Porous plasma protein matrices and methods for preparation thereof

• US 20060019389A1
• Filed: 07/22/2004
• Published: 01/26/2006
• Est. Priority Date: 07/22/2004
• Status: Active Grant

• Current Assignee: ProChon Biotech Ltd. (Ocugen, Inc.)
• Original Assignee: ProChon Biotech Ltd. (Ocugen, Inc.)
• Inventors: Yayon, Avner, Barkay, Hilla, Azachi, Malkit
• Granted Patent: US 7,335,508 B2
• Time in Patent Office: Days
• US Class Current: 435/395
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0231 Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
• CPC Class Codes: A61L 27/227 Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: C12N 2533/56 Fibrin; Thrombin
• CPC Class Codes: C12N 2533/80 Hyaluronan
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present invention relates to porous freeze-dried plasma protein matrices having an open channel structure and a concentration gradient of plasma proteins crosslinked by the action of thrombin, and methods of producing said matrices. The compositions of the present invention are useful clinically, per se or as cell-bearing implants.

Claims

1. A porous, freeze-dried plasma protein matrix comprising plasma proteins and thrombin, having two opposing surfaces substantially parallel to the horizontal axis of the matrix and at least one additional surface extending along the periphery of the sponge substantially parallel to the vertical axis, wherein the plasma proteins crosslinked by the action of thrombin are present in a gradient having a higher concentration along one of the opposing surfaces and, wherein the average size of the pores in cross section is smaller along the surface of the matrix exposed to the higher concentration of thrombin.

2. The porous freeze-dried plasma protein matrix according to claim 1, wherein the thrombin is provided at a concentration of about 300 IU/ml to about 1,500 IU/ml.

3. The porous freeze-dried plasma protein matrix according to claim 1, wherein the thrombin is provided at a concentration of about 500 IU/ml to about 1000 IU/mi.

4. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins and thrombin are present in a ratio of about 5:

1 (v/v) to about 50;

1 (v/v).

5. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins and thrombin are present in a ratio of about 8:

1 (v/v) to about 30;

1 (v/v).

6. The porous freeze-dried plasma protein matrix according to claim 1, the pores having an average size of about 5 μ

m to about 30 μ

m in cross section in the fraction of the matrix exposed to the higher concentration of thrombin.

7. The porous freeze-dried plasma protein matrix according to claim 1, the pores having an average size of about 10 μ

m to about 20 μ

m in cross section in the fraction of the matrix exposed to the higher concentration of thrombin.

8. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins crosslinked by the action of thrombin are generated by a thrombin solution comprising at least one viscosity-enhancing agent selected from a protein, a glycosaminoglycan, a polysaccharide, disaccharide and a synthetic polymer.

9. The porous freeze-dried plasma protein matrix according to claim 8, wherein the protein is an extracellular matrix protein selected from collagen, fibronectin, elastin and laminin

10. The porous freeze-dried plasma protein matrix according to claim 9, wherein the protein is collagen.

11. The porous freeze-dried plasma protein matrix according to claim 9, wherein the polypeptide is fibronectin.

12. The porous freeze-dried plasma protein matrix according to claim 8, wherein the glycosaminoglycan is selected from crosslinked hyaluronic acid and non-crosslinked hyaluronic acid.

13. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins crosslinked by the action of thrombin are generated by a thrombin solution comprising a transglutaminase.

14. The porous freeze-dried plasma protein matrix according to claim 13, wherein the transglutaminase is factor XIII.

15. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins are substantially devoid of plasminogen.

16. The porous freeze-dried plasma protein matrix according to claim 1, wherein the plasma proteins are substantially devoid of organic chelating agents.

17. The porous freeze-dried plasma protein matrix according to claim 1, further comprising at least one additive selected from the group consisting of a polysaccharide, a glycosaminoglycan and a synthetic polymer.

18. The porous freeze-dried plasma protein matrix according to claim 17, wherein the glycosaminoglycan is selected from crosslinked hyaluronic acid, non-crosslinked hyaluronic acid and heparin and derivatives thereof.

19. The porous freeze-dried plasma protein matrix according to claim 1, further comprising at least one bioactive agent selected from the group consisting of growth factors, cytokines, platelets, platelet supernatant and platelet derived proteins, hormones, analgesics, anti-inflammatory agents, anti-microbials and enzymes.

20. The porous freeze-dried plasma protein matrix according to claim 19, wherein the growth factor is selected from a fibroblast growth factor and variant thereof.

21. The porous freeze-dried plasma protein matrix according to claim 1, further comprising cells.

22. The porous freeze-dried plasma protein matrix according to claim 21, wherein the cells are selected from stem cells, progenitor cells, chondrocytes, osteoblasts, hepatocytes, mesenchymal cell types, endothelial cell types, epithelial cell types, urothelial cell types, endocrinal cell types, neuronal cell types, pancreatic cell types, renal cell types and ocular cell types.

23. The porous freeze-dried plasma protein matrix according to claim 22, wherein the cells are chondrocytes.

24. The porous freeze-dried plasma protein matrix according to claim 22, wherein the cells are hepatocytes.

25. The porous freeze-dried plasma protein matrix according to claim 1, wherein at least one of the plasma proteins is autologous.

26. The porous freeze-dried plasma protein matrix according to claim 1, wherein at least one of the plasma proteins is recombinant.

27. The porous freeze-dried plasma protein matrix according to claim 1, further comprising a fibrin fiber-modifying agent.

28. A method of preparing the porous, freeze-dried plasma protein matrix of claim 1, comprising the steps:

(a) introducing a thrombin solution to a solid receptacle or mold;

(b) layering a plasma protein solution over the thrombin solution in the solid receptacle or mold;

(c) incubating under conditions appropriate to achieve clotting;

(d) freezing the clotted mixture; and

(e) lyophilizing the clotted mixture, to obtain a porous matrix.

29. The method according to claim 28, further comprising the steps of:

(f) seeding the porous matrix with cells; and

(g) implanting said cell-bearing porous matrix into an individual in need thereof.

30. The method according to claim 28, further comprising the step of:

(h) implanting the porous matrix per se into an individual in need thereof.

31. A method of treating diseased or injured tissue in an individual in need thereof, the method comprising implanting the freeze-dried plasma protein matrix of claim 1 at the site of diseased or injured tissue in the individual.

32. The method according to claim 31, wherein the diseased or injured tissue is selected from neural, muscular, skeletal, cartilaginous, tendonous, hepatic, pancreatic, renal, ocular, arteriovenous, mammary and urinary tissue types.

33. The method according to claim 32, wherein the diseased or injured tissue is cartilage tissue.

34. The method according to claim 32, wherein the diseased or injured tissue is hepatic tissue.