FIELD OF INVENTION

[001] The present invention relates to the field of bone, cartilage and hard tissue prosthetics, and more particularly to the use of cellular-based implants for the preparation of prosthetic implants for bone replacement and repair.

BACKGROUND OF THE INVENTION

[003] The repair of outsized deficiencies, typically defined as gaps of at least about 2.4 mm in size, in the diaphyseal, craniomaxillofacial and other skeletal bones is a considerable problem in orthopedic surgery.

[004] In 1998, about 300,000 bone-graft procedures were performed in the United States alone. This number increased to approximately 450,000 by the year of 2000, when the number of bone grafting procedures performed worldwide exceeded 2.2 million (Lewandrowski et al, 2000). Of the 300,000 procedures performed in 1998, 90% involved the use of either autologous grafts (i.e. using tissue from another part of the body of the patient), or of allografts (i.e. using tissue from a live human donor or cadaver). Therefore, a phase of tissue harvest from the patient or from a donor is required.

[005] The tissue harvesting is executed by a surgical procedure usually involved collecting tissue from the iliac crest, the distal femur, the proximal tibia, the fibula, or from other small bones. The harvested tissue is restructured and transplanted at the damaged site.

[006] However, the graft-harvesting procedures are associated with considerable morbidity and substantial pain. Tissue harvesting for an autologous grafts or from live donors for an allograft may also result in complications such as inflammation, infection, or even death.

[007] The limited supply and inherited harvesting complications have inspired the development of alternative strategies for the repair of significant bone defects.

[008] The use of 3-dimensional (3-D) bone substitutes such as bone extract, polymer or mineral scaffolds as implants has been investigated and porous biocompatible scaffolds have been used for the repair and regeneration of bone tissue.

[009] Early attempts at tissue repair have focused mainly on the use of amorphous, biocompatible foam as porous plugs to fill large voids in bone. U.S Patent No. 4,186,448 described the use of porous mesh plugs composed of polyhydroxy acid polymers, such as polylactide, for healing bone voids. Several different methods for making other scaffolds were also described (i.e. U.S Patent Nos. 5,133,755; 5,514,378; 5,522,895; 5,607,474; 5,677,355; 5,686,091; 5,716,413; 5,755,792; 5,769,899; 5,770,193; 6,333,029; 6,365,149 and 6,534,084).

[010] Bone marrow (BM) has been shown to contain population of cells that possess osteogenic potential. As such, an alternative to the scaffold-osteoinductive approach is to transplant into patients living cells that possess this capacity. Cytokine-manipulated, naive autologous and allogeneic BM cells have successfully healed diffracted or resorbed bones in experimental models (Werntz et al, 1996; Lane et al, 1999; Nilsson et al, 1999; Kawaguchi et al, 2004) and human patients (Horwitz et al, 1999; Horwitz et al 2001, 2004). Progenitor cells of the osteogenic lineage are seeded onto biocompatible (biodegradable or nonbiodegradable) scaffolds in the presence or absence of growth promoting factors (U.S. Patent Nos. 6,541,024; 6,544,290; 6,852,330). Transplantation into affected patients is performed following an ex-vivo expansion phase of the cells on the given scaffold. Using this approach, either primary osteogenic cells or expanded Mesenchymal Stromal Cells (MSC) layered upon ceramic scaffolds was able to regenerate bone tissue (Kadiyala et al, 1997; Bruder et al, 1998a,b; Cinotti et al, 2004).

[011] Living bone is a continuously evolving organ and in the normal course of bone maintenance, a constant remodeling process is being employed. In those procedures, Old bone is being replaced by new bone and the organ responds to its environment changing requirements for strength and elasticity. Therefore, normal remodeling progression requires that the mechanical loading processes of bone resorption and bone formation procedures are tightly coordinated.

[012] In cellular terms, this depends on sequential functioning of osteoclasts (bone resorbing cells) and osteoblasts (bone forming cells). In addition, endothelial cell and endothelial cell precursors (angioblasts) are required to form the new blood vessels in the developed bone tissue. Yet, the various cell types participating in bone formation are of different lineages. It is now known that osteoblasts stalk from mesenchymal stem cells, while osteoclasts (directly originating from Hematopoietic Stem Cells (HSC)) and endothelial cells are descendents of a common blast colony-forming cell (Choi et al, 1998; Hamaguchi et al, 1999). As such, methodologies for ex-vivo production of bone-like material that rely on osteoblasts as the exclusive cellular component suffer from an inherited fault. SUMMARY OF THE INVENTION

[013] In one embodiment, the present invention provides a composition comprising, (a) a mineral particle, (b) endothelial cells and mesenchymal cells, and (3) hyaluronic acid. In another embodiment, the present invention provides a composition comprising, (a) a mineral particle, (b) adipose derived cells comprising mesenchymal stem cells and/or osteoprogenitor cells and (3) hyaluronic acid.

[014] In another embodiment, the present invention provides a kit comprising: a syringe, a mineral particle comprising endothelial cells and mesenchymal cells organized in 2 or more cell layers attached thereto, and hyaluronic acid.

[015] In another embodiment, the present invention provides a method for filling a gap in a bone of a subject in need thereof, comprising the step of contacting the gap with a composition comprising: (a) a mineral particle, (b) endothelial cells and mesenchymal cells, and (3) hyaluronic acid, thereby filling a gap in a bone of a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[016] FIG. 1 is a bar graph showing the Average σ bending of rat femur bones 8 weeks after injection. Results are expressed as mean + SEM.

[017] FIG. 2 is a scheme showing the automatic identification of the three compartments used for microCTanalysis. A) Femur is semi-transparent color with trabecular bone and cortical ring (yellow). B) Cortical mask is semi-transparent and bone within the mask is solid. C) Trabecular mask is semi-transparent and bone within the mask is solid.

[018] FIG. 3 are MicroCT scan micrographs of treated legs 16 weeks post injection: (A, B) Treated legs of scaffold only group. (C,D) Treated leg of scaffold (mineral particle) and 3 million Adipose Tissue derived Cells. (E, F) Treated legs of scaffold (mineral particle) and 5 million Adipose Tissue derived Cells.

[019] FIG. 4 is a bar graph showing full bone parameters - On an organ level (full compartment), the bone volume density (AVD) of group 5xl0 ⁶ cells appeared to be the highest and the values from group 3xl0 ⁶ appeared to be the lowest. Results are expressed as mean + SEM. [020] FIG. 5 are bar graphs showing the Trabecular bone parameters/measures - several micro structural bone parameters were measured, in all measurements the scaffold containing 5xl0 ⁶ cells group was superior to scaffold containing 3xl0 ⁶ group and to the scaffold only group. (A) Trabecular separation (B) Trabecular number (C) Trabecular connectivity (D) Bone surface / total volume (E) Bone volume / Total volume. Results are expressed as mean + SEM. * p<0.05, ** p<0.001, *** p<0.0001, the difference is compared to scaffold only group.

[021] FIG. 6 are bar graphs showing Cortical bone parameters/measures - MicroCT scan results show that the scaffold containing 5xl0 ⁶ cells group was superior to scaffold containing 3xl0 ⁶ group and to the scaffold only group. (A) Cortical thickness (B) Bone volume. Results are expressed as mean + SEM. * p<0.05, the difference is compared to scaffold only group.

[022] FIG. 7 is a micrograph and a bar graph showing Histology and morphometric analysis of both 8 and 16 weeks post transplantation groups. (A) 8 weeks post transplantation. (B) 16 weeks post transplantation. (C) Morphometric analysis of 3 slides from each femur in each group. Results are expressed as mean + SEM. #, * p<0.05, the difference is compared to scaffold only group.

[023] FIG. 8 is a bar graph and a micrograph showing seeded cells adhesion to scaffold. (A) Adipose tissue derived cells were seeded for 2 hours and cell numbers were quantified. (B) Gimsa Cell staining of seeded cells.

[024] FIG. 9 is a bar graph and 2 micrographs showing (A) Cultivated Adipose tissue derived cells propagate for up to 13 days. (B, C) Cells generate multi cellular layers onto mineral particles. Histological and scanning electron microscope analysis shows the formation of multicellular layers on top of the bone mineral particles. These layers generate a typical connective tissue structure containing collagen fibers and polysaccharides as seen in Picro Sirius Red stain and Toluidine Blue stain, respectively.

[025] FIG. 10 are micrographs showing scanning electron microscope and histological analysis of the adipose tissue derived cells cultivated on bone mineral particles (OG). (A) SEM (B) Hematoxiline & Eosin stain (C) Toluidine blue stain (D) Picro Sirius Red stain.

[026] Fig 11. Are micrographs showing the cells from the 3-D multi cell culture sprouting onto Matrigel after 72 hours culturing. DETAILED DESCRIPTION OF THE INVENTION

[027] The present invention, in some embodiments, provides a novel solution for the ex- vivo regeneration of remodeled bone, cartilage and other hard tissue applications. The background art describes bone substitutes made from cells of osteogenic lineage cultured on a scaffold. In contrast with this art, the present invention manipulates co-culture and multi-cell cultures made up of two or more independent cell types grown as multi-layered cell culture on mineral particles as scaffolds to optimize the bone regeneration and remodeling processes. In some embodiments, multi-layered cell culture is obtained by growing the culture in a flow system at a high density. In some embodiments, cells within one layer are in communication with cells within a second layer.

Multi-layer cell culture

[028] In some embodiments, a multi-layered cell culture is a heterogeneous cell culture composed of at least two cell types. In another embodiment, a multi-layered cell culture is a heterogeneous cell culture composed of at least three cell types. In another embodiment, a multi-layered cell culture is a heterogeneous cell culture composed of at least four cell types. In another embodiment, a multi-layered cell culture comprises mesenchymal cells. In another embodiment, a multi-layered cell culture comprises osteogenic cells. In another embodiment, a multi-layered cell culture comprises osteoprogenitor cells.

[029] In another embodiment, a multi-layered cell culture comprises a bottom layer of cells and a top layer of cells. In another embodiment, a multi-layered cell culture comprises a bottom layer of cells, a middle layer of cells and a top layer of cells. In another embodiment, a multi-layered cell culture is a 3D cell culture (as opposed to a single layer of cells that is termed a 2D cell culture). In another embodiment, a 3D cell culture consists cells and extra cellular matrix. In another embodiment, a 3D cell culture consists cells and extra cellular matrix. In another embodiment, a 3D cell culture is grown on the surface of a mineral particle as described herein. In another embodiment, a 3D cell culture consists a biotic matter. In another embodiment, a 3D cell culture of 2 or more cell layers is attached to the mineral particle. In another embodiment, a 3D cell culture of 2 or more cell layers is operably attached to the mineral particle.

[030] In some embodiments, a multi-layered cell culture or a 3D cell culture includes at least 2 layers of cells, wherein at least 10% of the cells in one layer are in contact with at least 10% of the cells in another layer. In some embodiments, a multi-layered cell culture or a 3D cell culture includes at least 3 layers of cells.

[031] In some embodiments, at least 10% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 10% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In some embodiments, at least 20% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 20% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In some embodiments, at least 30% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 30% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In some embodiments, at least 40% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 40% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In some embodiments, at least 50% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 50% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In some embodiments, at least 60% of the cells in one layer within a multi-layered cell culture or a 3D cell culture are in contact with at least 60% of the cells in another layer within the same multi-layered cell culture or 3D cell culture. In another embodiment, the phrase "in contact" is in physical contact. In another embodiment, the phrase "in contact" is in cell to cell interaction.

[032] In another embodiment, the phrase "3D culture (three dimensional culture)" refers to a culture in which the cells are disposed to conditions which are compatible with cell growth while allowing the cells to grow in more than one layer. In another embodiment, cells within the 3D cell culture are held in a complex network of extra cellular matrix nanoscale fibers that allows the establishment of various local microenvironments. In another embodiment, extra cellular ligands within the ECM mediate not only the attachment to the basal membrane but also access to a variety of vascular and lymphatic vessels. In another embodiment, cells within the 3D cell culture are exposed to oxygen, hormones and nutrients. In another embodiment, a 3D cell culture is characterized by cell-cell and cell-ECM interactions.

The cells [033] In another embodiment, the invention provides a composition comprising, (a) a mineral particle, (b) endothelial cells and mesenchymal cells, and (c) hyaluronic acid. In another embodiment, the invention provides a composition comprising, (a) a mineral particle, (b) a 3D cell culture, and (c) hyaluronic acid. In another embodiment, the invention provides a composition comprising, (a) a mineral particle, (b) osteoprogenitor cells and (c) hyaluronic acid. In another embodiment, a 3D cell culture comprises endothelial cells and mesenchymal cells. In another embodiment, a 3D cell culture comprises adipose tissue derived cells. In another embodiment, adipose tissue derived cells comprise adipose-derived stem cells (ASC) (CD34- CD45- CDl lb-, CD19, HLA-DR-, CD105+, CD73+, CD90+). In another embodiment, adipose tissue derived cells comprise endothelial progenitor cells (CD31+ CD34+ CD45- CD144+ CD146+ CD102+). In another embodiment, adipose tissue derived cells comprise mature endothelial cells (CD31+ CD34+ CD45- CD90- CD144+ CD 146+ CD105+). In another embodiment, adipose tissue derived cells comprise vascular smooth muscle cells (Smooth muscle alpha-actin positive, Desmin positive, h-caldesmon positive, Smooth muscle myosin heavy chain positive). In another embodiment, adipose tissue derived cells comprise cells capable of differentiating into adipogenic and osteogenic cells. In another embodiment, a 3D cell culture comprises: osteoprogenitor cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, osteoclasts, or any combination thereof. In another embodiment, mesenchymal cells are mesenchymal stem cells. In another embodiment, mesenchymal cells comprise mesenchymal stem cells and/or osteoprogenitor cells. In another embodiment, a 3D cell culture comprises Hematopoietic CD34+ cells. In another embodiment, a 3D cell culture comprises osteoprogenitor cells. In another embodiment, adipose derived cells comprise endothelial cells, mesenchymal cells, mesenchymal stem cells, or any combination thereof.

[034] In another embodiment, Adipose derived cells were obtained from adipose tissue (such as of liposuction procedures). In another embodiment, endothelial cells were collected from peripheral blood. In another embodiment, cells of the invention are maintained and grown at 37°C in a tissue culture incubator under humidified condition with 5% C0 ₂

[035] In another embodiment, mineral particles carrying a multi-layered heterogeneous cell culture are subjected to osteogenic culture differentiation conditions comprising: osteogenic culture differentiation medium composed of one or more of the following molecules in preferred concentration: dexamethasone (10-200 nM) (Sigma), sodium .beta.-glycerophosphate (5-25 mM) (Sigma), 1,25 dihydroxycholecalciferol (calcitriol: 1- 50 nM) (Sigma),L-ascorbic acid-2-phosphate (0.05-500 mM) (Sigma)and BMP-2 (lOng/ml-lOug/ml).

[036] In another embodiment, mineral particles carrying a multi-layered heterogeneous cell culture are subjected to Chondrocyte differentiation conditions comprising: medium with chondrogenic cocktail: DMEM HG, BMP-6 [500 ng/ml] (Sigma), TGF-b3 [10 ng/ml] (Sigma), ITS and premix [dil. :20 of the dil. Stock(:100)] (Sigma), Dexamethasone [100 nM] (Sigma), L-ascorbic acid 2-phosphate [50 mg/ml] (Sigma), Sodium pyruvate [100 mg/ml] (Sigma), Proline [40 mg/ml] (Sigma), Pen/Strep/Nys 1% , Glutamine 1% .

[037] In another embodiment, a multi-layered heterogeneous cell culture comprises at least two cell types selected from the group consisting of: osteoprogenitor cells, osteoblasts, mesenchymal cells, embryonic mesenchymal cells, osteoclasts, osteocytes, hematopoietic progenitor cells, chondrocytes, chondroblasts and endothelial cells. In another embodiment, cells as described herein are derived from mesenchymal stem cells and/or osteoprogenitor cells. In another embodiment, cells as described herein are derived from an autologous source, a syngeneic source and an allogeneic source. In another embodiment, cells as described herein are derived from bone marrow, placenta, adipose tissue, cord blood, peripheral blood, mobilized peripheral blood, embryonic stem cells, or any combination thereof.

Cell density

[038] In another embodiment, a composition as described comprises at least lxlO ² cells as described herein per 1 mg of mineral particle. In another embodiment, a composition as described comprises at least lxlO ³ cells as described herein per 1 mg of mineral particle. In another embodiment, a composition as described comprises at least lxlO ^{2 t0} lxlO ⁶ cells as described herein per 1 mg of mineral particle. In another embodiment, a composition as described comprises at least lxlO ^{2 t0} lxlO ⁴ cells as described herein per 1 mg of mineral particle. In another embodiment, a composition as described comprises at least 5x10 2 to 5x104 cells as described herein per 1 mg of mineral particle. [039] In another embodiment, a composition consisting particles and cells include 2% to 60% v/v cells as described herein. In another embodiment, a composition consisting particles and cells include 20% to 60% v/v cells as described herein. In another embodiment, a composition consisting particles and cells include 2% to 10% v/v cells as described herein. In another embodiment, a composition consisting particles and cells include 20% to 40% v/v cells as described herein. In another embodiment, a composition consisting particles and cells include 30% to 75% v/v cells as described herein. In another embodiment, a composition consisting particles and cells include 40% to 80% v/v cells as described herein.

Bio tic components

[040] In another embodiment, the invention provides that the composition further comprises albumin. In another embodiment, the invention provides that the composition further comprises an extra-cellular matrix (ECM) protein. In another embodiment, the invention provides that the composition further comprises fibrin. In another embodiment, the invention provides that the composition further comprises fibronectin. In another embodiment, the invention provides that the composition further comprises collagen type I. In another embodiment, the invention provides that the composition further comprises laminin. In another embodiment, the invention provides that the composition further comprises vitronectin.

[041] In another embodiment, the invention provides that the composition further comprises a bone morphogenetic protein. In another embodiment, the invention provides that the composition further comprises insulin like growth factor. In another embodiment, the invention provides that the composition further comprises interleukin-1, interleukin-6, a tumor necrosis factor, RANKL, or any combination thereof. In another embodiment, a composition includes an autologous multicellular 3D cell culture suspended in Human Serum Albumin (HSA) containing medium. In another embodiment, a composition as described herein further comprises an anti-inflammatory agent. In another embodiment, a composition as described herein further comprises an antibiotic.

[042] In another embodiment, the invention provides that the composition further comprises a biocompatible binder. In another embodiment, the biocompatible binder are one or more selected from the group consisting of fibrin adhesive, fibrinogen, thrombin, mussel adhesive protein, silk, elastin, collagen, casein, gellatin, albumin, keratin, chitin and chitosan. In another embodiment, the biocompatible binder are one or more selected from the group consisting of starch, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polydioxanone, polycaprolactone, polycarbonate, polyoxoester, polyamino acid, poly- anhydride, polyhydroxybutylate, polyhydroxyvalerate, poly(propylene glycol -co -fumaric acid), tyrosine-based-polycarbonate, polyvinylpyrrolidone, cellulose, ethyl cellulose and carboxy methyl cellulose.

[043] In another embodiment, the invention provides that the composition further comprises vitamins. In another embodiment, the invention provides that the composition further comprises a glucosamine. In another embodiment, the invention provides that the composition further comprises a cytokine. In another embodiment, the invention provides that the composition further comprises growth factors.

Biocompatible polymer for suspending cells

[044] In another embodiment, the invention provides that the composition comprises mineral particles and cells deposited or attached to the mineral particles wherein the particles carrying the cells are suspended in cell culture media comprising biocompatible polymer, or a mixture of cell culture media and biocompatible polymer. The biocompatible polymers can be a natural polymer or a synthetic polymer. The phrase "synthetic polymer" refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, and combinations thereof.

[045] Naturally occurring biocompatible polymerinclude include, but are not limited to, fibrinogen, fibrin, thrombin, chitosan, collagen, alginate, poly(N-isopropylacrylamide), hyaluronate, albumin, collagen, synthetic polyamino acids, prolamines, polysaccharides such as alginate, heparin, and other naturally occurring biodegradable polymers of sugar units. In another embodiment, ionic hydrogels may be used, for example, ionic polysaccharides, such as alginates or chitosan. Ionic hydrogels may be produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with ions, such as calcium cations. [046] In another embodiment, biocompatible polymer for suspending cells deposited or attached to the mineral particles, is a water-soluble. In another embodiment, biocompatible polymer for suspending cells deposited or attached to the mineral particles is a gel.

[047] In another embodiment, the biocompatible polymer is characterized as a viscous substance. Viscous materials resist shear flow and strain linearly with time when a stress is applied. The viscosity of a solution comprising biocompatible polymer can be higher by 10 %, by 20 %, by 50 %, by 80 %, or by 100 % of that of an aqueous medium not comprising biocompatible polymer, and can be even 3-folds, 4-folds, 5-folds, 10-folds, 20-folds, 30- folds, 50-folds, 60-folds, 60-folds and even 100-folds or 1000-folds higher.

[048] Exemplary biocompatible polymers that are suitable for use in the context of embodiments of the present invention are polysaccharides. The term "polysaccharide" as used herein is meant to include compounds composed of 10 saccharide units and up to hundreds and even thousands of monosaccharide units per molecule, which are held together by glycoside bonds and range in their molecular weights from around 5,000 and up to millions of Daltons.

[049] In another embodiment, the polysaccharide is a glycosaminoglycan (GAG). In another embodiment, the GAG is Hyaluronic acid (HA), as described hereinbelow. HA is a high molecular weight unsulfated glycosaminoglycan (GAG), composed of repeating disaccharide units composed of (P-l,4)-linked D-glucuronic acid and (P-l,3)-linked N-acetyl- D-glucosamine.

[050] Additional suitable polysaccharides include, but are not limited to, agar, alginate, laminarin and pectin, as long as these polymers exhibit the desired physical characteristic.

[051] In another embodiment, the biocompatible polymer comprises at least 50% porosity. In another embodiment, the biocompatible polymer comprises at least 60% porosity. In another embodiment, the biocompatible polymer comprises at least 70% porosity. In another embodiment, the biocompatible polymer comprises at least 75% porosity. In another embodiment, the biocompatible polymer comprises at least 80% porosity. In another embodiment, the biocompatible polymer comprises at least 85% porosity. In another embodiment, the biocompatible polymer comprises at least 90% porosity. In another embodiment, the biocompatible polymer comprises at least 92% porosity. In another embodiment, a porous sponge comprises at least 95% porosity. [052] In another embodiment, the biocompatible polymer comprises pores having a diameter of at least 100 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of at least 120 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of at least 150 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 100-900 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 120-900 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 120-850 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 150-800 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 200-800 μηι. In another embodiment, the biocompatible polymer comprises pores having a diameter of 220-750 μηι.

[053] In another embodiment, the porosity of the scaffold (e.g., comprising the mineral particle) is controlled by a variety of techniques known to those skilled in the art. In another embodiment, as the porosity is increased, use of polymers having a higher modulus, addition of suffer polymers as a co-polymer or mixture, or an increase in the cross-link density of the polymer are used to increase the stability of the scaffold with respect to cellular contraction.

[054] In another embodiment, the choice of polymer and the ratio of polymers in a co-polymer scaffold of the invention is adjusted to optimize the stiffness/porosity of the scaffold. In another embodiment, the molecular weight and cross-link density of the scaffold is regulated to control both the mechanical properties of the scaffold and the degradation rate (for degradable scaffolds). In another embodiment, the mechanical properties are optimized to mimic those of the tissue at the implant site. In another embodiment, the shape and size of the final scaffold are adapted for the implant site. In another embodiment, scaffold materials comprise natural or synthetic organic polymers that can be gelled, or polymerized or solidified (e.g., by aggregation, coagulation, hydrophobic interactions, or cross-linking) into a 3-D open-lattice structure that entraps water and/or other molecules, e.g., to form a hydrogel.

[055] In another embodiment, the choice of polymer and the ratio of polymers in a copolymer scaffold of the invention is adjusted to optimize the stiffness/porosity of the scaffold. In another embodiment, the molecular weight and cross-link density of the scaffold is regulated to control both the mechanical properties of the scaffold and the degradation rate (for degradable scaffolds). In another embodiment, the mechanical properties are optimized to mimic those of the tissue at the implant site. In another embodiment, the shape and size of the final scaffold are adapted for the implant site. In another embodiment, scaffold materials comprise natural or synthetic organic polymers that can be gelled, or polymerized or solidified (e.g., by aggregation, coagulation, hydrophobic interactions, or cross-linking) into a 3-D open-lattice structure that entraps water and/or other molecules, e.g., to form a hydrogel.

[056] In another embodiment, polymers used in scaffold material compositions are biocompatible, biodegradable and/or bioerodible and act as adhesive substrates for cells. In another embodiment, the structural scaffold materials are non-resorbing or nonbiodegradable polymers or materials. The phrase "non-biodegradable polymer", as used herein, refers to a polymer or polymers which at least substantially (i.e. more than 50%) do not degrade or erode in- vivo. The terms "non-biodegradable" and "non-resorbing" are equivalent and are used interchangeably herein.

[057] In another embodiment, the phrase "biodegradable polymer" as used herein, refers to a polymer or polymers which degrade in-vivo, and wherein erosion of the polymer or polymers over time occurs concurrent with or subsequent to release of cells/tissue. The terms "biodegradable" and "bioerodible" are equivalent and are used interchangeably herein.

Hyaluronic acid

[058] In another embodiment, the invention provides that the composition comprises mineral particles and cells deposited or attached to the mineral particles wherein the particles carrying the cells are suspended in cell culture media, hyaluronic acid, or a mixture of cell culture media and hyaluronic acid. In another embodiment, the term "hyaluronic acid (HA)" is synonymous with hyaluronan or sodium hyaluronate. In another embodiment, hyaluronic acid is within a composition comprising a physiological buffer. In another embodiment, hyaluronic acid has a molecular weight of 200,000 to 850,000 daltons. [059] The term "HA" is also meant to include its pure or salt form, and also all cross-linked, modified or hybrid forms of HA. Cross-linker agents for HA operating via the carboxylic groups or via the amine groups following HA deacetylation include, but are not limited to, aldehydes (e.g., Glutaraldehyde), Dialdehydes, Genipin, Cinnamic acid, or derivatives thereof. Hybrid forms of HA include, without limitation, a protein or a carbohydrate polymer with HA, such as, diphenylalanin HA, Albumin HA, Fibrinogen HA, fibrin HA, and Chitosan HA.

[060] In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 0.5 mg to 50 mg Hyaluronic acid per 1 mL of solution (comprising a buffer). In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 0.5 mg to 5 mg Hyaluronic acid per 1 mL of solution (comprising a buffer). In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 5 mg to 20 mg Hyaluronic acid per 1 mL of solution (comprising a buffer). In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 10 mg to 30 mg Hyaluronic acid per 1 mL of solution (comprising a buffer). In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 10 mg to 25 mg Hyaluronic acid per 1 mL of solution (comprising a buffer). In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 0.05% to 5% by weight Hyaluronic acid. In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 0.1% to 1% by weight Hyaluronic acid. In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a composition comprising from 0.1% to 0.5% by weight Hyaluronic acid.

[061] In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a solution. In another embodiment, Hyaluronic acid composition for suspending cells deposited or attached to the mineral particles is a gel. The carrier

[062] In another embodiment, provided herein a bone-repair composition comprising a mineral particle. In another embodiment, mineral particles are in the form of a pulverized composition. In another embodiment, mineral particles are in the form of a micro -pulverized composition. In another embodiment, mineral particles comprise edges and grooves which provide more cell attachment sites.

[063] In another embodiment, a mineral particle is a bone fiber. In another embodiment, a bone fiber of the invention has enhanced cell -binding surface. In another embodiment, a bone fiber of the invention is derived from a bone tissue. In another embodiment, a bone tissue is cut along its length or along the grain direction of the bone tissue to form a bone fiber.

[064] In another embodiment, a mineral particle is a bone scaffold carrying a 3D cell culture. In another embodiment, a mineral particle is a bone mineral particle. In another embodiment, a mineral particle consists minerals. In another embodiment, a mineral particle comprises calcium phosphate. In another embodiment, a mineral particle comprises a calcium phosphate derivative. In another embodiment, a mineral particle comprises calcium sulfate. In another embodiment, a mineral particle comprises a calcium sulfate derivative. In another embodiment, a mineral particle comprises calcium hydroxyapatite. In another embodiment, a mineral particle comprises a silicate. In another embodiment, a mineral particle comprises a calcium sulfate derivative. In another embodiment, a mineral particle comprises a silicate mineral hydroxyapatite. In another embodiment, a mineral particle comprises beta-3 c alcium p hosphate. In another embodiment, a mineral particle comprises any combination of minerals known to one of skill in the art.

[065] In another embodiment, a mineral particle has a diameter of at least 50 microns. In another embodiment, a mineral particle has a diameter of at least 100 microns. In another embodiment, a mineral particle has a diameter in the range of 50 microns to 2000 microns. In another embodiment, a mineral particle has a diameter in the range of 100 microns to 1000 microns. In another embodiment, a mineral particle has a diameter in the range of 200 microns to 2000 microns.

[066] In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of at least a day. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of at least two days. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of 2 to 21 days. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of 5 to 21 days. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of 6 to 16 days. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of 2 to 16 days. In another embodiment, a composition comprising particles and cells is grown and/or maintained with cell culture media for a period of 8 to 14 days.

Kit

[067] In another embodiment, provided herein a kit comprising: a syringe, a mineral particle comprising endothelial cells and mesenchymal cells and/or osteoprogenitor cells organized in 2 or more cell layers attached thereto, and hyaluronic acid. In another embodiment, provided herein a kit comprising: a syringe, a mineral particle comprising Adipose Tissue derived Cells organized in 2 or more cell layers attached thereto, and hyaluronic acid. In another embodiment, provided herein a kit comprising: a syringe, a mineral particle comprising a 3D cell culture attached thereto, and hyaluronic acid. In another embodiment, provided herein a kit comprising: a syringe, a suspension comprising: mineral particles comprising a 3D cell culture attached thereto suspended in cell culture media, and hyaluronic acid. In another embodiment, a kit as described herein further comprises a composition comprising an antiinflammatory agent.

[068] In another embodiment, the pharmaceutical composition for filing a gap within a bone is produced by simply mixing hyaluronic acid and mineral particles comprising a 3D cell culture attached thereto. In another embodiment, the pharmaceutical composition for filing a gap within a bone is produced by simply mixing hyaluronic acid and a suspension comprising: mineral particles comprising a 3D cell culture attached thereto suspended in cell culture media.

[069] In another embodiment, a kit for filing a gap within a bone, comprises a first part that contains an effective amount of hyaluronic acid, and a second part that contains an effective amount of a suspension comprising: mineral particles comprising a 3D cell culture attached thereto suspended in cell culture media. In another embodiment, the kit is for injection, and the first and second parts can be in solution form and are separately placed in independent packs (such as plastic bottles or glass bottles like ampoules). In another embodiment, each pack can comprise multiple dosages, but preferably a single dosage, of the first or second part. In another embodiment, prior to injection, the two parts are put into the injection syringe according to the information in the instruction (comprising the information such as the operation method of the kit, the mixing ratio of the solutions, etc.) to apply the formulation. In another embodiment, prior to injection, the two parts are put into a mixing means inside or outside the syringe. In another embodiment, prior to injection, the two parts are mixed by a mixing means inside or outside the syringe.

[070] The proposed product contains 20-60% v/v Adipose Tissue derived Cells seeded on bone mineral particles for about 3-14 days.

[071] The proposed product is based on a previous study which was based on the same materials; the difference is that the cells were not cultivated on the scaffold prior

transplantation. The next section will detail results obtained in this study.

Treatment

[072] In another embodiment, provided herein a method for filling a gap in a bone of a subject in need thereof, comprising the step of contacting the gap with a composition comprising: (a) a mineral particle, (b) endothelial cells and mesenchymal cells, and/or osteoprogenitor cells, and (3) hyaluronic acid, thereby filling a gap in a bone of a subject in need thereof. In another embodiment, provided herein a method for filling a gap in a bone of a subject in need thereof, comprising the step of contacting the gap with a composition comprising: a suspension comprising: mineral particles comprising a 3D cell culture attached thereto suspended in cell culture media and mixed in hyaluronic acid. In another embodiment, provided herein a method for filling a gap in a bone of a subject in need thereof, comprising the step of contacting the gap with a composition comprising: a suspension comprising: mineral particles comprising a 3D cell culture attached thereto suspended in hyaluronic acid. In another embodiment, contacting the gap is filling the gap with a composition as described herein. In another embodiment, contacting the gap is injecting a composition as described herein into the gap. In another embodiment, after contacting the gap and/or injecting a composition as described herein into the gap, the gap is further sealed. In another embodiment, composition for sealing a gap in a bone are known to one of average skill in the art. [073] In another embodiment, a composition of the invention suspended in hyaluronic acid is prepared for application directly on location. In another embodiment, a composition of the invention suspended in hyaluronic acid is injected under radiological control (such as but not limited to x-ray control). In another embodiment, a composition of the invention suspended in hyaluronic acid is injected into the center of the bone shaft.

[074] In another embodiment, a gap in a bone is the result of a fracture. In another embodiment, the method of the invention is concerned with expediting bone repair and/or de- novo bone formation within a gap. In another embodiment, the method of the invention is concerned with repairing the periosteum. In another embodiment, the method of the invention is concerned with a vascular damage in proximity to the gap. In another embodiment, the method of the invention is concerned with inhibiting necrosis in the site of bone fracture or gap and inducing bone and vascular repair. In another embodiment, the method of the invention provides for bridging the fracture gap. In another embodiment, the method of the invention is directed to reinforcing and/or adding bone material to a trabecular zone.

[075] In another embodiment, the method of the invention further provides minimizing motion by internal or external fixation. In another embodiment, the method of the invention further provides rigid fixation where there is absolutely no motion at the fracture site. In another embodiment, the method of the invention further provides bone gap healing under rigid fixation. In another embodiment, the method of the invention includes filling of the fracture gap with a composition as described herein.

[076] In another embodiment, the method of the invention further provides repairing a fracture within a bone. In another embodiment, the method of the invention further provides repairing a gap within a bone. In another embodiment, the method of the invention further provides repairing a gap within a fractured bone. In another embodiment, the term "gap" is interchangeable with lesion. In another embodiment, a gap is at least 0.05 mm wide. In another embodiment, a gap is from 0.05 to 5 mm wide. In another embodiment, a gap is from 0.1 to 1 mm wide. In another embodiment, a gap is at least 0.05 mm deep. In another embodiment, a gap is from 0.05 to 20 mm deep. In another embodiment, a gap is from 0.1 to 15 mm deep. In another embodiment, a gap is from 0.5 to 10 mm deep. In another embodiment, a gap is from 0.5 to 5 mm deep.

[077] In another embodiment, a gap is from 5 mm to 10 cm wide. In another embodiment, a gap is from 5 mm to 0.1 cm wide. In another embodiment, a gap is from 50 mm to 10 cm wide. In another embodiment, a gap is from 1 to 10 cm wide. In another embodiment, a gap is from 5 to 10 cm wide. . In another embodiment, a gap is from 10 to 80 mm deep. . In another embodiment, a gap is from 1 to 50 mm deep. . In another embodiment, a gap is from 50 mm to 1 cm deep.

[078] In another embodiment, the present invention provides a method that enhances, induces, and/or increases bone repair and restoration in a fractured bone as described herein. In another embodiment, the present invention restores bone density at a site formerly characterized by a gap. In another embodiment, the present invention restores the joint surface at the gap site.

[079] In another embodiment, the present invention provides a method that is used for all kinds of bone fracture, bone-necrosis disease or bone repair. In another embodiment, the present invention provides a method wherein the bone-repair composition is directly injected into the region of bone loss/gap.

[080] In another embodiment, the compositions of the invention comprise liquid solutions, emulsions, suspensions, gels and the like. In some embodiments, pharmaceutically- acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, injectable compositions, of the invention are formulated in aqueous solutions, gels or suspensions. In one embodiment, injectable compositions of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[081] In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.

[082] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active composition. In one embodiment, the pack, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

Process of making the bone repair composition

[083] The present invention also provides, in at least some embodiments, a method of producing the prosthetic implant described above, the method comprising the steps of isolation, expansion and co-cultivation of at least two types of cells onto mineral particles.

[084] In another embodiment, each type of cells is first cultivated and expanded separately. Next the various types of cells are cultivated and co-expanded ex vivo under sterile conditions on the mineral particles, using conventional culture medium, such as DMEM, RPMI, with supplements of human serum (from autologous or allogeneic sources) or animal serum, or in serum-free media that allows the attachment and growth of adherent cells. In some embodiments, culture medium that supported the initial growth and expansion phase of these cells may optionally be replaced by another cell culture formula that supports the differentiation of these cells and bone formation.

[085] In another embodiment, cells are expanded and co-cultivated in a dedicated bioreactor system. In another embodiment, a dynamic flow system, such as a bioreactor for example, stimulates optimal cell density and cell viability of a 3D cell culture.

[086] In another embodiment, the bioreactor is described in detail in U.S. Pat. No. 6,911,201, which is incorporated by reference as if fully set forth herein. In this patent, a plug flow bioreactor system which allows the growth and prolonged maintenance of high density cell cultures that closely mimics the bone marrow microenvironment. In another embodiment, the bioreactor comprises a medium reservoir; gas mixture container; gas filters; injection points; plugs or containers of various sized plugs containing the mineral particles; flow monitors; flow valves; conditioned medium collecting and separating container; container for medium exchange; peristaltic pump; sampling point; container for medium exchange; monitor; steering device; and a pH probe. In another embodiment, a bioreactor that can be used according to the invention is described in United States Patent Publication No. US20120003185 which is hereby incorporated by reference in its entirety. In another embodiment, a dynamic flow bioreactor ensures the growth, longevity and differentiation of cell within the 3D cell culture.

[087] In another embodiment, a 3D heterogeneous cell culture grown on a mineral particle is obtained by subjecting the cells attached to the mineral particles to flow- through bioreactor system. In another embodiment, the growth medium (cell media) is supplemented with growth factors and cytokines, such as, for example, one or more of: transforming growth factor beta (TGF beta), insulin-like growth factor- 1 (IGF-1), osteogenic protein-1 (OP-1), fibroblast growth factor (FGF) members like FGF-2, FGF-9 and FGF- 10 and members of bone morphogenic proteins (BMP) especially BMP-2, BMP-4 and BMP-7.

[088] In another embodiment, a mineral particle covered by a 3D heterogeneous cell culture is transplanted into a pre-determined site of bone loss or gap.

[089] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[090] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

EXAMPLE 1

In vivo studies using first generation Adipose Tissue derived Cells based product Intra femoral in jection rat model

[091] Adipose Tissue derived Cells were evaluated for their ability to form new bone after femoral injection into the femur shaft. An injectable formulation containing the following components was prepared; the ingredients were 20% PBS containing Adipose Tissue derived Cells and lug/ml rhBMP-2 (R&D Systems, US), 20mg OraGraft mineral particles (freeze - dried bone allograft obtained from human bone, LifeNet Health, Inc. VA, USA), 60% hyaluronic acid (Bio-Technology General LTD, Israel). Sprague-Dawley rats were anaesthetized. Under aseptic conditions, a longitudinal incision was made over the knee. Skin and muscles were held aside to expose the femoral condyle. Using a trephine bit drill a hole was drilled into the inter-condylar notch and through the trabecular area of the femur into the femoral shaft. Flushing of the rat's bone marrow with saline was done using an 18 gauge needle until no bone marrow was left. 150 microlitters of the injection mixture was injected to fill the shaft. The hole in the condyle was blocked using bone wax and the muscles and skin were sutured. All animals recovered well from the surgery with no visible restrictions in their movements. Animals were weighed before and at the end of the experiment and no body weight loss was observed as a result of the surgery.

[092] The experiment included two time points, 8 and 16 weeks. At first we evaluated the effect of 3 or 5xl0 ⁶ Adipose Tissue derived Cells per leg in comparison to non-treated leg and to vehicle group containing no cells (control). At 8 weeks, mechanical testing using the 4 point bending method was used. The results clearly demonstrated that femurs treated with Adipose derived cells endured more mechanical force until they broke as compared to untreated leg. The difference was not significant (Figure 1).

[093] At 16 weeks (the end of the study), treated femurs were subjected to microCT scan and analysis. The analysis was done on three areas of the femur as illustrated in the image bellow: full bone, the metaphyseal trabecular bone and a cortical ring in the diaphysis (Figure 2).

[094] Scan and 3D images of treated legs clearly show bone fill-up with dense material. Bone fill-up was seen in legs treated with Scaffold (mineral particle) and 3 or 5 million cells and minimally seen in Scaffold only treated legs (Figure 3).

[095] MicroCT analysis revealed that on an organ level (full compartment), the bone volume density (AVD) of the Scaffold (mineral particle) and Adipose Tissue derived Cells (5 and 3 million cells) groups were higher than AVD value in the Scaffold only group.

[096] Furthermore, the AVD value in the 5 million Adipose Tissue derived Cells group was higher than the AVD value in the 3 million Adipose Tissue derived Cells group. However, the difference between the groups was not significant (Figure 4).

[097] On a micro -structural level it was found that in the trabecular region (TRAB) the scaffold only group had significantly lower BS/TV value, significantly higher trabecular separation (Tb.Sp) value and significantly lower trabecular number (Tb.N) value compared to the scaffold (mineral particle) and Adipose Tissue derived Cells (3 or 5 million) groups. Additionally, connectivity density (Conn.D) was lower in Scaffold only group compared to Scaffold (mineral particle) and 3 or 5 million Adipose Tissue derived Cells groups and BV/TV values were higher in scaffold (mineral particle) and Adipose Tissue derived Cells (3 or 5 million) groups compared to scaffold only group (Figure 5). [098] In the cortex region, the cortical thickness (Ct. Th) values of scaffold (mineral particle) and Adipose Tissue derived Cells (5 and 3 million cells groups) were higher than the values of the scaffold (mineral particle) only group. The difference between the scaffold (mineral particle) and 5xl0 ⁶ group and the Scaffold only group was significant.

[099] Bone volume (BV) values were higher in the scaffold (mineral particle) Adipose Tissue derived Cells (5 and 3 million cells) groups compared to scaffold only group (Figure 6).

[0100] Femurs used for microCT and mechanical bending tests were taken to histological processing and analysis. Femurs sections were stained with picro serious red stain to highlight new formed bone (Figure 7A, B). The histological sections were subjected to histomorphometry and measurements are shown and summarized in Figure 7, Histomorphometry data reveals that the amount of new formed bone is with concordance to cell number and to the time from transplantation. Scaffold (30 mg of mineral particle) and 5xl0 ⁶ cells group surpassed Scaffold (30 mg of mineral particle) and 3xl0 ⁶ cells group and significantly surpassed the Scaffold only group in terms of the amount of new bone formed. Moreover, it can be seen that after 16 weeks the amount of grown bone was higher than of the other two groups.

Second generation Adipose Tissue derived Cells based product

[0101] As previously detailed the proposed product is based on bone mineral particles seeded with multicell Adipose Tissue derived Cells and suspended in hyaluronic acid.

Cell attachment and cultivation on bone mineral particles

[0102] 2.5xl0 ⁵ Cells were suspended in ΙΟΟμΙ medium and carefully added to 30mg of bone mineral particles, in ventilated tubes (TRP, Innovation in plastic, Switzerland). The tubes were incubated for lh, in 37°C to allow cell adhesion to the construct. Additional culture medium (2.9 mL) was then added to the tube. Following predetermined time points a quantitative analysis of cell attachment was conducted by nucleus cell counting. Cells were further stained with Gimsa in order to evaluate cell morphology and seeding homogeneity.

[0103] Results show that above 90% of the Adipose Tissue derived Cells attached to two different types of scaffolds after only 2 hours. Further incubation did not improve cell attachment. Gimsa stain showed that the cells spread and were homogenously distributed (Figure 8). [0104] Further cultivation in 50ml ventilated tubes showed that cells maintain their viability and proliferate. Furthermore, cells generated multi cellular layers onto the mineral particles (Figure 9).

[0105] Histological and scanning electron microscope analysis shows the formation of multicellular layers on top of the bone mineral particles. These layers generate a typical connective tissue structure containing collagen fibers and polysaccharides as seen in Picro Sirius Red stain and Toluidine Blue stain, respectively (Figure 10).

[0106] Both Adipose derived endothelial cells and mesenchymal stem cells were seeded as a co-culture on the scaffold. For evaluation of endothelial cell functionality, seeded particles were placed in Matrigel for predetermined time points. Adipose Tissue derived endothelial cells were shown to be able to attach to the Matrigel and to form sprouts out of the scaffold and out to the surrounding (Figure 11).