BONE TISSUE ENGINEERING BY EX VIVO STEM CELLS ONGROWTH INTO THREE-DIMENSIONAL TRABECULAR METAL

Abstract

Adult autologous stem cells cultured on a porous, three-dimensional tissue scaffold-implant for bone regeneration by the use of a hyaluronan and/or dexamethasone to accelerate bone healing alone or in combination with recombinant growth factors or transfected osteogenic genes. The scaffold-implant may be machined into a custom-shaped three-dimensional cell culture system for support of cell growth, reservoir for peptides, recombinant growth factors, cytokines and antineoplastic drugs in the presence of a hyaluronan and/or dexamethasone alone or in combination with growth factors or transfected osteogenic genes, to be assembled ex vivo in a tissue incubator for implantation into bone tissue.

Claims

1. (canceled)

2. A method of generating tissue, comprising: providing or obtaining a tissue scaffold implant comprising a shaped, porous three-dimensional tissue scaffold with an inert biocompatible metal film present on surfaces of the porous three-dimensional tissue scaffold and with living cells in pores of the porous three-dimensional tissue scaffold, said porous three-dimensional tissue scaffold having an interconnected porosity for facilitating nutrient diffusion and media circulation throughout the porous three-dimensional tissue scaffold; and placing said tissue scaffold implant in an ex-vivo bioreactor for generating tissue on said porous three-dimensional tissue scaffold.

3. The method of claim 2, wherein said tissue scaffold implant is shaped and sized as an acetabular cup implant.

4. The method of claim 2, wherein said porous three-dimensional tissue scaffold has a porosity of 50% to 90%.

5. The method of claim 2, wherein said inert biocompatible metal film is a chemical vapor deposited metal film.

6. The method of claim 2, wherein said living cells comprise mesenchymal stem cells.

7. The method of claim 2 further comprising at least one substance on surfaces of the inert biocompatible metal film that is present on surfaces of the porous three-dimensional tissue scaffold with said living cells on said at least one substance, said at least one substance selected from the group consisting of a hyaluronan, dexamethasone, protein, peptide, transcript factor, cytokine, therapeutic agent, chitosan, polymer, osteogenic gene and growth factor.

8. The method of claim 2, wherein said porous three-dimensional tissue scaffold is a porous three-dimensional metallic tissue scaffold.

9. The method of claim 2 further comprising at least one substance having been applied ex-vivo with said living cells, said at least one substance selected from the group consisting of a hyaluronan, dexamethasone, protein, peptide, transcript factor, cytokine, therapeutic agent, chitosan, polymer, osteogenic gene and growth factor.

10. A tissue scaffold implant for implantation in a patient, comprising: a shaped, porous three-dimensional tissue scaffold that is effective to receive tissue ingrowth upon implantation in a patient; an inert biocompatible metal film present on surfaces of the porous three-dimensional tissue scaffold; and ex-vivo applied, living cells in pores of the porous three-dimensional tissue scaffold.

11. The tissue scaffold implant of claim 10 shaped and sized as an acetabular cup implant.

12. The tissue scaffold implant of claim 10, wherein said inert biocompatible metal film is a chemical vapor deposited metal film.

13. The tissue scaffold implant of claim 10, wherein said living cells comprise mesenchymal stem cells.

14. The tissue scaffold implant of claim 10, wherein said porous three-dimensional tissue scaffold has a porosity of 50% to 90%.

15. The tissue scaffold implant of claim 10 further comprising at least one substance on surfaces of the inert biocompatible metal film that is present on surfaces of the porous three-dimensional tissue scaffold with said living cells on said at least one substance, said at least one substance selected from the group consisting of a hyaluronan, dexamethasone, protein, peptide, transcript factor, cytokine, therapeutic agent, chitosan, polymer, osteogenic gene and growth factor.

16. The tissue scaffold implant of claim 10, wherein said porous three-dimensional tissue scaffold is a porous three-dimensional metallic tissue scaffold.

17. The tissue scaffold implant of claim 10 further comprising at least one substance having been applied ex-vivo with said living cells, said at least one substance selected from the group consisting of a hyaluronan, dexamethasone, protein, peptide, transcript factor, cytokine, therapeutic agent, chitosan, polymer, osteogenic gene and growth factor.

18. A tissue scaffold implant for implantation in a patient, comprising: a shaped, porous three-dimensional tissue scaffold fabricated as a single implant piece and effective to receive tissue ingrowth upon implantation in a patient, said porous three-dimensional tissue scaffold having an interconnected porosity for facilitating nutrient diffusion and media circulation throughout the porous three-dimensional tissue scaffold; and ex-vivo cultured tissue on the porous three-dimensional tissue scaffold.

19. The tissue scaffold implant of claim 18 further comprising an inert biocompatible metal film present on surfaces of the porous three-dimensional tissue scaffold.

20. The tissue scaffold implant of claim 19, wherein said inert biocompatible metal film is a chemical vapor deposited metal film.

21. The tissue scaffold implant of claim 18, wherein the tissue scaffold implant is shaped and sized as an acetabular cup implant.

22. A method of implantation, comprising: providing or obtaining a tissue scaffold implant that is effective to receive tissue ingrowth upon implantation in a patient, said tissue scaffold implant comprising a shaped, porous three-dimensional tissue scaffold with ex-vivo cultured tissue on the porous three-dimensional tissue scaffold; and implanting said tissue scaffold implant in a patient.

23. The method of claim 22, wherein tissue scaffold implant further comprises an inert biocompatible metal film present on surfaces of the porous three-dimensional tissue scaffold.

24. The method of claim 23, wherein said inert biocompatible metal film is a chemical vapor deposited metal film.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a flow chart depicting an embodiment of the method of the present invention.

[0017] FIG. 2 is a bar graph quantifying stern cells binding to a coralline hydroxyapatite disc and uncoated and coated porous tantalum discs, after a 24-hour incubation at 37° C. and normalized to the uncoated TA disc,

[0018] FIGS. 3A and 3B are Hoechst stained fluorescent micrographs at 50× original magnification and 20× original magnification respectively, showing the growing stem cells in the pores of porous tantalum after 7 days of incubation.

[0019] FIGS. 4A and 4B are histological micrographs at 20× original magnification and 6.25× original magnification respectively, after 8 weeks of implantation in pigs.

[0020] FIG. 5 is a scanning electronic micrograph of the three-dimensional tissue scaffold-implant.

[0021] FIG. 6 is a histological micrograph of the three-dimensional tissue scaffold-implant after 12 weeks of implantation in a pig.

DETAILED DESCRIPTION

[0022] The tissue formation method of the present invention utilizes an ex vivo cell culture system and a porous, three-dimensional metallic structure or tissue scaffold of a desired shape and size, which will be implanted into the body of an animal or human being (hereinafter scaffold-implant). The cell culture system induces early stage cell proliferation and differentiation on and in the tissue scaffold-implant, resulting in tissue generation. The tissue formation method of the present invention is especially useful for generating bone tissue. The method of the invention may also be used to generate connective tissue and hematopoietic tissue.

[0023] The flow chart of FIG. 1 depicts an embodiment of the tissue formation method of the present invention. In step 10 of the method, a porous, three-dimensional tissue scaffold-implant is fabricated in a desired shape and size, e.g., hip implant, spinal implant, knee implant, etc. For example, the scaffold-implant may be shaped and sized as a prosethetic acetabular cup such as the one disclosed in U.S. Pat. No. 5,443,519 entitled “Prosthetic Ellipsoidal Acetabular Cup,” issued to Averill et al. In another example, the scaffold-implant may be shaped and sized as a prosethetic femoral component such as the one disclosed in U.S. Pat. No. 5,702, 487 entitled “Prosethetic Device” issued to Averill et al. in one embodiment, the scaffold-implant may be fabricated as a single unitary member. In an alternative embodiment, the scaffold-implant may be fabricated as a single, integral member formed by two or more separately fabricated sections which are mechanically assembled together in a conventional manner. In still another embodiment, the scaffold-implant may be fabricated as an assembly of two or more cooperating, unitary and/or integral members (e.g., acetabular cup and femoral stem/ball assembly).

[0024] The porous, three-dimensional tissue scaffold-implant may comprise a carbon lattice having a strut or ligament skeleton which forms a three-dimensional network of continuously interconnected channels or pores each roughly approximating a dodecahedron, which create a series of continuous microniches and form surfaces of the lattice in three dimensions; and a thin film of an inert, bio-compatible metal or other bio-compatible material, which covers the surfaces.

[0025] The carbon lattice may be formed as a single, unitary member of a desired shape and size, or in sections of desired shapes and sizes to be mechanically assembled. The carbon lattice is substantially rigid, therefore, it may be machined into a bone regeneration tool of a desired shape and size using conventional machining methods.

[0026] The inert, bio-compatible metal or other bio-compatible material may be applied to the surfaces of the carbon lattice using conventional vapor depositing and infiltrating methods. In a preferred embodiment, the inert, biocompatible metal comprises tantalum. In other embodiments, the inert, biocompatible metal may comprise niobium or alloys of tantalum and niobium.

[0027] The completed porous, three-dimensional tissue scaffold-implant forms a three-dimensional network of continuously interconnected, channels or pores which define a three-dimensional porosity (volume porosity). In one embodiment, the tissue scaffold implant may comprise channels or pores having an average diameter of 400 to 500 μm and a volume porosity ranging from about 50 to about 90%. The geometry of the interconnected pores and surface texturing arising from the metal vapor deposition process produce high surface area-to-volume ratio.

[0028] The large pores and surfaces allow attachment of proteins, peptides and differentiated and undifferentiated cells. After fabrication, the scaffold-implant may be coated with substrate molecules such as fibronectin and collagens which aid in the attachment of the proteins, peptides and differentiated and undifferentiated cells.

[0029] In step 20, a hyaluronan (also referred to as hyaluronic acid or sodium hyaluronate) or a hyaluronan, dexamethasone, one or more growth factors and/or osteogenic genes is applied to the surfaces of the tissue scaffold-implant to stimulate early cell proliferation and differentiation, therefore accelerating tissue generation, Sodium hyaluronate is a natural high-viscosity anionic mucopolysaccharide with alternating beta (1-3) glucuronide and beta (1-4) glucosaminidic bonds. It is commonly found in the umbilical cord, in vitreous humor, in synovial fluid, in pathologic joints, in group A and C hemolytic streptococci, and in Wharton's jelly. Dexamethasone is a synthetic steroid compound. In one embodiment, the tissue scaffold-implant may be treated with a low concentration (4 mg/mL) of sodium hyaluronate to induce in-vitro, early stage stern cell proliferation and differentiation on and in the tissue scaffold-implant (after performing steps 30 and 40 to be described further on). In another embodiment, the tissue scaffold-implant may be treated with a high concentration (10-20 mg/mL) of sodium hyaluronate which forms a hydro gel with the stem cells in the tissue scaffold-implant intraoperatively.

[0030] In step 30 of the method, a cell transplantation process is performed on the porous, three-dimensional tissue scaffold-implant. In an embodiment of the cell transplantation process, the tissue scaffold-implant is seeded with living cells, which may comprise differentiated, undifferentiated or gene transfected cells. Examples of differentiated or undifferentiated cells include without limitation bone marrow cells, osteoblasts, mesenchymal stem cells, embryonic stem cells, endothelial cells. In another embodiment of the cell transplantation process, the tissue scaffold-implant is seeded with living cells and proteins, peptides, transcript factors, osteogenic genes, cytokines, therapeutic agents, and growth factors.

[0031] The living cells and other factors can be entrapped and delivered in the tissue scaffold-implant by means of a versatile self-assembly method. In this self-assembly method cellular matrix fibrils are formed with methylated collagen (type 1) and hyaluronic acid, or chitosan, which entrap and deliver living cells and other factors. The cellular matrix fibrils are then combined with an outer-layer membrane comprising a polymer such as alginate, hydroxylethyl methacrylate (HEMA), or a terpolymer of hydroxylethyl methacrylate (HEMA), methy methacrylate (MMA) and methylacric acid (MAA) by complex sandwich conjugation achieved, for example, using a complex coacervation process, to protect transplanted allogeneic cells from immune attacks and to sustain release of the stimulating factors and therapeutic agents. In one embodiment, the membrane may be several micrometers to about 100 micrometers thick. The thickness of the membrane may be adjusted by controlling the concentrations and contact time of polyelectrolytes in the complex sandwich conjugation process.

[0032] The surface features (the texture on the surface of the metal resulting from the CVD of the metal) and the open, highly interconnected pores of the tissue scaffold-implant readily facilitate nutrient diffusion and media circulation and thus will operate as conduits for cell infusion, adhesion, mass transfer, or to stimulate angiogenesis for blood flow.

[0033] In an alternate embodiment, the application of the hyaluronan or the hyaluronan, dexamethasone, one or more growth factors and/or osteogenic genes, to the surfaces of the tissue scaffold-implant (step 20) may be performed during the cell transplantation process of step 30.

[0034] In step 40, after seeding, the scaffold-implant is cultured in a bioreactor to generate the desired tissue. In one embodiment, the culturing step is an ex-vivo process. Ex-vivo culturing may be performed in a broth medium, e.g., Dulbecco's modified Eagle's medium (DMEM) available from HyClone, plus 10% fetal calf serum, which is placed in an incubator e.g., perfusion or spiner flask bioreactor or a rotating bioreactor. The broth medium and incubator operate as an in-vitro bioreactor. In one embodiment, the incubator may provide a humidified atmosphere of 95% air and 5% CO.sub.2 at 37° C. In addition, the incubator may be of the type which provides static, dynamic medium flow, pulsatile air flow, microgravity and multidirectional gravity culturing conditions. The scaffold-implant may then be implanted (in-vivo) into an animal or patient's body.

[0035] In another embodiment, the culturing step is an in-vivo process. In-vivo culturing may be performed in an animal or a patient by directly implanting the scaffold-implant in the animal or the patient. In this embodiment, the animal or the patient' body operates as an in-vivo bioreactor.

[0036] In still an alternate embodiment, the culturing step can be performed intraoperatively. In this embodiment, cells are taken from the animal or patient and applied to the scaffold-implant. The scaffold-implant is then implanted in the animal or patient.

[0037] FIG. 2 is a bar graph quantifying stem cells binding to 1) a coralline hydroxyapatite (HA) disc; 2) an uncoated porous, tantalum-based, three dimensional tissue scaffold-implant (TA) configured as a disc; 3) a TA disc coated with gelatin; 4) a TA disc coated with type I collagen; and 5) a TA disc coated with fibronectin, n=9 (repeated test), after a 24-hour incubation at 37° C. and normalized to the uncoated TA disc. In the graph, the stem cells are .sup.3H-thymidine labeling cells.

[0038] FIGS. 3A and 3B are fluorescent micrographs showing the growing cells in the pores of a porous, tantalum-based, three-dimensional tissue scaffold-implant configured as a disc after 7 days of incubation (Hoechst staining). As can be seen in FIG. 3A, at day 7, porcine bone marrow stem cells depicted funicular proliferations of spindle cells on the pore surface and within the pores. As shown in FIG. 3B, growing stem cells in the pores mainly distributed on the surface areas of disc (superior) where the cells were loaded on. Only a few stem cells had grown into the central pores and down to other surface areas of the disc (inferior) where the disc was seated on a well.

[0039] FIGS. 4A and 4B are histological micrographs which show, after 8 weeks of implantation in pigs, ectopic bone formation after autologous bone marrow stem cells cultured with a tantalum-based, three-dimensional tissue scaffold-implant for 7 days of incubation. Basic fuchsin and light green staining revealed the bone is green G and fibrous tissue is red R. The black structure B is porous tantalum strut. Specifically, FIG. 4A shows bone forming in the pore surface and the pores and FIG. 4B shows a layer of de novo bone formation at the surface area of the scaffold-implant.

[0040] FIG. 5 is a scanning electronic micrograph of the three-dimensional tissue scaffold-implant. As can he seen, the scaffold-implant has a volume porosity of about 50% to about 90% with interconnecting pores, allowing approximately 2-3 times greater bone ingrowth compared to conventional porous coatings.

[0041] FIG. 6 is a histological micrograph of the three-dimensional tissue scaffold-implant after 12 weeks of implantation in a pig. As can be observed, there is bone formation from intraoperative conjugation of autologous bone marrow stem cells and hyaluronic acid gel in the tissue scaffold-implant. Basic fuchsin and light green staining revealed the bone is green G and fibrous tissue is red R. The black structure B is porous tantalum strut.

[0042] While the foregoing invention has been described with reference to the above, various modifications and changes can be made without departing from the spirit of the invention. Accordingly, all such modifications and changes are considered to be within the scope of the appended claims.