showed up-regulation. Similarly two major pro-apoptotic signaling genes TIMP3 and CD28 were observed to be repressed at the transcriptomic level in cells treated with ColxDxpep. Taken together, RNA sequencing data suggest slow release of EPO, BMP2, and FGF2 signaling led to an increase in pro-survival and proliferative pathways and a repression of apoptosis at 96h post-treatment, consistent with observations from functional studies described in the other examples.

EXAMPLE 6

[00124] Characterization of the collagen linked peptide factors by Size exclusion chromatography (SEC): The SEC (FIG. 11) indicates that the Col, CoED and CoEDxpep elute at low retention time as high molecular weight region. The low molecular weight species are either absent or present at very low concentrations at above the retention time 20 min. The results indicate that the amount uncrosslinked peptide is very low (<5%) or non- detectable. The peak shift in CoED indicates that it forms a compact structure due to the presence of multiple charged dendrimers. A peak shoulder observed ~6 min indicate the presence of collagen as a dimer that can be due to crosslinking.

EXAMPLE 7

[00125] Characterization of the collagen linked factors and CoEDxpep by Raman spectroscopy at different regions: Investigation of the region, characteristic of the amide I band (1620-1720 cm-l) (FIG. 13) shows that grafting of PAMAM dendrimers and linking to peptides both decreases the intensity of the peaks responsible for the secondary structure. However, no peak shift was observed towards any other conformation indicating the triple helix was preserved. The investigation of the region, characteristic of C-H vibrations, demonstrates a strong peak around 2995 cm-l for the dendrimer-functionalized collagens, which is attributed to NH₃ ⁺ group in the material.

EXAMPLE 8

[00126] Characterization of the collagen linked factors and ColxDxpep by Dynamic Light Scattering (DLS): Even when largest clumps in collagen solutions have precipitated, the remaining seemingly clear solution still contained very large particles, as can be seen from DLS results (FIG. 15). The shortest relaxation times for two samples in particular, BMP2 and EPO, are governed by a single exponential process. If it is assumed that the particles have the shape of a sphere, their diameter may be in several microns. DLS relaxation curves do not show a plateau even for times approaching 1 second, which is indicative of very large aggregates present in solution. For very short times, however, a certain plateau exists for all of the samples up to 1000 microseconds. Lack of a relaxation below this time proves there are no individual small particles in solution, which is to be expected for a gel.

EXAMPLE 9

[00127] Characterization of ColxDxpep by Atomic Force Microscopy (AFM):

AFM images (FIG. 16) show aggregates that lack of collagen fibrils. All of the features are approximately 1 nm in height, which excludes the possibility of collagen fiber bundles. For ColxDxBMP2, grafted dendrimers are visible as small grains on the strands.

EXAMPLE 10 [00128] Characterization of the peptides released from ColxDxpep analyzed by MALDI-TOF and electrophoresis: The ColxDxpep biomaterial was taken in a dialysis tube (25 kDa MWCO) and then dialyzed for 48 hr at room temperature. The released peptide was also characterized Mass Spectrometry (MALDI) as well as SDS-PAGE. In order to visualize the weak band in the Coomassie staining, the released peptides and ColxDxpep were

fluorescently labeled using the click chemistry between the triple bond in peptides and the fluorescein azide. The results indicate that the peptides are released in the intact form (FIG. 19).

EXAMPLE 11

[00129] In vivo proof of biomaterial’s slow release delivery system (SRDS):

BLI was performed to evaluate the bioavailability of collagen crosslinked luciferin (SRDS-Luc), compared to luciferin alone I PBS (Luc) and collagen mixed with luciferin (uncrosslinked) (Collagen-Luc). The luciferase proteins expressed by the reporter gene generate a signal once interaction occurs with the reporter probe D-Luciferinl6. This signal is detectable by a charge- coupled device (CCD) camera over time. With a stable gene expression and subsequent luciferase expression, BLI signal measurements represent the bioavailability of free Luc.

[00130] Directly after injection, BLI signals significantly differed among groups, with a significantly higher mean BLI signal in the Luc group of l,590xl07±140xl07 (or 1.59c1010±0.14x1010) p/s/cm2/sr compared to the Collagen-Luc group with BLI measurements of l,070xl07±134xl07 (or 1.07c1010±0.134x1010) p/s/cm2/sr and compared to the SRDS-Luc group with BLI measurements of 3 l lxl07±12.2xl07 (or 3.l lxl09±0.l 22x 109) p / s/ cm2/ sr, p<0.05 between groups. This correlates with a higher bioavailability of free Luc directly at injection, compared to Luc mixed with collagen or to SRDS-Luc. A collagen network retains the active, free Luc resulting in the lower baseline signal in the Collagen-Luc group 17. The SRDS- Luc group showed a lower BLI signal at baseline because of the crosslinked Luc, which prevents initial availability for the luciferase-luciferin reaction.

[00131] Because of the short half-life of D-Luciferin, BLI signals decrease dramatically within hours after injection in the Luc group. At 3 hr after injection, a significantly higher BLI signal was seen in the SRDS-Luc group of 13.0c107±2.5c107 (or

L3xl08±0.25x108) p/s/cm2/sr compared to control groups with a BLI signal of

1.1 x 107±0.69x 107 p/s/ cm2/ sr in the D-Luc group and 0.66x 107±0.27x 107 (or 6.6x 106±2.7x106) p/s/cm2/sr in the Collagen-Luc group. Collagen alone, without the SRDS showed no effect on prolonging bioavailability of D-Luciferin in this study, as BLI signals were comparable to the Luc control group after 1 hr following injection.

[00132] The sustained bioavailability was seen in the SRDS-Luc group up to 14 days following injection with significantly higher mean BLI signals of 17.0xl03±4.6x103 p/s/cm2/sr compared to Collagen-Luc and Luc controls with BLI signals of 2.7xl03±0.33x103 p/s/cm2/sr and 3.7xl03±0.34x103 p/s/cm2/sr respectively, p<0.05 compared to control groups. A BLI signal was measurable in the SRDS-Luc group up to 21 days following injection, with a mean BLI signal of 5.9xl03±1.4xl03. At 24 days after injection, all groups showed BLI signals comparable to background signals (FIG. 22).

[00133] Further, ColxDxpep is crosslinked and cannot form fibrils, because the crosslinked dendrimers reduce collagen-collagen interaction that promote self-assembly. Hence the gelation process is inhibited. A strong contributing factor to the gel remaining at the injection site is that the crosslinked complex has significantly increased molecular weight and reduced mobility. The collagen component itself binds with the following elements of the extracellular matrix (ECM) to remain in place: (a) collagen in the extracellular components, (b) fibronectin heparin complex, and (c) glycosaminoglycans such as heparin and heparin sulfate that are present at the injection site of the gel 18. In addition, other studies have shown positively charged dendrimer components bind with moderate affinity negatively charged protein glycanl8. These interactions help retain the injected complex at the site of injection (FIGs. 23, 24).

EXAMPLE 12

[00134] Collagen-extracellular matrix binding studies: Extracellular fluid flow could wash away the collagen-linked/encapsulated factors considering the“gel solubility” data indicated by AFM (FIG. 16). Several cytokines, and growth factors are known to bind to glycosaminoglycans (GAG) such as heparin sulfate and chondroitin sulfate. Trinucleotides (CAGs) are produced by the cells and surrounds the cells in the pericellular envelope, and in the extracellular matrix (ECM) and also accumulate lipoproteins lipase, thrombomodulin, fibronectin. These secondary interactions are likely to increase the temporal and spatial resident time of these factors that are binding to the CAG. Hence if the soluble collagen linked factors have moderate binding to the CAG, they could get retained to the place of injection by the same mechanism. However, when they are released as peptides their diffusion should not be impaired because they lack the GAG binding sites binding sequences as their full-length counterparts.

[00135] Collagen component itself binds with (a) collagen in the extracellular components (b) with fibronectin heparin complex (c) glycosaminoglycans such as heparin and heparin sulfate are primary components of the extracellular matrix (ECM) that constitute the injection site of the gel. Early researchers have shown positively charged dendrimer components binds with moderate affinity negatively charged proteoglycanl8. These interactions will help retain the injected complex. To study the binding of collagen with components of extracellular matrix, different concentration of ColxDxpep biomaterial was titrated against chondroitin sulfate, heparin sulfate, hyaluronic acid, collagen-IV, collagen-I and fibronectin. Concanavalin, a non-ECM protein was used as a negative control. The results (FIG. 24) indicate that the

ColxDxpep biomaterial interacts and binds to ECM components.

EXAMPLE 13

[00136] Effect of needle gauge on the release profile: The results (FIG. 26) indicate that the collagen materials after passing through 5 different needles gauge (18G, 23G, 25G, 27G and 30G needles) 10 times each showed no significant difference in the release profile.

[00137] TABLE 1 Pro-survival factors tested in the initial screen

[00138] Table 2A-C: List of the biomaterials indicating crosslinking chemistry strategies used for each of the biomaterials for the growth factors/growth factors analogs and small molecules, A. EPO; B. FGF2; C. BMP2. Macromolecules used in these formulations are Collagen I, Hyaluronic acid, PLGA and PLA. Footnotes indicate details of the peptide sequence.

[00139] TABLE 2A

rhEPO = recombinant human Erythropoietin

EPO peptide analog 1= GGTYSCHFGPLTWVCKPQGG, disulfide: C6-C15 (peptide sequence; 1 -letter aminoacid code; SEQ ID: 1)

EPO peptide analog 2= TYSCHFGPLTWVCKPQGG, disulfide: C4-C13 (peptide sequence; 1- letter aminoacid code; SEQ ID: 3)

EPO peptide analog 3 =GGQEQLERALNSS (peptide sequence; 1 -letter aminoacid code; SEQ ID: 5)

[00140] TABLE 2B

RKRLDRIAR-NH2 (SEQ ID: 6)

[00141] TABLE 2C

BMP2 peptide analog 1 : KIPKASSVPTELSAISTLYL (peptide sequence; l-letter aminoacid code; SEQ ID: 7)

BMP2 peptide analog 2: CGKIPKASSVPTELSAISTLYL (peptide sequence; l-letter aminoacid code; SEQ ID: 8)

METHODS

[00142] Preparations of peptide crosslinked collagen: BMP2 mimetic peptide (5Hexynoic-KIPKASSVPTELSAISTLYL), EPO mimetic peptide (5Hexynoic- GGTYSCHFGPLTWVCKPQGG, disulfide:C6-Cl5) and FGF2 mimetic peptide (5Hexynoic- YRSRKYS SWYVALKRK(YRSRKY S S W YVALKR)- Ahx- Ahx- Ahx-RKRLDRIAR-NH₂) were obtained from CS Bio (Menlo Park, CA). Acid-soluble collagen I from rat tail (100 mg in 0.02 N acetic acid, -10 mg/ml) (BD) was dialyzed using 10K MWCO dialysis cassettes (Thermo Scientific) against 50 mM 2-(N-morpholino) ethanesulfonic acid (MES buffer), pH 5.0 at 4 °C. To increase amine functionality, 2.7 ml (molar excess) of PAMAM dendrimers (Sigma- Aldrich) mixed with 2.0 ml of MES buffer (pH adjusted to 7.0) was then added to 3 ml of collagen in MES buffer, and was mixed using a 18G needle by gently drawing up and then expelling repeatedly for 10 min at 4 °C. The addition of excess amount of dendrimers also helped minimize self-crosslinking of collagen. To this mixture 2 mg of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific) and N-hydroxy- sulfosuccinimide (Sulfo-NHS) (Thermo Scientific) were added and mixed using a 18G needle for 30 min, then overnight with a stir bar at 4 °C. The above mixture was then diluted with 3.2 ml of 50 mM MES buffer pH 7.0 and forced through 21G, 26G, and 30G needles. Importantly, passing through the needles with different gauges did not affect the crosslinking or the release profile of the material (FIG. 26). The treated collagen was then dialyzed against 50 mM MES buffer pH 7.0 to remove unreacted dendrimers and crosslinking reagents. For crosslinking of BMP2 and EPO, 3 mg of peptides were dissolved in 0.3 ml dimethyl formamide (DMF) and activated by mixing with EDC (1 mg) and Sulfo-NHS (2 mg) dissolved in 60 mΐ 50 mM MES pH 5.0 for 30 min at room temperature. After activation, the peptides were purified on a PD mini trap G10 column (GE Healthcare Life Sciences) and then mixed with 3 ml of dendrimerized- collagen at 4 °C for 30 min using a syringe with 18G needle, and overnight by a stir bar. Peptide- linked collagen was then dialyzed against 50 mM MES buffer pH 6.0 to remove unreacted peptides and crosslinking reagents.

[00143] Quantification of free amines by TNBSA assay: Primary amine groups of unmodified and crosslinked collagen were determined using TNBSA assay (Thermo

Scientific) according to the manufacturer’s instructions. The free amine groups were quantified by comparison to a standard curve of known concentrations of glycine.

[00144] Click chemistry and fluorescence assays to quantify peptide using coumarin azide: To quantify the amount of pro-survival peptides crosslinked to the collagen, the N-terminus of the peptides were modified with hexynoic acid, which can be quantitatively measured by an azide fluorescent probe using the click chemistry. A coumarin azide derivative was used as the probe, which by itself was non-fluorescent due to the quenching effect from the electron-rich a-nitrogen of the azido group. Upon click reaction, the quenching effect is released, yielding a fluorescent signal proportional to the amount of peptide. 12.5 mΐ of 2 mM 3-azido-7- hydroxycoumarin (Glen Research) dissolved in DMF, a premixed solution of 12.5 mΐ of 5 mM CuS0₄ in H₂0 and 12.5 mΐ of 5 mM tris(benzyltriazoylmethyl)amine (TBTA) (Sigma) dissolved in DMSO, and 25 mΐ of 100 mM sodium ascorbate (Sigma) in H₂0 were added to the free and crosslinked peptide samples. The total volume was then brought up to 250 mΐ. The pH was adjusted to 4.0 using 1N HC1. The reaction mixture was incubated at room temperature in the dark for 1 hr. Fluorescence intensity was measured using a GloMax-Multi micro-plate reader (Promega) with a UV optical kit (excitation 365 nm, emission 410-460 nm). The concentration of peptides was calculated by comparison to a standard curve of known concentrations of propargyl alcohol.

[00145] In vitro peptide release assay: 200 mΐ of collagen with crosslinked or uncrosslinked peptides was deposited at the bottom of a 1.5 ml micro centrifuge tube. After incubation at 37 °C for 15 min, 500 mΐ of 1 / PBS was carefully layered on top of the collagen. The mixture was then incubated at 37 °C for up to 15 days. 100 mΐ of solution was withdrawn from the PBS layer at different time points and replenished with 100 mΐ of fresh 1 xPBS. Peptides released into the supernatant were quantified by fluorescent labeling using click chemistry as described above.

[00146] In vivo peptide release assay: The hind limb gastrocnemius muscle of SCID mice was explanted at the various time points after the injection of coUD/pep. The tissue was homogenized in 300 mΐ of PBS to extract the peptides into the solution. The homogenate was centrifuged at 5000 g for 10 min. The peptides present in the supernatant were quantified by fluorescent labeling using click chemistry as described above.

[00147] Isolation and culture of CPCs: Animal protocols were approved by the Stanford University Animal Care and Use Committee. CPCs were isolated from the L2G85 transgenic mice of FVB background with b-actin promoter driving FLuc-eGFP as previously described. Briefly, hearts were explanted, cut into 1-2 mm pieces, and digested with 0.1% collagenase II for 30 minutes at 37 °C while on a shaker. Cells were then filtered through a 100- pm strainer and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 0.1 mM nonessential amino acids, 100 U/ml Penicillin G, 100 pg/ml streptomycin, 2 mM glutamine, and 0.1 mM b-mercaptoethanol. After 3 weeks of culture, a population of phase-bright cells was observed to appear over the adherent cells. Phase-bright cells were collected by light digestion with a cell dissolution buffer (Life Technologies, Carlsbad, CA) at room temperature under microscope monitoring, and sub- cultured in poly-lysine coated plates (BD Biosciences) with the same medium.

[00148] Myocardial infarction and cell delivery: SCID Beige mice (Charles River Laboratories, MA.) were stratified into one of six groups (n = 12 per group), each receiving an intramyocardial injection of CPCs suspended in different solutions as listed: 30 pl of PBS, 30 pl of unmodified collagen I (5 mg/ml in 50 mM MES pH 6.0), 30 pl of a mixture of BMP2, EPO, and FGF2 (0.3 mg/ml each in a 1 : 10 solution of DMSO: PBS), 30 pl of an admixture of collagen and peptides (same concentrations as above), or 30 pl of 1 :1

colxD pepxol (at a final concentration of 5 mg/ml in MES pH 6.0 for each). Heparin (Sigma,

St. Louis) was also added 1 : 100 to the groups that contain collagen. For the study of cardiac function, myocardial infarction was induced by aseptic lateral thoracotomy and ligation of the left anterior descending coronary artery, as previously described.

[00149] Bioluminescence imaging to monitor survival of transplanted cells in living mice: BLI was performed using the Xenogen IVIS 200 in vivo imaging system (Alameda, CA). After intraperitoneal injections of reporter probe /4-Luciferin (250 mg of luciferin/kg), animals were imaged with exposure times ranging from two seconds to two minutes pre-surgery and followed at days 1, 2, 4, 7, 14, and weekly thereafter to day 56 post-surgery (n = 6 per group). Immunocompetent FVB animals were imaged weekly until day 42 post-surgery (n = 6 per group). Imaging signals were quantified in units of maximum photons per second per square centimeter per steradian (p/s/cm²/sr), as previously described.

[00150] Immunofluorescence staining: Immunofluorescence stains were performed using primary antibodies phospho-AKT and phospho-ERKl/2 (Cell Signaling Technology, Boston, MA) and AlexaFluor conjugated secondary antibodies (Invitrogen) as previously described. DAPI was used for nuclear counterstaining.

[00151] Hind limb ischemia model: Hind limb ischemia was induced in SCID and immunocompetent FVB animals as previously described. Briefly, mice were anesthetized with 1.5% isoflurane and the right hind limb was opened to expose the femoral artery for ligation, after which lxlO⁶ BMMNCs were delivered into the gastrocnemius muscle using a 29 gauge Hamilton syringe (n = 5 per group). Control animals received PBS alone. Skin was closed using 6-0 silk sutures. Following surgery, cell survival and revascularization was monitored by BLI and revascularization, respectively. The appropriate authorities approved animal studies.

[00152] Measurement of hind limb blood flow by laser Doppler imaging: Animals were knocked down using 1.5% isoflurane in oxygen, and hind limb vascularization was monitored by laser Doppler perfusion imaging using a PeriScan PIM3 laser Doppler system (Perimed AB, Sweden) as described previously for both SCID and immunocompetent FVB animals (n ^::: 5 per group). Temperature variability was maintained at constant levels by keeping animals on heat pads set to 37 °C during measurement. Non-ligated contra-lateral hind limbs served as controls. Perfusion was calculated as the ratio of the flow in the ischemic to non ischemic limbs.

[00153] Left ventricular functional analysis with echocardiogram:

Echocardiography was performed using a Siemens-Acuson Sequoia C512 system (Malvern, PA) equipped with a multi -frequency (8-14 MHz) 15L8 transducer. SCID mice were assessed at days 2, 14, 28, and 56 post-surgery (n = 6 per group). Immunocompetent FVB mice were assessed at days 2, 14, 28, 42, and 56 (n = 6 per group). Briefly, animals were knocked down using 1.5% inhaled isoflurane and imaged in the supine position. The following formula was used to calculate fractional shortening (FS) from M-Mode short axis images of the left ventricle: FS = [Left ventricular end diastolic diameter (LVEDD) - Left end-systolic diameter (LVESD)] /

[LVEDD]

[00154] Assessment of cardiac contractility and LV remodeling with magnetic resonance imaging (MRI): A subset of SCID mice was assessed by MRI for cardiac function (n = 6 per group) at days 2 and weeks 2, 4, 8 post-surgery using a Signa 3.0T Excite HD scanner (GE, Milwaukee, Wisconsin) equipped with a Mayo Clinic T/R MRI coil (Mayo Clinic Medical Devices, Rochester, Minnesota) as previously described. Briefly, mice were knocked down using 1.5% isoflurane with 1 ml/min of oxygen, and murine electrocardiogram, respiration, and body temperature were monitored (Small Animal Instruments, Stony Brook, New York). Gradient recalled echo (GRE) was employed for cardiac localization, after which 20 short axis cine frames were acquired using fast spoiled GRE (FSPGR) over 1 complete cardiac cycle. The following imaging parameters were applied: TR = 10 ms, TE = 4.6 ms, number of excitations (NEX) = 10, field of view (FOV) = 40 x 40 mm, matrix = 256 x 256, flip angle (FA) = 45°, slice thickness 1.5 mm, spacing = 0 mm, imaging voxel size: 1.57x1.57 mm. A commercial contour analysis program (Osirix Version 3.81) was used to calculate ejection fraction by tracing the endocardial border of the left ventricle (LV) at end diastole and end systole.

[00155] Statistical Analysis: Continuous variables with normal distribution were expressed as mean ± SD. Differences in continuous variables were compared using a one-way ANOVA or two-way repeated ANOVA, followed by a Students’ t test. A post-hoc Sidak- Bonferroni correction was performed if needed to adjust for multiple comparisons. Statistical analysis was performed using GraphPad Prism (La Jolla, CA). Tests had an alpha level for significance set at p <0.05 were considered significant.

[00156] Data availability: RNA sequencing data have been deposited into the Sequence Read Archive (SRA): https://www.ncbi.nlm.nih.gov/bioproject/PRJNA4l2785.

[00157] Bone marrow mononuclear cell (BMMNC) collection and

characterization: L2G transgenic mice (Stanford ETniversity, Stanford, USA) were used as donors for cell survival evaluationl. L2G mice are bred on a FVB background and ubiquitously express green fluorescent protein (GFP) and firefly luciferase (FLuc) reporter genes driven by a b-actin promoter. BMMNCs were harvested as described previously2,3. Briefly, mice long bones were explanted, washed, and flushed with PBS using a 25-gauge needle to collect bone marrow. After passing through a 70 pm strainer, the isolate was centrifuged at 1200 rpm for 5 minutes. The bone marrow isolate was centrifuged for 30 minutes at 1900 rpm using a 15 ml tube with 3 ml Ficoll-Paque Premium (GE Healthcare, Piscataway, NJ, USA) gradient and 4 ml cell/saline suspension. BMMNCs were prepared freshly before application. Directly after isolation, the BMMNC were re-suspended in PBS. Cell suspensions were placed in a 6-well plate in known concentrations (1 x 105; 2x 105; 4x 105; 6x 105; 8x 105; and l0x l05cells). After administration of Luc (4.5pg/ml), peak signal expressed as photons per second per centimeter square per steridian (photons/s/cm2/sr) was measured using a charged coupled device bioluminescence camera by Xenogen In Vivo Imaging System (IVIS 200, Xenogen, Alameda, CA, USA) as described.

[00158] Pro-survival factors used in the initial screening in vivo: Pro-survival growth factors used for evaluation included: human Bcl-XL BH44-23 (Bcl-XL; Merck,

Darmstadt, Germany); human bone morphogenetic protein 2 (BMP2; R&D Systems,

Minneapolis, MN, USA); cyclosporin A (CsA, Wako Chemicals, Richmond, USA); mouse erythropoietin (EPO; R&D Systems); human basic fibroblast growth factor (FGF; R&D

Systems); human insulin-like growth factor I (IGF-l; R&D Systems); pinacidil monohydrate (pinacidil; Sigma-Aldrich, St. Louis, MO, USA); human transforming growth factor beta 1 (TGF; R&D Systems); vascular endothelial growth factor (VEGF; R&D Systems); and caspase inhibitor 1, Z-VAD (OMe)-FMK (ZVAD; Merck).

[00159] Screening of pro-survival factors in vivo with bioluminescence imaging: The collection and characterization of BMMNCs are described in detail in the SI. Adult SCID mice (20-25 gm, 8 weeks old, Charles River) received isoflurane (2%) for general anesthesia and were subcutaneously injected at individual dorsal sites with 5x l05/site BMMNC. BMMNCs were co-injected with individual pro-survival factors (detailed information of all the factors tested is provided in the SI) or control (medium only) in a total volume of 50 mΐ

Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco), supplemented with 1% penicillin/streptomycin (Gibco; n=3 per group). After transplantation, the mice received intraperitoneal injections of the reporter probe D-luciferin (250 mg/kg body weight) and were imaged repeatedly using the Xenogen In Vivo Imaging System (IVIS 200; Xenogen). BLI was performed at baseline, 24 hr, days 2, 5, 7, and 14, and then weekly until only background signals could be measured.

[00160] In vitro evaluation of pro-survival factors: Freshly harvested BMMNCs were incubated (1x105 cells/well) in DMEM supplemented with 10% fetal bovine serum (FBS) (Hy clone) and 1% penicillin/streptomycin at 37 °C with either 5% C02 in air, or under hypoxic conditions at 1% 02, for 12 hr. Before incubation, the selected individual pro-survival factors were added to the medium and compared to control (medium only). Additionally, a mixture of all 3 selected factors was added to cells and analyzed. Cell injury and apoptosis were assessed using 1) lactate dehydrogenase (LDH) release assay (Cytotoxicity kit, Roche Diagnostic) and 2) annexin-V detection (annexin-V-PE apoptosis detection kit, BD Biosciences), according to the manufacturer’s instruction.

[00161] Hydroxyproline assay: Collagen samples were diluted 10 times with 6 N HC1 and hydrolyzed in a glass Pasteur pipet sealed at both ends at 120 °C overnight. The hydroxyproline content was determined based on a previously reported method using a commercial kit (Chondrex)4.

[00162] Turbidity assay: Collagen samples were diluted in l xPBS to a final concentration of 0.5 mg/ml. 0.5 ml of diluted collagen solution was placed in a semi-micro quartz cuvette (10 mm light path) heated to 37 °C in the temperature controlled holder of NanoDrop 2000c (NanoDrop/Thermo Scientific). Turbidity was recorded continuously for 5 min at the optical density of 313 nm. [00163] Size exclusion chromatography (SEC): SEC was performed using a Shimadzu HPLC, LC-ADvp pumps, CBM 20A module, SPD M20A diode array detector, RF lOA xl fluorescence detector, SIL 20 AC-HT auto sampler and LC Solutions software. BioSep- SEC-s3000 (Phenomenex), 300 x 7.8 mm column was used with isocratic flow of lmL/min in MES buffer. Col, colxD and colxDxpep were labeled with Rhodamine fluorescent dye using Rhodamine-B isothiocyanate (Sigma-Aldrich). The fluorescence emission was measured at 590 nm, exciting at 550 nm during elution.

[00164] Atomic force microscopy (AFM): Samples were prepared for AFM by drop-casting and drying under vacuum of 10 pL droplets of collagen solutions (1 pg/mL) on the surface of clean silicon wafers. AFM imaging was performed with Park Systems NX10 (Suwon, Korea) instrument. Samples were imaged in semi-contact mode with standard commercial cantilevers (k = 5-9 N/m, R < 10 nm), at 0.8-1 Hz scan speed and -30% oscillation damping.

[00165] Dynamic light scattering (DLS): DLS measurements were performed on collagen samples (1 mg/mL) using Brookhaven 90 plus DLS nanosizer (Brookhaven Instruments Corporation, Holtsville, NY) in transmission geometry. The autocorrelation function was approximated manually with a single-relaxation process using second-order term for the cumulant analysis of particle polydispersity.

[00166] Raman spectroscopy: Raman measurements of collagen samples were performed on“just dried” samples on the surface of Ti substrates using 10X (0.17 NA) objective in backscattering geometry with NTEGRA Spectra Raman spectrometer from NT-MDT. Laser power at sample was less than 10 mW. The curves have been baseline-corrected in Origin (OriginLab, Northhampton, MA) using either first or second order curves. [00167] Creation of the collagen-based biomaterial slow release delivery system (SRDS): Analogs of peptides mimicking full-length pro-survival growth factors were made and linked with collagen using dendrimer chemistry protocols as described previously5-8. Acid soluble rat-tail collagen type 1 (BD Biosciences) was dialyzed against 50 mM MES of pH 6.5. Peptide derivatives are activated using 1 -Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide EDC and N-hydroxysulfosuccinimide (Sulfo-NHS) in DMF; Dimethylformamide for 30 minutes. The activated peptides were mixed with collagen solution using syringes using repeated dispensing the viscous liquid thru the syringe. After incubation at 4°C for 12 hr, the solution was dialyzed against MES buffer and phosphate buffer subsequently. The linked collagen was collected from the dialysis cartridges were used without further purification.

[00168] In order to test the SRDS system in vivo , firefly D-luciferin (Luc) was linked to the SRDS according to the protocol as mentioned above (SI Fig. S13). D-luciferin was activated with 4-nitrophenylchloroformate (PNF) as described5-8. D-luciferin 0.274g

(0.89mmol) and a catalytic amount of 4-dimethylaminopyridin (l.25g) were dissolved in 12.5ml Tetrahydrofuran (THF). The solution was cooled to 0 °C and PNF 0.268g (l.335mmol) dissolved in 5ml THF was added dropwise. The reaction was then stirred at room temperature for 3 hr until complete as monitored by Thin-layer chromatography (TLC). The solution was then diluted with Ethyl acetate (EtOAc) and washed with saturated Ammonium chloride. This step was repeated three times and the solvent was then filtered over Magnesium sulfate. Finally, the solvent was removed under reduced pressure to give 2ml of compound 1 in the form of yellowish oil. The product was further purified over column chromatography (24/40, Purasil 230-400 mesh) in EtO Ac/Hexane (1 : 19). Bovine Collagen 1 (Purecol, Advanced Biomatrix, San Diego, CA, EISA) 9mg was dialyzed against 1M HEPES of pH 7 for 12 hr at 4°C. The activated luciferin (40mg) was dissolved in 300 mΐ DMF, mixed with collagen and dialyzed as described above.

[00169] In vivo evaluation of the kinetics of the slow release delivery system (SRDS): BLI was performed to evaluate the in vivo effect of the slow delivery release system, as described previously9. 8-10 weeks old transgenic L2G mice, ubiquitously expressing the firefly luciferase (FLuc) reporter gene driven by a b-actin promoter, were used. Because of the short half-life of free Luc (t½=3.54 minutes), this in vivo model was used to assess the effect of the SRDS on sustained peptide release.

[00170] The animals (n=6) were anesthetized with isoflurane and 100 pl of 0.2 mg/ml SRDS-D-Luciferin (SRDS-Luc) was injected in the left gastrocnemius muscle. As a control, 20 pg of Luc was reconstituted in 100 mΐ of PBS and injected in the right gastrocnemius muscle to monitor the local bioluminescence (luciferase-luciferin) reaction over time. Additional animals were injected with 100 mΐ of the 0.2 mg/ml SRDS-Luc in the left gastrocnemius muscle and as a control, 20 pg of Luc in 50 mΐ was mixed with 50 mΐ of rat-tail collagen type I (Collagen- Luc) and injected in the right gastrocnemius muscle. Bioluminescence imaging (BLI) was performed at baseline, 30 min, 1 hr, 3 hr, 6 hr and 24 hr. Additional imaging was performed at day 2, 4, 6, 7, 14, and weekly until only background signals were measured. At the time of imaging, the animal was placed in a light-tight chamber, and photons emitted from luciferase expression were collected with integration times of 1 sec to 5 min, depending on the intensity of the bioluminescence emission. BLI peak signals (p/s/cm2/sr) from a fixed region of interest (ROI) were quantified and analyzed.

[00171] Binding of ColxDxpep to extracellular matrix components (ECM) by enzyme-linked immunosorbent assay (ELISA) method: Binding of ColxDxpep to chondroitin sulfate, heparin sulfate, hyaluronic acid, collagen-IV, collagen-I and fibronectin. Concanavalin, a non-ECM protein, was used a negative control to measure non-specific binding. The

concentration dependent response of ColxDxpep indicates the binding to ECM components. Binding of ColxDxpep was measured by a colorimetric biotin-streptavidin HRP reaction.

MaxiSorp plates (96 well) were coated overnight at 4°C (without shaking) with each of ECM components separately collagen I, IV, hyaluronic acid, heparin sulfate, chondroitin sulfate, fibronectin (F), and concanavalin A (Neg con) (100 mΐ/well at 5 pg/ml) diluted in 100 mM carbonate buffer, pH 9.3 (except for collagen I, diluted in 250 mM MES buffer pH 6). The plates were rinsed three times with HPBS pH 7.4, containing 0.05% Tween-20 (PBST), which was used for all subsequent rinses and dilutions. The wells were then blocked with 1% bovine serum albumin in PBST (340 mΐ/well) at room temperature for 1 h without shaking. The plates were rinsed three times and subsequently incubated with biotin-labeled ColxDxpep diluted in PBST (100 pl/well, 0, 100, 250 and 500 ng/ml) at room temperature for 3 h on a shaker. After rinsing with PBST for four times, ColxDxpep was detected with streptavidin-HRP (100 pl/well, 200x, R&D SYSTEMS lot 296124) at room temperature for 1 h on a shaker. After rinsing with PBST for four times, each well was incubated with 100 pl ultra- TMB for 10 min without shaking. The reaction was stopped by adding 100 mΐ of 2M sulfuric acid and the absorbance was measured at 450 nm using a plate reader. The results (SI Fig. S16) represent mean ± SD (n = 4) and P < 0.02 (ECM components compared to baseline). Concanavalin was not statistically different compared to baseline.

[00172] Effect of needle gauge on the release profile: In order to study the effect of the needle gauge on the release profile, 5 different needles gauge (18G, 23G, 25G, 27G and 30G needles) were used to pass-through the crosslinked gel. Collagen was crosslinked with dendrimers and then to a fluorescein molecule using the method mentioned in the manuscript. After the preparation, the gel was passed through needles with different gauges for 10 times before placing it in a 10,000 MWCO dialysis membrane. The release of the fluorescein was monitoring by fluorescence spectroscopy. The results are shown in FIG. 26.

[00173] RNA-sequencing: Total RNA was extracted using Qiagen RNeasy from the mouse CPCs (passage 7) treated with collagen (COL), peptides (PEP) and the linked matrix (MAT). The extracted RNA was sequenced with Illumina Hi-Seq (strand-specific l50bp paired- end, 100 Gb total raw reads, ~40M mapped fragments each). RNA-Seq sequencing data were mapped against the mml0/GrcM38 reference genome with the help of hisat2 v.2.0.512. Number of reads overlapping features and meta-features were counted using RSubRead (v.1.26.1)13 and R/Bioconductor (v.3.2.1)14, requiring strand specific paired end reads to Ensembl gene body coordinates. Differential gene expression was analyzed with the aid of DESeq2l5 using default settings. RNA sequencing data is available on the Sequence Read Archive (Bioproject):

(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA4l2785).

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[00174] This invention was made with LIS Government support under contract EB009689 awarded by the National Institutes of Health. The US Government has certain rights in the invention.

Claims

CLAIMS What is claimed is:

1. An implantable biomaterial comprising:

a scaffold comprising a matrix and one or more dendrimers crosslinked to the matrix; and one more or peptides or peptide analogs crosslinked to the dendrimers or the matrix, or both; wherein the matrix comprises a collagen, a hyaluronic acid, a chondroitin sulfate, an extracellular matrix component, or combinations thereof.

2. The biomaterial of claim 1, wherein the matrix comprises a collagen and a hyaluronic acid.

3. The biomaterial of claim 1, wherein the one or more peptides or peptide analogs promote cell survival, cell growth or both.

4. The biomaterial of claim 1, wherein the peptide or peptide analog is selected from one or more of a bone morphogenetic protein-2 peptide (BMP2) or BMP2 analog, a erythropoietin peptide (EPO) or EPO analog, or a fibroblast growth factor-2 peptide (FGF2) or FGF2 analog, or combinations thereof.

5. The biomaterial of claim 4, wherein the EPO comprises a recombinant human

Erythropoietin (rhEPO) peptide.

6. The biomaterial of claim 4, wherein the FGF2 comprises a recombinant human fibroblast growth factor-2 (rhFGF2) peptide.

7. The biomaterial of claim 4, wherein the BMP2 comprises a recombinant human bone morphogenetic protein-2 (rhBMP2).

8. The biomaterial of claim 4, wherein the EPO peptide analog comprises EPO peptide analogs comprising SEQ ID: l, or SEQ ID:2, or SEQ ID:3, or SEQ ID:4, or SEQ ID:5, or combinations thereof.

9. The biomaterial of claim 4, wherein the FGF2 analog comprises SEQ ID: 6.

10. The biomaterial of claim 4, wherein the BMP2 analog comprises SEQ ID: 7, or SEQ ID: 8, or combinations thereof.

11. The biomaterial of claim 1, further comprising molecules or drugs that induce cell growth-promoting or cell-survival promoting peptides.

12. The biomaterial of claim 11, wherein the molecules comprise EPO-inducing, BMP2- inducing, or FGF2-inducing molecules.

13. The biomaterial of claim 12, wherein the FGF2 inducing molecule comprises

Amitriptyline.

14. The biomaterial of claim 12, wherein the BMP2 inducing molecule comprises

Tacrolimus.

15. The biomaterial of claim 12, wherein the EPO inducing molecule comprises a EPO mimicking molecule PAMAM-HMB linked methyl (2-(2-([l,l'-biphenyl]-4-yl)-6-chloro-5- methyl-lH-indol-3-yl)acetyl)-L-lysinate.

16. The biomaterial of claim 1, further comprising stem cells.

17. The biomaterial of claim 16, wherein the stem cells are cord blood stem cells, mesenchymal stem cells, endothelial stem cells, progenitor cells, bone marrow stem cells or combinations thereof.

18. The biomaterial of claim 16, wherein the stem cells are co-implantable with the biomaterial to a wound or injury site.

19. The biomaterial of claim 16, wherein the stem cells are embedded, or adsorbed, or attached, or seeded, or incorporated into the biomaterial.

20. The biomaterial of claim 1, wherein the biomaterial is injectable or implantable in form of a gel, or a patch, or combinations thereof.

21. The biomaterial of claim 1, wherein the one or more peptide analogs is present at a concentration of from about 5% to about 75% of wild type levels of the corresponding peptide in an animal onto which the biomaterial is transplanted.

22. The biomaterial of claim 1, wherein the collagen comprises telopeptides.

23. The biomaterial of claim 1, wherein the dendrimers comprise a Poly(amidoamine) (PAMAM).

24. The biomaterial of claim 23, wherein the PAMAMs are selected from one or more of first, second or third generation PAMAMs.

25. The biomaterial of claim 1, wherein the crosslinking chemistry is selected from one or more of 1 -Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), Bis-N-PEG-carbamoylated lysine, and Boc-B-alanine-N-diacetic diacid.

26. A method for transplantation comprising:

preparing a scaffold comprising a matrix and one or more dendrimers crosslinked to the matrix, wherein the matrix comprises one or more of a collagen, a hyaluronic acid, a chondrointin sulfate, or an extracellular matrix component;

crosslinking one more or peptides or peptide analogs to the dendrimers or the matrix, or both, to yield an implantable biomaterial; and

administering the biomaterial to a tissue or an organ site.

27. The method of claim 26, wherein administering is carried out on in vivo on an animal.

28. The method of claim 26, wherein administering is carried out in vitro or ex vivo, on experimental models.

29. The method of claim 26, wherein administering is carried out to treat or investigate a disease condition.

30. The method of claim 26, further comprising providing stem cells for transplantation.

31. The method of claim 30, wherein the stem cells are cord blood stem cells, mesenchymal stem cells, endothelial stem cells, progenitor cells, bone marrow stem cells, or combinations thereof.

32. The method of claim 30, wherein the stem cells are co-injectable or co-implantable with the biomaterial to the injury site or wound site.

33. The method of claim 30, wherein the stem cells are provided at a concentration of from about 1 million cells/milliliter to about 25 million cells/milliliter.

34. The method of claim 26, wherein administering is carried out in an exosome or with a poly(lactic-co-glycolic acid) (PLGA) or a poly lactic acid (PLA) encapsulation.

35. The method of claim 26, where administering is intranasal, delivering the biomaterial to the brain.

36. The method of claim 26, wherein the one or more peptides or peptide analogs promote cell survival, cell growth or both.

37. The method of claim 26, wherein the peptide or peptide analog is selected from one or more of a bone morphogenetic protein-2 peptide (BMP2) or BMP2 analog, a erythropoietin peptide (EPO) or EPO analog, or a fibroblast growth factor-2 peptide (FGF2) or FGF2 analog, or combinations thereof.

38. The method of claim 37, wherein the EPO comprises a recombinant human

Erythropoietin (rhEPO) peptide.

39. The method of claim 37, wherein the FGF2 comprises a recombinant human fibroblast growth factor-2 (rhFGF2) peptide.

40. The method of claim 37, wherein the BMP2 comprises a recombinant human bone morphogenetic protein-2 (rhBMP2).

41. The method of claim 37, wherein the EPO peptide analog comprises SEQ ID:l, or SEQ ID: 2, or SEQ ID:3, or SEQ IDA, or SEQ ID: 5, or combinations thereof.

42. The method of claim 37, wherein the FGF2 analog comprises SEQ ID: 6.

43. The method of claim 37, wherein the BMP2 analog comprises SEQ ID: 7, or SEQ ID: 8, or combinations thereof.

44. The method of claim 26, further comprising molecules or drugs that induce cell growth- promoting or cell-survival promoting peptides.

45. The method of claim 44, wherein the molecules comprise EPO-inducing, BMP2- inducing, or FGF2-inducing molecules.

46. The method of claim 44, wherein the FGF2 inducing molecule comprises Amitriptyline.

47. The method of claim 44, wherein the BMP2 inducing molecule comprises Tacrolimus.

48. The method of claim 44, wherein the EPO inducing molecule comprises a EPO mimicking molecule PAMAM-HMB linked methyl (2-(2-([l,l'-biphenyl]-4-yl)-6-chloro-5- methyl-lH-indol-3-yl)acetyl)-L-lysinate.

49. The method of claim 26, wherein the one or more peptide analogs is present at a concentration of from about 5% to about 75% of wild type levels of the corresponding peptide in an animal onto which the biomaterial is transplanted.

50. The method of claim 26, wherein the collagen comprises telopeptides.

51. The method of claim 26, wherein the dendrimers comprise a Poly(amidoamine)

(PAMAM).

52. The method of claim 51, wherein the PAMAMs are selected from one or more of first, second or third generation PAMAMs.