Bioengineered anterior cruciate ligament

• US 20020062151A1
• Filed: 09/10/2001
• Published: 05/23/2002
• Est. Priority Date: 05/14/1999
• Status: Abandoned Application

• Current Assignee: David Kaplan, Gordana Vunjak-Novakovic, Gregory Altman, Ivan Martin
• Original Assignee: David Kaplan, Gordana Vunjak-Novakovic, Gregory Altman, Ivan Martin
• Inventors: Altman, Gregory, Vunjak-Novakovic, Gordana, Kaplan, David, Martin, Ivan
• Application Number:
  US09/950,561
• Publication Number:
  US 20020062151A1
• Time in Patent Office: Days
• US Class Current: 623/13.17
• CPC Class Codes: A61F 2/08 Muscles; Tendons; Ligaments sutures A61B17/04
• CPC Class Codes: A61L 2430/10 for reconstruction of tendons or ligaments
• 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 27/3608 Bone, e.g. demineralised bone matrix [DBM], bone powder
• CPC Class Codes: A61L 27/3691 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: Y10S 623/901 Method of manufacturing prosthetic device

Abstract

The present invention provides a method for producing an anterior cruciate ligament ex vivo. The method comprises seeding pluripotent stem cells in a three dimensional matrix, anchoring the seeded matrix by attachment to two anchors, and culturing the cells within the matrix under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors. Bone marrow stromal cells are preferably used as the pluripotent cells in the method. Suitable matrix materials are materials to which cells can adhere, such as a gel made from collagen type I. Suitable anchor materials are materials to which the matrix can attach, such as Goinopra coral and also demineralized bone. Some examples of tissue which can be produced include other ligaments in the body (hand, wrist, elbow, knee), tendon, cartilage, bone, muscle, and blood vessels.

Claims

1. A method for producing an anterior cruciate ligament ex vivo, comprising the steps:

a) providing pluripotent cells, a 3-dimensional matrix of cylindrical form comprised of collagen, and two cylindrically shaped anchors suitable for attachment to the matrix;

b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;

c) attaching a face of each respective anchor to either end of the seeded matrix so that the entire surface of each face of the seeded matrix of step b) contacts the face of the respective anchors; and

d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors.

2. The method of claim 1 wherein the pluripotent cells are bone marrow stromal cells.

3. The method of claim 1 wherein the seeded matrix has a concentration of collagen type I ranging from 2 mg/ml to 6 mg/ml.

4. The method of claim 3 wherein the seeded matrix has a final concentration of collagen type I of 2 mg/ml.

5. The method of claim 3 wherein the collagen is not cross linked.

6. The method of claim 3 wherein the collagen is cross-linked .

7. The method of claim 1 wherein the anchors are comprised of Goinopra coral with pore size 500 μ

m, the coral having been treated by means to convert the calcium carbonate to calcium phosphate.

8. The method of claim 7 wherein the Goinopra coral is further infused with fibronectin.

9. The method of claim 1 wherein the anchors are comprised of demineralized bone.

10. The method of claim 9 wherein the bone is further infused with fibronectin.

11. The method of claim 1 wherein the magnitude, duration and combination of mechanical forces are changed over the period of culture to approach that which is experienced by a native ACL in vivo.

12. The method of claim 1 wherein the mechanical forces mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.

13. The method of claim 13 wherein the anchored matrix is further cultured under conditions which mimic the chemical stimuli experienced by an anterior cruciate ligament in vivo.

14. The method of claim 12 wherein the mechanical force is tension and compression.

15. The method of claim 12 wherein the mechanical force is torsion.

16. The method of claim 12 wherein the mechanical force is shear.

17. The method of claim 12 wherein a combination of mechanical forces are applied to simulate knee joint extension.

18. The method of claim 17 wherein the motion of knee joint extension is in the coronal plane.

19. The method of claim 17 wherein the motion of knee joint extension is in the sagittal plane.

20. The method of claim 12 wherein a combination of mechanical forces are applied to simulate knee joint flexion.

21. The methods of claim 12 wherein a combination of mechanical forces are applied which simulate a combination of flexion and extension, the combination of mechanical forces being applied over time to produce an anterior cruciate ligament which has helically organized fibers.

22. A bioengineered anterior cruciate ligament produced by the method comprising the steps:

a) providing pluripotent cells, a 3-dimensional matrix of cylindrical form comprised of collagen, and two cylindrically shaped anchors suitable for attachment to the matrix;

b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;

c) attaching a face of each respective anchor to either end of the seeded matrix so that the entire surface of each face of the seeded matrix of step b) contacts the face of the respective anchors; and

d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors.

23. The bioengineered ligament of claim 22 wherein the pluripotent cells are bone marrow stromal cells.

24. The bioengineered ligament of claim 23 which is characterized by cellular orientation and/or matrix crimp pattern in the direction of the applied mechanical forces of step d).

25. The bioengineered ligament of claim 24 which is further characterized by the production of collagen type I, collagen type III, and fibronectin proteins along the axis of mechanical load produced by the mechanical forces of step d).

26. The bioengineered ligament of claim 23 wherein the mechanical forces of step d) mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.

27. The bioengineered ligament of claim 26 wherein the ligament fiber bundles are arranged into a helical organization.

29. The method of claim 28 wherein the pluripotent cells are bone marrow stromal cells.

30. The method of claim 28 wherein the matrix has a cylindrical form and is attached to the respective anchor faces at each face of the cylinder.

31. The method of claim 28 wherein the matrix is comprised of collagen.

32. The method of claim 31 wherein the matrix has a concentration of collagen type I ranging from 2 mg/ml to 6 mg/ml.

33. The method of claim 32 wherein the collagen is not cross-linked.

34. The method of claim 32 wherein the collagen is cross-linked.

35. The method of claim 28 wherein the anchors are further infused with a factor which promotes matrix adhesion to the anchor.

36. The method of claim 28 wherein the anchors are comprised of Goinopra coral with pore size 500 μ

m, wherein the coral has been treated by means to convert the calcium carbonate to calcium phosphate.

37. The method of claim 28 wherein the anchors are comprised of demineralized bone.

38. The method of claim 28 wherein the ligament produced is an anterior cruciate ligament.

39. The method of claim 28 wherein the magnitude, duration and combination of mechanical forces are changed over the period of culture to approach that which is experienced by a native ligament in vivo.

40. The method of claim 28 wherein the anchored matrix is further cultured under conditions which mimic the chemical stimuli experienced by a native ligament in vivo.

41. The method of claim 28 wherein the mechanical force is tension-compression.

42. The method of claim 28 wherein the mechanical force is torsion.

43. The method of claim 28 wherein the mechanical force is shear.

44. The method of claim 28 wherein a combination of mechanical forces are applied to simulate extension of the joint in which the ligament is located in vivo.

45. The method of claim 28 wherein a combination of mechanical forces are applied to simulate flexion of the joint in which the ligament is located in vivo.

46. The methods of claim 28 wherein a combination of mechanical forces are applied which simulate a combination of flexion and extension, the combination of forces being applied over time to produce a ligament which has helically organized fibers.

48. The bioengineered ligament of claim 47 wherein the pluripotent cells are bone marrow stromal cells.

49. The bioengineered ligament of claim 48 which is characterized by cellular orientation in the direction of the applied mechanical forces of step d).

50. The bioengineered ligament of claim 49 which is further characterized by collagen III fiber production and fibronectin fiber production along the axis of mechanical load produced by the mechanical forces of step d).

51. The bioengineered ligament of claim 47 which is an anterior cruciate ligament, wherein the mechanical forces of step d) mimic mechanical stimuli experienced by an anterior cruciate ligament in vivo.

52. The bioengineered ligament of claim 51 wherein the ligament fiber bundles are arranged into an helical organization.

28. A method for producing a predetermined type of ligament ex vivo, comprising the steps:

a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and two anchors each having a face which is suitable for attachment to the matrix;

b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;

c) attaching the face of each respective anchor to opposite ends of the seeded matrix; and

d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors, wherein the mechanical forces mimic one or more mechanical forces experienced by the ligament in vivo.

47. A bioengineered ligament produced by the method comprising the steps:

a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and two anchors each having a face which is suitable for attachment to the matrix;

b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;

c) attaching the face of the respective anchors to opposite ends of the seeded matrix; and

d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or both of the attached anchors, wherein the mechanical forces mimic forces experienced by the ligament in vivo.

53. A method for producing a specific tissue type ex vivo, comprising the steps:

a) providing pluripotent cells, a 3-dimensional matrix to which cells are able to adhere, and a plurality of anchors each having a face which is suitable for attachment to the matrix;

b) seeding the cells in the matrix, either pre- or post-matrix formation, by means to uniformly immobilize the cells within the matrix;

c) attaching the faces of the respective anchors to the seeded matrix at the appropriate positions; and

d) culturing the cells in the anchored matrix of step c) under conditions appropriate for cell growth and regeneration, while subjecting the matrix to one or more mechanical forces via movement of one or more of the attached anchors, wherein the mechanical forces mimic stresses experienced by the specific tissue type in vivo.

54. The method of claim 53 wherein the cells in the anchored matrix are further cultured under conditions which mimic chemical stimuli experienced by the tissue in vivo.

55. The method of claim 53 wherein the cells in the anchored matrix are further cultured under conditions which mimic the electromagnetic stimuli experienced by the tissue in vivo.

56. The method of claim 53 wherein the tissue type is cartilage.

57. The method of claim 53 wherein the tissue type is bone.

58. The method of claim 53 wherein the tissue type is blood vessel.

59. The method of claim 53 wherein the tissue type is tendon.

60. The method of claim 53 wherein the tissue type is muscle.