FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
TITLE OF THE INVENTION
NANOCOMPOSITE BONE REGENERATIVE MATERIAL COMPRISING METAL SILICATE
APPLICANT
INDIAN INSTITUTE OF TECHNOLOGY, BOMBAY POWAI, MUMBAI400076 MAHARASHTRA, INDIA
INVENTORS
Prof. Jayesh Bellare of Department of Chemical Engineering,
Prof. Vivek P Soni and Amit Kumar Jaiswal of Department of Biosciences and
Bioengineering, Indian Institute of Technology, Bombay, Powai,
Mumbai-400 076, Maharashtra, India; all Indian
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to a nanocomposite scaffold useful for bone regeneration comprising a polymer component and a ceramic component, and a process for its preparation. In particular, the present invention relates to a nanocomposite scaffold wherein the ceramic component is a metal silicate. The said scaffold displays enhanced cell proliferation, osteogenic, in-vitro cellular activity and bioactivity.
BACKGROUND OF THE INVENTION
Scaffold mediated bone regeneration offers an exciting approach to cure bone defects occurred due to trauma, rumor, disease and biochemical disorders. Synthetic bone grafts have gained popularity in bone defect treatment due to the limited availability of auto graft tissue and also the associated adverse immune reactions that can occur with allograft approach. The ideal bone graft should possess adequate mechanical properties, porosity, biocompatibility, degradability and be osteoconductive [1, 2]. Unfortunately, available plans to treat bone defects could not meet all the desired requirements and still there is a need to develop a suitable synthetic bone graft for bone tissue engineering that meets all of the above success criteria.
Important considerations for scaffold preparation are material selection and fabrication technology. There are several methods for scaffold fabrication such as phase separation, solvent casting, particulate leaching and fiber bonding. Electro spinning has also emerged as an efficient technique to form nanoflbrous scaffolds which closely mimic the nanometer scale feature of the extracellular matrix. The large surface area to volume ratio, high porosity and nano-sized features of electro spun scaffolds provided better cell-biomaterial interaction as compared to macroporous scaffolds.
Hydroxyapatite (HA) is the most investigated ceramic material for creating bone tissue scaffolds as it is the major inorganic component of natural bone. Several polymers with HA have been studied for bone tissue engineering including poly-L-lactic acid/HA (PLLA/HA), poly-L-glycolic acid/HA (PLGA/HA), poly (3-hyroxybutyrate)/nano-hydroxyapatite (PHB/nHA), cellulose/HA, and polycaprolactone (PCL)/HA.

Considerable research has been reported over the last decade over the use of polymeric and ceramic biomaterials for producing scaffolds, WO2009021209 discloses biomedical scaffolds that may be used for treatment of bone diseases and bone repair, US20100272826 discloses a biocompatible inorganic porous material having a three-dimensional coexistent network of interconnected macro-pores and nanopores which may be suitable for use as a bone tissue scaffold, US20110144765 discloses a process for producing a porous glass construct with interconnected porosity, WO2008118943 discloses an artificial bone composite structure which includes a fibrous matrix that itself includes a plurality of fibers and a plurality of hydroxyapatite (HA) particles which are. dispersed within the fibrous matrix.
Various scaffold architectures and manufacturing processes have also been disclosed in journal publications such as M.V. Jose, V. Thomas, K.T. Johnson, D,R. Dean, E. Nyairo, Aligned PLGA/HA nanofibrous nanocomposite scaffolds for bone tissue engineering, Acta Biomater. 5 (2009) 305-315, J.R; Venugopal, S Low, A.T. Choon, A.B. Kumar, S. Ramakrishna, Nanobioengineered electrospun composite nanofibers and osteoblasts for bone regeneration, Artif.Organs 32 (2008) 388-397; K. Fujihara, M. Kotaki, S. Ramakrishna, Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers, Biomaterials 26 (2005) 41394147; M.P. Prabhakaran, J. Venugopal, S. Ramakrishna, Electrospun nanostructured scaffolds for bone tissue engineering, Acta Biomater. 5 (2009) 2884-2893 and others.
Most of the journals and patent documents disclose the use of polymers such as polycaprolactone (PCL) and other polymers blended with hydroxyapatite (HA) and natural polymer gelatin. The architecture of the scaffold prepared is also different. However, an ideal material and fabrication technique for optimal bone tissue regeneration is yet to be identified.
Accordingly what is needed is an improved bone graft material of sound mechanical

integrity, flexibility, and handling properties; exceptional biocompatibility and osteoconductive properties; and superior resorption properties that overcomes the problems associated with the currently available bone graft materials. The present invention fulfills this need. Superior physiochemical and biological response of the scaffolds of the present invention make them a potential applicant for bone tissue engineering.
The present inventors have have fabricated nanofibrous composite scaffolds combining PCL and pure hardystonite and compared the same with PCL-hydroxyapatite scaffolds of similar architecture. The present inventors have used process of electrospinning for the fabrication of the same.
Other features and advantages will be apparent from the following more detailed description of some Example embodiments.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a nanocomposite scaffold comprising a polymer component and a ceramic component, characterized in that the ceramic component comprises a metal silicate.
In another aspect, the present invention provides a process for the preparation of a nanocomposite scaffold, comprising:
a) synthesizing the ceramic component by sol-gel process;
b) dispersing the ceramic component obtained from step (a) and the polymer component individually in a solvent;
c) blending said ceramic component with said polymer component to form a solution; and
d) electroprocessing said solution to form said scaffold thereby depositing a three-dimensional matrix of nano sized elecrroprocessed fibers comprising the ceramic-polymer composite on a collector.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the morphology of electrospun (a) PCL, (b) PCL-HA20, (c) PCL-HA40, (d) PCL-HS20 and (e) PCL-HS40 nanofibers (Scale bar = 20 μm).
Figure 2 shows transmission Electron Microscopy (TEM) images of electrospun nanofiber of (a) PCL, (b) PCL-HA20, (c) PCL-HA40, (d) PCL-HS20 and (e) PCL-HS40 showing HA and HS particle on sub-surface of nanofibers (Scale bar=200 nm).
Figure 3(a) shows X-ray diffraction (XRD) partem of nanofibrous scaffolds showing HA and HS scaffold peaks and figure 3(b) shows FTTR spectra of PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 nanofibers.
Figure 4(a) shows in-vitro degradation of electrospun PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds. In-vitro degradation is represented as a function of mass loss over time and figure 4(b) shows stress-strain curve of PCL, PC-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 nanofibers.
Figure 5(a) is bar graph showing percentage hemolysis for PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds (no significant difference observed between groups)
Figure 5(b) shows an amount of protein adsorbed on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds and figure 5(c) shows proliferation of murine adipose-tissue derived stem cells (mE-ASCs) on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 after 1,4 and 7 days of culture.
Figure 6 shows scanning electron microscopy (SEM) micrograph of mE-ASCs attached on PCL(a & f), PCL-HA20(b & g), PCL-HA40(c & h) and PCL-HS20(d & i) and PCL-HS40(e & j) scaffolds after 1 and 7 days of culture respectively.
Figure 7 shows confocal microscope z-stack images which shows deeper cell migration into electrospun nanofibrous scaffolds after 7 days of culture on PCL (a), PCL-HA20 (b), PCL-HA40

(c) and PCL-HS20 (d) and PCL-HS40 (e) nanofibers. Series of optical sections through the entire cell-scaffold construct was acquired at 1.5 urn intervals in the axial (z) dimension. Nucleus (blue) were stained with 4', 6-diamidino-2-phenylindole (DAPI) are shown.
Figure 8(a) shows ALP activity of mE-ASCs on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCLHS40 scaffolds after 1,4 and 7 days of culture and figure 8(b) shows quantitative analysis of mineralization by measuring the amount of ahzarin on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds after 14 and 21 days of culture.
Figure 9(a) shows TEM image, figure 9(b) shows electron diffraction, figure 9(c) shows EDAX spectra and figure 9(d) shows XRD of HS powders.
Figure 10 shows water contact angles of PCL, PC-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 nanofibers.
Figure 11 shows thermogram of PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds and HS showing the percentage mass change with temperature.
DETAILED DESCRIPTION OF THE INVENTION
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual components, substituents, and ranges are for illustration only; they do not exclude other defined values or other values within defined ranges for the components and substituents.

As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
The terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
As used herein, the terms "comprising," "including," "having," "containing," "involving," and the like are to be understood to be open-ended, i.e. to mean including but not limited to.
Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or process parameters that may of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner.
As used herein, the terms "scaffold" or "matrix" can be used interchangeably and they relate to a semi-solid structure comprising a three-dimensional network of one or more species of polysaccharide chains. Depending on the properties of the polysaccharide (or mixtures of polysaccharides) used, as well as the nature and density of the network, such structures in equilibrium can comprise various amounts of water. The scaffolds of the present invention may be cut and shaped to take a desired size and form. The term scaffold also refers to a structure, comprising a biocompatible material that provides a surface suitable for adherence, proliferation, and differentiation of cells.
As used herein, the terms "bioactive" and "bioactivity" can be used interchangeably and they refer to any effect on, interaction with, or response from living tissue.

As used herein, the term "biocompatible" refers to any material which when introduced into an individual, does not provoke an adverse immune response. A biocompatible material is capable of implantation in biological systems, for example, tissue implantation, without causing excessive fibrosis or rejection reactions.
As used herein, the term "biodegradable" refers to the ability of a substance or material to break down into harmless substances by the action of living organisms.
As used herein, the term "osteoinductive" refers to a material having the ability to serve as a scaffold on which bone cells may attach, migrate and form new bone.
As used herein, the term "osteoinductive" refers to a material having the ability to induce bone to grow.
As used herein, the term "polymer" refers to a macromolecule formed by the chemical union of five or more identical combining units called monomers. In most cases, the number of the monomer is quite large and often is not precisely known. In synthetic polymers, this number may be controlled to a predetermined extent. Combinations two, three, or four monomers are called, respectively, dimmers, trimers, and tetramers, and are known collectively as oligomers. Polymers may be inorganic or organic, biodegradable and/or non-biodegradable. Organic polymers may be natural, synthetic, thermosetting and semi-synthetic.
As used herein, the terms "polycaprolactone", "6-Caprolactone polymer" and the abbreviation "PCL" can be used interchangeably and refers to a biodegradable polyester having the molecular formula (CsHioCh)*, and having a molecular weight of 80,000 daltons. PCL may be obtained commercially, for example, from Sigma - Aldrich.
As used herein, the term "ceramic" or "bioceramic" refers to materials having a crystalline or at least partially crystalline structure formed essentially from inorganic and non-metallic compounds and is intended to include all ceramics which may be formed from metal silicates.

The term "metal silicate" is to be interpreted broadly to include any compounds having at least one metal cation forming a bond with a silicate anion. The term "silicate" is to be interpreted broadly to include any anion in which one or more central silicon atoms are surrounded by electronegative ligands such as oxygen.
As used herein, the term "hardystonite" and the abbreviation "HS" refers to calcium zinc silicate (Ca2ZnSi2O7) with a melting temperature of 1425°C and a density of 3.4 g/cm3.
As used herein, the term "hydroxyapatite" and the abbreviation "HA" refers to hydroxylated calcium phosphate [Ca10(PO4)6(OH)2], that is the major constituent of bone and tooth mineral.
As used herein, the term "stem cells" refers to undifferentiated cells having high proliferative potential with the ability to self-renew that may migrate to areas of injury and may generate daughter cells that may undergo terminal differentiation into more than one distinct cell phenotype. These cells have the ability to differentiate into various cell types and thus promote the regeneration or repair of a diseased or damaged tissue of interest. The term "cellular differentiation" refers to the process by which cells acquire a cell type.
As used herein, the term "proliferation" refers to the reproduction or multiplication of similar forms, especially of cells. That is, proliferation encompasses production of a greater number of cells.
As used herein, the term "osteoblast" or "osteoblasts" refers to cells that arise when osteoprogenitor cells or mesenchymal cells, which are located near all bony surfaces and within the bone marrow, differentiate under the influence of growth factors. Osteoblasts, which are responsible for bone matrix synthesis, secrete a collagen rich ground substance essential for mineralization of bone. Once osteoblasts become trapped in the matrix they secrete, they become osteocytes. From least to terminally differentiated, the osteocyte lineage is (i) Colony-forming unit fibroblast (CFU-F); (ii) mesenchymal stem cell I

marrow stromal cell (MSC); (iii) osteoblast; (iv) osteocyte.
The term "osteogenesis" refers to the formation of new bone from bone forming or osteocompetent cells.
As used herein, the term "nanofibers" refers to fibers whose diameter ranges from about 1 nanometer (10-9 m) to about 1000 nanometers.
As used herein, the term "osteogenic" refers to a material that induces the development of some or all of the characteristics of an osteoblast or osteocyte in a stem cell, adipose- . derived stem cell or other such progenitor cell that is not fully differentiated.
As used herein, the term "seeding" refers to plating, placing and/or dropping the cells on the scaffold of the present invention. It will be appreciated that the concentration of cells which are seeded on the scaffold depends on the type of cells used and the composition of the scaffold.
As used herein, the term "nanocomposite" refers to a composite material preferably at a nanometer level. The term "composite" refers to a complex material, in which two or more distinct substances combine to produce structural or functional properties not present in any individual component. The composite of the present invention is prepared by blending a ceramic component with a polymer component.
As used herein, the term "electroprocessing" shall be defined broadly to include all methods combinations of two or more such methods, and any other method wherein materials are streamed, sprayed, dripped or sputtered across an electric field and toward a target. The electroprocessed material can be electroprocessed from one or more grounded reservoirs in the direction of a charged substrate or from charged reservoirs towards a grounded target. In preferred embodiments, the term electroprocessing includes electrospinning. "Electrospinning" or "electrospun" means a process in which fibers are formed from a solution or melt by streaming an electrically charged solution or melt through an orifice.

As used herein, the term "solution" refers to the liquid in the reservoirs of the electroprocessing method. The term is defined broadly to include any liquids that contain materials to be electroprocessed. It is to be understood that any solutions capable of forming a material during electroprocessing are included within the scope of the present invention. "Solutions" can be in organic or biologically compatible forms. This broad definition is appropriate in view of the large number of solvents or other liquids (polar and non-polar) and carrier molecules that can be used in the many variations of electroprocessing.
As used herein, the term "subject" refers to mammals, including humans. Preferably, this term encompasses individuals who suffer from pathologies as described hereinabove.
The present invention provides a nanocomposite scaffold comprising a polymer component and a ceramic component, characterized in that the ceramic component comprises a metal silicate.
In an embodiment of the present invention, the said metal silicate is selected from the group consisting of calcium silicate, calcium zinc silicate (hardystonite), calcium magnesium silicate, zinc silicate, magnesium silicate, magnesium zinc silicate, strontium silicate, strontium calcium zinc silicate (strontium-hardystonite), strontium calcium silicate, zirconium calcium silicate, calcium zirconium silicate (Ca3ZrSi2O9), magnesium calcium zinc silicate (magnesium-hardystonite), barium calcium zinc silicate (barium-hardystonite) and combinations thereof.
In an embodiment of the present invention, the said polymer component is selected from the group consisting of polyesters such as polycaprolactone or derivatives thereof. Other than polycaprolactone, poly(14actic acid), polyglycolic acid and polylactic acid/polyglycolic acid copolymers can also be used to prepare polymer component of the composite scaffold of the present invention.
In a preferred embodiment, the polymer component comprises polyesters such as

polycaprolactone or derivatives thereof and the ceramic component comprises a metal silicate such as calcium zinc silicate (hardystonite) and strontium calcium zinc silicate (strontium-hardystonite).
In an embodiment of the present invention, the nanocomposite scaffold comprises of from 70% to 85% by weight of the polymer component and at least 15% to 30% by weight of the ceramic component. More preferably, it comprises of at least 84% to 72% by weight of the polymer component and 16% to 29% by weight of the ceramic component
The present invention also relates to the process for the preparation of a nanocomposite scaffold, comprising:
a) Synthesizing the ceramic component by sol-gel process;
b) dispersing the ceramic component obtained from step (a) and the polymer component individually in a solvent;
c) blending said ceramic component with said polymer component to form a solution; and
d) electroprocessing said solution to form said scaffold thereby depositing a three-dimensional matrix of nano sized electroprocessed fibers comprising the ceramic-polymer composite on a collector,
The sol-gel method offers specific advantages in preparation of the ceramic component. The early formation of a gel provides a high degree of homogeneity.
Solvents useful for dissolving, dispersing or suspending a material or a substance depend on the material or substance to be electroprocessed. In a preferred embodiment, for example, 1,1,1,3,3, 3-hexafluoro-2-propanol (HFP) is used as a solvent. Other non limiting examples of the solvent used for electroprocessing include trifluoroethanol (TFE), chloroform, dichloromethane (DCM), and mixture of formic acid/acetic acid.
In preferred embodiments, the nanocomposite scaffold of the present invention is prepared by electrospinning. Electrospinning is a process that uses an electric field to draw a solution comprising, for example, a polymer component and a ceramic component from the tip of the capillary to a collector. A high voltage DC current is applied to the solution which causes a jet of

the solution to be drawn towards the grounded collector screen. Once ejected out of the capillary orifice, the charged solution jet gets evaporated to form fibers and the fibers get collected on the collector. Electrostatic spinning is a process by which fibers of nanometer to micrometer size in diameters and lengths up to several kilometers can be produced using an electrostatically driven jet of solution or melt.
In an embodiment, the scaffolds of the present invention can be prepared by uniaxial and multiaxial electrospining. However, while preparing the same scaffolds with multiaxial electrospinning, the process parameters need to be optimized. The electroprocessing step may be affected by varying the electric potential, flow rate, solution concentration, surface tension, and viscoelasticity of the solution, dielectric properties of the solution, molecular weight, nature of the polymer and ceramic, capillary-collector distance, diameter of the needle, other parameters regarding the electroprocessing apparatus and ambient parameters like temperature. Therefore, it is possible to manipulate the porosity, surface area, fineness and uniformity, diameter of fibers, and the pattern thickness of the scaffold.
In an embodiment of the present invention, the nanocomposite scaffold comprises randomly oriented, uniform, non-woven fibers with interconnected pores. The fiber diameter range of the said scaffold is between 200nm - 650nm. Furthermore, the scaffold may comprise any shape including, without limitation, a round shape or a cube shape. However, aligned fibers of the same composition can also be prepared by varying the setup of electrospinning.
In an embodiment of the present invention, the nanocomposite scaffold has improved mechanical properties. The improved mechanical properties are obtained by blending the said ceramic component with the said polymer component. The mechanical properties include improved tensile strength, improved toughness, improved stiffness or any combinations thereof.
The stress/force needed to break the sample by increasing the amount of force, and stress on the sample is the "tensile strength" of the material. Stress and strength are measured in units of force divided by units of area, usually N/m2 also called Pascal. Stress and strength can also be measured in megapascals (MPa) or gigapascals (GPa). In an

embodiment of the present invention, the tensile strength of said scaffold is between 7.50 MPa to l0.60MPa.
In an embodiment of the present invention, the nanocomposite scaffold may be implantable.
In a preferred embodiment of the present invention, the nanocomposite scaffold may be osteogenic, osteoinductive and osteoconductive.
The present invention particularly provides a nanocomposite scaffold comprising hardystonite which is developed by blending hardystonite and a polymer polycaprolactone followed by electrospinning. The said scaffold displays enhanced cell infiltration, cell proliferation, osteogenic, in vitro cellular activity and bioactivity. The scaffold provides mechanical support for cells to attach and proliferate. It provide a conducive environment for cells to grow and also provides guidance. It creates an artificial niche so that cells feel 'home like'.
In an embodiment of the present invention, the process for the preparation of the nanocomposite scaffold further comprises:
e) Seeding the said scaffold with a cell; and
f) Culturing the cell on the scaffold so that lhe cell differentiates into a mature cell phenotype.
Following generation by electroprocessing, the scaffolds of the present invention are typically sterilized, for example, by alcohol and UV, following which they are seeded with cells.
The cells may comprise a heterogeneous population of cells or alternatively the cells may comprise a homogeneous population of cells. Such cells can be for example, stem cells (such as embryonic stem cells, bone marrow stem cells, cord blood cells, mesenchymal stem cells, adult tissue stem cells), progenitor cells (e.g. progenitor bone cells), or differentiated cells such as chondrocytes, osteoblasts, connective tissue cells (e.g., fibrocytes, fibroblasts and adipose cells), endothelial and epithelial cells. Furthermore, the cells may be of autologous origin or non-autologous origin. Typically the cells are selected according to the tissue being generated.
In a preferred embodiment, the cells suitable for seeding comprises stem cells such as murine

adipose-tissue derived stem cells (mE-ASCs)
The cells may be seeded, inoculated or contacted directly onto the scaffold. Following seeding, the scaffolds are routinely examined using a microscope (e.g., an inverted microscope, an axioplan light microscope or an electronic microscope) for evaluation of cell growth, spreading and tissue formation. The cells are seeded on the surface of the scaffold but eventually they migrate deep into the scaffold after getting attached to the surface of the scaffold.
It will be appreciated that to support cell growth, the cells are seeded on the scaffold in the presence of a culture medium. In some embodiments, it is important to re-create in culture the cellular microenvironment found in vivo. The culture medium used by the present invention may be any liquid medium which allows at least cell survival. Such a culture medium can include, for example, salts, sugars, amino acids and minerals in the appropriate concentrations and with various additives. Non-limiting examples of such culture medium include, phosphate buffered saline, DMEM, MEM, RPMI1640, McCoy's 5A medium, medium 199 and IMDM. The culture medium may be supplemented with various antibiotics, hormones, specific amino acids, cytokines and the like. In addition, growth factors such as osteogenic inducing agents (β-glycerophosphate, dexamethasone, ascorbic acid), and/or angiogenic foctors may be added to the culture medium prior to, during, or subsequent to seeding/inoculation of the cells to trigger differentiation and tissue formation by the cells.
In some embodiments of the present invention, the cells adhere to the scaffold. In one embodiment of the present invention, the scaffold is cultured with cells ex vivo. The seeded scaffold may be introduced into the subject at any time point in the culturing process, in any way known in the art, including but not limited to implantation, injection, surgical attachment, transplantation with other tissue, injection, and the like.
In an embodiment of the present invention, therapeutic compounds or agents that modify cellular activity may also be incorporated (e.g. attached to, coated on, embedded or impregnated) into the scaffold. In addition, agents that act to increase cell attachment, cell spreading, cell proliferation, cell differentiation and/or cell migration in the scaffold may also be incorporated into the scaffold. Such agents can be biological agents such as an amino acid, peptides, polypeptides, proteins, DNA,

RNA, lipids and/or proteoglycans. The cells to be seeded onto the scaffolds may be genetically engineered to express therapeutic or biological agents.
The nanocomposite scaffold of the present invention may be used for cell-based therapy, surgical repair, tissue engineering, and wound healing. Preferably, the scaffold is used to repair bone and/or cartilage at a target tissue site, e.g., one resulting from injury, defect brought about during the course of surgery, infection, malignancy, genetic disease or developmental malformation. The scaffold of the present invention can be utilized in a wide variety of orthopedic, periodontal, neurosurgical, craniomaxillofacial, oral and maxillofacial surgical procedures such as the repair of simple and/or compound fractures and/or nonunions; external and/or internal fixations; joint reconstructions such as arthrodesis; general arthroplasty; cup arthroplasty of the hip; femoral and humeral head replacement; femoral head surface replacement and/or total joint replacement; repairs of the vertebral column including spinal fusion and internal fixation; tumor surgery, e.g., deficit filling; discectomy; laminectomy; excision of spinal cord tumors; anterior cervical and thoracic operations; repairs of spinal injuries; scoliosis, lordosis and kyphosis treatments; intermaxillary fixation of fractures; mentoplasty; temporomandibular joint replacement; alveolar ridge augmentation and reconstruction; inlay implantable matrices; implant placement and revision; sinus lifts; cosmetic procedures; etc. Specific bones which can be repaired or replaced with the implantable scaffold herein include the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and/or metatarsal bones.
The nanocomposite scaffold of the present invention may be suitable for ex vivo or in vivo tissue formation.
In conclusion, a biocompatible, biodegradable, cell proliferative, nanofibrous, membranous scaffold has been developed, characterized and its superiority with established hydroxyapatite scaffold and the process of preparing the same have been disclosed. PCL-HS scaffolds at two different HS concentrations have been prepared by blending followed by electrospinning. Polycaprolactone-hydroxyapatite (PCL-HA) scaffold also developed in similar way. The physico-chemical properties viz. XRD, FTIR, tensile strength, contact angle and biological properties such

as cell proliferation, cell infiltration, mineralization level and ALP are evaluated on both kinds of scaffolds. An in-vitro cellular behavior was evaluated by growing mE-ASCs over the scaffolds and found that the mechanical properties and in-vitro bioactivity of PCL-HS scaffolds are superior to PCL-HA scaffolds. PCL-HS scaffolds exhibited greater cell proliferation, ALP activity and mineralization than PCL-HA scaffolds. Superior physiochemical and biological response of PCL-HS scaffolds make them a potential applicant for bone tissue engineering. PCL-HS scaffolds presents itself as a cost-effective and potential biomaterial for bone tissue engineering application where it could be used safely, economically and effectively for repairing the bone defects.
MATERIALS USED IN THE PRESENT INVENTION
Polycaprolactone (PCL) with an average molecular weight of 80,000 daltons, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP), trypsin-EDTA, penicillin- streptomycin, triton-X, Dimethyl sulfoxide (DMSO) and ((3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (MIT) were purchased from Sigma-Aldrich (St Louis, MO, USA).
Fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (PBS) and Dulbecco's modified eagle's medium (DMEM) were purchased from Gibco-BRL (Grand Island, NY).
Hydroxyapatite powders were a kind gift from Plasma Biotal Ltd, UK.
Tetraethy! orthosilicate (TEOS) - (C2H5O)4 zinc nitrate hexahydrate (ZnfNC^-^O), calcium nitrate tetrahydrate (Ca(NO3)2-4H2O) and nitric acid (HNO3) were purchased from Merck, India.
Water was distilled and deionized (DDW) using a MilH-Q system (Millipore, MA, USA).
EXPERIMENTAL EXAMPLES
The present invention will now be further described with reference to the following examples. It is being understood that these are intended to illustrate the invention and in no manner to limit its scope.
Synthesis of handy stonite (HS) powders

Hardystonite powders were prepared by the sol-gel process described by Wu et al., [3]. Briefly, TEOS, DDW and 2M HNO3 were st
irred for 30 minutes (mins) in a molar ratio of TEOS, DDW and HNO3 equivalent to 1,8 and 0.16 respectively. After that Zn(NO3)2-6H2O and Ca(NO3)2.4H2O were added into the mixture of TEOS/Zn(NO3)26H2O/Ca(NO3)2-4H2O in a molar ratio of 2,1,2 respectively and the reactants were stirred for 5 hours at room temperature. The solution was kept at 60°C for 24 hours and further dried at 120°C. After drying, the gel was ground and finally calcined at 1250°C in a glass furnace for 2 hours.
Preparation of scaffolds
Scaffolds were prepared by the process of electrospinning. For the preparation of pure polymeric PCL scaffold, a 15% w/v polymer solution was prepared in HFP by stirring overnight at room temperature. To prepare PCL-HA (HA in 20 and 40% of PCL, w/w) and PCL-HS (HS in 20 and 40% of PCL, w/w) electrospun scaffolds, HA and HS powders were weighed, dispersed in HFP, sonicated for 30 minutes, and then mixed with the 15% w/v PCL solution. For electrospinning, the polymer solution was loaded into a 5mL plastic syringe connected to a blunt end stainless steel hypodermic needle (24 Gauge) (BD, India). The polymer filled syringe was loaded onto a syringe pump (New Era Pump System Inc., USA) with the syringe needle tip connected to the positive output of a high voltage power supply (Gamma High Voltage, USA) while the alurninum foil . covered collector was grounded. The parameters used for the electrospininng process were as follows: 12 kV voltages, 12.5 cm tip to collector distance, lmL/h flow rate and 55-58% relative humidity.
Characterization of scaffold
Physico-chemical Analysis
i) Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)
The morphologies of the electrospun pure polymeric and composite scaffolds were examined by scanning electron microscope (Hitachi, S-3400 N, UK) at an accelerating voltage of 15kV. For SEM, the samples were cut into 5x5 mm squares, mounted on to sample stubs, and sputter coated with gold using a SC7640 Sputter Coater (Quorum Technologies Ltd, UK). The fiber diameter range of the scaffolds was determined from the SEM micrographs using image analysis software (Image J, National Institutes of Health, Bethesda, USA). Microstructures of the fibers were studied by TEM (Tecnai 20, Philips FEL Netherlands) at a voltage of 100 kV. Fibers of PCL, PCL-

HA and PCL-HS were collected on TEM grids during the process of electrospinning.
The morphology of electrospun scaffolds studied by SEM has shown in fig. I. All scaffolds exhibited randomly oriented, uniform, non-woven morphology with interconnected pores which resemble the extracellular matrix (ECM) of bone. The composite fibers were prepared by blending hydroxyapatite (HA) and hardystonite (HS) particles in PCL solution. The ceramic particles were rarely visible on PCL fibers when observed under SEM. The reason for this may be the embedding of HA and HS particles into the polymer matrix so they were not widely present on the surface of the fibers. The fiber diameter range was found to be 200-650 nm in all scaffolds.
TEM images of PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 fibers confirmed the presence of ceramic particles internalized/or on the sub-surface of PCL fibers (Fig. 2b to Fig. 2e).
Agglomeration of HA particles were observed in the case of PCL-HA20 and PCL-HA40 nanofibers.
ii) X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy
Hardystonite mineral and electrospun scaffolds were characterized by X-ray diffraction using a PANalytical X'Pert Pro X-ray diffractometer. The operating conditions were 40kV and 30mA by using copper Ka monochromatic radiation. The XRD patterns were recorded between 20° to 60° (20) in step of 0.010 intervals with counting time of 1 second at each step.
FTIR spectra were recorded for hardystonite powders and all scaffolds using Nicolet Magna-IR FTIR 550 spectrophotometer (Nicolet Instrument Corporation, Madison, WI, USA). The spectra were obtained with 30 scans per sample ranging from 4000 to 400cm-1.
The XRD and FTIR studies of scaffolds were done to confirm the presence of inorganic components in the scaffolds after the electrospinning process. Figure 3 shows the XRD and FTIR spectra obtained from scanning of PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCU1S40 scaffolds. The characteristic XRD peaks of HA at 31.7 [211] were observed in PCL-HA20, PCL-HA40 scaffolds (Figure 3(a)). Peaks observed at 28.8 [201], 31.0 [211] and 36.2 [310] correspond to HS in PCL-HS20 and PCL-HS40 scaffolds (Figure 3(a)). All the diffraction patterns were

compared to International Centre for Diffraction Data (ICDD) database (ICDD Powder Diffraction File No. 00-24-0033 for HA and 75-0916 for HS).
FTIR scans of the scaffolds are shown in Figure 3(b). The spectra shows a carbonyl stretch from PCL (C=0) at 1730 cm-1 in all the scaffolds. Other major PCL peaks were observed at 1290cm-1 (OO-C stretching), 1242cm-1 (asymmetric C-O-C stretching) and at 1170cm-1 (symmetric CO-C stretching). Peaks at 603cm-1 and 576cm-1 correspond to P043- from HA in PCL-HA20 and PCL-HA40 scaffolds. The OH- group peaks were observed in the region of 3250-3570cm-1 in composite scaffolds.. The spectra of PCL-HS20 and PCL-HS40 scaffold showed peaks near 617 and 680 cm'1 which correspond to SiO-Si and Si-O- vibration respectively.
To study the degradation profile, scaffolds were immersed in phosphate buffer saline (PBS) solution for two months at 37°C and the percentage mass loss of the scaffolds were measured at 7, 28,42 and 60 days. Figure 4(a) clearly indicates that up to 42 days all scaffolds showed very less % mass loss. At the end of 60 days, composite scaffolds showed significant degradation while the mass of pure PCL scaffold remained almost consistent (only 1.54% mass loss). Among the composite scaffolds, PCL-HS scaffolds exhibited a significant mass loss compared to their PCL-HA counterpart PCL-HS20 and PCL-HS40 scaffolds displayed 45.1 and 49.9% degradation respectively at the end of 60 days while, PCL-HA20 and PCL-HA40 scaffolds showed 16.3% and 41.5% mass loss respectively. The mechanical properties of electrospun scaffolds were measured as ultimate tensile stress. Figure 4(b) shows a typical stress-strain curve of PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40. All composite scaffolds exhibited better tensile strength than pure PCL scaffold. The ultimate tensile stress of PCL was 6.21±0.81 MegaPascals (MPa), which significantly increased to 8.3±0.46 MPa after addition of 40% HA into the PCL scaffold. The ultimate tensile stress of PCL-HS20 (9.23±1.28 MPa) and PCL-HS40 (9.89±0.52 MPa) was found to be significantly higher as compared to PCL-HA20 (6.6±0.52 MPa) and PCL-HA40 (8,3±0.46 MPa), respectively. This might be due to presence of Si in HS containing scaffolds which reinforced the PCL nanofibers and resulted into mechanically stronger nanofibers. This indicates that HS containing scaffolds are mechanically more stable than HA containing scaffolds and provide strength to the resultant nanofiber.
iv)In-vitro degradation of scaffolds

In-vitro degradation of all the scaffolds was measured from their percentage mass loss over a period of time after incubation in PBS. All the scaffolds were cut into 1 x1 cm2 squares, weighed and immersed into test tubes containing 30mL PBS (pH 7.2) at 37°C. Samples were kept in a rotating shaker at the speed of 125 rpm and were observed for a maximum of 60 days. The PBS solution was changed after every 7 days. At the end of 7, 28, 42 and 60 days samples were removed, washed with deionized distilled water three times and freeze dried for 24 hours. The samples were then weighed and degree of degradation was determined using following equation

where Mf is the final mass at each time point and Mi is the initial mass.
Tensile properties of PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 were measured with a table top tensile tester (Material Testing Machines, Tinius Olsen H1KT) with 50 N load cells. A standard specimen shape of 10 mm x 50 mm rectangular specimenswas used. Test samples were vertically mounted with two mechanical gripping units of the tensile tester at its ends leaving a 30mm gauge length for mechanical loading. Load deformation data were recorded at a crosshead speed of 5rnnVmin and ultimate tensile strength measurement were calculated from the stress-strain curve.
Biological Analysis
i) Blood compatibility of scaffolds
The hemocompatibility of scaffolds was evaluated by hemolysis assay described elsewhere [4]. Briefly, 20ml of whole blood was collected from a healthy individual, as per institutional ethical guidelines in place, into two 10ml BD Vacutainer® Plus plastic plasma tubes containing 150 USP units sodium heparin (spray-coated) anticoagulant. The heparinized whole blood was immediately centrifuged for 15 mins at 1200rpm to pack erythrocytes. The platelet-rich plasma was removed from the centrifuge tubes and the packed erythrocytes were washed three times with normal saline solution (NSS) (0.9% w/v NaCI). A 50% hematocrit solution was prepared by adding NSS to the erythrocytes. Scaffolds were equilibrated in normal saline for 30 minutes at 37°C and all samples were incubated with lmL of blood for 1 hour at 37°C in a water bath. Thereafter, blood was centrifuged (lOOOrpm, l0mins) and the hemolytic percentage was determined by photometric analysis of supernatant at 545 nm. DDW and normal saline were used as positive and negative control respectively. The hemolysis ratio (HR) was obtained by the equation


where AS, AN and AP are absorbance of sample supernatant, negative control and positive control respectively.
In-vitro hemolysis assay was performed on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds. Figure 5(a) shows the percentage hemolysis of all scaffolds. The PCL, PCLHA20 and PCL-HA40 scaffolds showed 0.48%, 0.42% and 0.42% hemolysis, respectively while PCL-HS20 and PCL-HS40 showed 0.65% and 0.48%, respectively. The percentage hemolysis of all scaffolds was under the permissible limit of 5% as per Zhou et al., [19]. This result confirms that addition of HA or HS does not compromise the hernocompatibility of the scaffolds.
ii) Protein adsorption
For the measurement of protein adsorption, PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 were placed in 24 well tissue culture plates (TCP). Each scaffold was pre-washed with PBS, air dried and equilibrated at 37°C. To each well, 500 ul of fetal bovine serum (FBS) was added and the plate was incubated for 2 hours at 37°C. Scaffolds were rinsed gently with PBS to remove any unattached proteins. Aqueous solution of sodium dodecyl sulphate (SDS, lwt %) was used to remove adsorbed proteins on the surface. After adding SDS, the TCP were shaken for 60 mins. The amount of proteins adsorbed (ug/cm) on the surface was calculated from the concentration of proteins in the SDS solution using a protein analysis kit (Micro BCA Protein Assay Kit, Pierce Biotechnology, IL, USA). The protein concentration was calibrated with bovine serum albumin (BSA) standard solutions included with the test kit. Data are expressed as the means ± SD of four independent measurements.
Figure 5(b) shows protein adsorption on PCL, PCL-HA20, PCL-HA40, PCL-HS20, PCL-HS40 and TCP. Despite showing similar contact angle (Figure 9), the amount of protein adsorbed on composite scaffolds was found to be significantly higher than pure polymeric PCL scaffold. This might have occurred due to the presence of HA or HS particles, increasing the surface area and roughness of the scaffold. Also the calcium ions present in HA and HS might have acted as

a chelator allowing increased protein adsorption. TCP showed the least protein adsorption compared to all the scaffolds.
iii) Cell culture and seeding
mE-ASCs were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% (v/v) FBS, 1% penicillin-streptomycin, 1% L-glutamine at 37°C and 5% CO2 in a humidified atmosphere. For in-vitro culture, scaffolds were sterilized by immersion in 70% ethanol followed by washing with sterile PBS twice and irradiated by short-UV-light for 30 mins.
For cell seeding, sterile scaffolds were placed in 24-well TCP, completely covering the bottom of the TCP. mE-ASCs cells were seeded on these sterile scaffolds at a seeding density of 1x104 cells/scaffold. The medium was replenished every 48 hours. Cells seeded on the TCP without scaffold was also maintained simultaneously as a TCP control. These cell seeded scaffolds were then used for determination of cell proliferation and cell adhesion assay. For osteogenic differentiation experiments (ALP assay and mineralization), the medium was replenished after 1 day with osteogenic induction medium (2.0 mM β-glycerophosphate, 0.1 mM dexamethasone, and 0.2 mM ascorbic acid) (Sigma-Aldrich).
iv) Cell adhesion: Scanning Electron Microscopy (SEM)
The cell attachment on to the scaffold was characterized by SEM. Sterilized scaffolds (1cm x 1cm) were kept in 24-well TCP and 1xlO4 mE-ASCs were seeded on all scaffolds for a period of 1 and 7 days. At each time point, the cells on the samples were washed twice with PBS and fixed in 2.5% glutaraldehyde (Merck) for 2 hours at room temperature, washed with PBS and dehydrated with graded ethanol from 10% to 100%. Samples were dried overnight, sputter coated with gold (Quorum Technologies Ltd, UK) and observed under SEM (Hitachi, S-3400 N, UK) at an accelerating voltage of 15kV.
Adhesion of mE-ASCs to scaffolds was analyzed by SEM on day 1 and day 7 of cell culture (Figure 6). SEM micrographs of mE-ASCs cultured on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 exhibit the cells adhesion on all scaffolds. Initially at day 1, PCL-HA20 scaffold showed less number of cells attached as compared to other scaffolds (Figure 6). However, on day 7 all scaffolds were covered by a confluent layer of cells, indicating the efficiency of the

scaffolds in supporting cell proliferation and growth (Figure 6(f) - Figure 6(j)).
v) Cell infiltration: Confocal laser scanning microscopy (CLSM)
Cell infiltration inside the scaffolds was assessed by fluorescence microscopy after 7 days of culture. Scaffolds containing cells were washed twice with PBS. Cells were fixed in a 3.7% formaldehyde solution (Sigma-Aldrich) in PBS for 30mins at room temperature, washed with PBS twice and then dyed with 4'-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for lhour. After staining, samples were washed with PBS and mounted on to a glass-slide with vectashield mounting medium (Vector laboratories, Burlingame, CA). Samples were subjected to optical sectioning at 1.5um increments in the axial (z) dimension using a confocal laser scanning microscope (CLSM) (Olympus Fluoview, FV500, Tokyo, Japan).
These images were captured along the z axis at every 1.5 um up to 30 urn (Figure 7). Although, many published literature indicates that small pore size promotes the growth of a cell monolayer on the scaffold, our obtained results contradicts them with cell infiltration occurring inside the scaffold in spite the smaller pore size of the scaffolds. Cell infiltration plays an important role in tissue engineered scaffolds and is governed by biochemical as well as biophysical cues provided by the surrounding scaffold. From figure7 it is clear that the PCL scaffold showed enhanced cell infiltration which could be due to the superior porosity of this scaffold as compared to others. Cell infiltration as well as cell density was found to be improved in HS containing scaffolds as compared to HA composite scaffolds. It may be due to the stimulatory action of Zn and Si as observed in the MTT assay, which also showed facilitated cell infiltration into the scaffolds. PCL-HS20 scaffold showed similar cellular infiltration (20 urn) as compared to PCL-HS40 (18μm) scaffold.
vi) Cell proliferation assay
Proliferation of mE-ASCs on PCL, PCL-HA20, PCL-HA40, PCL-HS20 and PCL-HS40 scaffolds were assessed as a function of time by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Yellow MTT is reduced to purple formazon by mitochondrial dehydrogenase activity in living cells. mE-ASCs (1 x104) were seeded over scaffolds for different time periods (day 1,4 and 7) and at each time point, 100 μl of MTT (lmg/mL) (Sigma-Aldrich) was added to each well of 24 well TCP. Cells were incubated for 4 hours at 37°C in a humidified C02 incubator and

then 900 ul of DMSO (Sigma-Aldrich) was added followed by shaking the plate for 15 mins to dissolve the formazan crystals. Afterward, 200 ul of sample was pipetted into a 96-well tissue culture plate and the absorbance was measured at 560nm using a plate reader (Multiskan EX, Thermo Fisher Scientific Inc., USA).
The proliferation of mE-ASCs cells on different scaffolds was checked by MTT assay. MTT, a quantitative method for measuring cell proliferation, is based on reduction of MTT to purple formazon by mitochondrial dehydrogenase enzyme. Results in figure 5(c) demonstrated that the addition of HA and HS to PCL did not show any inhibitory effect on the cytocompatibility aspect of the scaffolds. All the scaffolds supported cell proliferation as the absorbance was found to have increased from day 1 to day 4 and then to day 7. Figure 5(c) clearly showed that on day 7, cell proliferation was significantly higher on PCL-HS scaffolds as compared to hydroxyapatite composite scaffolds, increased cell proliferation was likely promoted by the presence of Zn and Si in HS. Zn is known to have a positive effect on protein synthesis and secretion of bone growth factors which enhance cell proliferation and inhibit osteoclast cell formation.
vii) ALP assay
To evaluate ALP activity, 1x104 mE-ASCs were seeded on all scaffolds and cultured for 1,4 and 7 days. At each time point, medium was decanted and scaffolds were washed with PBS. Following this, cell lysis buffer containing 0.1% Triton X-100 was added and cell lysate was centrifuged at 2500g for lOmins. ALP activity of the supernatant was measured according to the manufacturer's instructions (Sensolyte, Anaspec). The cell lysate (50uL) was incubated with para-nitro phenyl phosphate (PNPP) solution (50uL) at 37°C for 60mins. After incubation, the reaction was stopped with lOOuL IN NaOH. ALP activity was determined from the absorbance of p-nitrophenol at 405nm using an ELISA reader. The ALP activity was calculated from a standard curve and normalized with total protein content. The total protein was determined using a Pierce BCA Protein Assay Kit with bovine serum dburnin as a standard.
To assess the functionality of the scaffolds towards bone tissue formation, ALP assay was performed. ALP enzyme is produced by differentiating osteoblast cells and is known to be necessary for bone matrix construction. The ALP activity of the cells seeded over scaffolds is

compared in figure 8(a). All scaffolds showed increase in ALP activity as the time of culture progressed from day 1 to day 7. TCP was used as control and showed minimum ALP activity. At day 1 and day 4, no significant difference was observed in ALP activity among all the scaffolds. At day 7, composite scaffolds exhibited better ALP activity as compared to pure PCL scaffold. These results are consistent with various studies which showed that the addition of ceramic components such as hardystonite, hydroxyapatite, bioglass to polymeric scaffold increased the functionality of scaffolds towards an osteoblastic lineage. No difference in ALP activity was seen with the increase in concentration of either HA or HS. However, ALP activity of HS scaffolds significantly increased as compared to HA containing scaffolds, indicating the positive role of Zn and Si in bone metabolism. Zn is an essential trace element and is cofactor of various enzymes including alkaline phosphatase which plays an important role in the mineralization process of extracellular matrix for bone formation. Si is a constituent of some glycosaminoglycans and help in stabilization of the collagen matrix. The ALP results also suggest the superiority of hardystonite over hydroxyapatite for bone tissue engineering.
viii) Mineralization assay
Mineralization deposition by mE-ASCs on different scaffolds was assessed by alizarin red-S (ARS) dye, which binds to the calcium salt selectively and forms an alizarin red S-calcium complex in a chelation process. After 14 and 21 days of culture, cells were fixed with 10% formaldehyde for 30mins. The cell scaffold constructs were washed twice with DDW and then incubated with ARS (2mM) for 20mins with shaking to facilitate staining. After that, excess dye was removed and scaffolds were washed four times with DDW, For calcium quantification, the supernatant was extracted with 10% acetic acid. After 30mins incubation with acetic acid, the scaffold with acetic acid was placed into a micro centrifuge tube, heated at 85°C for lOmins, cooled and centrifuged at 10,000g for 15mins. 500μl of above solution was taken and neutralized with 10% ammonium hydroxide. An aliquot of 150 pi of this solution was transferred to a 96 well plate and absorbance was measured at 405nm. The concentration of alizarin was calculated from the alizarin standard curve.
To assess the deposited calcified matrix by differentiating cells, alizarin red S staining was performed after 14 and 21 days of culture. This method measures the amount of calcium present in the matrix as ARS binds to calcium specifically. Figure 8(b) clearly exhibits the proficiency of

composite scaffolds in matrix mineralization as compared to pure PCL scaffold. The PCL scaffold showed a similar alizarin level as TCP which was used as control. The addition of a ceramic component of either HA or HS to the polymeric scaffold provided enhanced biochemical cues to mesenchymal stem cells towards osteogenic differentiation because they better mimic the native bone environment. PCL-HS scaffolds showed a significant increase (more than two fold) in alizarin as compared to HA based scaffolds; however the effect of concentration was not observed with either ceramic composite HA or HS. These results are in good agreement with the ALP results obtained in this study. HS based scaffolds showed higher ALP activity at day 7 and then exhibited better mineralization at later time points. As mesenchymal stem cells differentiated towards an osteoblastic lineage, they express ALP which is an early marker of osteoblasts and further secrete calcium rich ECM. The better mineralization by HS based scaffolds is attributed to the presence of Zn and Si in hardystonite. The PCL-HS nanofibrous scaffolds fabricated via electro spinning demonstrate the superior mechanical strength of PCL-HS scaffolds as compared to PCL-HA scaffolds. Further, proliferation and cellular infiltration of mE-ASCs were also found to be better on PCL-HS scaffolds. ALP activity and mineralization were found to be significantly higher in PCL-HS scaffolds as compared to PCL-HA scaffolds.
References
1. M. Navarro, C. Aparicio, M. Charles-Harris, M.P. Ginebra, E. Engel, J.A. Planell, Development of a biodegradable composite scaffold for bone tissue engineering: Physicochemical, topographical, mechanical, degradation, and biological properties, Adv. Polym. Sci. 200 (2006) 209-231.
2. D.W. Hutmacher, Scaffolds in tissue engineering bone and cartilage, Biomaterials 21 (2000) 2529-2543.
3. C. Wu, J. Chang, W. Zhai, A novel hardystonite bioceramic: Preparation and characteristics, Ceram. Int. 31 (2005) 27-31.
4. C. Zhou, Z. Yi, Blood-compatibility of polyurethane/liquid crystal composite membranes, Biomaterials 20 (1999) 2093-2099.

We claim
1. A nanocomposite scaffold comprising a polymer component and a ceramic component, characterized in that the ceramic component comprises a metal silicate.
2. The nanocomposite scaffold as claimed in claim 1, wherein the polymer component is selected from polycaprolactone, poly(l-lactic acid), poiyglycolic acid, polylactic acid/polyglycolic acid copolymers or derivatives thereof.
3. The nanocomposite scaffold as claimed in claim 1, wherein the polymer component is polycaprolactone.
4. The nanocomposite scaffold as claimed in claim 1, wherein the metal silicate is selected form a group consisting of calcium silicate, calcium zinc silicate (hardystonite), calcium magnesium silicate, zinc silicate (hardstonite), magnesium silicate, magnesium zinc silicate, strontium silicate, strontium calcium zinc silicate (strontium-hardystonite), strontium calcium silicate, zirconium calcium silicate, calcium zirconium silicate (Ca3ZrSi209), magnesium calcium zinc silicate (magnesium-hardystonite), barium calcium zinc silicate (barium-hardystonite) and combinations thereof.
5. The nanocomposite scaffold as claimed in claim 1, wherein said scaffold comprises at least 72 to 84 % by weight of the polymer component.
6. The nanocomposite scaffold as claimed in claim 1, wherein said scaffold comprises at least 16 to 29 % by weight of the ceramic component.
7. The nanocomposite scaffold as claimed in claim 1, wherein the tensile strength of said scaffold is between 7.50 MegaPascals (MPa) to 10.60 MegaPascals (MPa).

8. A process for the preparation of a nanocomposite scaffold, comprising:
a) synthesizing the ceramic component by sol-gel process;
b) dispersing the ceramic component obtained from step (a) and the polymer component individually in a solvent;
c) blending said ceramic component with said polymer component to form a solution; and
d) electroprocessing said solution to form said scaffold thereby depositing a three-dimensional matrix of nano sized electroprocessed fibers comprising the ceramic-polymer composite on a collector.
9. The process as claimed in claim 8, wherein it comprises:
e) seeding the said scaffold with a cell; and
f) culturing the cell on the scaffold so that the cell differentiates into a mature cell phenotype; wherein the cell is selected from a group consisting of an osteoprogenitor cell, a mesenchymal cell, a stem cell, an osteoblast, an osteocyte, and any combinations thereof.

10. The process as claimed in claim 8, wherein said electroprocessing comprises electrospinning.
11. The process as claimed in claim 8, wherein the solvent is selected from the group consisting of 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFP), trifluoroethanol (TFE), chloroform, dichloromethane (DCM), or a mixture of formic acid/acetic acid.
12. The process as claimed in claim 8, wherein the solvent is 1,1,1,3,3,3-hexafluoro-2-propanol (HFP).
13. The process as claimed in claim 8, wherein the fiber diameter of said scaffold is between 200nm - 650nm,

14. The process as claimed in claim 8, wherein the fibers are randomly oriented fibers.