Materials and methods for nerve grafting, selection of nerve grafts, and in vitro nerve tissue culture

• US 20040180434A1
• Filed: 03/29/2004
• Published: 09/16/2004
• Est. Priority Date: 08/13/2001
• Status: Active Grant

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Muir, David F.
• Granted Patent: US 7,732,200 B2
• Time in Patent Office: Days
• US Class Current: 435/380
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61L 2430/32 for nerve reconstruction
• 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 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3675 Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
• CPC Class Codes: A61L 27/3683 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• CPC Class Codes: A61L 27/3687 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• CPC Class Codes: A61L 27/383 Nerve cells, e.g. dendritic cells, Schwann cells
• CPC Class Codes: A61L 27/3878 Nerve tissue, brain, spinal cord, nerves, dura mater
• CPC Class Codes: A61L 31/047 Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

The subject invention pertains to compositions and methods for culturing nerve tissue in vitro and nerve grafts produced using such methods. The compositions and methods of the subject invention can be employed to restore the continuity of nerve interrupted by disease, traumatic events or surgical procedures. The invention also concerns methods for promoting repair of damaged nerve tissue using the present compositions and nerve tissue treated according to such methods.

Claims

1. A method for preparing nerve tissue for use as nerve grafts, wherein said method comprises culturing the nerve tissue in vitro under conditions that permit the nerve tissue to grow in vitro and increase the neurite-promoting activity of the nerve tissue when subsequently implanted.

2. The method according to claim 1, wherein the increase in neurite-promoting activity is as determined by an in vitro neurite outgrowth assay of the nerve tissue.

3. The method according to claim 2, wherein the in vitro neurite outgrowth assay comprises a cryoculture bioassay.

4. The method according to claim 1, wherein the increase in neurite-promoting activity is as determined by an in vivo neurite outgrowth assay of the nerve tissue.

5. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is for a period of time that allows pre-degeneration of the nerve tissue in vitro.

6. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is for a period of time that achieves an increase in axon ingress and extent of growth within the nerve tissue when subsequently implanted.

7. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is for a period of time within the range of about 24 hours to about 96 hours.

8. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is for a period of time within the range of about 24 hours to about 72 hours.

9. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is for a period of time of about 48 hours.

10. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 10°

C. to about 37°

C.

11. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 30°

C. to about 37°

C.

12. The method according to claim 1, wherein said culturing of the nerve tissue in vitro is conducted at a temperature of about 37°

C.

13. The method according to claim 1, wherein the nerve tissue is an explant.

14. The method according to claim 1, wherein the nerve tissue is mammalian tissue.

15. The method according to claim 1, wherein the nerve tissue is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.

16. The method according to claim 1, wherein the nerve tissue is human tissue.

17. The method according to claim 1, wherein the nerve graft is an autograft.

18. The method according to claim 1, wherein the nerve graft is an allograft.

19. The method according to claim 1, wherein the nerve graft is a xenograft.

20. The method according to claim 1, wherein the nerve tissue comprises living nerve tissue, and wherein said method further comprising rendering the living nerve tissue acellular after said culturing.

21. The method according to claim 1, wherein the nerve tissue comprises living nerve tissue, and wherein said method further comprises freeze-killing the living nerve tissue after said culturing.

22. The method according to claim 1, wherein said method further comprises freezing the nerve tissue for storage.

23. The method according to claim 22, wherein said freezing is carried out after said culturing in vitro.

24. The method according to claim 1, wherein said method further comprises applying at least one chondroitin sulfate proteoglycan-degrading enzyme to the nerve tissue.

25. The method according to claim 24, wherein the chondroitin sulfate proteoglycan-degrading enzyme is applied to the nerve tissue during said culturing.

26. The method according to claim 24, wherein the chondroitin sulfate proteoglycan-degrading enzyme is applied to the nerve tissue after said culturing.

27. The method according to claim 24, wherein said method further comprises freeze-killing the nerve tissue after said culturing in vitro, and wherein the chondroitin sulfate proteoglycan-degrading enzyme is applied to the nerve tissue after said freeze-killing.

28. The method according to claim 24, wherein the chondroitin sulfate proteoglycan-degrading enzyme is selected from the group consisting of chondroitinase, hyalurondiase, and matrix metalloproteinase, or a combination thereof.

29. The method according to claim 24, wherein the chondroitin sulfate proteoglycan-degrading enzyme is a chondroitinase.

30. The method according to claim 1, wherein the nerve tissue comprises peripheral nerve tissue.

31. The method according to claim 1, wherein said culturing comprises placing the nerve tissue in contact with culture medium.

32. The method according to claim 31, wherein the culture medium comprises a defined medium.

33. The method according to claim 31, wherein the culture medium comprises a defined medium supplemented with serum.

34. The method according to claim 31, wherein the culture medium comprises undefined medium.

35. The method according to claim 31, wherein the culture medium comprises dulbecco'"'"'s modified eagles'"'"' medium.

36. The method according to claim 1, wherein said method further comprises isolating the nerve tissue from a mammal prior to said culturing of the nerve tissue in vitro.

37. The method according to claim 1, wherein said method further comprises applying a tissue adhesive to the nerve tissue.

38. A method for enhancing the regenerative potential of nerve tissue for use as nerve grafts, wherein said method comprises culturing the nerve tissue in vitro for a period of time within the range of about 24 hours to about 96 hours.

39. The method according to claim 38, wherein said culturing of the nerve tissue in vitro is for a period of time within the range of about 24 hours to about 72 hours.

40. The method according to claim 38, wherein said culturing of the nerve tissue in vitro is for a period of time of about 48 hours.

41. The method according to claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 10°

C. to about 37°

C.

42. The method according to claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 30°

C. to about 37°

C.

43. The method according to claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature of about 37°

C.

44. The method according to claim 38, wherein said culturing comprises placing the nerve tissue in contact with culture medium.

45. The method according to claim 44, wherein the culture medium comprises defined medium.

46. The method according to claim 44, wherein the culture medium comprises defined medium supplemented with serum.

47. The method according to claim 44, wherein the culture medium comprises undefined medium.

48. The method according to claim 38, wherein the nerve tissue comprises living nerve tissue, and wherein said method further comprises rendering the living nerve tissue acellular after said culturing.

49. The method according to claim 38, wherein the nerve tissue comprises living nerve tissue, and wherein said method further comprises freeze-killing the living nerve tissue after said culturing.

50. The method according to claim 38, wherein the nerve tissue is mammalian tissue.

51. The method according to claim 38, wherein the nerve tissue is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.

52. The method according to claim 48, wherein the nerve tissue is human tissue.

53. The method according to claim 38, wherein the nerve tissue comprises peripheral nerve tissue.

54. The method according to claim 38, wherein the nerve graft is an autograft.

55. The method according to claim 38, wherein the nerve graft is an allograft.

56. The method according to claim 38, wherein the nerve graft is a xenograft.

65. The nerve graft of claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 10°

C. to about 37°

C.

66. The nerve graft of claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature within the range of about 30°

C. to about 37°

C.

67. The nerve graft of claim 38, wherein said culturing of the nerve tissue in vitro is conducted at a temperature of about 37°

C.

57. A nerve graft comprising nerve tissue prepared by a method comprising culturing the nerve tissue in vitro under conditions that permit the nerve tissue to grow in vitro and increase the neurite-promoting activity of the nerve tissue when subsequently implanted.

58. The nerve graft of claim 57, wherein the increase in neurite-promoting activity is as determined by an in vitro neurite outgrowth assay of the nerve tissue.

59. The nerve graft of claim 58, wherein the in vitro neurite outgrowth assay comprises a cryoculture bioassay.

60. The nerve graft of claim 58, wherein the increase in neutrite-promoting activity is as determined by an in vivo neurite outgrowth assay of the nerve tissue.

61. The nerve graft of claim 57, wherein said culturing of the nerve tissue in vitro is for a period of time that achieves an increase in axon ingress and extent of growth within the nerve tissue when subsequently implanted.

62. The nerve graft of claim 57, wherein said culturing of the nerve tissue in vitro is for a period of time within the range of about 24 hours to about 96 hours.

63. The nerve graft of claim 57, wherein said culturing of the nerve tissue in vitro is for a period of time within the range of about 24 hours to about 72 hours.

64. The nerve graft of claim 57, wherein said culturing of the nerve tissue in vitro is for a period of time of about 48 hours.

68. The nerve graft of claim 57, wherein said nerve tissue comprises living nerve tissue, and wherein said method further comprises rendering the living nerve tissue acellular.

69. The nerve graft of claim 57, wherein said nerve tissue comprises living nerve tissue, and wherein said method further comprises freeze-killing the living nerve tissue.

70. The nerve graft of claim 57, wherein said method further comprises freezing the nerve tissue for storage.

71. The nerve graft of claim 70, wherein said freezing is carried out after said culturing in vitro.

72. The nerve graft of claim 57, wherein said culturing comprises placing the nerve tissue in contact with culture medium.

73. The nerve graft of claim 72, wherein the culture medium comprises defined medium.

74. The nerve graft of claim 72, wherein the culture medium comprises defined medium supplemented with serum.

75. The nerve graft of claim 72, wherein the culture medium comprises undefined medium.

76. The nerve graft of claim 57, wherein the nerve tissue is mammalian tissue.

77. The nerve graft of claim 57, wherein the nerve tissue is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.

78. The nerve graft of claim 57, wherein the nerve tissue is human tissue.

79. The nerve graft of claim 57, wherein the nerve tissue comprises peripheral nerve tissue.

80. The nerve graft of claim 57, wherein the nerve graft is an autograft.

81. The nerve graft of claim 57, wherein the nerve graft is an allograft.

82. The nerve graft of claim 57, wherein the nerve graft is a xenograft.

83. A method for promoting repair of a damaged nerve comprising implanting a nerve graft at the site of damage, wherein the nerve graft has been prepared by a process comprising culturing the nerve graft in vitro under conditions that permit the nerve graft to grow in vitro and increase the neurite-promoting activity of the nerve graft when subsequently implanted.

84. The method according to claim 83, wherein said culturing of the nerve graft in vitro is for a period of time that achieves an increase in axon ingress and extent of growth within the nerve graft when subsequently implanted.

85. The method according to claim 83, wherein the increase in neurite-promoting activity is as determined by an in vitro neurite outgrowth assay of the nerve graft.

86. The method according to 85, wherein the in vitro neurite outgrowth assay comprises a cryoculture assay.

87. The method according to 85, wherein the increase in neurite-promoting activity is as determined by an in vivo neurite outgrowth assay of the nerve graft.

88. The method according to claim 83, wherein said culturing of the nerve graft in vitro is for a period of time within the range of about 24 hours to about 96 hours.

89. The method according to claim 83, wherein said culturing of the nerve graft in vitro is for a period of time within the range of about 24 hours to about 72 hours.

90. The method according to claim 83, wherein said culturing of the nerve graft in vitro is for a period of time of about 48 hours.

91. The method according to claim 83, wherein said culturing of the nerve graft in vitro is conducted at a temperature within the range of about 10°

C. to about 37°

C.

92. The method according to claim 83, wherein said culturing of the nerve graft in vitro is conducted at a temperature within the range of about 30°

C. to about 37°

C.

93. The method according to claim 83, wherein said culturing of the nerve graft in vitro is conducted at a temperature of about 37°

C.

94. The method according to claim 83, wherein said nerve graft comprises living nerve tissue, and wherein said process further comprises rendering the living nerve tissue acellular.

95. The method according to claim 83, wherein said nerve graft comprises living nerve tissue, and wherein said process further comprises freeze-killing the living nerve tissue.

96. The method according to claim 83, wherein said process further comprises freezing the nerve graft for storage.

97. The method according to claim 96, wherein said freezing is carried out after said culture in vitro.

98. The method according to claim 83, wherein said culturing comprises placing the nerve graft in contact with culture medium.

99. The method according to claim 83, wherein the culture medium comprises defined medium.

100. The method according to claim 83, wherein the culture medium comprises defined medium supplemented with serum.

101. The method according to claim 83, wherein the culture medium comprises undefined medium.

102. The method according to claim 83, wherein the nerve graft is mammalian tissue.

103. The method according to claim 83, wherein the nerve graft is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.

104. The method according to claim 83, wherein the nerve graft is human tissue.

105. The method according to claim 83, wherein the nerve graft comprises peripheral nerve tissue.

106. The method according to claim 83, wherein the damaged nerve comprises nerve tissue of the central nervous system.

107. The method according to claim 83, wherein said method further comprises applying a tissue adhesive to the damaged nerve, or to the nerve graft, or to both the damaged nerve and the nerve graft.

108. The method according to claim 83, wherein the nerve graft is a terminal nerve graft.

109. The method according to claim 83, wherein the nerve graft is an interpositional nerve graft.

110. The method according to claim 83, wherein said method further comprises applying at least one chondroitin sulfate proteoglycan-degrading enzyme to the damaged nerve, or the nerve graft, or both the damaged nerve and the nerve graft.

111. The method according to claim 110, wherein the chondroitin sulfate proteoglycan-degrading enzyme is applied to the nerve graft after said culturing in vitro.

112. The method according to claim 83, wherein said method further comprises resecting the damaged nerve prior to said applying of the nerve graft.

113. The method according to claim 83, wherein the nerve graft is autologous with the damaged nerve.

114. The method according to claim 83, wherein the nerve graft is allogeneic with the damaged nerve.

115. The method according to claim 83, wherein the nerve graft is xenogeneic with the damaged nerve.