Plasma protein matrices and methods for their preparation
A freeze dried biocompatible matrix comprising plasma proteins, useful as implants for tissue engineering as well as in biotechnology, and methods of producing the matrix are provided. Mechanical and physical parameters can be controlled by use of auxiliary components or additives which may be removed after the matrix is formed in order to improve the biological properties of the matrix. The matrices according to the present invention may be used clinically per se, or as a cell-bearing implant.
1. An elastic freeze-dried biocompatible porous fibrin matrix useful as a scaffold for growing cells, wherein the matrix has substantially regular pores and a residual moisture below 3% and is obtained by mixing lplasma proteins comprising fibrinogen and Factor XIII with thrombin and at least one anti-fibrinolytic agent in substontial absence of organic chelating agents.
2. The matrix according to claim 1 wherein the plasma proteins are present with at least 0.5 units of thrombin per milligram of protein.
3. The matrix according to claim 1 wherein at least one of the plasma proteins is autologous to a patient in need of the matrix.
4. The matrix according to claim 1 wherein all the plasma proteins are autologous to a patient in need of the matrix.
5. The matrix according to claim 1 wherein the anti-fibrinolytic agent is tranexamic acid.
6. The matrix according to claim 1 further comprising at least one auxiliary component selected from the group consisting of polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers.
7. The matrix according to claim 6 wherein the auxiliary component is selected from a group consisting of hyaluronic acid, pectin, alginate, galactans, galactomannans, glucomannans, polyuronic acids, heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hexuronyl hexosaminoglycan sulfate, inositol hexasulfate, sucrose octasulfate and polyethylene glycol.
8. The matrix according to claim 7 wherein the wherein the auxiliary component is dextran sulfate or hyaluronic acid.
9. The matrix according to claim 1 wherein the cells are stem cells or progenitor cells.
10. The matrix according to claim 1 wherein the cells are selected from the group consisting of chondrocytes, osteocytes, hepatocytes and mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal and ocular cell types.
11. The matrix according to claim 1 wherein the cells attain a density of at least 10 4 cells per cm 3 .
12. The matrix according to claim 1 further comprising at least one bioactive agent, selected from the group consisting of growth factors, cytokines, enzymes, anti-microbials, and anti inflammatory agents.
13. The matrix according to claim 1 having pores in the size range of 50–300 microns.
14. The matrix of claim 1 , wherein the plasma proteins are mixed with the thrombin in the presence of the calcium ions and the at least one anti-fibrinolytic agent under conditions suitable for clotting, optionally with adding of at least one auxiliary component thereto; and the mixture of plasma protiens, thrombin, anti-fibrinolytic agent and optional auxiliary agent are cast upon a solid support prior to clotting; the clotted mixture is frozen; and the clotted mixtire is lyophilized to obtain the matrix.
15. The matrix according to claim 14 wherein the plasma proteins comprise at least fibrinogen and factor XIII.
16. The matrix according to claim 14 wherein at least one of the plasma proteins is autologous.
17. The matrix according to claim 14 wherein all the plasma proteins are autologous.
18. The matrix according to claim 14 wherein the plasma proteins are mixed with at least 0.5 units of thrombin per mg protein.
19. The matrix according to claim 14 wherein the anti-fibrinolytic agent comprises tranexamic acid in an amount of at least 5%.
20. The matrix according to claim 14 wherein the at least one auxiliary component is present and is selected from the group consisting of polysaccharides, anionic polysaccharides, glycosaminoglycans, and synthetic polymers.
21. The matrix according to claim 14 wherein the at least one auxiliary component is present and is selected from the group consisting of hyaluronic acid, pectin, alginate, galactans, galactomannans, glucomannans, polyuronic acids, heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hexuronyl hexosaminoglycan sulfate, inositol hexasulfate, sucrose octasulfate and polyethylene glycol.
22. The matrix according to claim 20 wherein the auxiliary component is present and is dextran sulfate or hyaluronic acid.
23. The matrix of claim 1 , wherein the plasma proteins with thrombin in the presence of calcium ions and at least one anti-fibrinolytic agent under conditions suitable for clotting, optionally with adding of at least one auxiliary component; and the mixture of plasma proteins, thrombin, anti-fibrinolytic agent and optional auxiliary agent are cast upon a solid support prior to clotting; the clotted mixture is frozen; and the clotted mixture is lyophilized to obtain a sponge; and the sponge is cut into sections of desired shape; to obtain the matrix; and further the sections are seeded with cells; the cells are grown on the sections until the cells reach a density of at least 104 cells per cm 3 ; and the seeded sections are implanted in vivo.
24. The matrix according to claim 23 wherein the plasma proteins comprise at least fibrinogen and factor XIII.
25. The matrix according to claim 23 wherein at least one of the plasma proteins is autologous.
26. The matrix according to claim 23 wherein all the plasma proteins are autologous.
27. The matrix according to claim 23 wherein the plasma proteins are mixed with at least 0.5 units of thrombin per mg protein.
28. The matrix according to claim 23 wherein the anti-fibrinolytic agent comprises tranexamic acid in an amount of at least 5%.
29. The matrix according to claim 23 wherein the at least one auxiliary component is present and is selected from the group consisting of polysaccharides, anionic polysaccharides, glycosaminoglycans, and synthetic polymers.
30. The matrix according to claim 23 wherein the auxiliary component is present and is selected from the group consisting of hyaluronic acid, pectin, alginate, galactans, galactomannans, glucomannans, polyuronic acids, heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hexuronyl hexosaminoglycan sulfate, inositol hexasulfate, sucrose octasulfate and polyethylene glycol.
31. The matrix according to claim 23 wherein the auxiliary component is present and is dextran sulfate or hyaluronic acid.
32. The matrix according to claim 23 wherein the at least one auxiliary component is present and is a bioactive agent selected from the group consisting of growth factors, cytokines, enzymes, anti microbials, and anti-inflammatory agents.
33. The matrix according to claim 23 wherein the cells are selected from the group consisting of chondrocytes, hepatocytes, and osteocytes, mesenchymal, epithelial, urothelial, neuronal, pancreatic, renal and ocular cell types.
34. The matrix of claim 1 , wherein the plasma proteins are mixed with the thrombin in the presence of the calcium ions and at least one anti-fibrinolytic agent under conditions suitable for clotting, optionally with adding of at least one auxiliary component; and the mixture of plasma proteins, thrombin, anti-fibrinolytic agent and optional auxiliary agent are cast upon a solid support prior to clotting; the clotted mixture is frozen; and the clotted mixture is lyophilized to obtain a sponge having no more than 3% residual moisture; the sponge is optionally washed to remove soluble auxiliary components; optionally the washed sponge is re-lyophilized to reduce the residual moisture to no more than 3%; the sponge is cut into sections of desired shape to obtain the matrix; and the sections of matrix are implanted in situ.
35. The matrix according to claim 34 wherein the plasma proteins comprise at least fibrinogen and factor XIII.
36. The matrix according to claim 34 wherein at least one of the plasma proteins is autologous.
37. The matrix according to claim 34 wherein all the plasma proteins are autologous.
38. The matrix according to claim 34 wherein the plasma proteins are mixed with at least 0.5 units of thrombin per mg protein.
39. The matrix according to claim 34 wherein the anti-fibrinolytic agent comprises tranexamic acid in an amount of at least 5%.
40. The matrix according to claim 34 wherein the at least one auxiliary component is present and is selected from the group consisting of polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers.
41. The matrix according to claim 34 wherein the at least one auxiliary component is present and is selected from the group consisting of hyaluronic acid, pectin, alginate, galactans, galactomannans, glucomannans, polyuronic acids, heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hexuronyl hexosaminoglycan sulfate, inositol hexasulfate, sucrose octasulfate and polyethylene glycol.
42. The matrix according to claim 34 wherein the at least one auxiliary component is present and is dextran sulfate or hyaluronic acid.
43. The matrix according to claim 34 wherein the at least one auxiliary component is present and is a bioactive agent selected from the group consisting of growth factors, cytokines, enzymes, anti microbials, and anti-inflammatory agents.
44. The matrix according to claim 34 wherein the cells are selected from the group consisting of chondrocytes, osteocytes, hepatocytes, and epithelial, urothelial, neuronal, mesenchymal, pancreatic, renal and ocular cell types.
45. The matrix according to claim 34 which further comprises seeding the sections with cells and growing the cells on the sections until the cells reach a density of at least 104 cells per cm 3 .
46. The matrix according to claim 34 which further comprises seeding the sections with cells in vivo at a site of treatment.
47. An implant comprising the matrix according to claim 14 .
48. A method for treating injured tissue, the method comprising the step of implanting into an injury site an implant according to claim 47 .
49. The method according to claim 48 wherein the injured tissue to be treated is skeletal tissue.
50. A porous coating comprising the matrix according to claim 1 .
51. The coating according to claim 50 wherein the matrix further comprises at least one auxiliary component selected from the group consisting of polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers.
52. The matrix according to claim 1 wherein the plasma proteins and thrombin is mixed in the presence of calcium ions.
53. The matrix according to claim 1 wherein the matrix has a tensile strength of at least about 0.2 kPa.