Composite scaffold for cartilage regeneration and its process thereof

Abstract

The present invention relates to composite scaffold for cartilage regeneration and its process thereof. The composition of composite scaffold is comprised of 7.5-15wt% PGS, 7.5-15wt% PCL, 6-8wt% of protein / growth factor loaded SGNP, and 50-100µg of protein (BSA) / growth factor (GDF-5) offering excellent protein encapsulation efficiency into fibrous matrix, excellent protection of structural and functional properties of encapsulated proteins against process related stresses and capable of delivering proteins in a sustained therapeutic dosing manner for a prolonged period of time.

Description

[0001] COMPOSITE SCAFFOLD FOR CARTILAGE REGENERATION AND ITS PROCESS THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention relates to composite scaffold for cartilage regeneration and its process thereof. The composite scaffold composition is comprised of 7.5-15 wt% PGS, 7.5-15 wt% of PCL, 6-8wt% of protein / growth factor loaded SGNP, and 50-100 pg of protein (BSA) / growth factor (GDF-5). The work is focused on developing a unique regenerative strategy by combining biomaterials science and cell therapy in which a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based electrospun composite fibrous scaffold has been fabricated by conjugating with therapeutic growth factor (namely Growth and differentiation factor-5, GDF-5) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration. In particular, the novel scaffold composition mechanically stabilize the damaged cartilage as well as deliver therapeutic growth factor (Growth and differentiation factor-5, GDF-5) from the sugar-glass nanoparticles (GF-SGnP) reservoir that can increase [bone marrow derived mesenchymal stem cells (MSCs)] enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, reinstate extra cellular matrix (ECM) production, enhance proteoglycan content and maintain cartilage cell population and phenotype towards cartilage regeneration. More specifically, the developed novel multifunctional biodegradable electrospun fibrous scaffold conjugated with therapeutic growth factor loaded sugar-glass nanoparticles nanocarriers (GF-SGnP) can deliver the therapeutic protein sustainably while retaining the structural integrity, bioactivity and bioavailability properties of the therapeutic proteins. Overall, this invention elucidates the composition and fabrication procedure of developing a multifunctional tissue engineered 3D electrospun scaffold that showed tremendous potential in inducing chondrogenesis in bone marrow derived mesenchymal stem cells (MSCs) for restoration of healthy cartilage tissues for treatment of osteoarthritis (OA) in articulating joints and low back pain (LBP) caused by the degeneration of intervertebral disc (IVD).

[0004] BACKGROUND OF INVENTION

[0005] Osteoarthritis (OA), a highly prevalent destructive joint disease produces progressive pathological changes in the articular hyaline cartilage lining of synovial joints, as well as the subjacent bone and surrounding joint tissues. Globally, hip and knee osteoarthritis was ranked as the 11thhighest contributor towards disability with a prevalence of 3.8% and 0.85% for knee and hip, respectively. Degeneration of cartilage tissue is often found in both young and elderly patients. It is also expected that with increase in the aging and obese population, there exists large increase in the healthcare costs associated with treating these OAs. The hallmark of OA is its origin from progressive degeneration of articular cartilage, which is still an unmet need of great healthcare significance. Currently the treatment of cartilage damage or injuries is highly challenging and no licensed drugs are available. The avascular multilayer structure of the cartilage tissue comprises low number of cells with proteoglycan rich ECM. Majority of the cartilage tissue degeneration arising due to the aging, heavy overload during the intense physical activities and it resulted in severe inflammation which leads to the state of osteoarthiritis. Because of this reason, treatment of degenerated cartilage is challenging, and procedures like debridement, abrasion arthroplasty, micro fracturing, and autologous chondrocyte implantation have been followed to restore the functional cartilage. However, these techniques could not restore the biochemically and biomechanically appropriate cartilage tissue, and the poor integration of the implanted material these techniques with the surrounding cartilage tissue leads to inferior tissue formation. Therefore, two major techniques involving increasing the number of cells in the defect region through cell implantation or migration are popular to treat cartilage defects. When the cartilage damage is small procedures such as autologous chondrocyte implantation (ACI), bone marrow stimulation (BMS) are used to treat cartilage, and for severe damage often total joint replacement (TJR) is performed. In these treatments, except TJR, often mechanically and tribologically weak fibrocartilage forms and its integration with surrounding tissue is always very poor. In order to alleviate the symptoms and improve quality of patient's life there is growing demand for more efficient methods to restore damaged articular cartilage which has no intrinsic capacity to regenerate on its own. The biggest challenge in the treatment of damaged articular cartilage is to develop the optimal method of cartilage repair, capable of regenerating long lasting hyaline cartilage.

[0006] On the other hand, low back pain (LBP) is the second leading cause of disability and a common reason for lost work days primarily caused by the degeneration of intervertebral disc (IVD) resulting in the compression of the spinal nerves and adjacent vertebrae. Over 60% of the Indian population and 84% of US citizens experience low back pain due to the degenerative IVDs at some point in their lives. The pathophysiologies of degenerative IVD have a remarkable socioeconomic impact and associated healthcare expenditure estimated over 100 billion dollars annually. The prevalence of back pain will increase substantially in the coming years due to the ageing demographic. The IVD is confined by the two cartilage endplates and is composed of two distinct structures: a gelatinous nucleus pulposus (NP) center and several surrounding coaxial lamellae that form the inner and outer annulus fibrosus (AF). This unique structural feature allows the IVD to constrain motion at high loads and provide flexibility at low loads. Degenerative disc disease (DDD) is defined as an “aberrant, cell-mediated response to progressive structural failure”, a structural disorganization of cartilage tissues leading to a loss of disc height and herniation causing an overall effect on the biomechanics of the spinal column with age. The exact pathophysiology of DDD has not yet been completely delineated, however it is known that disc degeneration is induced in different stages by the interaction between various mechanical, biochemical, genetic, and nutritional factors. However, despite the prevalence of DDD and its enormous socioeconomic impact current treatment options are limited because they can ease the pain but are unable to completely restore normal functions or promote tissue regeneration. IVD replacement prostheses such as total disc replacement implants facilitate the preservation of motions and disc space height, however; are unable to sustain compressive forces due to their lack of elasticity. In addition; their longevity is unknown, but inherently limited due to their inability to biointegrate and subsequently remodel. Therefore, the inherent limitation of cartilage tissue replacement prostheses emphasizes the importance of regenerative approaches that will enable / stimulate the repair of the ruptured cartilage tissues in the articulating joints or IVD in spine for the treatment of Osteoarthritis (OA) or degenerative disc diseases (DDD) causing low back pain.

[0007] Herein, regenerative strategies are increasingly focusing on development of a unified approach for regeneration of cartilage tissues by utilizing tissue engineering techniques approaches that can address the cartilage degeneration by providing tissue specific therapeutic growth factors (GF), and delivery of stem cells (MSCs) using suitable biodegradable scaffold. These constructs are designed to treat cartilage degeneration on a molecular level by correcting the biochemical imbalance, maintaining disc cell population, phenotype, proteoglycan content, increase the potential of cartilage regeneration as well as mechanically stabilize the damaged native cartilage tissues.

[0008] Importantly, therapeutic molecules like proteins are the essential extracellular signaling molecules that interact with the specific parts of the extracellular matrix and regulate the cells' anabolic / catabolic gene expression. Growth and differentiation factor - 5 (GDF-5) is a member of bone morphogenic protein identified for its involvement in the regulation and development of joint and chondrogenic activity during the embryonic development of mesenchymal stem cells. Because the in-situ hybridization and immuno staining of cartilaginous tissue of mice bone elucidated the prominent presence of GDF-5 and null mutation in the GDF-5 shown to disorganize the joint development up to 30%. The homodimer GDF-5 protein interacts through type I and type II serine / threonine receptor kinases to activate Smad proteins to regulate the target gene expression. However, it is important to note here that there is a strong need to develop an efficient biochemical functionalization technique for the delivery of therapeutic growth factors for tissue regeneration. For optimal therapeutic application with the GDF-5, the delivery system needs to keep up with GDF-5's physiological conformation and bioactivity, which is liable to environmental factors like pH, protease enzymes, polarity of the solution and temperature. So, emulsification-based microspheres were utilized to deliver the therapeutic molecules but encapsulate the protein in a conventional process into a non-polar region which further deteriorates the physiology of the protein. So, the poor encapsulation efficiency, burst release profile, reduced bioactivity and bioavailability made the pathway to identify a better carrier system for delivering therapeutic molecules. Recently, a new class of nano -reservoir systems has been developed to efficiently encapsulate therapeutic growth factors or proteins in the core of sugar-glass nanoparticles (GF-SGnP) for delivering therapeutic protein. Our earlier report demonstrated that the encapsulated proteins were protected from the synthesis related and environmental stresses, by retaining bioactivity, structural integrity and bioavailability. Also, the SGnP has delivered the protein in a prolonged manner without the initial burst release. So, in this invention we have formulated a novel composition of multifunctional PGS-PCL based electrospun fibrous scaffold conjugated with GDF-5 loaded SGnP system for the delivery of GDF-5 growth factor.

[0009] It is well recognized in the field that electrospun fibrous scaffold have been recognized as a potential construct that closely mimic the architectural scale and morphology of native fibrillar collagen, including that in the native disc ECM. The combined features of 3D structure, high porosity, and their ability to encapsulate different therapeutic agents make fibrous scaffold a promising and effective component for functional regeneration. For this purpose different fibrous scaffold made from synthetic thermoplastic polymers such as PGS, PCL, PLA and their copolymers have been recently studied for cartilage tissue regeneration. Ideally, the scaffold used in cartilage regeneration should show optimum elasticity, to favor rhythmic contraction and relaxation of the disc. Therefore, an elastomer like PGS which is a long chain viscoelastic polymer having low young's modulus and high failure strain that are capable of sustaining and recovering from deformation under cyclic strain and is being investigated for use in dynamic environments like cardiac tissues compared with other thermoplastic polymers can be used as a potential alternative scaffold material. Poly (glycerol sebacate) (PGS) is a biodegradable and elastomeric polymer which can be synthesized using glycerol and sebacic acid through a polycondensation reaction. Since it is impossible to electro spun the PGS alone, poly(caprolactone) is combined with PGS because it is a widely used polymer in medical devices and drug delivery applications. Hence, in this invention a novel formulation of PGS- PCL composite electrospun fibrous scaffold have been fabricated after successful conjugation with therapeutic GDF-5 loaded SGnPs for cartilage regeneration applications. No such study has been yet conducted to use our unique elastomeric multifunctional scaffold systems for cartilage regeneration

[0010] There is also increasing evidence that progenitor cells with characteristics of mesenchymal stem cells (MSCs) shows cartilage regeneration potential. A recent clinical study transplanting autologous bone marrow derived MSCs has shown promising initial results in terms of improvement of pain and disability. However, cell leakage is one of the problems of injecting cell suspension. A study recently reported that one of the possible downside of mishandling of MSC, where the leaking of MSC in the disc surrounding has led to osteophyte formation. Hence, cells need to be embedded in a carrier to protect MSC from leaking and to provide a framework for supporting growth and differentiation of MSC.

[0011] We have hypothesized that our developed multifunctional GF-SGnP conjugated PGS-PCL fibrous scaffold can satisfy the requirement of total disc regeneration by providing the appropriate microenvironment of biophysical (structure and mechanical support) and biochemical cues (delivery of therapeutic growth factor, and MSCs) that will up-regulate tissue specific ECM components to replenish the ECM proteins, proteoglycan towards cartilage regeneration. To test our hypothesis we have selected human recombinant growth and differentiation factor-5 (GDF-5) protein as a therapeutic growth factor and human bone marrow derived MSCs which have shown potential for secreting the relevant paracrine factors that are beneficial for the treatment of damaged cartilage in both in vitro and in vivo models. Further, GDF-5 has reportedly shown to enhance ECM production in vivo. It is also known that reduction in cell number in cartilage during ageing is an important factor for cartilage degeneration. Therefore, delivery of MSCs will increase the cell density in the damaged disc and can potentially increase the paracrine signaling effect and hence lead to restore the function of degenerated articulating cartilages including IVD. For GDF-5 delivery, the protein will be initially encapsulated into trehalose-based sugar-glass matrix of nanoparticles system (GF- SGnP). Then the protein containing nanoparticles will be incorporated into PGS-PCL fibrous matrix (i.e., core functionalization) by electrospinning technique. The in vitro cartilage regeneration potential of the developed novel multifunctional PGS-PCL fibrous mat was finally demonstrated through comprehensive in vitro assessments after culturing with bone marrow derived mesenchymal stem cells (MSCs). So, overall, the present invention focuses on developing a unique regenerative strategy by combining biomaterials science and cell therapy in which a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional elctrospun fiber has been fabricated by conjugating with therapeutic growth factor (GF) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration.

[0012] Reference may be made to the research article (Poulomi Polley et al. Molecular Pharmaceutics 2020, 17 (1), 284-300), wherein the group has fabricated Poly (lactic acid-co-glycolic acid) (PLGA) based biodegradable microparticle or microsphere depot wherein the protein is encapsulated into sugar glass nanoparticles (SGnP) to provide a burst-free sustained release of protein. The microparticle depot demonstrated to encapsulate Bovine serum albumin (BSA), Horseradish Peroxidase (HRP), Fibroblast growth factor -2 (FGF-2) Epidermal growth factor (EGF), or combinations into a SGnP core with protein to sugar ratio ranging from about 1 :20 to about 1 :200. Developed microsphere depot system for biomolecule delivery is advantageous in terms of biocompatibility, biodegradability and effective over burst free protein release. Whereas, the present invention has reported the polymeric scaffold which are fabricated by electrospinning technique using PGS-PCL based polymeric composition. Moreover, the present invention has encapsulated the therapeutic growth factors (GDF-5) and / or model protein bovine serum albumin (BSA) into the core of SGnP nanoparticles with protein to sugar ratio ranging from about 1 :2000 to about 1 :2666 (Table 1). More importantly, the prior arts did not encapsulate the protein nanoparticles loaded microparticles inside a fibrous scaffold for cartilage regeneration applications. Whereas, present invention work demonstrated successful conjugation of SGnP loaded therapeutic growth factors (GDF-5) and / or model protein bovine serum albumin (BSA) into the PGS-PCL fiber matrix by mixing and subsequent electrospinning to develop novel range of compositions for fabricating protein (i.e., PGSpP:PCL:SGnP-BSA50, PGSPP:PCL:SGnP-BSA75, and PGSPP:PCL:SGnP-BSA100 for BSA conjugated) and / or therapeutic growth factor (PGSpp:PCL:SGnP-GDF5 for GDF-5 conjugated) conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-3, Figure 1 and 2).

[0013] Reference may be made to the research articles (Aniruddha Pal et al. 2022 Nano Futures 6 025008), wherein the group has demonstrated the synthesis and characterization of a sugar- glass nanoparticle (SGnP) based reservoir type protein delivery system pertinent to tissue engineering applications. One model protein (bovine serum albumin, BSA) and one therapeutic potein [growth and differentiation factor-5 (GDF-5)] were successfully encapsulated into SGnP Nano-reservoir system to achieve excellent encapsulation efficiency, sustain release of protein and preservation of bioavailability / bioactivity of proteins. Moreover, a novel PGS based copolymer was prepared by polycondensation reaction between glycerol and sebacic acid and subsequently conjugated with methyl acrylate (MA) by atom transfer radical polymerization (ATRP) technique to synthesize electro spinnable PGS-pMA copolymers with enhanced molecular weight and viscosity. The fluorescent dye (Rhodamine B) encapsulated sugar-glass nanoparticles (SGnP-RB) was also demonstrated to loaded into PGS-pMA fibers matrix. However, in this prior art growth factors, proteins or therapeutic agents loaded SGnP was not conjugated into the electrospun fibers matrix. Whereas, present invention provides a pathway to utilizing the SGnP to load therapeutic proteins or growth factor into 3D fibrous scaffold that can be used for tissue engineering applications. In particular, this present invention is focused on developing a develop novel range of compositions for fabricating protein (i.e., PGSpP:PCL:SGnP-BSA50, PGSPP:PCL:SGnP-BSA75, and PGSPP:PCL:SGnP-BSA100 for BSA conjugated) and / or therapeutic growth factor (PGSpp:PCL:SGnP-GDF5 for GDF-5 conjugated) conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-4, Figure 1-3).

[0014] Reference may be made to the research article {Liu Y et al. J Mater Sci Mater Med. 2019 Apr 29;30(5):53), wherein the prior art has proposed a porous 3D scaffold of PGS and PCL cultured with BMSC for cartilage tissue engineering. The scaffold was fabricated using thermal crosslinking followed by the salt leaching method. The drawback is that the fabrication process requires 150 °C, which makes it difficult to be utilized in the protein delivery system. Because the proteins are liable above 40 °C, the present invention addressed this issues by utilizing the SGnP carrier system to encapsulate the proteins and safely pack them inside a fibrous scaffold for prolonged release with better bioavailability (Table 1-4, Figure 1-3).

[0015] Reference may be made to the research article (K. Andreas et al. Acta Biomaterialia 2011, 7 (4), 1485-1495), wherein the group has explored the formulation of insulin-loaded PLGA microspheres using three different emulsification techniques and utilizing the microspheres for cartilage tissue engineering. The aim was to investigate comparatively three different microencapsulation techniques: solid-in-oil-in-water (s / o / w), water-in-oil-in-water (w / o / w), and oil-in-oil-in-water (o / o / w), for the fabrication of insulin-loaded PLGA microspheres about protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. The drawback of the study is that the nanoparticle has shown a lower encapsulation efficiency in the range of 25.2 % to 79.9 %. Also, the initial burst release of 32% was identified within 24 hours from the s / o / w microsphere. Whereas, present invention investigated these factors in a detailed manner to prove the efficacy and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-3, Figure 1 and 2).

[0016] Reference may be made to the research article (F. Buket Basmanav et al. Biomaterials 2008, 29 (31), 4195-4204), wherein the prior art aimed to fabricate a tissue-engineering scaffold comprising microspheres of polyelectrolyte complexes of poly (4-vinyl pyridine) (P4VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7. This construct was intended to use a regenerative scaffold for bone tissue engineering. Entrapment efficiency tends to control the system's efficiency, so the system exhibited between 7.66 % and 11.07 %. Release kinetics was studied using albumin as the model drug, and it confirmed the initial burst release within five days. This is attributed to drawbacks in the efficacy of the system. Neither BMP-2 nor BMP-7 delivery directly affected proliferation with the present doses. However, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration. Whereas, present invention investigated these factors in a detailed manner to prove the efficacy and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-3, Figure 1 and 2).

[0017] Reference may be made to the research article (L. Berten-Schunk et al. Pharmaceutics 2023, 15(4), 1303), wherein the prior art optimized an implantable scaffold for regenerative therapy specific to cartilage degeneration. So, the electrospun fiber mats of poly(s-caprolactone) (PCL) loaded with Chitosan / tripolyphosphate (CS / TPP) nanoparticles were used to encapsulate transforming growth faclor-Ps (TGF-P3) with increasing loading concentrations for the regeneration of the cartilage zone within direct entheses. This study proposed a loading concentration of 20 pg / ml for optimal chondrogenesis. This loading concentration exhibited increased chondrogenic marker genes (SOX9, COL2A1, COMP). These data were further supported by an increase in the cell pellets' glycosaminoglycan (GAG)-to-DNA ratio. But the drawbacks are listed here. The release study has shown a burst release profile of 95% to 99% of total protein from the fibrous mat, which was released within 4 - 5 days. Also, the retention of bioactive protein is not mentioned throughout the study, making it difficult to understand the system's efficiency. Whereas, present invention investigated these factors in a detailed manner to prove the efficacy and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-3, Figure 1 and 2).

[0018] Reference may be made to the research article (C. Wang et al. Materials Science and Engineering: C 2017, 79, 507-515), wherein the prior art proposed Poly (1-lactic acid)-co- poly(s-caprolactone) (PLLA-CL) electrospun fiber scaffold and chitosan nanoparticles as a dual release system to sustain release Nel-like molecule- 1 (Nell-1) growth factor for inducing the human bone MSCs (hBMSCs) differentiate toward chondrocytes. The study confirmed a burst release profile of Nell-1. At the same time, it is incorporated in the scaffold alone, and the Nell-1 into chitosan nanoparticles significantly extended the release time and increased the released Nell-l's bioactivity than directly incorporating Nell-1 into the scaffold. Furthermore, Nell-1 promotes hBMSCs in vitro chondrogenic differentiation by increasing the expression of chondrogenic -related genes and proteins. It may be taken as a drawback from the study where the authors did not talk about the actual release every day, which is crucial for the drug delivery system that needs to be used in regenerative therapy. Whereas, present invention investigated these factors in a detailed manner to prove the efficacy and experimentally demonstrated excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc towards successful in vitro cartilage regeneration (Table 1-4, Figure 1 and 2).

[0019] Reference may be made to the article (Kalvand E et al. Journal of Biomedical Materials Research: Part A 2023, 1-12.), wherein the prior art proposed a transforming growth factorbeta 1 (TGF-pi) delivery system for cartilage tissue engineering. This delivery system was formulated using the electrospinning technique using the Poly-s-caprolactone (PCL) / poly- L - lactic acid (PLLA). This 3D scaffold is entrapped with chitosan nanoparticles loaded with TGF- pi. The scaffold retained good mechanical swelling properties, and the TGF-pi differentiated the adipose-derived mesenchymal stem cells (ASC) into the chondrocyte-like lineage, confirmed by the qRT-PCR results. However, the drawback of the system is that here, the system shows an initial burst release of 83% in 5 days, and the retention of bioactive TGF- pi is not studied well. This present work carried out in a comprehensive manner to address the issues of burst release of protein in conventional approach by encapsulating the therapeutic protein in a reservoir of sugar-glass nanoparticles to achieve excellent protein encapsulation efficiency, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-4, Figure 1 and 2).

[0020] Reference may be made to the article (Selcan G et al. Journal of Biomedical Materials Research: Part A 2014, 102A, 1897-1908), wherein the prior art proposed a double-layer nanofibrous scaffold loaded with fibroblast Growth Factor - 2 (FGF-2) encapsulated gelatin microspheres for tissue engineering applications. The upper layer was fabricated using PCL- gelatin, and the bottom layer was fabricated using PCL-PLLA. The gelatin microspheres were placed between the layers for a sustained release profile. However, the drawback of the scaffold is that it exhibited a burst release profile, and the total amount of loaded molecules was released within 48 hours. This present work carried out in a comprehensive manner to address the issues of burst release of protein in conventional approach by encapsulating the therapeutic protein in a reservoir of sugar-glass nanoparticles to achieve excellent protein encapsulation efficiency, sustained delivery of protein or Growth factors at a therapeutic daily dosage, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-4, Figure 1 and 2).

[0021] Reference may be made to Patent No. IN201941049522A (Assignee: Eaffocare Innovation Pvt. Ltd., Indian Institute of Technology, Room No. B408, Kandi, Sangareddy, Telangana, India, Publication date: 02.12.2020), wherein the inventors fabricated Poly (lactic acid-co-glycolic acid) (PLGA) and PCL based biodegradable microparticle or microsphere depot wherein the protein is encapsulated into sugar glass nanoparticles (SGnP) to provide a burst-free sustained release of protein. The microparticle depot demonstrated to encapsulate Bovine serum albumin (BSA), Horseradish Peroxidase (HRP), Fibroblast growth factor -2 (FGF-2) Epidermal growth factor (EGF), or combinations into a SGnP core with protein to sugar ratio ranging from about 1 :20 to about 1 :200. The microsphere depot was fabricated by double emulsion method for for fabrication of polymeric scaffold. The microsphere depot has demonstrated to deliver the protein and preserved the bioactivity till 7 days. Developed microsphere depot system for biomolecule delivery is advantageous in terms of biocompatibility, biodegradability and effective over burst free protein release, whereas, the present invention relates to composite scaffold for cartilage regeneration and its process thereof. Moreover, the present invention has encapsulated the therapeutic growth factors (GDF-5) and / or model protein bovine serum albumin (BSA) into the core of SGnP nanoparticles with protein to sugar ratio ranging from about 1 :2000 to about 1 :2666 (Table 1). More importantly, the prior arts did not encapsulate the protein nanoparticles loaded microparticles inside a fibrous scaffold for cartilage regeneration applications. Whereas, present invention demonstrated successful conjugation of SGnP loaded therapeutic growth factors (GDF-5) and / or model protein bovine serum albumin (BSA) into the PGS-PCL fiber matrix by mixing and subsequent electrospinning to develop novel range of compositions for fabricating protein (i.e., PGSpp:PCL:SGnP-BSA50, PGSPP:PCL:SGnP-BSA75, and PGSPP:PCL:SGnP-BSA100 for BSA conjugated) and / or therapeutic growth factor (PGSpp:PCL:SGnP-GDF5 for GDF-5 conjugated) conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold comprising of 7.5- 15 wt% PGS, 7.5-15 wt% of PCL, 7.56 wt% of protein / growth factor loaded SGnP, and 50-100 pg of protein (BSA) / growth factor (GDF-5) loading exhibiting a unique combination of relevant properties (as presented in Table 3) like excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosages, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. (Table 1-3, Figure 1 and 2). The prior art has demonstrated to deliver the protein and preserved the bioactivity till 7 days, whereas the present invention showed sustained release and preservation of bioactivity upto 21 days for protein (BSA) towards actual release of 38-240 ng protein / day wise release and in the range of 62.28% during the period from 1-23 days for growth factor (GDF-5) towards actual release of 29.64-438.89 ng protein / day wise release ensuring good therapeutic dosing pertinent to cartilage regeneration avoiding burst release kinetics of the compositions reported in prior art. In contrast to prior art that did not show any specific tissue regeneration potential of their developed SGnP -protein loaded microsphere system, CSIR-CGCRI work demonstrated successful in vitro cartilage regeneration properties of novel multifunctional biodegradable 3D electrospun fibrous composite scaffold [i.e., PGSpp:PCL:SGnP-GDF5 (Example 4, Figure 3 ) (Table 4).

[0022] Reference may be made to Patent No. IN201841034177A (Assignee: Virtis Bio Labs Pvt Ltd, 39 / 3 A2 Easwari garden, Kesavan Nagar, Kannankuruchi, Salem-636008, Tamil Nadu (India) and Vellore Institute of Technology, Near Katpadi road, Vellore- 632014, Tamil Nadu (India)), wherein the prior art invented a novel hybrid nanofibrous scaffold composition, comprises, a resorbable synthetic polymer (PVA (polyvinyl alcohol, 5-10 wt%), a bioceramic derivative nano hydroxyapatite (nHA, 3.5 to 5 wt%) and a coating agent (human platelet lysate either commercially available or freshly prepared, 5 to 10 molar %) to form a nanofibrous material with average porosity in the range of 60% to 80% for potential bone regeneration applications. Whereas, the present invention is focused on fabricating of novel PGSpp:PCL:SGnP-GDF5 composition based fibrous scaffold for cartilage tissue regeneration where the therapeutic growth factor (GDF-5) or protein (BSA) initially encapsulated into trehalose-based sugar-glass matrix of nanoparticles system (GF-SGnP). Then the protein containing nanoparticles has been incorporated into PGSpp-PCL fibrous matrix (i.e., core functionalization) by electrospinning technique. Another major limitation of this prior art is that the cocktail of growth factors from the platelet lysate was not reported in terms of the release profile, bioavailability, and bioactivity and the intended application is also towards bone regeneration. Whereas, present invention investigated these factors in a detailed manner to prove the efficacy and experimentally demonstrated in vitro cartilage regeneration potential through comprehensive in vitro assessments after culturing with bone marrow derived mesenchymal stem cells (hBMSCs) (Table 3 and 4, Figure 3).

[0023] Reference may be made to Patent No. IN201721031262A (Assignee: Meril Life Sciences Pvt. Ltd., an Indian company, of the address Bilakhia House Muktanand Marg, Chala, Vapi, Gujarat - 396191 disclosed, Publication date: 02.09.2017), wherein the prior art developed a scaffold graft is made of a biodegradable polymer [poly-L-5 lactic acid (PLLA)] to be used for the treatment of arterial perforations and condition of aneurysm. Then the first scaffold is further coated with a biodegradable layer of polymer [second coating scaffold is made of an antiproliferative drug formulation of Sirolimus and biodegradable poly-DL-lactide (PDLLA)] on an outer surface of first scaffold by means of a spray coating technique. So, the prior art has utilized different composition of biodegradable polymers in comparison to present invention and utilized the double layer scaffold structure in the form of stents; whereas, the present invention provides polymeric scaffold fabricated by direct mixing of two polymers. Furthermore, the prior art’s scaffold graft is made of a cross-linker (includes at least one of hexamethylene diisocyanate (HDI), Lysine diisocyanate (LDI)), wherein the present invention has used PGS-PCL polymers directly without any crosslinkers. Finally, the scope of prior art is completely different from the present invention. Notably, the present invention focuses on developing a unique regenerative strategy by combining biomaterials science and cell therapy in which a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun fiber has been fabricated by conjugating with therapeutic growth factor (GDF5) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration (Table 3 and 4, Figure 3).

[0024] Reference may be made to Patent No. US11124897B1 (Assignee: King Abdulaziz University, Publication date: 21.09.2021), wherein the prior art invented controlled-drug release-based wound dressing scaffold by co-axial electrospinning, comprising core-shell nanofibers wherein an outer shell coating layer of the scaffold comprises polycaprolactone (PCL) and a core gel layer of the scaffold comprises a mixture of chitin, lignin, polyethylene oxide (PEO) and polyglycerol sebacate (PGS), to the method of drug encapsulation in a core-shell fibrous scaffold. Core-shell electrospinning is the fabrication method utilized in the prior art. So, the prior art has utilized the double layer scaffold structure in the form of gel and fibers; whereas, in present invention utilized the PCL and PGS-based polymer blend and the therapeutic growth factor (GDF5) loaded sugar-glass nanoparticles (SGnP) were mixed together with PCL-PGS solution in optimized formulation to fabricate protein loaded nanoparticles conjugated into the matrix of electrospun multifunctional PGS-PCL nanofirous matrix for cartilage regeneration (Table 3 and 4, Figure 3). the disclosed polymeric scaffold were fabricated by direct mixing of two polymers. Notably, the prior art has incorporated the drugs responsible for repairing of chronic wounds and diabetic ulcers and elucidated the antibacterial activity wherein the outer shell coating layer is configured to provide adhesion to the wound by treating the scaffold with polydopamine, whereas the scope of present invention is completely different which is focused on developing a unique regenerative strategy by combining biomaterials science and cell therapy in which a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun fiber has been fabricated by conjugating with therapeutic growth factor (GDF5) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration (Table 3 and 4, Figure 3).

[0025] Reference may be made to Patent No. CN201810102243 (Assignee: Shanghai Normal University University of Shanghai for Science and Technology, Publication date: 09.04.2021), wherein the prior art invented an oriented porous composite electrospun fiber scaffold having a bionic surface, characterized in that it is composed of a composite fiber having a nanoporous surface distributed in a single orientation, in which mesopores are distributed, and the composite fibers are arranged in a single orientation The surface is distributed with nanoporous L-polylactic acid (PLLA) electrospun fiber as matrix, the surface of the substrate is modified with polydopamine, or the surface-modified polydopamine grafting cartilage repairing drug. Whereas present invention is different in comparison to this prior art both in terms of polymer composition as well as method of encapsulating the proteins for therapeutic application. The present invention is focused on developing a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun fiber has been fabricated by conjugating with therapeutic growth factor (GF) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration (Table 3 and 4, Figure 3).

[0026] Reference may be made to Patent No. US20230148084A1 (Assignee: Scripps Health; Publication date: 11.05.2023), wherein the prior art elucidated a meniscal implant comprising a plurality of cross-linked polymer fibers, the polymer fibers of the plurality of cross-linked polymer fibers having a structure comprising of a shell comprising a first polymer (first polymer is selected from the group consisting of: collagen, methacrylated collagen, and both) and a core comprising a second polymer (second polymer is selected from the group consisting of: polylactic acid, polycaprolactone, a copolymer thereof, and a blend thereof) construct of a biodegradable electrospun polymeric meniscal implant loaded with a plurality of cells (hondrocytes, chondroblasts, cartilaginous cells, mesenchymal stem cells, fibre cartilaginous cells, meniscal cells, and a combination thereof) for cartilage regeneration. The construct has a similar compressive strength to native articular cartilage, which helps in the better adoption of the mechanical load of the joint. Whereas present invention is different in comparison to this prior art as present invention utilized different polymer composition such as PCL and PGS- based polymer blend and the therapeutic growth factor (GDF5) loaded sugar-glass nanoparticles (SGnP) were mixed together with PCL-PGS solution in optimized formulation to fabricate protein loaded nanoparticles conjugated into the matrix of electrospun multifunctional PGS-PCL nanofirous matrix for cartilage regeneration (Table 3 and 4, Figure 3). Moreover, no cross-linking agent was also used in present invention.

[0027] Reference may be made to Patent No. US20100061962A1 (Assignee: US Department of Health and Human Services, Publication date: 19.06.2012), wherein the prior art elucidated tissue engineered cartilage comprising a nanofibrous biocompatible polymer support having chondocytes dispersed therein, which has compressive strength equal to natural cartilage, methods of fabricating tissue engineered cartilage by culturing a mixture of stem cells or chondocytes and a electrospun nanofibrous polymer substrate in a suitable bioreactor and methods of treatment comprising implantation of tissue engineered cartilage into a patient. Whereas present invention is completely different which is focused on developing a unique regenerative strategy by combining biomaterials science and cell therapy in which a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional elctrospun fiber has been fabricated by conjugating with therapeutic growth factor (GDF-5) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration (Table 3 and 4, Figure 3).

[0028] Reference may be made to Patent No. EP4119169 (Al) (Assignee: Lietuvos Sveikatos Mokslu Universitetas Kauno Technologijos Universitetas, Publication date: 18.01.2023), wherein the prior art invented a method of preparing an implantable polymeric construct for regenerating articular cartilage damaged by trauma or chronic inflammatory-degenerative process. The scaffold consist of two layers for cartilage and subchondral bone tissue formation. So, the blend of biodegradable polymer (PCL) and cellulose fabricates layer 1. Like this, the second layer comprises biodegradable polymer (PCL) and cellulose along with bioactive substance (hydroxyapatite) to mimic the bone microenvironment. The prior art does not directly correlate with the current invention, which is a drawback. Whereas present invention is completely different in terms of composition and scaffold fabrication methodology. Present work is focused on developing a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun fiber by conjugating with therapeutic growth factor (GDF-5) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for cartilage regeneration (Table 3 and 4, Figure 3 ).

[0029] Reference may be made to Patent No. US2020246126A1 (Assignee: University of Pittsburgh, USA; Publication date: 2020-08-06), wherein the prior art invented a method of preparing a vascular graft, comprising of a biodegradable scaffold comprising a biodegradable polyester tubular core comprising poly(glycerol sebacate) (PGS) and having small pores of about 1 pm to about 500 pm and comprising an inner lumen surface and an outer surface comprises poly(caprolactone) (PCL). Whereas present invention is not a core-shell fibrous structure as the PGS-PCL electrospun fibers were produced by by direct mixing of two polymers. Moreover, therapeutic protein loaded SGnP nanoparticles were encapsulated into PGS-PCL nanofibers matrix to render support in cartilage regeneration (Table 3 and 4, Figure 3).

[0030] OBJECTIVES OF THE INVENTION

[0031] The main objective of the present invention is to provide novel growth and differentiation factor-5 protein encapsulated sugar-glass nanoparticles (GDF5-SGnP)) conjugated poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun composite fibrous scaffold for cartilage regeneration and its process thereof which obviates the drawbacks of the known prior art as detailed above.

[0032] Another objective of the present invention is to provide a novel multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) electrospun composite fibrous scaffold compositions containing BSA (model protein; bovine serum albumin) or GDF-5 (therapeutic growth factors; Growth & Differentiation Factor - 5) proteins loaded sugar-glass nanoparticles (GDF-SGNP) reservoirs in it’s matrix which offers excellent protein encapsulation efficiency into 3D fibrous matrix at optimized parameters (unique protein to sugar loading concentration, polymer to SGnP loading concentration, electro -spinning parameters) and also capable of delivering proteins in a sustained manner and thus, eliminate or minimize the problems described in the background and prior art section.

[0033] Still another objective of the present invention is to provide novel multifunctional biodegradable electrospun composite fibrous scaffold compositions which show considerably higher retention of proteins (BSA; model protein i.e., bovine serum albumin or GDF-5; therapeutic growth factors i.e., Growth & Differentiation Factor - 5) bioactivity, structural integrity and bioavailability.

[0034] Yet another objective of the present invention is to provide novel multifunctional biodegradable electrospun composite fibrous scaffold compositions that can increase enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhance extra cellular matrix (ECM) production and proteoglycan content for the regeneration of cartilage tissues with the conjugation of bone marrow derived mesenchymal stem cells (MSCs).

[0035] Another objective of the present invention is to provide an easy and economic regenerative therapy for cartilage regeneration by using a novel multifunctional biodegradable electrospun 3D fibrous scaffold.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] The present invention is illustrated in Figure 1 to Figure 4 of the drawing(s) accompanying this specification. In the drawings like reference numbers / letters indicate corresponding parts in the various figures.

[0038] Fig. 1 represents (a) SDS PAGE (Lane 1 - Molecular weight marker, lane 2 - BSA, Lane 3- SGnP-BSA50, Lane 4 - SGnP-BSA75, Lane 5 - SGnP-BSAlOO) and (b) NATIVE PAGE (Lane 1 - BSA, Lane 2- SGnP-BSA50, Lane 3 - SGnP-BSA75, Lane 4 - SGnP-BSAlOO) analysis for assessing the protein intergrity of BSA from SGnP-BSA; SEM images of (c) PGSpP:PCL:SGnP-BSA50 (d) PGSPP :PCL:SGnP-BSA75 (e) PGSPP :PCL:SGnP-BSA75; (d) Cumulative release profile and (g) Day wise release profile for the different grades of PGSpp :PCL:SGnP-BSA.

[0039] Fig. 2 represents (a) SDS PAGE analysis for assessing the GDF5 integrity, (Molecular weight marker, lane 2 - Bare GDF5 and Lane 3 - SGnP-GDF5); (b) SEM image of SGnP-GDF5 encapsulated PGS copolymer fibrous patch; (c) Cumulative release profile of GDF-5 and (d) Day wise release profile of different grades of GDF-5 from SGnP-GDF5 encapsulated PGSpp:PCL-SGnP-GDF5; (e) Estimation of Bioactive GDF5 released from SGnP-GDF5 encapsulated PGS copolymer fibrous patch. [One-way ANOVA was carried out and the statistical significance is mentioned as ns = p> 0.05 - statistically insignificant] Fig. 3 represents in vitro assays were performed for the following groups; Only PGSpp:PCL based fibrous scaffold cultured in hBMSC expansion medium (PGSpp:PCL + EM), PGSpp:PCL based fibrous scaffold cultured in chondrogenic medium (PGSpp:PCL + CM), PGSpp:PCL based fibrous scaffold cultured in GDF-5 supplemented hBMSC expansion medium (PGSpp:PCL + GDF5), and PGSpp:PCE based fibrous scaffold conjugated with SGnP-GDF5 (PGSpp:PCE-SGnP-GDF5). (a) In vitro cytotoxicity and cell proliferation assessment (MTT assay); (b) Quantification of sulphated glycosaminoglycans (sGAG); (c) Quantification of total collagen; (d) Quantification of double standard DNA (dsDNA); (e) Ratio of sGAG / dsDNA; (f) Ratio of Total collagen / dsDNA; (g) Alcian blue staining to visualize the ECM remodeling ; (h) Fluorescence cell morphology (DAPI and FITC staining) [# = P>0.05 statistically insignificant, * = P<0.05 statistically significant, statistical analysis was done by one-way ANOVA analysis and pair comparison was made between PGSpp:PCE + EM vs. remaining sample groups].

[0040] The drawings are included to provide better understanding of the invention, illustrate the embodiments of the invention and together with the description serve to explain the importance of the invention.

[0041] SUMMARY OF THE INVENTION

[0042] Accordingly, the present invention relates to composite scaffold for cartilage regeneration and its process thereof. The present invention discloses novel range of compositions for fabricating protein (i.e., PGSPP:PCE:SGnP-BSA50, PGSPP:PCE:SGnP-BSA75, and PGSPP:PCE:SGnP- BSA100 for BSA conjugated) and / or therapeutic growth factor (PGSpp:PCE :SGnP-GDF5 for GDF-5 conjugated) conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold via commercially viable electrospinning technique. The composite scaffold composition is comprised of 7.5-15 wt% PGS, 7.5-15 wt% of PCE, 6-8wt% of protein / growth factor loaded SGnP, and 50-100 pg of protein (BSA) / growth factor (GDF-5). The assessed characteristics of these developed multifunctional scaffold offers excellent protein encapsulation efficiency into 3D fibrous matrix, unique advantage of protecting the encapsulated proteins structural and functional properties against the process and environment related stresses and also capable of delivering proteins in a sustained therapeutic dosing manner for a prolonged period of time eliminating / minimizing the problems described in the background and prior art. Additionally, the invented novel compositions of composite fibrous scaffold exhibit excellent in vitro cartilage regeneration capabilities as confirmed by the enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhanced cartilage-like extra cellular matrix (ECM) protein (collagen) and ECM substances (sGAG) production, upregulating anabolic genes pertinent to regeneration of cartilage tissues. Overall, this invention is anticipated to be greatly useful as an efficient regenerative and tissue engineering strategy for regeneration of damaged cartilage tissues in treatment of osteoarthritis (OA) in articulating joints and low back pain (LBP) caused by the degeneration of intervertebral disc (IVD) via degenerative disc diseases (DDD).

[0043] In an embodiment, the present invention discloses a composite scaffold for cartilage regeneration comprising of: a. poly(glycerol sebacate) [PGS]; b. poly(caprolactone) [PCL]; c. protein and growth factors with protein to sugar ratio ranging from 1:2000 to 1:2666; d. protein and growth factor loaded SGnP and; e. optionally, stem cells wherein the poly(glycerol sebacate) [PGS] and poly(caprolactone) [PCL] is in the range of 7.5 - 15 wt . %; wherein the protein and growth factors is in the range of 50-100 pg; wherein the protein and growth factor loaded SGnP is in the range of 6-8 wt%; wherein the stem cells in the range of 10000 - 50000 cells loaded / transplanted with composite scaffold.

[0044] In another embodiment of the present invention discloses the protein is bovine serum albumin (BSA).

[0045] In yet another embodiment of the present invention discloses the growth factors are selected from the group comprising of differentiation factor 5 (GDF-5), transforming growth factor - pi (TGF- pi), growth and differentiation factor 6 (GDF-6) and bone morphogenic protein - 6 (BMP - 6). In other embodiment of the present invention discloses the stems cell is selected from the group comprising of human bone marrow derived mesenchymal stem cells (hBMSCs), human chondrocytes, human adipose derived stem cells.

[0046] In yet other embodiment of the present invention discloses a process for preparing the composite scaffold, wherein the process comprising the steps of: a. preparing the protein encapsulated sugar-glass nanoparticles (SGnP); b. adding the protein encapsulated sugar-glass nanoparticles (SGnP) to a solvent followed by electrospinning to obtain SGnP-BSA solution; c. preparing the PCL- PGS solution; d. adding the SGnP-BSA solution of step (b) to the PCL- PGS solution of step (c) to obtain the final composite solution; e. loading of the final composite solution as obtained in step (d) in a syringe fitted with stainless steel needle to obtain a fibrous scaffold; f. collecting, drying and storing the fibrous scaffold as obtained in step e to obtain processed scaffold.

[0047] In yet another embodiment of the present invention discloses the process for preparing the protein encapsulated sugar-glass nanoparticles, wherein the process comprising the steps of:

[0048] I. providing 50-100 pg protein and 200 mg of trehalose dissolving in 0.8 mL of deionised (DI) water to get aqueous phase;

[0049] II. providing 1.6 g of surfactant dioctyl sulfosuccinate (AOT) and dissolving in 14 mL of isooctane to obtain organic phase;

[0050] III. mixing the aqueous phase as obtained in step (I) with organic phase as obtained in step (II) to obtain protein and sugar laden inverse micelle suspension and then slowly spraying into a container containing liquid nitrogen to get flash frozen protein and sugar laden inverse micelles followed by lyophilizing for 48h to obtain lyophilized nanoparticles;

[0051] IV. washing the lyophilized nanoparticles as obtained in step (III) 4-5 times in isooctane and subsequently centrifuging at 2500 rpm for 10 min to obtain protein conjugated sugar-glass nanoparticles (BSA-SGnP, GDF5-SGnP) exhibiting protein encapsulation efficiency is in the range of 89 - 94 %. 7. A process for preparing the protein encapsulated sugar-glass nanoparticles conjugated poly(glycerol sebacate) (PGS)- poly(caprolactone) (PCL) composite fibrous scaffold comprising the steps of:

[0052] I. providing a blend of 7.5 - 15 wt. % poly(glycerol sebacate) (PGS) in form of PGS prepolymer (PGSpp) and 7.5-15 wt. % poly(caprolactone) (PCL) in 5 mL of chloroform and methanol (3: 1 ratio) solution and overnight mixing to obtain homogenous solution;

[0053] II. mixing of 6-8 wt% protein conjugated sugar-glass nanoparticles (BSA-SGnP or GDF5- SGnP) in 5 mL of Poly(glycerol sebacate) (PGS)- Poly(caprolactone) (PCL) homogeneous solution for 2 hours to obtain final solution mixture;

[0054] III. electrospinning the final solution mixture as obtained in step (II) at a feeding rate of 0.5 mL / h, 11 cm distance between the needle tip and collector with applied voltage in the range of 12.5 - 20 kV to fabricate composite fibrous scaffold;

[0055] In another embodiment of the present invention discloses the process of preparing the scaffold composition, is having an in vitro biodegradation of 27.60 ± 0.16 wt% after 28 days and is having bioavailability of 93.31 ± 0.88 % and is capable of delivering proteins in a sustained manner for a prolonged period of time in the range of 59.95% - 76.43 %.

[0056] In yet another embodiment of the present invention discloses a kit for cartilage regeneration comprising the components of:

[0057] (a) composite fibrous scaffold;

[0058] (b) stem cells;

[0059] (b) an instructions manual;

[0060] In an embodiment of the present invention, the invention provides a process as wherein, the scaffold compositions show a unique combination of properties which make them more suitable and advantageous for enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhance extra cellular matrix (ECM) protein production.

[0061] In an embodiment of the present invention, the invention provides a novel compositions of poly(glycerol sebacate) and poly(caprolactone) electrospun composite fibrous scaffold containing model protein (BSA) or therapeutic growth factor (GDF-5) loaded sugar-glass nanoparticles (GF-SGnP) reservoirs exhibit excellent protein encapsulation efficiency in the range of 89.16% - 94.83% and conjugated protein / growth factor amount in the range of 50- 100 pg resulting in actual protein / growth factor loading in the range of 952 - 3516 ng per unit of multifunctional 3D composite scaffold surface area overcoming the problem into 3D fibrous matrix at optimized parameters (protein to sugar loading concentration ~ 1:2666 to 1:4000; PGS:PCL ~ 15:7.5 (w / w); % Mass loading of SGnP-BSA or SGnP-GDF5 to polymer solution ~ 7.5 wt%, electro- spinning parameters ~ flow rate of 0.5 ml / h, voltage of 17.5 kW, distance between needle to collector plate ~ 11 cm) [presented in Table 1 & Table 2]. The invented novel composite fibrous scaffold compositions are also capable of delivering protein (BSA) or growth factor (GDF-5) in a highly sustained manner from the multifunctional biodegradable 3D electrospun fibrous composite scaffold in the range of 59.95% - 76.43 % during the period from 1-21 days for protein (BSA) towards actual release of 38-240 ng protein / day wise release and in the range of 62.28% during the period from 1-23 days for growth factor (GDF-5) towards actual release of 29.64-438.89 ng protein / day wise release ensuring good therapeutic dosing pertinent to cartilage regeneration avoiding burst release kinetics of the compositions reported in prior art.

[0062] In another embodiment of the present invention, the invented novel compositions of poly(glycerol sebacate) and poly(caprolactone) electrospun composite fibrous scaffold containing model protein (BSA) or therapeutic growth factor (GDF-5) loaded sugar-glass nanoparticles (GF-SGnP) reservoirs exhibit excellent retention of proteins / Growth factors bioactivity (2738.65 - 2988.63 ng / area), structural integrity (as per the SDS-PAGE and NATIVE PAGE) and bioavailability (92.43 % - 94.19 % / area which presented in Figure 1, Figure 2, table 1 and table 2) overcoming the problem of synthesis related and environmental stresses of the compositions reported in prior art.

[0063] In yet another embodiment of the current invention, the invented novel compositions of poly(glycerol sebacate) and poly(caprolactone) electrospun composite fibrous scaffold containing therapeutic growth factor (GDF-5) loaded sugar-glass nanoparticles (SGnP-GDF5) reservoirs (i.e., PGSpp:PCL-SGnP-GDF5 group) exhibit in vitro cartilage regeneration capabilities evidently confirmed by the enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhanced extra cellular matrix (ECM) protein production (total collagen production upto 0.73 ± 0.20 pg at 28 day), enhanced agreecan content (agreecan production upto 22.34 ± 2.07 pg at 28 day) during culturing with human bone marrow derived mesenchymal stem cells (hBMSCs) as presented in Figure 3. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention proposes four different nanofibrous scaffold made up of biodegradable poly(glycerol sebacate) (PGSpp) and poly(caprolactone) (PCL) electrospun composite fibrous scaffold containing BSA (model protein; bovine serum albumin) or GDF-5 (therapeutic growth factors; Growth & Differentiation Factor - 5) proteins loaded sugar-glass nanoparticles (GDF- SGNP) reservoirs in its matrix. a) PGSPP:PCL: SGnP-BSA50 b) PGSPP:PCL: SGnP-BSA75 c) PGSPP:PCL: SGnP-BSAlOO d) PGSPP:PCL: SGnP-GDF5

[0065] The invention is comprised of four different SGnP formulations which were synthesized by following the synthesis parameters from below Table 1.

[0066] Table 1: Synthesis parameters and encapsulation efficiency of different grades of BSA- SGnP and GDF5-SGnP The synthesised BSA / GDF5-SGnP were further loaded into the PGSpp: PCL fibrous scaffold to formulate the four different scaffold. These scaffold were fabricated using electrospinning by following the parameters in Table 2. a) PGSPP:PCL: SGnP-BSA50 b) PGSPP:PCL: SGnP-BSA75 c) PGSPP:PCL: SGnP-BSAlOO d) PGSPP:PCL: SGnP-GDF5

[0067] Table 2: Composition, synthesis parameters, fiber diameter and protein loading of different grades of BSA-SGnP and GDF5-SGnP loaded PGSPP:PCL fibrous scaffold

[0068] The schematics of how the protein encapsulated SGnP has been fabricated [claimed 4 nos. composition of BSA (model protein ~ Bovine serum albumin) or GDF-5 (therapeutic protein ~ Growth and Differentiation Factor 5) encapsulated SGnP (Sugar-Glass Nano particles)] and also claimed 4 nos. composition of BSA / GDF-5 encapsulated SGnP conjugated PGS-PCL composite electrospun fibrous patch / scaffold. Details about the batch wise composition has been provided in Table 1 and 2.

[0069] Detailed process flow chart is given below to summarize the process step by step. Electrospinning parameters and compositions of proteins encapsulated SGnP loaded PGS-PCL electrospun composite fibrous scaffold have also mentioned the Table 2.

[0070] Flowchart represents the fabrication procedure of the fibrous scaffold

[0071] According to the present invention, novel range of compositions for fabricating protein (i.e., PGSPP:PCL:SGnP-BSA50, PGSPP:PCL:SGnP-BSA75, and PGSPP:PCL:SGnP-BSA100 for BSA conjugated) and / or therapeutic growth factor (PGSpp:PCL:SGnP-GDF5 for GDF-5 conjugated) conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold comprising of 7.5-15 wt% PGS, 7.5-15 wt% of PCE, 6-8wt% of protein / growth factor loaded SGNP, and 50-100 pg of protein (BSA) / growth factor (GDF-5) loading exhibiting a unique combination of relevant properties (as presented in Table 3) like excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of protein or Growth factors at a therapeutic daily dosages, considerably higher retention of protein or Growth factors bioactivity, structural integrity and bioavailability etc. Table 3: Various Groups of multifunctional biodegradable 3D electrospun fibrous composite scaffold compositions: Nomenclature and Properties More specifically, the present invention develops a unique regenerative process by combining biomaterials science and cell therapy in which a novel composition of multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) based multifunctional electrospun fiber has been fabricated by conjugating with therapeutic growth factor (GF) loaded sugar-glass nanoparticles (SGnP) into the matrix of electrospun fibrous scaffold for regeneration of cartilage tissues and it’s processes thereof for cartilage tissue engineering. The novel multifunctional biodegradable 3D electrospun fibrous composite scaffold (i.e., PGSpp:PCL :SGnP-GDF5) belonging to this composition range 7.5-15 wt% PGS, 7.5 wt% of PCL, 7.56 wt% of growth factor loaded SGnP (SGnP-GDF5), and 75 pg of growth factor (GDF-5) loading that exhibit exhibit a unique combination of properties which make them more suitable and advantageous for enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhance extra cellular matrix (ECM) production pertinent to regeneration of cartilage tissues with the conjugation of bone marrow derived mesenchymal stem cells (MSCs) compared to the compositions reported in prior art. The in vitro cartilage regeneration properties of novel multifunctional biodegradable 3D electrospun fibrous composite scaffold (i.e., PGSpp:PCL:SGnP-GDF5) are presented in Table 4.

[0072] Table 4: In vitro cartilage regeneration properties of novel multifunctional biodegradable 3D electrospun fibrous composite scaffold [i.e., PGSpp:PCL:SGnP-GDF5 (Example 4)

[0073] The present invention relates to composite scaffold for cartilage regeneration and its process thereof. The present invention discloses a novel composition of fibrous composite scaffold comprising of 7.5-15 wt% PGS, 7.5-15 wt% of PCL, 6-8wt% of protein / growth factor loaded SGnP, and 50-100 pg of protein (BS A) / growth factor (GDF-5) and fabricated via commercially viable electrospinning technique. The novel composite exhibit unique combination of relevant properties like excellent protein encapsulation efficiency into 3D fibrous matrix, sustained delivery of proteins / Growth factors at therapeutic daily dosages, considerably higher retention of proteins / Growth factors bioactivity, structural integrity and bioavailability which make them more suitable and advantageous for enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhance extra cellular matrix (ECM) production pertinent to regeneration of cartilage tissues with the conjugation of stem cells compared to the compositions reported in prior art. The examined properties which make these 3D electrospun fibrous composite scaffold stand out are:

[0074] Protein (BSA) or growth factor (GDF-5) encapsulation efficiency into multifunctional biodegradable 3D electrospun fibrous composite scaffold matrix in the range of 89.16% - 94.83% and conjugated protein / growth factor amount in the range of 50-100 pg resulting in actual protein / growth factor loading in the range of 952 - 3516 ng per unit of multifunctional 3D composite scaffold surface area overcoming the problem of low protein / growth factors encapsulation efficiency of the compositions reported in prior art. Excellent sustained release profile of protein (BSA) or growth Factor (GDF-5 from the multifunctional biodegradable 3D electrospun fibrous composite scaffold in the range of 59.95 % - 76.43 % during the period from 1-21 days for protein (BSA) towards actual release of 38- 240 ng protein / day wise release and in the range of 62.28% during the period from 1-23 days for growth factor (GDF-5) towards actual release of 29.64-438.89 ng protein / day wise release ensuring good therapeutic dosing pertinent to cartilage regeneration avoiding burst release kinetics of the compositions reported in prior art.

[0075] Considerably higher retention of proteins / growth factor bioactivity (2738.65 - 2988.63 ng / area), structural integrity (as per the SDS-PAGE and NATIVE PAGE) and bioavailability (92.43 % - 94.19 % / area) at optimal composition (SGnP-BSA75) and electrospinning parameters of the PGSpp: PCL-SGnP-GDF5 overcoming the problem of synthesis related and environmental stresses of the compositions reported in prior art.

[0076] In vitro cartilage regeneration capabilities of optimized composition of multifunctional biodegradable 3D electrospun fibrous composite scaffold evidently confirmed by the enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhanced extra cellular matrix (ECM) protein production (total collagen production upto 0.73 ± 0.20 pg at 28 day), enhanced agreecan content (agreecan production upto 22.34 ± 2.07 pg at 28 day) during culturing with human bone marrow derived mesenchymal stem cells (hBMSCs).

[0077] Such unique combination of properties ensures an easy and economic regenerative therapy for cartilage regeneration by using a novel multifunctional biodegradable electrospun 3D fibrous scaffold with the ability to restore the biomechanical functionality of damaged cartilage tissues for treatment of osteoarthritis (OA) in articulating joints and low back pain (LBP) caused by the degeneration of intervertebral disc (IVD). A commercially viable and simple process for fabricating the off-the shelf compositions of multifunctional biodegradable 3D electrospun fibrous composite scaffold is also described. The scaffold has been fabricated by commercially viable and robust electrospinning technique resulting in protein / growth factors conjugated multifunctional biodegradable 3D electrospun fibrous composite scaffold that exhibit tremendous promises and opportunities for the treatment of the patients suffering from highly prevalent destructive joint disease joint osteoarthritis (OA) and spinal joint impairments like degenerative disc diseases (DDD). The process parameters involved in protein / growth factor encapsulation and subsequent conjugation in PGS-PCL scaffold (protein to sugar ratio, protein loading amount, solvent composition, PGS-PCL mixing ratio) and electrospinning parameters (voltage, flow rate, distance between needle and deposition plate) and in vitro cell-scaffold interactions during the culturing with human bone marrow derived mesenchymal stem cells (hBMSCs) are very crucial since the desired cartilage regeneration capabilities can only be obtained by strictly adhering to the optimized process parameters.

[0078] EXAMPLES

[0079] The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way.

[0080] Example 1

[0081] The PGSPP:PCL:SGnP-BSA50 is consisting of 15 wt% PCL, 7.5 wt% PGSPP, 7.56 wt% of BSA-SGnP and 50 pg of BSA. The BSA encapsulated SGnP is synthesized by a two-step process which starts with flash freezing and followed by lyophilization of the BSA and sugar (i.e. trehalose) laden inverse micelle. This procedure was adopted from previous literatures (Giri et al., Adv Mater. 2011 November 9; 23(42): 4861-4867, Aniruddha Pal et al 2022 Nano Futures 6 025008) and modified to check with the hypothesis. Initially, the aqueous phase is formulated by solublizing 200 mg of trehalose and 50 pg of BSA in 0.8 ml of deionized water (DI) water in a 15 ml falcon tube. Subsequently, 1.6 g of surfectant dioctyl sulfosuccinate (AOT) is dissolved in 14 ml isooctane in a 50 ml falcon tube to prepare the organic phase. Afterwards, the aqueous phase was added slowly into the predefined organic phase and continuously vortexed until a clear suspension was observed. Then, the protein and sugar laden inverse micelle suspension was flash-frozen by slowly spraying into liquid nitrogen in a 50 ml falcon tube. Further, the flash frozen nanoparticles lyophilized for 48 h and then washed 4-5 times by resuspending in isooctane and subsequently centrifuging at 2500 rpm for 10 min. Finally, the different grades of BSA loaded SGnP were stored under desiccation at -20 °C for future use. The PGSpp is prepared by polycondensation of glycerol and sebacic acid. Here, 4.60 g (0.05 mol) of glycerol was mixed with 8.357 g (0.05 mol) of sebacic acid in a 3-necked round bottom (RB) flask, attached to a Dean-Stark, fitted with condenser. The reaction was proceeded at 120-125 °C for 72 h in presence of inert atmosphere. After completion of the reaction, the yellowish, highly viscous product was collected and stored under vaccum for further use. The PGSpp:PCL:SGnP-BSA50 composite scaffold is prepared by following the upcoming procedure. Throughout the invention, chloroform: methanol is used as a solvent for electrospinning. Further, 1.5 g of PCL and 0.75 g of PGSpp (total 2250 mg of polymer solute) is added to the premixed solvent in the glass vials and it is mixed to get a homogenous solution. After that, the polymer blend solution is mixed in a 25 ml glass vial to get a good blending and it is continued to stir overnight in a magnetic stirrer. In the next step, SGnP-BSA50 (powder form weighing around 170 mg of weight) is added to another 5 mL of 3: 1 ratio of chloroform: methanol solution) and stirred for few minutes. And finally this 5 mL of SGnP-BSA50 solution was added to earlier mixed PCL-PGSpp solution and continued to stir for 2 more hours to get the final mixture. And then desired volume of this final composite solution is loaded in a 5 ml syringe fitted with a stainless-steel needle (21G). At the time of electrospinning process, the feeding rate of 0.5 mL / h and the distance between the needle tip and collector covered by aluminum foil of 11 cm and the applied voltage is 17.5 kV is maintained to produce the fibrous scaffold. After completion of electrospinning process, each of the fibrous mat was collected from the collector, dried in a vaccum oven at 40 °C and stored in a desiccator. After drying, the fibrous scaffold is peeled off carefully from the aluminium foil. The processed scaffold exhibits a protein loading of 1489.334 ± 538.920 ng / area, cumulative release profile of 76.43 ± 1.28 % at 21 days towards the actual release of 38.81 ± 21.96 till 248.46 ± 61.14 ng / day.

[0082] Example 2

[0083] The PGSPP: PCL:SGnP-BSA75 is consisting of 15 wt% PCL, 7.5 wt% PGSPP, 7.56 wt% of BSA-SGnP and 75 pg of BSA. The BSA encapsulated SGnP is synthesized by a two-step process which starts with flash freezing and followed by lyophilization of the BSA and sugar (i.e. trehalose) laden inverse micelle. Initially, the aqueous phase is formulated by solublizing 200 mg of trehalose and 75 pg of BSA in 0.8 ml of deionized water (DI) water in a 15 ml falcon tube. Subsequently, 1.6 g of surfactant dioctyl sulfosuccinate (AOT) is dissolved in 14 ml isooctane in a 50 ml falcon tube to prepare the organic phase. Afterwards, the aqueous phase was added slowly into the predefined organic phase and continuously vortexed until a clear suspension was observed. Then, the protein and sugar laden inverse micelle suspension was flash-frozen by slowly spraying into liquid nitrogen in a 50 ml falcon tube. Further, the flash frozen nanoparticles lyophilized for 48 h and then washed 4-5 times by re-suspending in isooctane and subsequently centrifuging at 2500 rpm for 10 min. Finally, the different grades of BSA loaded SGnP were stored under desiccation at -20 °C for future use. The PGSpp is prepared by polycondensation of glycerol and sebacic acid. Here, 4.60 g (0.05 mol) of glycerol was mixed with 8.357 g (0.05 mol) of sebacic acid in a 3-necked round bottom (RB) flask, attached to a Dean-Stark, fitted with condenser. The reaction was proceeded at 120-125 °C for 72 h in presence of inert atmosphere. After completion of the reaction, the yellowish, highly viscous product was collected and stored under vaccum for further use. The PGSpp: PCL:SGnP- BSA75 composite scaffold is prepared by following the upcoming procedure. Throughout the invention, chloroform: methanol is used as a solvent for electrospinning. In individual 10 ml glass vial, 5 ml of 3: 1 ratio of chloroform: methanol was added. Further, 1.5 g of PCL and 0.75 g of PGSpp (total 2250 mg of polymer solute) is added to the premixed solvent in the glass vials and it is mixed to get a homogenous solution. After that, the polymer blend solution is mixed in a 25 ml glass vial to get a good blending and it is continued to stir overnight in a magnetic stirrer. In the next step, SGnP-BSA75 (powder form weighing around 170 mg of weight) is added to another 5 mL of 3: 1 ratio of chloroform: methanol solution) and stirred for few minutes. And finally this 5 mL of SGnP-BSA75 solution was added to earlier mixed PCL- PGSpp solution and continued to stir for 2 more hours to get the final mixture. And then desired volume of this final composite solution is loaded in a 5 ml syringe fitted with a stainless-steel needle (21G). At the time of electrospinning process, the feeding rate of 0.5 mL / h and the distance between the needle tip and collector covered by aluminum foil of 11 cm and the applied voltage is 17.5 kV is maintained to produce the fibrous scaffold. After completion of electrospinning process, each of the fibrous mat was collected from the collector, dried in a vaccum oven at 40 °C and stored in a desiccator. After drying, the fibrous scaffold is peeled off carefully from the aluminium foil. The processed scaffold exhibits a protein loading of 1706.092 ± 282.99 ng / area, cumulative release profile of 68.74 ± 1.092 % at 21 days towards the actual release of 34.71 ± 4.65 till 171.73 ± 58.23 ng / day.

[0084] Example 3

[0085] The PGSPP:PCL:SGnP-BSA100 is consisting of 15 wt% PCL, 7.5 wt% PGSPP, 7.56 wt% of BSA-SGnP and 100 pg of BSA. The BSA encapsulated SGnP is synthesized by a two-step process which starts with flash freezing and followed by lyophilization of the BSA and sugar (i.e. trehalose) laden inverse micelle. Initially, the aqueous phase is formulated by solublizing 200 mg of trehalose and 100 pg of BSA in 0.8 ml of deionized water (DI) water in a 15 ml falcon tube. Subsequently, 1.6 g of surfectant dioctyl sulfosuccinate (AOT) is dissolved in 14 ml isooctane in a 50 ml falcon tube to prepare the organic phase. Afterwards, the aqueous phase was added slowly into the predefined organic phase and continuously vortexed until a clear suspension was observed. Then, the protein and sugar laden inverse micelle suspension was flash-frozen by slowly spraying into liquid nitrogen in a 50 ml falcon tube. Further, the flash frozen nanoparticles lyophilized for 48 h and then washed 4-5 times by re-suspending in isooctane and subsequently centrifuging at 2500 rpm for 10 min. Finally, the different grades of BSA loaded SGnP were stored under desiccation at -20 °C for future use. The PGSpp is prepared by polycondensation of glycerol and sebacic acid. Here, 4.60 g (0.05 mol) of glycerol was mixed with 8.357 g (0.05 mol) of sebacic acid in a 3-necked round bottom (RB) flask, attached to a Dean-Stark, fitted with condenser. The reaction was proceeded at 120-125 °C for 72 h in presence of inert atmosphere. After completion of the reaction, the yellowish, highly viscous product was collected and stored under vaccum for further use. The PGSpp: PCL:SGnP- BSA100 composite scaffold is prepared by following the upcoming procedure. Throughout the invention, chloroform: methanol is used as a solvent for electrospinning. In individual 10 ml glass vial, 5 ml of 3: 1 ratio of chloroform: methanol was added. In individual 10 ml glass vial, 5 ml of 3: 1 ratio of chloroform: methanol was added. Further, 1.5 g of PCL and 0.75 g of PGSpp (total 2250 mg of polymer solute) is added to the premixed solvent in the glass vials and it is mixed to get a homogenous solution. After that, the polymer blend solution is mixed in a 25 ml glass vial to get a good blending and it is continued to stir overnight in a magnetic stirrer. In the next step, SGnP-BSAlOO (powder form weighing around 170 mg of weight) is added to another 5 mL of 3: 1 ratio of chloroform: methanol solution) and stirred for few minutes. And finally this 5 mL of SGnP-BSAlOO solution was added to earlier mixed PCL-PGSpp solution and continued to stir for 2 more hours to get the final mixture. And then desired volume of this final composite solution is loaded in a 5 ml syringe fitted with a stainless-steel needle (21G). At the time of electrospinning process, the feeding rate of 0.5 mL / h and the distance between the needle tip and collector covered by aluminum foil of 11 cm and the applied voltage is 17.5 kV is maintained to produce the fibrous scaffold. After completion of electrospinning process, each of the fibrous mat was collected from the collector, dried in a vaccum oven at 40 °C and stored in a desiccator. After drying, the fibrous scaffold is peeled off carefully from the aluminium foil. The processed scaffold exhibits a protein loading of 3317.46 ± 199.85 ng / area, cumulative release profile of 59.95 ± 3.15 % at 21 days towards the actual release of 50.46 ± 9.5 Itill 372.70 ± 39.59 ng / day.

[0086] Example 4

[0087] The PGSpP:PCL:SGnP-GDF5 is consisting of 15 wt% PCL, 7.5 wt% PGSPP, 7.56 wt% of GDF5-SGnP and 75 pg of GDF5. The BSA encapsulated SGnP is synthesized by a two-step process which starts with flash freezing and followed by lyophilization of the BSA and sugar (i.e. trehalose) laden inverse micelle. Initially, the aqueous phase is formulated by solublizing 200 mg of trehalose and 75 pg of GDF5 in 0.8 ml of deionized water (DI) water in a 15 ml falcon tube. Subsequently, 1.6 g of surfactant dioctyl sulfosuccinate (AOT) is dissolved in 14 ml isooctane in a 50 ml falcon tube to prepare the organic phase. Afterwards, the aqueous phase was added slowly into the predefined organic phase and continuously vortexed until a clear suspension was observed. Then, the protein and sugar laden inverse micelle suspension was flash-frozen by slowly spraying into liquid nitrogen in a 50 ml falcon tube. Further, the flash frozen nanoparticles lyophilized for 48 h and then washed 4-5 times by re-suspending in isooctane and subsequently centrifuging at 2500 rpm for 10 min. Finally, the different grades of GDF5 loaded SGnP were stored under desiccation at -20 °C for future use. The PGSpp is prepared by polycondensation of glycerol and sebacic acid. Here, 4.60 g (0.05 mol) of glycerol was mixed with 8.357 g (0.05 mol) of sebacic acid in a 3-necked round bottom (RB) flask, attached to a Dean-Stark, fitted with condenser. The reaction was proceeded at 120-125 °C for 72 h in presence of inert atmosphere. After completion of the reaction, the yellowish, highly viscous product was collected and stored under vaccum for further use. The PGSpp:PCL:SGnP- GDF5 composite scaffold is prepared by following the upcoming procedure. Through out the invention, chloroform: methanol is used as a solvent for electrospinning. In individual 10 ml glass vial, 5 ml of 3: 1 ratio of chloroform: methanol was added. Further, 1.5 g of PCL and 0.75 g of PGSpp (total 2250 mg of polymer solute) is added to the premixed solvent in the glass vials and it is mixed to get a homogenous solution. After that, the polymer blend solution is mixed in a 25 ml glass vial to get a good blending and it is continued to stir overnight in a magnetic stirrer. In the next step, SGnP-GDF5 (powder form weighing around 170 mg of weight) is added to another 5 mL of 3: 1 ratio of chloroform: methanol solution) and stirred for few minutes. And finally this 5 mL of SGnP-GDF5 solution was added to earlier mixed PCL-PGSpp solution and continued to stir for 2 more hours to get the final mixture. And then desired volume of this final composite solution is loaded in a 5 ml syringe fitted with a stainless-steel needle (21G). At the time of electrospinning process, the feeding rate of 0.5 mL / h and the distance between the needle tip and collector covered by aluminum foil of 11 cm and the applied voltage is 17.5 kV is maintained to produce the fibrous scaffold. After completion of electrospinning process, each of the fibrous mat was collected from the collector, dried in a vaccum oven at 40 °C and stored in a desiccator. After drying, the fibrous scaffold is peeled off carefully from the aluminium foil. The processed scaffold exhibits a protein loading of 3068.79 ± 110.841 ng / area, cumulative release profile of 62.33 ± 0.67 % at 23 days towards the actual release of 29.64 ± 10.95 till 438.89 ± 41.40 ng / day. Also 2863.63 ± 125 ng / area of GDF5 is bioactive which is 93.31 ± 0.88 % of the total protein in that area so it is structurally confined in its native form. Table 5: Procurement details of biological materials and assay kits used in this invention

[0088] ADVANTAGES OF THE INVENTION

[0089] The main advantages of the present invention are:

[0090] 1. The proposed biodegradable 3D electrospun fibrous composite scaffold will be useful in regeneration of cartilage tissues and it’s processes thereof for cartilage tissue engineering.

[0091] 2. The described process is a commercially viable, simple and economic regenerative therapy that can be utilized for cartilage regeneration to restore the biomechanical functionality of damaged cartilage tissues for treatment of osteoarthritis (OA) in articulating joints and low back pain (LBP) caused by the degeneration of intervertebral disc (IVD) via degenerative disc diseases (DDD).

[0092] 3. The novel compositions of multifunctional biodegradable poly(glycerol sebacate) and poly(caprolactone) electrospun composite fibrous scaffold compositions containing BSA (model protein; bovine serum albumin) or GDF-5 (therapeutic growth factors; Growth & Differentiation Factor - 5) proteins loaded sugar-glass nanoparticles (GDF- SGnP) reservoirs in it’s matrix offer excellent protein encapsulation efficiency into 3D fibrous matrix and also capable of delivering proteins in a sustained manner for a prolonged period of time and thus eliminate or minimize the problems described in the background and prior art. The invented novel compositions of composite fibrous scaffold has the unique advantage of protecting the encapsulated proteins structural and functional properties against the process (i.e., synthesis) and environment related stresses (i.e., physiological agents like proteolytic enzymes via sugar-glass nanoreservoir based encapsulation platform and thus exhibit excellent retention of proteins / Growth factors bioactivity, structural integrity and conformation, and associated bioavailability overcoming the problem of the compositions reported in prior art. This invention discloses the tunable biodegradability nature of fabricated multifunctional electrospun composite fibrous scaffold that slowly desorbs via body metabolism and hydrolytic degradation in physiological environment and thus will be useful in delivering proteins in a sustained manner for a prolonged period of time at therapeutic dosage. The invented novel compositions of composite fibrous scaffold exhibit excellent in vitro cartilage regeneration capabilities as confirmed by the enhanced cartilage specific ECM proteoglycan deposition and paracrine signaling of MSCs, enhanced extra cellular matrix (ECM) protein production (collagen), enhanced formation of cartilagelike ECM substances (sGAG)pertinent to regeneration of cartilage tissues during culturing with human bone marrow derived mesenchymal stem cells (hBMSCs).

Claims

We claim:

1. A composite scaffold for cartilage regeneration comprising of f. poly(glycerol sebacate) [PGS]; g. poly(caprolactone) [PCL]; h. protein and growth factors with protein to sugar ratio ranging from 1:2000 to 1:2666; i. protein and growth factor loaded SGnP and; j. optionally, stem cells wherein the poly(glycerol sebacate) [PGS] and poly(caprolactone) [PCL] is in the range of 7.5 - 15 wt . %; wherein the protein and growth factors is in the range of 50-100 pg; wherein the protein and growth factor loaded SGnP is in the range of 6-8 wt%; wherein the stem cells in the range of 10000 - 50000 cells loaded / transplanted with composite scaffold.

2. The composite scaffold as claimed in claim 1, wherein the protein is bovine serum albumin (BSA).

3. The composite scaffold as claimed in claim 1, wherein the growth factors are selected from the group comprising of differentiation factor 5 (GDF-5), transforming growth factor - pi (TGF- pi), growth and differentiation factor 6 (GDF-6) and bone morphogenic protein - 6 (BMP - 6).

4. The composite scaffold as claimed in claim 1, wherein the stems cell is selected from the group comprising of human bone marrow derived mesenchymal stem cells (hBMSCs), human chondrocytes, human adipose derived stem cells.

5. A process for preparing the composite scaffold as claimed in claim 1, wherein the process comprising the steps of: a. preparing the protein encapsulated sugar-glass nanoparticles (SGnP);b. adding the protein encapsulated sugar-glass nanoparticles (SGnP) to a solvent followed by electrospinning to obtain SGnP-BSA solution; c. preparing the PCL- PGS solution; d. adding the SGnP-BSA solution of step (b) to the PCL- PGS solution of step (c) to obtain the final composite solution; e. loading of the final composite solution as obtained in step (d) in a syringe fitted with stainless steel needle to obtain a fibrous scaffold; f. collecting, drying and storing the fibrous scaffold as obtained in step e to obtain processed scaffold.

6. The process for preparing the protein encapsulated sugar-glass nanoparticles as claimed in claim 5, wherein the process comprising the steps of:I. providing 50-100 pg protein and 200 mg of trehalose dissolving in 0.8 mL of deionised (DI) water to get aqueous phase;II. providing 1.6 g of surfactant dioctyl sulfosuccinate (AOT) and dissolving in 14 mL of isooctane to obtain organic phase;III. mixing the aqueous phase as obtained in step (I) with organic phase as obtained in step (II) to obtain protein and sugar laden inverse micelle suspension and then slowly spraying into a container containing liquid nitrogen to get flash frozen protein and sugar laden inverse micelles followed by lyophilizing for 48h to obtain lyophilized nanoparticles;IV. washing the lyophilized nanoparticles as obtained in step (III) 4-5 times in isooctane and subsequently centrifuging at 2500 rpm for 10 min to obtain protein conjugated sugar-glass nanoparticles (BSA-SGnP, GDF5-SGnP) exhibiting protein encapsulation efficiency is in the range of 89 - 94 %.

7. A process for preparing the protein encapsulated sugar-glass nanoparticles conjugated poly(glycerol sebacate) (PGS)- poly(caprolactone) (PCL) composite fibrous scaffold comprising the steps of:I. providing a blend of 7.5 - 15 wt. % poly(glycerol sebacate) (PGS) in form of PGS prepolymer (PGSpp) and 7.5-15 wt. % poly(caprolactone) (PCL) in 5 mL of chloroform and methanol (3: 1 ratio) solution and overnight mixing to obtain homogenous solution;II. mixing of 6-8 wt% protein conjugated sugar-glass nanoparticles (BSA-SGnP or GDF5- SGnP) in 5 mL of Poly(glycerol sebacate) (PGS)- Poly(caprolactone) (PCL) homogeneous solution for 2 hours to obtain final solution mixture;III. electrospinning the final solution mixture as obtained in step (II) at a feeding rate of 0.5 mL / h, 11 cm distance between the needle tip and collector with applied voltage in the range of 12.5 - 20 kV to fabricate composite fibrous scaffold;8. The process of preparing the scaffold composition as claimed in claim 5, is having an in vitro biodegradation of 27.60 ± 0.16 wt% after 28 days and is having bioavailability of 93.31 ± 0.88 % and is capable of delivering proteins in a sustained manner for a prolonged period of time in the range of 59.95% - 76.43 %.

9. A kit for cartilage regeneration comprising the components of:(a) composite fibrous scaffold as claimed in claim 1;(b) stem cells of claim 1 ;(b) an instructions manual;

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