Methods and compositions for the repair of articular cartilage defects in mammals

• US 5,786,217 A
• Filed: 04/14/1997
• Issued: 07/28/1998
• Est. Priority Date: 05/05/1994
• Status: Expired due to Fees

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Tubo, Ross A., Barone, Leesa M., Wrenn, Courtney A.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US08/834,150
• Time in Patent Office: 470 Days
• Field of Search: 435/402, 435/325, 435/383, 435/384
• US Class Current: 435/402
• CPC Class Codes: A61B 17/00491 Surgical glue applicators surgical adhesives A61L24/00; two-component delivery syringes A61M5/19
• CPC Class Codes: A61F 2/30756 Cartilage endoprostheses A61F2/3603 takes precedence
• CPC Class Codes: A61F 2/3094 Designing or manufacturing processes
• CPC Class Codes: A61F 2002/2817 Bone stimulation by chemical reactions or by osteogenic or biological products for enhancing ossification, e.g. by bone morphogenetic or morphogenic proteins [BMP] or by transforming growth factors [TGF]
• CPC Class Codes: A61F 2002/30125 elliptical or oval
• CPC Class Codes: A61F 2002/30762 Means for culturing cartilage
• CPC Class Codes: A61F 2002/30764 Cartilage harvest sites
• CPC Class Codes: A61F 2002/30971 Laminates, i.e. layered products
• CPC Class Codes: A61F 2230/0008 elliptical or oval
• CPC Class Codes: A61F 2240/001 Designing or manufacturing processes
• CPC Class Codes: A61F 2310/00365 Proteins; Polypeptides; Degradation products thereof
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• CPC Class Codes: A61L 27/3654 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Provided are methods and compositions for the repair of articular cartilage defects in a mammal. Denuded chondrogenic cells are proliferated ex vivo as monolayer cultures in order to expand the pool of available chondrogenic cells. During proliferation the chondrogenic cells stop secreting the extracellular matrix components, type II collagen and sulfated proteoglycans. The proliferated cells then are seeded into a pre-shaped well having a cell contacting, cell abhesive surface. The cells cultured in the well redifferentiate and begin to secrete cartilage-specific extracellular matrix again. Accordingly, essentially unlimited amounts of synthetic cartilage may be prepared from small samples of biopsy tissue. Also provided are methods for surgically repairing articular cartilage defects in mammals using the synthetic cartilage prepared in accordance with the invention.

Claims

1. A method for preparing a multi cell layered synthetic cartilage patch, the method comprising the steps of:

(a) proliferating denuded chondrogenic cells in vitro, wherein said cells are undifferentiated;

(b) seeding said cells into a pre-shaped well having a cell contacting surface which prevents attachment of the cells; and

(c) culturing said cells for a time sufficient for said cells to differentiate and form a multi cell-layered synthetic cartilage and to endogenously produce an extracellular matrix.

5. The method of claim 1, 2, 3, or 4, wherein said surface comprises a surface coated with silicon or polytetrafluoroethylene.

6. The method of claim 1, 2, 3, or 4, wherein said well is defined by a material selected from the group consisting of agarose, glass, untreated cell culture plastic and polytetrafluoroethylene.

7. The method of claim 1, 2, 3 or 4, wherein said chondrogenic cells are derived from human tissue.

8. The method of claim 1, 2, 3 or 4, wherein said chondrogenic cells are derived from cartilage.

9. The method of claim 8, wherein said cartilage is articular cartilage.

10. The method of claim 8, wherein said cartilage is selected from the group consisting of costal cartilage, nasal cartilage, auricular cartilage and cricoid cartilage.

11. The method of claim 1, 2, 3, or 4, wherein said extracellular matrix comprises type II collagen.

12. The method of claim 1, 2, 3, or 4, wherein said extracellular matrix comprises a proteoglycan.

13. The method of claim 12, wherein said proteoglycan comprises chondroitin-6-sulfate or keratan sulfate.

14. The method of claim 1, 2, 3, or 4, wherein said synthetic cartilage comprises synthetic articular cartilage.

15. The method of claim 1, 2, 3 or 4, wherein said well is dimensioned to produce synthetic cartilage that interfits with a mammalian cartilage defect.

16. The method of claim 1, further comprising an additional step of adding a polypeptide growth factor to the cells cultured in step (b).

20. The method of claim 16, 17, 18, or 19, wherein said growth factor is selected from the group consisting of transforming growth factor-β

, platelet derived growth factor, insulin-like growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, hepatocytic growth factor, keratinocyte growth factor, and bone morphogenic protein.

21. The method of claim 1, further comprising an additional step of adding ascorbate to the cells cultured in step (b).

25. A synthetic cartilage patch produced by the method of claim 1, 2, 3, or 4.

2. A method for preparing a multi cell-layered synthetic cartilage, the method comprising the steps of:

(a) providing a tissue comprising interconnected chondrogenic cells;

(b) disaggregating said tissue to release denuded chondrogenic cells;

(c) proliferating said denuded chondrogenic cell in vitro, wherein said cells are undifferentiated;

(d) seeding said cells into a pre-shaped well having a cell contacting surface which prevents attachment of the cells; and

(e) culturing said cells for a time sufficient for said cells to differentiate and form a multi cell-layered synthetic cartilage and to endogenously produce an extracellular matrix.

17. The method of claim 2, further comprising an additional step of adding a polypeptide growth factor to the cells cultured in step (e).

22. The method of claim 2, further comprising an additional step of adding ascorbate to the cells cultured in step (e).

3. A method for preparing a multi cell-layered synthetic cartilage patch of pre-determined volume, the method comprising the steps of:

(a) proliferating denuded chondrogenic cells in vitro, wherein said cells are undifferentiated;

(b) seeding denuded chondrogenic cells into a pre-shaped well, said well having a cell contacting surface which prevents attachment of the cells and being dimensioned to determine the volume of the resulting synthetic cartilage patch; and

(c) culturing said cells for a time sufficient for said cells to differentiate and form a multi cell-layered synthetic cartilage and to endogenously produce an extracellular matrix of pre-determined volume.

18. The method of claim 3, further comprising an additional step of adding a polypeptide growth factor to the cells cultured in step (b).

23. The method of claim 3, further comprising an additional step of adding ascorbate to the cells cultured in step (b).

4. A method for preparing a multi cell-layered synthetic cartilage patch of pre-determined volume, the method comprising the steps of:

(a) providing a tissue comprising interconnected chondrogenic cells;

(b) disaggregating said tissue to release denuded chrondrogenic cells;

(c) proliferating said denuded cells in vitro, wherein said cells are undifferentiated;

(d) seeding said cells into a pre-shaped well, said well having a cell contacting surface which prevents attachment of the cells and being dimensioned to determine the volume of the resulting synthetic cartilage; and

(e) culturing said cells for a time sufficient for said cells to differentiate and form a multi cell-layered synthetic cartilage and to endogeneously produce an extracellular matrix of pre-determined volume.

19. The method of claim 4, further comprising an additional step of adding a polypeptide growth factor to the cells cultured in step (e).

24. The method of claim 4, further comprising an additional step of adding ascorbate to the cells cultured in step (e).

26. A multi cell-layered synthetic articular cartilage patch comprising:

chondrogenic cells dispersed within an endogenously produced extracellular matrix, wherein said synthetic articular cartilage is prepared by proliferating denuded chondrogenic cells in vitro, wherein said cells are undifferentiated, seeding chondrogenic cells into a pre-shaped well having a cell contacting surface which prevents attachment of the cells, and culturing said cells in said well for a time sufficient to differentiate and induce formation of synthetic articular cartilage for repairing the cartilage defect in said mammal.