Peptide mimetic conjugated 3D porous polymer scaffold

Abstract

The invention relates to biocompatible and biodegradable 3D porous polymer scaffolds-conjugated with peptidomimetic chain and their use as cell delivery vehicles of stem cells for directing their fate into differentiated cells thereby accelerating the tissue regeneration processes, in particular to chronic non-healing diabetic wounds, diseased related to impaired tissue regeneration-induced degenerative diseases.

Description

[0001] PEPTIDE MIMETIC CONJUGATED 3D POROUS POLYMER SCAFFOLD

[0002] FIELD OF THE INVENTION

[0003] The invention relates to biocompatible and biodegradable 3D porous polymer scaffolds- conjugated with keratinocyte growth factor mimetic peptide (KGFp) and their use as cell delivery vehicles of stem cells for directing their fate into differentiated cells thereby accelerating the tissue regeneration processes, in particular to chronic non-healing diabetic wounds, diseased related to impaired tissue regeneration-induced degenerative diseases. The invention also relates to the synthesis of these 3D porous polymer scaffolds-conjugated with keratinocyte growth factor mimetic peptide (KGFp) under controlled conditions.

[0004] BACKGROUND OF THE INVENTION

[0005] Skin wound healing is a complex biological process involving several factors such as soluble mediators, extracellular matrix components, and resident cells (keratinocytes, fibroblasts, endothelial cells, nerve cells, and leukocyte subtypes) (Life 2021, 11(7), 665). Skin tissue regeneration is a challenging clinical problem, especially in patients with severe bums, accidents, and chronic non-healing wounds like diabetic foot ulcers, pressure ulcers, and venous ulcers (Advance Wound Care (New Rochelle) 2015, 4(9), 560-582). The process of wound healing involves four stages, hemostasis, inflammation, proliferation, and remodeling, which becomes highly dysregulated in chronic wounds (Pharmaceutics 2020, 12(8), 735). Prolonged nonhealing wounds become infected and often lead to limb amputation or mortality due to sepsis. The process of cutaneous tissue repair involves the interaction of several growth factors, cytokines, and their cognate receptors. Topical application or subcutaneous injection of several growth factors like platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), granulocyte macrophage-colony stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF) have been used as a treatment strategy for chronic and acute skin wounds (Wound Repair Regeneration 2014, 22(5), 569-578). However, due to the proteolytic microenvironment of the wound, these growth factors are often rapidly degraded. Additionally, the safety of systemic administration of growth factors and cytokines is questionable due to non-specific cell mobilization and the associated risk of malignancy (Cytotherapy 2019, 21(11),1137-1150; Chronic Wound Care Management and Research 2014, 11-14). Regranex (becaplermin), an FDA-approved drug containing recombinant platelet-derived growth factor-BB (PDGF-BB) has been used for treating lower extremity ulcers in diabetic patients, but in 2009 a black box warning was issued by the FDA due to the risk of potential malignancy (Wound Care Management and Research 2014, 1, 11-14). Autologous and allogeneic cell transplantation can serve as a promising therapeutic approach that can improve patient survival. Cultured keratinocyte sheets obtained from patient skin biopsies have been used in bum wound victims (Transplantation 2000, 70(11), 1588-1598). However, autologous stem cells are compromised in patients with comorbidities like diabetes. The use of allogeneic mesenchymal stem cells (MSCs) provides great promise as an efficient therapy for wound healing. MSCs possess unique immunomodulatory and immunosuppressive properties. Allogeneic MSCs can evade the recognition by the host CD4+ T cells due to the absence of MHC class II antigens and costimulatory molecules (Frontiers in Immunology 2020, 11, 54; Journal of Inflammation, 2005, 2, 1-11). Additionally, MSCs also possess a unique plasticity phenotype which allows them to transdifferentiate into multiple non-obvious cell lineages other than the MSC-specific tri-lineage - Osteoblast, Adipocyte, and Chondrocyte (Stem Cells Translational Medicine 2017, 6(12), 2173-2185). However, the hostile microenvironment of the wound bed owing to an increased level of reactive oxygen species (ROS) compromises the current treatment strategies including stem cell transplantation therapy due to poor survivability, low engraftment, and delayed differentiation. In a previous invention, a castor oil-based biodegradable, biocompatible porous polymer scaffold that possesses antioxidant properties. The polyethylene glycol-polyurethane scaffolds significantly reduced oxidative stress-mediated cellular apoptosis and enhanced the engraftment of MSCs at the wound site which accelerated wound healing was synthesized. (US9925298B2, Biomaterials 2016, 77, 1-13).

[0006] The epidermal layer of the skin is majorly composed of keratinocytes. Therefore, efficient skin tissue regeneration requires transdifferentiation of the transplanted MSCs towards the keratinocyte lineage. Keratinocyte growth factor (KGF) is involved in re-epithelialization which promotes the migration and proliferation of keratinocytes (Nucleic Acid Therapeutics, 2018, 28(6), 335-347). Multiple therapeutic strategies were explored to deliver KGF at the wound site for targeted re-epithelialization. The clinical application of growth factors for regenerative therapy has several limitations such as protein instability, low expression yield, and high production cost (ACS Applied Materials and Interfaces, 2019, 11(4), 3771-3780; Materials Science and Engineering: C, 2019, 103,109815). Thus, a combinatorial approach of engineering 3D polymer scaffolds and growth factor mimetics can overcome such limitations. In the present invention, a KGF mimetic peptide (KGFp) that promotes the transdifferentiation of MSCs towards keratinocyte-like cells (KLCs) is designed. This KGFp was chemically conjugated and grafted on the 3D polymer scaffolds and comprehensively characterized. Finally, MSC transplantation at the chronic type 2 diabetic non-healing wounds using these KGFp-conjugated 3D polymeric scaffolds enhanced the epithelialization-mediated skin regeneration and accelerated the wound closure.

[0007] Our prior reports have established the viability of employing biodegradable polyurethane-based porous polymer scaffolds for on-site delivery of stem cells and its efficacy in wound healing applications (US Patent: US9925298B2; Indian Patent, 3470 / DEL / 2015; Biomaterials 2016, 77, 1-13; Data in Brief, 2016, 6:221-228). The present investigation extends the hypotheses that suitable peptide mimetics appropriately grafted on such scaffolds can effectively direct the stem cell fate, a key challenge for tissue engineering applications. The polyurethane polymer scaffolds were considered as a starting point of the strategy. Structural modification of the parent scaffold is essential to achieve a polymer matrix with grafted peptide units. Towards this end, the synthetic strategy was modified to incorporate active functional groups on the polymer matrix, which can provide an option to graft / anchor suitable peptide chains of interest. Of the several approaches that can possibly be employed towards generating active functional groups on the polymer scaffolds, an alkyne-terminated small alcohol, i.e., propargyl alcohol was selected to introduce extensive alkyne functionality on the parent polymer matrix. This apart, castor oil and polyethylene glycol remained the starting materials of choice owing to the beneficial properties that they impart to these scaffolds.

[0008] The concept of using vegetable oils has increasingly gained footing in renewable polymer research (Angew. Chem. Int. Ed.2000, 39, 2206 - 2226). Nonetheless, only a few studies have been reported on its biodegradability under physiological conditions (Polym. Degrad. Stab.2007, 92, 480 - 489), and almost none on the use of such polyurethanes as tissue regeneration scaffolds. Castor oil is a bio-based polyol, an important renewable bio-resource, a locally abundant precursor offering wide versatility, and sustainability, and is economical. It is a vegetable triglyceride with a major constituent as ricinoleic acid, a tri-hydroxyl-containing fatty acid. (J. Chem. Eng. Data 2002, 47, 1502 - 1505) Bioavailability from renewable agricultural resources, low cost, low toxicity, and traditional medicinal use as a laxative and antioxidant make it an attractive starting material for biomedical polyurethanes under discussion. Long chains can potentially add to the flexibility in a network, ester groups as labile hydrolyzable groups, and inherent double bonds in combination provide for anti-oxidative properties while the free trihydroxyl functionality can be used as such for urethanation. While biodegradation in polyurethanes can be easily achieved by incorporating hydrolyzable moieties like esters in the polymeric chain, control on occasional cytotoxicity of cleaved fragments post-degradation remains the key area of concern and challenge. Additionally, the hydrophobicity of castor oil was effectively counterbalanced by the incorporation of exceedingly hydrophilic polyethylene glycols as chain extenders in the framework.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides a peptide-mimetic conjugated porous polymer scaffold, comprising: a peptidomimetic chain; a porous polymer matrix; and at least one linker moiety, wherein, the peptidomimetic chain is covalently grafted through at least one linker moiety onto the functionalized porous polymer matrix; the peptidomimetic chain is an azide or alkyne end functionalized peptidomimetic chain of 8 - 20 amino acids that mimics KGF (FGF7), VEGF, FGF, FGF4, EGF, bFGF, BDNF, GM-CSF, HGF, BMP2, TGF-P, IGF-I, GDF-8, AEDG, AEDP, AEDL, AED, KE, KED, EDA, IRW, GRGDS, GLP-1, CLE, CLV3, RGD, HFL-1, P-15 or combinations thereof; the porous polymer matrix having a pore size in the range of 50 nm - 5 pm comprises, alkynyl alcohol or alkynyl amine units, castor oil as crosslinker, a polyether chain extender and a diisocyanate containing compound.

[0011] In a preferred embodiment of the present invention, the peptidomimetic chain is an azide or alkyne end functionalized amino acids chain of sequence ID 1, that mimics KGF.

[0012] In an aspect of the present invention, the porous polymer matrix is synthesized from a semiinterpenetrated polymer network which contains a non-reactive polyethylene glycol dimethyl ether polymer of molecular weight in the range of 250 to 5000 Daltons entangled within the network, which is sacrificially lost to produce alkyne functionalized porous polymer matrix.

[0013] In a preferred embodiment of the present invention, the crosslinker is a triglyceride of castor oil.

[0014] In a preferred embodiment of the present invention, the alkynyl alcohol or alkynyl amine is selected from a group consisting of propargyl alcohol, propargyl amine, alkyne-PEG2-amine and alkyne-PEG3-amine.

[0015] In a preferred embodiment of the present invention, the alkynyl alcohol is propargyl alcohol and the functional end groups of the propargyl grafts are chemically modified to achieve chain extension and free terminal azide groups. In a preferred embodiment of the present invention, the polyether chain extender is selected from the group consisting of di-hydroxyl, di-amine, and di-carboxyl terminated compounds.

[0016] In a preferred embodiment of the present invention, the polyether chain extender is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), poly tetramethylene glycol (PTMG), block copolymers thereof, branched / graft copolymers thereof, and combinations thereof.

[0017] In a preferred embodiment of the present invention, the diisocyanate containing compound is selected from the group consisting of methylene diphenylene diisocyanate (MDI), polymeric methylene diphenylene diisocyanate (p-MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HMDI), dicyclohexane methylene diisocyanate (H12MDI), isophorone diisocyanate (IPDI), xylene diisocyanate, hydrogenated xylene diisocyanate, and Desmodur-N.

[0018] The present invention provides a process to prepare the peptide-mimetic conjugated porous polymer scaffolds as mentioned above, wherein the process comprising the steps of:

[0019] (a) reacting 10 wt % to 60 wt % Castor oil with diisocyanate compound with total — NCO / — OH ratio in the range of 0.8-2.5 in tetrahydrofuran (THF) solvent to form a pre-polymer;

[0020] (b) charging the pre-polymer as obtained in step (a) with polyether macromonomer, alkynyl alcohol or alkynyl amine in THF solvent to obtain charged pre-polymer;

[0021] (c) adding N, N-dimethylaniline as catalyst to the charged pre-polymer obtained in step (b) to initiate the polyurethane reaction to form a growing polymer network;

[0022] (d) adding a non-reactive polyethylene glycol dimethyl ether (PEGDME) polymer of molecular weight in the range of 250 to 5000 Daltons to the growing polymer network of step (c) to obtain a viscous reaction mixture;

[0023] (e) degassing and vigorously mixing the viscous reaction mixture obtained in step (d) under inert atmosphere to obtain a uniformly homogeneous viscous mix;

[0024] (f) casting the uniformly homogeneous viscous mix as obtained in step (e) onto a teflon petri-dish to obtain a polymeric product;

[0025] (g) curing the polymeric product as obtained in step (f) at room temperature for 24 h followed by curing at higher temperature and inert atmosphere at 60-90° C for 48 h-96 h forming a semi-interpenetrating polymer network with the non-reactive polyethylene glycol dimethyl ether polymer entangled within the network matrix, to obtain a freestanding film; (h) wrapping the free-standing film obtained in step (g) in Whatman filter paper bag and treating to a repeated soxhlet extraction process to obtain processed film;

[0026] (i) subjecting the processed film obtained in step (h) to repeated swelling and drain cycles for 4-7 days against THF to extract out the PEGDME from the polymer network matrix completely, leaving behind an alkyne terminated porous polymer network;

[0027] (j) extracting the porous polymer network using deionized millipore water to obtain an impurity free and sterile alkyne terminated porous polymer matrix;

[0028] (k) reacting the alkyne terminated porous polymer matrix swelled in THF with 1,4- diazidobutane and N,N-Diisopropylethylamine (DIPEA) in the presence of Copper iodide (Cui) under nitrogen atmosphere and 80 °C for 24h leading to the formation of azide terminated-porous polymer scaffolds; and

[0029] (l) reacting the azide-terminated porous polymer scaffolds with an azide or alkyne end functionalized peptidomimetic chain in presence N,N-Diisopropylethylamine (DIPEA) in and Copper iodide (Cui) for 48h at room temperature and soxhlet extraction using deionized millipore water to obtain impurity free peptide-mimetic conjugated porous polymer, wherein peptidomimetic chain mimics KGF (FGF7), VEGF, FGF, FGF4, EGF, bFGF, BDNF, GM-CSF, HGF, BMP2, TGF-P, IGF-I, GDF-8, AEDG, AEDP, AEDL, AED, KE, KED, EDA, IRW, GRGDS, GLP-1, CLE, CLV3, RGD, HFL-1, P-15 or combinations thereof.

[0030] In the present invention, a KGF-mimetic peptide (KGFp, 13mer-terminally modified with propargyl moiety) was designed based on the receptor interaction sites in murine KGF. The role of KGFp in enhancing the migration and transdifferentiation of mouse bone-marrow-derived MSCs towards keratinocytes-like cells (KLCs) was evaluated. Further, KGFp-conjugated 3D porous polymer scaffolds were designed, synthesized, and physico-chemically characterized. In- vitro evaluation of the KGFp-conjugated polymer scaffolds depicted these to be biocompatible, biodegradable, and efficient in transdifferentiation of MSCs into KLCs. Transplantation of allogenic MSCs using KGFp-conjugated 3D polymer scaffolds in chronic non-healing type 2 diabetic wounds (db / db transgenic, 50-52 weeks old male mice) enhanced re-epithelialization- mediated accelerated wound closure as evident from the morphological, histological, immunohistochemical, and molecular analysis.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1. KGFp enhanced the migration and differentiation but not the proliferation of MSCs. Figure 2. KGFp enhances the expression of the keratinocyte markers and activates downstream signaling in MSC-derived KLCs.

[0033] Figure 3. KGFp enhances ERK1 / 2 and STAT3 downstream signaling in KLCs.

[0034] Figure 4. Physico-chemical characterization of KGFp-grafted 3D polymer scaffolds.

[0035] Figure 5. KGFp grafted polymer scaffolds are stable, biocompatible, and biodegradable.

[0036] Figure 6. Evaluation of the stability, biodegradability, and biocompatibility of the unconjugated and KGFp conjugated 3D polymer scaffolds in increasing concentration of glucose.

[0037] Figure 7. MSCGFFtransplantation with KGFp-grafted 3D polymer scaffold accelerated wound closure in type 2 diabetic (db / db) mice.

[0038] Figure 8. Transplanted MSCG / / ’-3D polymer scaffold-KGFp transdifferentiates into epithelial cells.

[0039] Figure 9. Co-localization of GFP and CK10 in MSCGFF-3D polymer scaffold and MSCGFF- 3D polymer scaffold-KGFp transplanted group.

[0040] Figure 10. An enhanced colocalization of GFP and IVL in MSCGFF-3D polymer scaffold- KGFp transplanted group.

[0041] Figure 11. An activation of FGFR2IIIB in MSCGFF-3D polymer scaffold-KGFp transplanted group.

[0042] Figure 12. KGFp-mediated activated molecular signaling in regenerated skin tissues.

[0043] Figure 13. An enhanced activation of AKT signaling in MSCGFF-3D polymer scaffold-KGFp transplanted group.

[0044] Figure 14. Enhanced expression of ECM-related, neural, and vascularity markers in MSCGFF- 3D polymer scaffold-KGFp transplanted group.

[0045] Figure 15. Illustrates the scheme 1 depicting scheme for achieving the 3D-porous polymer scaffolds in Stage-I and subsequent modification followed by grafting of the KGF mimetic peptide on the scaffolds (Stage-II).

[0046] The accompanying drawings, which are incorporated into and constitute a part of these specifications, illustrate one or more embodiments, and serve to explain the principles and implementations of the invention. The foregoing aspects together with the detailed description will be readily appreciated by the skilled artisan from the illustrative embodiments when read in conjunction with the drawings.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] Reference will now be made in detail to specific embodiments of the invention. Examples of the specific embodiments are illustrated in the accompanying drawings. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. The present invention may be practiced without some or all these specific details. In other instances, well-known processes have not been described in detail to not unnecessarily obscure the present invention.

[0049] It will, of course, be appreciated that in the development of any actual implementation of the invention, numerous implementation-specific decisions must be made to achieve the developer’s specific goals, such as compliance with application-, performance- and business-related constraints, and that these specific goals will vary from one developer to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming but would nevertheless be a routine undertaking of the chemistry and engineering for those of ordinary skill in the art having the benefit of this disclosure.

[0050] In the present invention, provides the use of biodegradable porous polymer scaffolds conjugated with mimetic peptides to drive the fate of the stem cells towards a specific lineage for tissue regeneration. Detailed studies on these scaffolds depict not only the molecular mechanism occurring during the transdifferentiation of the MSCs towards keratinocyte-like cells (KLCs) but also their use for skin tissue regeneration in the type 2 diabetic non-healing wounds.

[0051] Cell culture studies

[0052] Mouse bone marrow-derived mesenchymal stem cells (MSCs) (Cyagen, USA) were cultured in a- MEM medium (HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) as described earlier (Molecular Therapy, 28(5), (2020) 1314-1326).

[0053] Physiological assays Proliferation analysis: MTT assay was performed to determine the rate of proliferation as described earlier (Cytotherapy, 21(2), (2019) 260-273). Briefly, MSCs were seeded in basal keratinocyte expansion medium (KEM) at a density of 5xl03cells per well in a 96-well plate and treated with an increasing concentration of KGFp (1, 10, and 100 ng / mL). After 48 h of incubation, the medium was replaced with 100 pL of MTT (5 mg of MTT dissolved in 10 mL complete growth medium). Post 90 min of incubation, the medium was carefully removed, and the formed formazan crystals were allowed to dry. After 150 min, 100 pL of DMSO was added to dissolve the crystals and the absorbance was measured at 570 nm.

[0054] Migration analysis: Chemotaxis was measured using Boyden chamber assay (8 pm pore size, Neuro Probes, Inc. Gaithersburg, MD, USA) (Diabetes, 69(6), (2020) 1232-1247). Briefly, the bottom wells of the chamber were filled with 100 pL of serum-free media (basal KEM) containing increasing concentrations (1, 10, and 100 ng / mL) of KGFp, and covered with a polycarbonate filter. MSCs (IxlO3) were added into the upper chamber. The cells were incubated for 12 h at 37°C. Migrated cells at the lower surface of filters were fixed and stained using Hema3 stain (Biochemical Sciences Inc., Swedesboro, NJ, USA). Cellular migration was determined by counting the number of stained cells on the membrane in the selected non-overlapping high- power fields.

[0055] Differentiation analysis: For transdifferentiation of MSCs into keratinocytes, MSCs were cultured for 14 days in the keratinocyte basal differentiation medium (HiMedia, Mumbai, India) without the growth factor supplement along with 2% FBS and 1% penicillin-streptomycin. The experimental groups were treated with an increasing concentration of KGFp (1, 10, and 100 ng / mL) every 48 h. MSCs cultured in a complete MSC expansion medium were used as control. Transdifferentiated cells, post-culturing for 14 days in the respective culture medium were subjected to RNA isolation followed by cDNA synthesis and evaluation of keratinocyte-specific and epithelial gene expressions.

[0056] Gene and protein expression studies

[0057] Quantitative real-time PCR (qPCR) analysis: qPCR was performed to evaluate the expression of keratinocyte-specific markers - Basonuclin, CK5, CK14, CK1, CK10, CK13, Involucrin, and epithelial markers - Ep-CAM, E-Cadherin, Claudin, and Mucin- 1 using the mouse gene-specific primers. Separately, the expression levels of the matrix metalloproteinases (MMP2, MMP9, and MMP13) and its tissue inhibitor of MMPs (TIMP1 and TIMP2) were also evaluated using the mouse gene-specific primers. The values were represented as fold-change relative to MSC as control and normalized to the housekeeping gene, Eukaryotic 18S rRNA as described earlier (Molecular Therapy, 28(5), (2020) 1314-1326).

[0058] Immunoblot analysis: Cellular protein extracted from transdifferentiated keratinocyte-like cells (KLCs) was subjected to immunoblotting with primary antibodies against the signaling molecules pSTAT3, STAT3, pERKl / 2, ERK1 / 2, pAKT, and AKT. MSCs cultured in basal KEM were used as a control (Cytotherapy , 21(2), (2019) 260-273).

[0059] Immunofluorescence analysis: Transdifferentiated keratinocyte-like cells (MSCs cultured in 10 ng / ml KGFp) were washed, fixed, and permeabilized before separate incubation with primary antibodies against Involucrin, CK5, and CK14 followed by Alexa Fluor 488 / 555 conjugated secondary antibodies and mounted with DAPI containing mounting media. Corrected total cell fluorescence (CTCF) was analyzed using ImageJ software (Cytotherapy, 21(2), (2019) 260-273).

[0060] Synthesis and conjugation of KGFp-grafted 3D porous polymer scaffolds'. The KGFp grafted- 3D porous polymer scaffolds were achieved following a two-stage synthesis procedure. The synthesis Stage-I adopted is a modification of the inventor’s earlier reported procedure to generate 3D-polymer scaffolds (Biomaterials, 11, (2016) 1-13).

[0061] Briefly, a typical reaction of Stage-I (Scheme 1, Figure 15) initially involved a pre-polymer formation by reacting castor oil (CO) and excess diphenylmethane-4,4’-diisocyante (MDI) in THF under an inert atmosphere. At the end of Ih, a macromonomer, polyethylene glycol (PEG, Mn~ 4000), propargyl alcohol (PA), and a room temperature catalyst, dimethylaniline (DMA) in appropriate stoichiometries dissolved in THF / AN (1: 1 v / v) was added. The pre-polymer along with these added constituents comprised Component-I of the targeted polymer networks. Additionally, another non-reactive polymer, polyethylene glycol dimethyl ether (PEGDME, Mn~ 500; Component-II) was also charged into the reaction vessel at this stage. Under vigorous stirring, the reaction was allowed to proceed for another 30 mins under an inert atmosphere before casting the viscous mix in a mold. This cast mix was cured at room temperature (25 - 30 °C) for 24h and subsequently at 80°C for 48h to yield free-standing semi-interpenetrating polymer networks (semi-IPN) films. Soxhlet extraction was carried out on these fully cured semi-IPN films first with THF for 48h and then with DI water for another 48h to leach out the PEGDME (Component-II) entangled / trapped within the semi-IPN matrix. Post this sacrificial treatment an alkyne terminated-3D porous polymer scaffold matrix was achieved. The progress of the reaction, curing, and the final product was comprehensively followed at each phase by mz -FTIR analysis. Stage II (Scheme 1, Figure 15) comprises initially modifying the alkyne-end groups of the 3D porous polymer scaffolds achieved in Stage-I by treating the swelled polymer matrix in THF with 1.3eq. of 1,4-diazidobutane and N,N-Diisopropylethylamine (DIPEA) in the presence of Copper iodide (Cui) under a nitrogen atmosphere and 80°C for 24h. The treatment led to the formation of azide terminated-3D polymer scaffolds, which was confirmed by mz -FTIR analysis. Post confirmation of azide-termination, the scaffolds were grafted with alkyne terminated-KGF peptide mimetic (Pra-KGFp) by repeating azide-alkyne Huisgen cycloaddition using the same catalysts / reactants in DI water for 48h at room temperature.

[0062] ^2\ Lys-Glu-Leu-!le-Leu-Glu-Asn^is Tyr-Asn-Thr-Tyr-A

[0063] (Pra-KGFp)

[0064] The successful grafting of the KGFp on the 3D-polymer scaffolds was confirmed by following the reaction with the progressive disappearance of the alkyne and azide peaks in the mz -FTIR region. Finally, the KGFp grafted-3D porous polymer scaffolds so obtained were comprehensively characterized employing optical microscopy, FESEM, DSC, and TG to ascertain their physicochemical properties before carrying them forward for bio-evaluation and stem cell transplantation.

[0065] Physico-chemical characterization of KGFp-grafted 3D polymer scaffold.'.

[0066] Fourier-transform infrared spectroscopy (FTIR): The successful formation of the targeted 3D scaffolds and subsequent chemical modification were followed at each stage of the reaction employing rnz -FTIR analysis carried out in the range of 4000 - 400 cm1employing a Bruker Alpha-T spectrometer. Typically, 2mg of the samples were mixed with FTIR grade KBr (Aldrich), ground, pressed into a transparent pellet, and dried under vacuum before each sample ran. The FTIR data on the samples were collected for the KBr pellets with 256 scans and a resolution of 2 cm1at ambient temperature.

[0067] Field-emission scanning electron microscopy (FESEM): The 3D scaffolds swelled in water were assessed employing an optical microscope (Leica, Model: DM4000 M). The morphology and porosity of the dried scaffolds were investigated in a field-emission scanning electron microscope (FESEM Model: 7610F; JEOL).

[0068] Thermo-mechanical properties - Differential scanning calorimetry (DSC), Dynamic mechanical analysis (DMA), and Thermo gravimetry (TG): The thermal properties of the polymer matrices were evaluated using both differential scanning calorimetry (DSC) and thermogravimetry (TG). Calorimetry experiments were typically run with samples (~ 10 mg) sealed in aluminum pans on DSC equipment (TA Instruments, Model: Q100) in the temperature range of -70°C - 150°C with a scan rate of 10°C / min under an N2 atmosphere and heat flow (W / g) was recorded for further analysis. The thermo-mechanical properties of the synthesized porous polymer scaffolds were assessed employing a dynamic mechanical analyzer (TA Instruments, Model Q800) with a cyclic strain applied at a constant frequency of 1Hz under tension mode within the temperature window -110°C - 120°C at a scan rate of 10°C / min and N2 flow. Thermogravimetry profiles were recorded on TG equipment (TA Instruments, Model Q50) in the temperature range RT - 800°C with a heating rate of 10°C / min under an N2 atmosphere. The heat flow profile and weight loss data were analyzed for the observed transition ranges and change in weight using Universal Analysis Software from TA Instruments.

[0069] The successful formation of the targeted 3D scaffolds and subsequent chemical modification were followed at each stage of the reaction employing rnz -FTIR analysis carried out in the range of 400 - 4000 cm1employing a Bruker Alpha-T spectrometer. Typically, 2mg of the samples were mixed with FTIR grade KBr (Aldrich), grinded, pressed into a transparent pellet, and dried under vacuum before each sample run. The FTIR data on the samples were collected for the KBR pellets with 256 scans and a resolution of 2 cm1at ambient temperature. The 3D scaffolds swelled in water were assessed employing an optical microscope (Leica, Model: DM4000 M). The morphology and porosity of the dried scaffolds were investigated in a field-emission scanning electron microscope (FESEM Model: 7610F; JEOL). The thermal properties of the polymer matrices were evaluated using both differential scanning calorimetry (DSC) and thermogravimetry (TG). Calorimetry experiments were typically run with samples (~ 10 mg) sealed in aluminum pans on DSC equipment (TA Instruments, Model: Q100) in the temperature range of -70°C - 150°C with a scan rate of 10°C / min under an N2 atmosphere and heat flow (W / g) was recorded for further analysis. Thermogravimetry profiles were recorded on TG equipment (TA Instruments, Model Q50) in the temperature range RT - 800°C with a heating rate of 10°C / min under an N2 atmosphere. The heat flow profile and weight loss data were analyzed for the observed transition ranges and change in weight using Universal Analysis Software from TA Instruments.

[0070] Stability, biodegradability, and biocompatibility studies:

[0071] Stability of KGFp-grafted 3D porous polymer scaffolds: 3D-polymer scaffold (control) and 3D- polymer scaffold-KGFp of 5 mm diameter were cut into 4 pieces and weighed. The pieces were then incubated in 100 pl of potassium phosphate buffer ranging from pH 5.8-7.8 as described earlier (Biomaterials, 77, (2016) 1-13). Biodegradability of KGFp-grafted 3D porous polymer scaffolds: The polymers (5 mm diameter cut into 4 pieces) were subjected to 20% Tri-Chloro-Acetic acid (TCA), and the enzymes Collagenase (1 mg / ml), and 0.25% Trypsin to determine the enzyme and / or acid-mediated degradation of the synthesized 3D-polymer scaffold Biomaterials, 77 , (2016) 1-13).

[0072] Biocompatibility of KGFp-grafted 3D porous polymer scaffolds: For biocompatibility, MSCs were cultured in the presence or absence of the 3D-polymer scaffold, and 3D-polymer scaffold- KGFp in alpha-MEM medium. 5 mm circular pieces of scaffolds were placed within each well of a 96 well-plate and 5xl03MSCs were seeded on the scaffolds. After 48h MTT assay was performed to determine the rate of proliferation as described previously {Biomaterials, 77 , (2016) l-13).Additionally, stability, biodegradability and biocompatibility studies were performed with an increasing concentration of glucose (0, 5, 10, 15, and 25 mM) to mimic the hyperglycemic condition in diabetic mice model.3D-polymer scaffold (control) and 3D-polymer scaffold-KGFp were cut into small pieces and weighed. The pieces were then incubated in 100 pl of potassium phosphate buffer ranging from pH 5.8-7.8 as described earlier {Biomaterials, 11, (2016) 1-13). Similarly, the polymers were also subjected to 20% Tri-Chloro-Acetic acid (TCA), and the enzymes Collagenase (Img / ml), and 0.25% Trypsin to determine the enzyme and / or acid- mediated degradation of the synthesized 3D-polymer scaffold. For biocompatibility, MSCs were cultured in the presence or absence of the 3D-polymer scaffold, and 3D-polymer scaffold-KGFp in alpha-MEM medium. After 48 h MTT assay was performed to determine the rate of proliferation as described previously {Cytotherapy, 21(2), (2019) 260-273).

[0073] In-vivo studies

[0074] Type 2 diabetic db / db transgenic mice were used in the study. The protocols for animal translational studies were approved by the Institutional Animal Ethics Committee (IICT / IAEC / 05 / 2021) and the Institutional Biosafety Committee (IICT / IBSC / 03 / 2019).

[0075] Excisional splint wound healing model: A full excisional splint wound healing model was generated in 50 - 52 weeks-old male db / db transgenic mice as described earlier {Diabetes, 69(6), (2020) 1232-1247). The consequence of delayed healing increases in aged db / db mice like that happens in humans. Briefly, two symmetrical wounds were created on the dorsum of each mouse using a 5 mm sterile biopsy punch. The wounds were splinted using a cyanoacrylate adhesive and sutured to prevent wound closure due to contraction. The mice were divided into three groups: UT (Untransplanted wound control), MSC + 3D-polymer scaffold, and MSC + 3D- polymer scaffold-KGFp. Before transplantation, control 3D polymer scaffolds, and 3D polymer scaffolds-KGFp of 5 mm diameter were cut into circular pieces using the same biopsy punch, and IxlO6MSCGf / >were seeded on the scaffolds separately and cultured as described previously

[0010] . After 24h, MSCGFP + 3D-polymer scaffold, and MSCGFP + 3D-polymer scaffold-KGFp were transplanted on the excisional wounds on day 0 of the surgery.

[0076] Morphometric wound healing analysis: The morphometric wound healing analysis was performed by capturing the images of the wounds on post-surgery days 0, 3, 5, 7, 10, 12, and 14. The rate of wound closure was determined using ImageJ analysis software as described earlier (Biomaterials, 77, (2016) 1-13; Diabetes, 69(6), (2020) 1232-1247). The mice were euthanized, and skin wound tissues were collected post-surgery day 14.

[0077] Molecular, histological, and immunohistochemical analysis: The regenerated wound tissue sections were subjected to RNA isolation followed by cDNA synthesis and qPCR analysis. The expression of keratinocyte and epithelial specific genes was evaluated. Additionally, qPCR analysis was also performed to evaluate the expression of ECM-related genes Fibronectin (Fnl), Asporin, Periostin, Activin (Inhba), and a-SMA, neural marker Neurofilament heavy polypeptide (Nefh) and vascularity marker CD31. Separately, the samples were processed for histological analysis with hematoxylin-eosin and Sirius red staining, and immunohistochemical analysis. The sections were immunostained with antibodies against pERK / ERK, pSTAT3 / STAT3, pTyr / FGFR2IIIB, and pAKT / AKT along with, and the fibrosis marker a-SMA, and endothelial marker CD31. Alexafluor-488 / 555 conjugated secondary antibodies were used followed by DAPI containing mounting medium as described above. Co-localization of GFP immuno staining and mouse- specific antibodies against keratinocyte differentiation markers CK5, CK14, CK10, and Involucrin was evaluated to determine the fate of the transplanted MSCs using confocal microscopy (Olympus F VI Oi, Olympus). Corrected total cell fluorescence (CTCF) and Pearson’s correlation coefficient for colocalization were analyzed using ImageJ and Fluo-view software, respectively (Diabetes, 69(6), (2020) 1232-1247).

[0078] Statistical analysis: Data from the experiments performed at least thrice were analyzed by taking the mean ± standard deviation (SD). GraphPad Prism version 9.5.1 was used to determine the statistical significance of the difference between the treatment and control groups with one-way or two-way ANOVA followed by Tukey’s multiple comparison test or Student’ s unpaired t-test. Pearson’s correlation coefficient was used to analyze the correlation between variables in immunofluorescence analysis. Immunoblots and photomicrographs represent experiments performed at least thrice with similar results.

[0079] EXAMPLES KGFp did not alter the rate of proliferation but enhanced the migratory potential ofMSCs: To evaluate the effect of KGFp on MSC physiology, MTT (proliferation) and Boyden Chamber (migration) assays were performed. The presence of KGFp did not alter the proliferation rate of cells as compared with the vehicle control (basal KEM) group [Fig. 1A]. However, a significantly higher migration potential was observed in the MSCs in the presence of the KGFp as compared with the vehicle control [Fig. IB]. Further, a dose -dependent effect of the KGFp was observed in this study with MSCs plated on the upper chamber and KGFp used as the ligand in the lower chamber of the Boyden apparatus suggesting its role in inducing the migration of MSCs. These observations led us to also evaluate the role of KGFp in inducing the transdifferentiation of MSCs.

[0080] KGFp enhanced the trans differentiation ofMSCs toward keratinocyte lineage: Interestingly, the epidermal layer of the skin is composed of four layers - stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum. Basonuclin (Bncl), Cytokeratin 5 (Ck5), and Ckl4 are expressed in the basal layer (stratum basale), Ckl, CklO, and Ckl 3 are expressed in the middle layers (stratum spinosum), and Involucrin (7vZ) is expressed in the terminal layer (stratum granulosum) of the epidermis. Thus, the effect of KGFp on the transdifferentiation of bone marrow-derived MSCs into keratinocyte-like cells (KLCs) was evaluated using molecular gene expression analysis of keratinocyte-specific genes such as Bncl, Ivl, Ckl, Ck5, CklO, Ckl 3, Ckl4 as well as epithelial cell-specific genes such as E-Cadherin (Cdhl), EpCam, Claudin (Cldnl ), and Mucin (Mud ) (Fig. 1C). A significant increase in the relative mRNA expression of the keratinocyte-specific markers Bncl (28.5-fold), Ck5 (14.6-fold), Ckl4 (26.1-fold), CklO (187.7-fold) and the epithelial-specific markers, EpCam (23.3-fold) Cdhl (64.2-fold) were observed in the presence of 10 ng / mL KGFp as compared with control (Fig. 1C). Since most of the keratinocyte-specific markers were significantly induced by KGFp at 10 ng / mL, the in vitro experiments were subsequently performed at this concentration. Immunofluorescence analysis depicted a marked increase in the expression of the keratinocyte markers IVL, CK5, and CK14 in the MSCs transdifferentiated in the presence of KGFp (10 ng / mL) (Fig. 2A-B). This observation confirms the efficacy of the KGFp in inducing the transdifferentiation of bone marrow derived MSCs into KLCs. Furthermore, the activation (phosphorylation) of the downstream signaling modulators STAT3, ERK1 / 2, and AKT suggested the role of pSTAT3, pERKl / 2, and pAKT molecular signaling during the transdifferentiation of MSCs into KLCs (Fig. 2C). The KGFp-induced increase in the expression of Bncl and Ivl was significantly reversed in the presence of both ERK1 / 2 and STAT3 inhibitors, while the expression of Ckl4, Ckl, and CklO was significantly downregulated in the presence of STAT3 inhibitor (Fig. 2D), suggesting the role of ERK1 / 2 and STAT3 signaling in regulating the transdifferentiation of MSCs into KLCs. This observation was further substantiated with the immunofluorescence analysis which revealed a marked increase in the KGFp-induced activation of ERK1 / 2 (p- ERK1 / 2) (Fig. 3A), and increased expression of IVL and CK14 (Fig. 3B) was reduced in the presence of ERK inhibitor, PD98059 (Fig. 3A-B). Similarly, the KGFp-induced activation of STAT3 (pSTAT3) (Fig. 3C) and enhanced expression of IVE and CK5 was reverted with STAT3 inhibitor, S3I-201 (Fig. 3C-D). These observations confirmed the role of ERK1 / 2, and STAT3 signaling in enhancing the KGFp-mediated transdifferentiation of MSCs towards KECs. Further, to enhance the stability of KGFp in an in vivo diabetic wound microenvironment, KGFp with a previously described biocompatible 3D polymer scaffold was conjugated, which can also act as an efficient delivery vehicle for MSCs.

[0081] KGFp-grafted 3D polymer scaffolds:

[0082] The successful formation of the initial alkyne terminated semi-IPNs in Stage-I, followed by first achieving the free azide grafts on the 3D scaffolds and finally realizing the KGFp grafted 3D- porous polymer scaffolds in Stage-II (refer to Scheme- 1, Figure 15) were evaluated at each stage by mz -FTIR. The representative stacked plots of mz -FTIR spectra are provided in Figure 4A; wherein the parent semi-IPN, optimized in the earlier efforts (Fig. 4A(a)) is provided as a control for comparison. The synthesis of castor oil crosslinked semi-IPNs is now well established (Biomaterials, 77, (2016) 1-13). The carbonyl peak indicative of castor oil ester linkage at -1746 cm1, while the prominent C-O-C symmetric stretch at -1110 cm1coupled with strong -OH stretching and bending modes at 3600-3200 cm1, are all characteristic vibrational modes of the macromonomer used and are noticeable in all the spectrum presented. In general, the urethanation chemistry involving the diisocyanate reaction with the diols / polyols is followed by monitoring the strong isocyanate (-NCO) stretching peak at ca. 2277 cm1, which gradually disappears as the reaction and curing progress to completion. Concurrently, an initial broadening followed by the gradual appearance of a peak at -1725 cm1overlapped as a shoulder to the 1746 cm1peak of castor oil ester carbonyl confirms urethane formation. The absence of the signature 2277 cm'1peak corresponding to the reactant N,N’ -diphenyl methane di-isocyanate is very evident in the spectra (Fig. 4A). Other major peaks identified in the spectra at ca. 3400 cm-1(b), 1520 cm-1(s), 2926 cm1, and 2859 cm'1are attributed to free N-H stretching and bending vibrational modes of urethane linkages along with C-H stretching of methylene and methyl groups, respectively. The modification realized in the parent semi-IPN with the introduction of propargyl alcohol in Stage - I is evident with the presence of a 2115 cm'1peak (Fig. 4A(b)), a signature of the alkyne bonds grafted onto the scaffolds. The follow-up treatment in Stage II on these alkyne-terminated 3D- scaffolds swelled in THF with 1,4-diazidobutane and N,N-Diisopropylethylamine (DIPEA) in the presence of Copper iodide (Cui) under nitrogen atmosphere and 80°C for 24h resulted in the formation of triazole moieties as linkers along with free-azide functional groups at the terminal ends. The signature peak of azide termination is evident with the appearance of a relatively stronger sharp peak at ca. 2098 cm1with concomitant disappearance of the 2115 cm1alkyne peak (Fig. 4A(c)). Upon confirmation of the availability of the free azide (-N3) grafts on the 3D scaffolds, the final step of coupling alkyne terminated-KGF peptide mimetic (Pra-KGFp) was accomplished in DI water following the azide-alkyne Huisgen cycloaddition using the same Cui catalyst and DIPEA as the promoter at room temperature. The success of the reaction was affirmed by the complete disappearance of the azide peak at ~ 2098 cm1coupled with an apparent increase in the stretching bands of the amide linkages of the peptide molecule grafted onto the 3D-polymer scaffolds (Fig. 4A(d)).

[0083] The morphology of the water-swelled polymer scaffolds revealed the presence and distribution of random co-continuous pores both on the film surface (Fig. 4B(a)) and its cross-sectional images (Fig. 4B(b)) under an optical microscope. The average pore diameter estimated for these swelled films was in the range of 1-5 pm and their interconnectivity was quite evident in the cross-sectional image. The surface topography of the dry scaffolds in FESEM images (Fig. 4B(c)-(d)) was noticeably not smooth and evidence of void spaces and ditches was reminiscent of the sacrificial extraction of component-II (PEGDME) from the synthesized semi-IPNs to induce porosity in the system. The cross-sectional imaging (Fig. 4B(e)-(f)) confirmed the presence of a co-continuous porous structure randomly distributed throughout the 3D-matrix of the polymer, albeit in collapsed form, understandably in the high vacuum condition maintained during imaging.

[0084] The key thermal properties of the polymer matrices were assessed by both DSC and TG-DTA. Calorimetry runs collected at each stage of the synthesis substantiate two critical parameters of these 3D scaffolds designed with the intention of stem cell delivery: (i)the glass transition temperature (Tg) of the systems post-modifications remained well below room temperature (ca. 25°C - 30°C; see Fig. 4C(a) - (d)); and (ii) the 3D polymer matrix was completely amorphous and exhibited no indication of crystallinity. Both these parameters signify excellent flexibility of the polymeric scaffolds and provide for a soft interface conducive to cell attachment and growth. Dimensional stability and biomechanical strength of the polymer scaffolds post-modifications were appraised under dynamic strain, which exhibits an appreciable elastic storage modulus (E’) for all the synthesized polymer samples realized at different stages of modifications and grafting. The relatively low cross-linked pure-polymer scaffolds with E’ ~ 400 MPa display a significant augmentation in its stiffness for the peptide conjugated polymers (ca. 700 MPa) at 25oC (Fig. 4D(a)). The elastic loss modulus profile as a function of progressive rise in temperature, Fig. 4D(b); apparently reveals two transition regions: Tp and T« characteristically assigned to an initial increase in the free volume followed by intensified enhancement in segmental motions, respectively implying a gain in material toughness. The thermogravimetric profile and their corresponding differential plots presented in Fig. 4E(a) - (b), also demonstrate excellent thermal stability and degradation profile of these scaffolds. The degradation onset temperature (To) was estimated to be ca. 220°C - 230°C for all the samples tested followed by a three-stage degradation profile typical of such polyurethane systems. Following the degradation onset, the first phase of degradation is predominantly assigned to the cleavage of urethane bonds and ester bonds (T~ 220°C - 350°C), followed by degradation of ester linkages in the second stage (T ~ 350°C - 450°C) which finally culminates with advanced degradation and fragmentation of the polymeric / oligomeric chain segments beyond 450°C. The appreciably high degradation onset temperature (To) underscores the suitability of these materials to be sterilized before their use in biomedical applications.

[0085] Stability, biodegradability, and biocompatibility of the KGFp-grafted 3D polymer scaffolds: The stability of the polymer scaffolds depicted a higher percent change in weight when subjected to acidic pH as compared to the physiological pH of 7.4 (Fig. 5A). The half-life period of 3D- polymer scaffold-KGFp in physiological pH was 7.35 days. The biodegradability of the unconjugated 3D-polymer scaffold (control) and the KGFp-grafted 3D-polymer scaffolds were analyzed by subjecting these to biocatalysts, which revealed a relatively higher percent change in weight of both 22.8% and 23.6%, respectively in the presence of collagenase (1 mg / mL) as compared to trypsin and TCA (Fig. 5B). MSCs were cultured in the presence of the KGFp-grafted 3D porous polymer scaffold and unconjugated 3D-polymer scaffold (control) which revealed the biocompatibility phenotype of the synthesized scaffolds. MTT assay depicted the insignificant change in the percent proliferation of the cells cultured in both the 3D-polymer scaffold and the KGFp-grafted 3D-polymer scaffold (Fig. 5C). These observations confirmed the biocompatibility and biodegradability phenotype of the synthesized 3D-polymer scaffolds that were further evaluated for their efficacy as an MSC delivery vehicle. Furthermore, to evaluate the effect of cellular proteinases on biodegradability, qPCR analysis of the matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMPs) was evaluated that revealed a significantly higher expression of MMP2 (233.9-fold) in the MSCs cultured in a-MEM growth medium containing 3D-polymer scaffold-KGFp as compared with the MSC cultured in the absence / presence of unconjugated 3D-polymer scaffold (control) groups (Fig. 5D) suggests a plausible increased cellular penetration of cultured MSCs within the 3D polymer network.

[0086] KGFp-grafted 3D polymer scaffolds enhanced the transdifferentiation of MSCs into KLCs: Next, the MSCs cultured with control 3D-polymer scaffolds and 3D-polymer scaffold-KGFp were subjected to keratinocyte differentiation followed by a differential gene expression analysis of keratinocyte and epithelial- specific markers as mentioned above. A significant increase was observed in the expression of the keratinocyte-specific markers Ckl3 (11.11 -fold) and CklO (18.34-fold) in MSCs cultured in the presence of 3D-polymer scaffold-KGFp as compared with the control 3D-polymer scaffold (2.40 and 0.62-fold, respectively) (Fig. 4F). These results suggest that the KGF-mimetic peptide grafted 3D-polymer scaffold enhanced the transdifferentiation potential of MSCs into KLCs. Since a high glucose environment is present in the db / dbm\cc, the stability, biodegradability, and biocompatibility of the 3D polymer scaffold and 3D polymer scaffold-KGFp in low to high glucose conditions in vitro was investigated. The presence of high glucose did not alter the stability and biodegradability of the polymers (Fig. 6A- B). Although, the proliferation of MSCs reduced significantly with an increasing concentration of glucose, MSCs cultured in the presence of 3D polymer scaffold and KGFp conjugated 3D polymer scaffold did not show any significant change in the proliferative potential, suggesting enhanced survival of the MSCs in high glucose environment (Fig. 6C).

[0087] Transplantation of MSC along with 3D-polymer scaffold-KGFp accelerated wound closure in type-2 diabetic mice

[0088] To evaluate the in-vivo engraftment and change in the fate of the transplanted GFP expressing MSCs (MSCGFF) in the absence or presence of unconjugated / KGFp-grafted 3D-polymer scaffolds, an excisional splint wound healing model was generated in db / db type 2 diabetic 50 - 52 weeks old mice, which correlated with the clinical settings as described in the methods. The morphometric wound healing qualitative (Fig. 7A) and quantitative (Fig. 7B) analysis depicted a significantly higher rate of wound closure in the MSCGFF-3D polymer scaffold-KGFp transplanted group as compared with both the un-transplanted and MSCGFF-3D polymer scaffold (control) transplanted group (Fig. 7A-B). The healing kinetics corroborated with the histological staining using hematoxylin-eosin (Fig. 7C-upper panel) and Sirius Red (Fig. 7C-lower panel) of the regenerated wound tissues, which depicted enhanced granulation tissue formation and collagen deposition in the MSCGFF-3D polymer scaffold-KGFp transplanted group as compared with the control groups (Fig. 7C). Histologically, the wound tissue was restored with a well- organized wound matrix and thicker epidermis in the MSCGFF-3D polymer scaffold-KGFp transplanted group as compared with the controls where the microarchitectures of the regenerated skin were disorganized and discontinuous epidermis was observed. Furthermore, the differential expression analysis of the keratinocyte and epithelial markers in the regenerated skin depicted a significant increase in the expression of Ckl4, CklO, and Claudin in the MSCG / / ’-3D polymer scaffold-KGFp transplanted group as compared with the controls suggesting enhanced re- epithelialization (Fig. 7D). Next, the engraftment and fate of the transplanted MSCs were evaluated using immunohistochemical co -immuno staining of GFP and the keratinocyte markers CK14 (Fig. 8A-B), CK5 (Fig. 8C-D), CK10 (Fig. 9A-B), and Involucrin (Fig. 10A-B). A significantly higher co-localization of GFP / CK14 (Fig. 8A-B) and GFP / CK5 (Fig. 8C-D) was observed in the MSCGfF-3D polymer scaffold-KGFp transplanted group as compared with the control (un-transplanted and MSCG / / ’-3D polymer scaffold) groups. These results suggest the enhanced regeneration of the stratum basale layer of the epidermis due to higher re- epithelialization by keratinocyte differentiation in the presence of KGFp. Although, the colocalization of GFP / CK10 (Fig. 9A-B) was significantly higher in both the MSCG / / Jtransplanted groups in the presence of unconjugated (control) / KGFp-conjugated 3D-polymer scaffolds groups as compared with the un-transplanted group, a significant increase in the colocalization of GFP / Involucrin in the KGFp-conjugated 3D-polymer scaffolds groups as compared with both the control groups (Fig. 10A-B)suggested enhanced regeneration of the intermediate and terminal epidermal layers was observed. Interestingly, the increased expression of keratinocyte-specific genes was associated with a significant increase fluorescence intensity of p-Tyr / FGFR2IIIB (Fig. 11A) in the regenerated tissue sections of MSCG / / ’-3D polymer scaffold-KGFp transplanted group as compared with the controls (Fig. 11B quantitation of staining intensity) suggesting activation of the KGF receptor, FGFR2IIIB (Fig. 11C). Likewise, immunohistochemical analysis also revealed an increase in the expression of p-ERKl / 2 (Fig. 12A-B), p-STAT3 (Fig. 12C-D), and p-AKT (Fig. 13A-B) in the regenerated skin tissue of MSCGfF-3D polymer scaffold-KGFp transplanted group suggesting in-vivo activation of the molecular signaling mediators ERK, STAT3, and AKT.

[0089] MSC transplantation with KGFp-conjugated 3D polymer scaffold enhanced skin stiffness and functionality of the regenerated skin at db / db wound bed: significant increase in the expression of the pro-fibrotic ECM-related genes Fnl ,Asporin, Periostin, Inhba, and a-SMA in the MSCG / / J- 3D polymer scaffold-KGFp transplanted group as compared to the control groups indicating increased ECM deposition and collagen maturation which in turn leads to enhanced skin stiffness (Fig. 14A) was also observed. Further, the MSCG / / ’-3D polymer scaffold-KGFp transplanted group also depicted a significant increase in the expression of the neural marker Nefh (Fig. 14B) as compared to the untransplanted (UT) and MSCG / / ’-3D polymer scaffold transplanted groups. The expression of the vascularity marker CD31 increased significantly in both MSCG / / ’-3D polymer scaffold and MSCG / / ’-3D polymer scaffold-KGFp transplanted groups (Fig. 14C). Further, the regenerated skin in the MSCG / / ’-3D polymer scaffold-KGFp group and the unwounded skin (UW) showed similar expressions of Nefli and CD31 (Fig. 14B-C). These observations suggested enhanced functional properties of the regenerated skin. Additionally, immunofluorescence analysis revealed enhanced a-SMA (green), and CD31 (red) staining in both the MSCG / / ’-3D polymer scaffold and MSCGfF-3D polymer scaffold-KGFp groups as compared to the un-transplanted group (Fig. 14D). a-SMA staining significantly increased in the MSCG / / ’-3D polymer scaffold-KGFp group as compared to the MSCG / / ’-3D polymer scaffold group further confirming enhanced wound healing (Fig 14E). However, an insignificant difference was observed in the expression of the vascularity-related factor CD31 between the MSCGfF-3D polymer scaffold and MSCGfF-3D polymer scaffold-KGFp groups (Fig. 14F) suggesting the role of KGFp in driving the fate of the transplanted MSCs towards epithelial lineage in the chronic type 2 diabetic wound bed.

[0090] Having now fully described this invention, it will be understood by those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the inventions is not to be limited to the details given herein but may be modified within the scope and equivalents of the appended claims.

Claims

We Claim:

1. A peptide-mimetic conjugated porous polymer scaffold, comprising: a peptidomimetic chain; a porous polymer matrix; and at least one linker moiety, wherein, the peptidomimetic chain is covalently grafted through at least one linker moiety onto the functionalized porous polymer matrix; the peptidomimetic chain is an azide or alkyne end functionalized peptidomimetic chain of 8 - 20 amino acids that mimics KGF (FGF7), VEGF, FGF, FGF4, EGF, bFGF, BDNF, GM- CSF, HGF, BMP2, TGF-P, IGF-I, GDF-8, AEDG, AEDP, AEDL, AED, KE, KED, EDA, IRW, GRGDS, GLP-1, CLE, CLV3, RGD, HFL-1, P-15 or combinations thereof; the porous polymer matrix having a pore size in the range of 50 nm - 5 pm comprises, alkynyl alcohol or alkynyl amine units, castor oil as crosslinker, a polyether chain extender and a diisocyanate containing compound.

2. The polymer scaffold as claimed in claim 1, wherein the peptidomimetic chain is an azide or alkyne end functionalized amino acids chain of sequence ID 1, that mimics KGF.

3. The polymer scaffold as claimed in claim 1, wherein the porous polymer matrix is synthesized from a semi-interpenetrated polymer network which contains a non-reactive polyethylene glycol dimethyl ether polymer of molecular weight in the range of 250 to 5000 Daltons entangled within the network, which is sacrificially lost to produce alkyne functionalized porous polymer matrix.

4. The polymer scaffold of claim 1, wherein the crosslinker is a triglyceride of castor oil.

5. The polymer scaffold of claim 1, wherein the alkynyl alcohol or alkynyl amine is selected from a group consisting of propargyl alcohol, propargyl amine, alkyne-PEG2-amine and alkyne-PEG3-amine.

6. The polymer scaffold of claim 5, wherein the alkynyl alcohol is propargyl alcohol and the functional end groups of the propargyl grafts are chemically modified to achieve chain extension and free terminal azide groups.

7. The polymer scaffold of claim 1, wherein the polyether chain extender is selected from the group consisting of di-hydroxyl, di-amine, and di-carboxyl terminated compounds.

8. The polymer scaffold of claim 1, wherein the polyether chain extender is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), poly tetramethylene glycol (PTMG), block copolymers thereof, branched / graft copolymers thereof, and combinations thereof.

9. The polymer scaffold of claim 1, wherein the diisocyanate containing compound is selected from the group consisting of methylene diphenylene diisocyanate (MDI), polymeric methylene diphenylene diisocyanate (p-MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HMD I), dicyclohexane methylene diisocyanate (H12MDI), isophorone diisocyanate (IPDI), xylene diisocyanate, hydrogenated xylene diisocyanate, and Desmodur- N.

10. A process to prepare the peptide-mimetic conjugated porous polymer scaffolds as claimed in claim 1, wherein the process comprising the steps of;(a) reacting 10 wt % to 60 wt % Castor oil with diisocyanate compound with total — NCO / — OH ratio in the range of 0.8-2.5 in tetrahydrofuran (THF) solvent to form a pre-polymer;(b) charging the pre-polymer as obtained in step (a) with polyether macromonomer, alkynyl alcohol or alkynyl amine in THF solvent to obtain charged pre-polymer;(c) adding N, N-dimethylanilineas catalyst to the charged pre-polymer obtained in step (b) to initiate the polyurethane reaction to form a growing polymer network;(d) adding a non-reactive polyethylene glycol dimethyl ether (PEGDME) polymer of molecular weight in the range of 250 to 5000 Daltons to the growing polymer network of step (c) to obtain a viscous reaction mixture;(e) degassing and vigorously mixing the viscous reaction mixture obtained in step (d) under inert atmosphere to obtain a uniformly homogeneous viscous mix;(f) casting the uniformly homogeneous viscous mix as obtained in step (e) onto a teflon petri-dish to obtain a polymeric product;(g) curing the polymeric product as obtained in step (f) at room temperature for 24 h followed by curing at higher temperature and inert atmosphere at 60-90° C for 48 h- 96 h forming a semi-interpenetrating polymer network with the non-reactivepolyethylene glycol dimethyl ether polymer entangled within the network matrix, to obtain a free-standing film;(h) wrapping the free-standing film obtained in step (g) in Whatman filter paper bag and treating to a repeated soxhlet extraction process to obtain processed film;(i) subjecting the processed film obtained in step (h) to repeated swelling and drain cycles for 4-7 days against THF to extract out the PEGDME from the polymer network matrix completely, leaving behind an alkyne terminated porous polymer network;(j) extracting the porous polymer network using deionized millipore water to obtain an impurity free and sterile alkyne terminated porous polymer matrix;(k) reacting the alkyne terminated porous polymer matrix swelled in THF with 1,4- diazidobutane and N,N-Diisopropylethylamine (DIPEA) in the presence of Copper iodide (Cui) under nitrogen atmosphere and 80 °C for 24h leading to the formation of azide terminated-porous polymer scaffolds; and(l) reacting the azide-terminated porous polymer scaffolds with an azide or alkyne end functionalized peptidomimetic chain in presence N,N-Diisopropylethylamine (DIPEA) in and Copper iodide (Cui) for 48h at room temperature and soxhlet extraction using deionized millipore water to obtain impurity free peptide-mimetic conjugated porous polymer, wherein peptidomimetic chain mimics KGF (FGF7), VEGF, FGF, FGF4, EGF, bFGF, BDNF, GM-CSF, HGF, BMP2, TGF-P, IGF-I, GDF-8, AEDG, AEDP, AEDL, AED, KE, KED, EDA, IRW, GRGDS, GLP-1, CLE, CLV3, RGD, HFL-1, P-15 or combinations thereof.

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