阅读说明：本技术 用于促进半月板修复的功能化支架 (Functional scaffold for promoting meniscus repair ) 是由 马丁·洛茨 李光一 于 2018-03-16 设计创作，主要内容包括：本文公开了包含脱细胞半月板组织的支架,其中所述支架与肝素和生长因子共价缀合。本文还提供了在有需要的对象中修复和/或治疗组织损伤的方法,其包括：提供包含脱细胞半月板组织的支架；和通过将支架植入撕裂处中来修复和/或治疗组织损伤,其中支架与肝素和生长因子共价缀合。(Disclosed herein are scaffolds comprising decellularized meniscal tissue, wherein the scaffolds are covalently conjugated to heparin and a growth factor. Also provided herein are methods of repairing and/or treating tissue damage in a subject in need thereof, comprising: providing a scaffold comprising decellularized meniscal tissue; and repairing and/or treating the tissue damage by implanting a scaffold into the tear, wherein the scaffold is covalently conjugated to heparin and a growth factor.)

1. A stent, comprising:

the tissue of the decellularized meniscus is,

wherein the scaffold is covalently conjugated to heparin and a growth factor.

2. The scaffold of claim 1, wherein said growth factor is selected from Platelet Derived Growth Factor (PDGF), transforming growth factor beta (TGF β), Vascular Endothelial Growth Factor (VEGF), Connective Tissue Growth Factor (CTGF), Fibroblast Growth Factor (FGF), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1.

3. The scaffold of claim 1, wherein the growth factor is Platelet Derived Growth Factor (PDGF).

4. The stent of claim 3, wherein PDGF is PDGF-AA, PDGF-BB, and/or PDGF-AB.

5. The scaffold of claim 1, wherein the scaffold further comprises stem cells.

6. The scaffold of claim 1, wherein the scaffold further comprises meniscal cells.

7. The scaffold of claim 1, wherein the acellular meniscal tissue comprises collagen fibers, and wherein the orientation of the collagen fibers matches the orientation of a meniscal defect.

8. The stent of claim 1, wherein the decellularized meniscal tissue comprises pores.

9. The scaffold of claim 8, wherein pores are formed in the decellularized meniscal tissue by collagenase digestion, mechanical puncture, and/or application of a laser.

10. The stent of claim 1, wherein the stent releases growth factors at substantially first order kinetics over a period of at least 10 days after administration.

11. The stent of claim 1, wherein the stent releases growth factors at substantially first order kinetics over a period of at least 20 days after administration.

12. The stent of claim 1, wherein the stent releases growth factors at substantially first order kinetics over a period of at least 30 days after administration.

13. The scaffold of claim 1, wherein the scaffold has a tensile strength that is at least two times that of a similar acellular meniscal tissue that is not covalently conjugated to heparin and growth factors.

14. The scaffold of claim 1, wherein the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated to heparin and growth factors.

15. The stent of claim 1, wherein the tensile modulus of the stent is greater than 0.6 young's modulus (MPa).

16. The scaffold of claim 1, wherein the growth factor is from 10ng/mL scaffold to 1mg/mL scaffold.

17. The scaffold of claim 1, wherein the decellularized meniscal tissue is substantially sheet-like.

18. The scaffold of claim 1, wherein the acellular meniscal tissue has a three-dimensional form.

19. The stent of claim 1, wherein the stent is in the form of a medical dressing.

20. The scaffold of claim 1, wherein the decellularized meniscal tissue is derived from a mammal.

21. The scaffold of claim 1, wherein the decellularized meniscal tissue is derived from a human.

22. The stent of claim 1, wherein the stent is in sterile conditions and packaged in a sterile container.

23. A method of repairing and/or treating tissue damage in a subject in need thereof, comprising:

providing a scaffold comprising decellularized meniscal tissue; and

repairing and/or treating tissue damage by implanting a stent at the tear,

wherein the scaffold is covalently conjugated to heparin and a growth factor.

24. The method of claim 23, wherein the tissue injury is a tear in tissue.

25. The method of claim 23, wherein the tissue is meniscal tissue.

26. The method of claim 23, wherein the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1.

27. The method of claim 23, wherein the growth factor is Platelet Derived Growth Factor (PDGF).

28. The method of claim 27, wherein the PDGF is PDGF-AA, PDGF-BB, and/or PDGF-AB.

29. The method of claim 23, wherein the scaffold recruits a new population of cells to initiate repair in an avascular region of meniscal tissue.

30. The method of claim 23, wherein the scaffold is optimized to achieve efficient cellular infiltration and migration from host cells to the scaffold.

31. The method of claim 23 wherein the acellular scaffold is implanted into the meniscal tear by arthroscopic surgery.

32. The method of claim 23, wherein the scaffold releases growth factors at substantially first order kinetics over a period of at least 10 days after administration.

33. The method of claim 23, wherein the scaffold releases growth factors at substantially first order kinetics over a period of at least 20 days after administration.

34. The method of claim 23, wherein the scaffold releases growth factors at substantially first order kinetics over a period of at least 30 days after administration.

35. The method of claim 23, wherein the scaffold has a tensile strength at least twice that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF.

36. The method of claim 23, wherein the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF.

37. The method of claim 23, wherein the tensile modulus of the scaffold is greater than 0.6 young's modulus (MPa).

38. The method of claim 23, wherein PDGF is between 10ng/mL scaffold to 1mg/mL scaffold.

39. The method of claim 23, wherein the method of repairing and/or treating a tear in a tissue further comprises a second treatment protocol.

40. The method of claim 39, wherein the second treatment protocol comprises non-surgical treatment, such as rest, ice therapy, compression, elevation, and/or physical therapy.

41. The method of claim 39, wherein the second treatment protocol comprises a surgical treatment, such as a surgical repair, a meniscal resection, and/or a total meniscectomy.

42. The method of claim 23, wherein the subject is a mammal.

43. The method of claim 23, wherein the subject is a human.

44. A kit, comprising:

a sterile container comprising a scaffold covalently conjugated to heparin and a growth factor; and

instructions for using the kit.

45. The kit of claim 44, wherein said growth factor is selected from PDGF (platelet derived growth factor), TGF β (transforming growth factor β), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1.

46. The kit of claim 44, wherein the growth factor is Platelet Derived Growth Factor (PDGF).

47. The kit of claim 44, wherein the PDGF is PDGF-AA, PDGF-BB, and/or PDGF-AB.

48. The kit of claim 44, wherein the kit further comprises means for delivering a stent to the damaged meniscus.

49. The kit according to claim 48, wherein the delivery means is a medical glue, a medical suture, a medical staple and/or a medical anchor.

50. The kit of claim 44, wherein the scaffold is a biological decellularized scaffold.

51. The kit of claim 44, wherein the scaffold is derived from decellularized natural meniscal tissue.

52. The kit of claim 44, wherein the decellularized scaffold recruits a new population of cells to initiate repair in an avascular region.

53. The kit according to claim 44, wherein heparin conjugation enables slow release of growth factors.

54. The kit of claim 44, wherein slow release is sustained for a period of up to 30 days.

55. An apparatus, comprising:

acellular scaffolds covalently conjugated to heparin and growth factors,

wherein the device is used to repair tissue.

56. The device of claim 55, wherein said growth factor is selected from PDGF (platelet derived growth factor), TGF β (transforming growth factor β), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1.

57. The device of claim 55, wherein the growth factor is Platelet Derived Growth Factor (PDGF).

58. The device of claim 55, wherein the decellularized scaffold is a biological decellularized scaffold.

59. The device of claim 55 in which the decellularized scaffold is derived from decellularized natural meniscal tissue.

60. The device of claim 55 in which the decellularized scaffold has biological and mechanical properties similar to natural meniscus.

61. The device of claim 55, wherein the growth factor recruits a new population of cells to initiate repair in an avascular region.

62. The device of claim 55, wherein the decellularized scaffold is optimized for efficient cellular infiltration and migration from host cells to the scaffold.

63. The device of claim 55, wherein the device enables slow release of growth factors.

64. The device of claim 55, wherein slow release is sustained for a period of up to 30 days.

65. A method of inducing cell migration, comprising:

providing a decellularized meniscal scaffold to immobilize one or more growth factors; and

inducing cell migration to the decellularized meniscal scaffold.

66. The method of claim 65, wherein said one or more growth factors is PDGF.

67. The method of claim 65, wherein heparin is used for fixation.

68. The method of claim 65 in which the decellularized meniscal scaffold is implanted directly into a subject.

69. The method of claim 65, wherein the subject is a mammal.

70. The method of claim 65, wherein the subject is a human.

Technical Field

The present disclosure relates to scaffolds and methods of using the same for tissue repair and/or regeneration.

Background

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Meniscal tears are one of the most common injuries to the knee. Tearing occurs due to strong twisting, rotation or excessive bending of the knee joint. Meniscal tears can result in knee pain, swelling, stiffness, and limited knee extension. Meniscal tears, especially the most common form occurring in the medial third, do not usually heal spontaneously and are a major risk factor for developing knee Osteoarthritis (OA). Thus, meniscal repair strategies are critical to prevent disability and pain associated with OA.

While several treatment options currently exist for meniscal damage, these treatment options do not result in meniscal repair or regeneration. Most meniscal lesions are treated by a meniscal partial resection. Although patients may respond well to this therapy in the short term, they often develop OA after several years of surgery. The amount of tissue removed is related to the degree and rate of cartilage degeneration. When most of the meniscal tissue is affected by injury, a total meniscectomy will be performed. If the patient experiences pain after a total meniscectomy without significant joint degeneration, a second treatment with a meniscal allograft is possible. However, the use of allografts is limited by tissue availability and limited indications.

Meniscal repair and regeneration is mediated by the migration and proliferation of fibroblasts from the adjacent synovium and joint capsule. These cells produce fibrovascular scar tissue that undergoes the process of fibrocartilage metaplasia under appropriate environmental conditions, such as oxygen concentration and hydrostatic pressure, resulting in the transformation of the fibrotissue into fibrocartilage. Fibroblasts do not resynthesize fibrocartilage tissue. Thus, an external environmental stimulus is required to convert fibrous connective tissue into fibrocartilage. Accordingly, there remains a need for new tissue repair devices that can promote meniscal tissue regeneration, and methods of using such tissue repair devices.

Disclosure of Invention

Various embodiments disclosed herein include a stent comprising: acellular meniscal tissue in which the scaffold is covalently conjugated to heparin and growth factors. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the scaffold further comprises stem cells. In one embodiment, the scaffold further comprises meniscal cells. In one embodiment, the decellularized meniscal tissue comprises collagen fibers, and wherein the orientation of the collagen fibers matches the orientation of the meniscal defect. In one embodiment, the decellularized meniscal tissue comprises pores. In one embodiment, the holes are formed in the decellularized meniscal tissue by collagenase digestion, mechanical puncture, and/or application of a laser. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 10 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 20 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 30 days after administration. In one embodiment, the scaffold has a tensile strength at least twice that of a similar decellularized meniscal tissue not covalently conjugated to heparin and growth factors. In one embodiment, the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated to heparin and growth factors. In one embodiment, the tensile modulus of the scaffold is greater than 0.6 young's modulus (MPa). In one embodiment, the growth factor is from 10ng/mL scaffold to 1mg/mL scaffold. In one embodiment, the decellularized meniscal tissue is substantially sheet-like. In one embodiment, the decellularized meniscal tissue has a three-dimensional form. In one embodiment, the stent is in the form of a medical dressing. In one embodiment, the decellularized meniscal tissue is derived from a mammal. In one embodiment, the decellularized meniscal tissue is derived from a human. In one embodiment, the scaffold is in sterile conditions and packaged in a sterile container.

Various embodiments disclosed herein also include methods of repairing and/or treating a tissue injury in a subject in need thereof, comprising: providing a scaffold comprising decellularized meniscal tissue; and repairing and/or treating the tissue damage by implanting a scaffold into the tear, wherein the scaffold is covalently conjugated to heparin and a growth factor. In one embodiment, the tissue injury is a tear in the tissue. In one embodiment, the tissue is meniscal tissue. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the scaffold recruits a new cell population to initiate repair in the avascular or vascular region of the meniscal tissue. In one embodiment, the scaffold is optimized to achieve efficient cell infiltration and migration from the host cells to the scaffold. In one embodiment, the acellular scaffold is implanted at the meniscal tear by arthroscopic surgery. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 10 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 20 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 30 days after administration. In one embodiment, the scaffold has a tensile strength at least twice that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF. In one embodiment, the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF. In one embodiment, the tensile modulus of the scaffold is greater than 0.6 young's modulus (MPa). In one embodiment, the PDGF is 10ng/mL/mL scaffold to 1mg/mL scaffold. In one embodiment, the method of repairing and/or treating a tear in a tissue further comprises a second treatment regimen. In one embodiment, the second treatment regimen comprises a non-surgical treatment, such as rest, ice therapy, compression, elevation, and/or physical treatment. In one embodiment, the second treatment protocol comprises a surgical treatment, such as a surgical repair, a meniscal resection, and/or a total meniscectomy. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a horse.

Other embodiments of the present disclosure include kits comprising: a sterile container comprising a scaffold covalently conjugated to heparin and a growth factor; and instructions for using the kit. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the kit further comprises means for delivering the scaffold to the damaged meniscus. In one embodiment, the delivery means is a medical glue, a medical suture, a medical staple and/or a medical anchor. In one embodiment, the scaffold is a biological decellularized scaffold. In one embodiment, the scaffold is derived from decellularized natural meniscal tissue. In one embodiment, the decellularized scaffold recruits a new population of cells to initiate repair in an avascular or vascular region. In one embodiment, heparin conjugation enables slow release of growth factors. In one embodiment, the slow release is sustained for a period of up to 30 days.

Embodiments of the present disclosure also include an apparatus comprising: an acellular scaffold covalently conjugated to heparin and a growth factor, wherein the device is used to repair tissue. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the decellularized scaffold is a biological decellularized scaffold. In one embodiment, the decellularized scaffold is derived from decellularized natural meniscal tissue. In one embodiment, the decellularized scaffold has biological and mechanical properties similar to those of a natural meniscus. In one embodiment, the growth factors recruit a new cell population to initiate repair in the avascular region. In one embodiment, the decellularized scaffold is optimized to achieve efficient cellular infiltration and migration from the host cells to the scaffold. In one embodiment, the device enables slow release of growth factors. In one embodiment, the slow release is sustained for a period of up to 30 days.

Embodiments of the present disclosure also include methods of inducing cell migration, comprising: providing a decellularized meniscal scaffold to immobilize one or more growth factors; and inducing cell migration to the decellularized meniscal scaffold. In one embodiment, the one or more growth factors is PDGF. In one embodiment, heparin is used for fixation. In one embodiment, the decellularized meniscal scaffold is implanted directly into the subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

Drawings

Exemplary embodiments are shown in the referenced figures. The embodiments and figures disclosed herein are intended to be considered illustrative rather than restrictive.

Fig. 1 depicts a method for fibrochondrogenic differentiation during healing of a meniscal tear according to embodiments herein.

Fig. 2 depicts a novel technique for integrated healing by cell recruitment, according to embodiments herein.

Fig. 3 depicts a Decellularized Meniscus Scaffold (DMS) from bovine meniscus according to embodiments herein.

Fig. 4 depicts a schematic of PDGF-BB immobilization on heparin-conjugated DMS according to embodiments herein.

Fig. 5 depicts decellularization and PDGF conjugation of bovine menisci according to embodiments herein. (A) Images of decellularized meniscal blocks; (B) DNA content; (C) images of toluidine blue stained DMS; (D) quantification of toluidine blue content.

Figure 6 depicts the kinetics of PDGF-BB release from DMS according to embodiments herein. PDGF-BB was conjugated to DMS or heparin-coated DMS and DMS was incubated at 37 ℃ for up to 16 days. Each type of DMS was conjugated with 100ng PDGF-BB. Supernatants were collected at the indicated time points and analyzed for PDGF-BB by ELISA. Results were from 3 independent experiments.

Fig. 7 depicts anti-PDGFR β immunohistochemistry of meniscal samples according to embodiments herein. (A) And (B) a human meniscus; (C) and (D) DMS inserted into bovine meniscal explants after 2 weeks of ex vivo culture, conjugating DMS with heparin and 50ng/ml PDGF-BB; (E) anti-PDGFR β positive cells (%) from (C) and (D).

Fig. 8 depicts a comparative DAPI image in which (a) natural bovine meniscus; (B) DMS inserted into a meniscal explant; (C) PDGF coated DMS inserted into meniscal explants. Staining images of PDGF-BB (50ng/ml) coated DMS inserted into bovine explants after 2 weeks of culture; (D) DAPI; (E) safranin O; (F) tianlang scarlet. The black arrows indicate newly generated ECMs.

FIG. 9 depicts DMS conjugated to heparin and PDGF-BB (200ng/ml) after 2 weeks of culture according to embodiments herein. (A) DAPI staining; (B) dyeing with safranin O; (C) polarized light view of sirius red staining.

Fig. 10 depicts tensile testing after 2 weeks and 4 weeks ex vivo culture, according to embodiments herein.

Fig. 11 depicts growth factor fixation by heparin conjugation according to embodiments herein.

Figure 12 depicts an ex vivo model according to embodiments herein.

Fig. 13 depicts a mechanical assay after ex vivo culture according to embodiments herein.

Fig. 14 depicts anti-PDGFR β in human and bovine menisci (2 weeks) according to embodiments herein.

Figure 15 depicts cell migration in injured meniscal explants cultured with inserted DMS according to embodiments herein. Explant DAPI stained sections (n-3 to 6, 40x per group) were cultured for 2 weeks. (a.) a natural undamaged meniscus; (b.) damaged menisci not cultured with DMS; (c.) damaged menisci cultured with DMS; (d.) damaged menisci cultured with PDGF-DMS; (e.) graph of number of migrated cells. Data represent the average of 6 to 8 values from 3 independent experiments.

Fig. 16 depicts safranin O and sirius red staining assays (2 and 4 weeks) according to embodiments herein.

Figure 17 depicts the mechanical properties of injured meniscal explants cultured with DMS according to embodiments herein. DMS or PDGF conjugated DMS was inserted into the wounded explants and cultured for 2 and 4 weeks. Tensile properties were measured by pulling to failure. Data represent the average of 7 to 10 values from 3 independent experiments.

Fig. 18 depicts ex vivo culture of DMS or PDGF-conjugated DMS according to embodiments herein. (A) DAPI staining and (B) quantification, (C) anti-PDGFR β IHC and (D) quantification, (E) histological images after 2 weeks, (F) tensile tests after 2 and 4 weeks.

Figure 19 depicts the preparation of PDGF-HEP-DMS according to embodiments herein: DMS is made from decellularized bovine meniscus. After 0.1% (weight/volume) heparin was conjugated to DMS, PDGF was bound to heparin-conjugated DMS.

Fig. 20 depicts a quantitative analysis of anti-PDGFR β positive cells according to embodiments herein: positive cells account for the percentage of the total number of cells in bovine meniscal explants. Bovine meniscal explants (a) with DMS were inserted after 2 weeks of ex vivo culture; bovine meniscal explants (B) into which 50ng/ml PDGF-BB coated DMS was inserted; bovine meniscal explants (C) into which 100ng/ml PDGF-BB coated DMS was inserted; bovine meniscal explants (D) into which 200ng/ml PDGF-BB coated DMS was inserted.

Figure 21 depicts safranin O and sirius red staining of bovine meniscal explants inserted with DMS after 2 weeks of ex vivo culture, according to embodiments herein: explants (A, B) into which 50ng/ml PDGF-BB coated DMS was inserted; explants (C, D) with 100ng/ml PDGF-BB coated DMS inserted; explants with 200ng/ml PDGF-BB coated DMS were inserted.

Figure 22 depicts immunohistochemistry of bovine meniscal explants inserted with DMS and PDGF-coated DMS after 2 weeks of ex vivo culture, according to embodiments herein: anti-aggrecan (a); anti-collagen type 1a1 (B); anti-mkx (c); anti-collagen type 2a1 (D).

Fig. 23 depicts PDGFR β positive cells in injured meniscal explants according to embodiments herein. anti-PDGFR β stained sections of explants cultured for 2 weeks (n ═ 3 to 6, 40x per group). (a.) a natural undamaged meniscus; (b.) damaged menisci not cultured with DMS; (c.) damaged menisci cultured with DMS; (d.) damaged menisci cultured with PDGF-DMS; (e.) graph of number of migrated cells. Data represent the average of 6 to 8 values from 3 independent experiments.

Fig. 24 depicts ECM formation in damaged meniscal explants according to embodiments herein. (a-b.) safranin O staining: 2 and 4 weeks of native intact meniscus; (c-d.) safranin O staining: injured menisci without DMS at 2 and 4 weeks; (e-f.) safranin O staining: culturing the damaged menisci for 2 and 4 weeks with DMS; (g-h.) safranin O staining: culturing the damaged menisci for 2 and 4 weeks with PDGF-DMS; (i-j.) sirius red staining: culturing the damaged menisci for 2 and 4 weeks with DMS; (k-l.) sirius red staining: culturing the damaged menisci for 2 and 4 weeks with PDGF-DMS; (m-n.) safranin O positive staining area (% of total area) and the% integration between DMS and explants assessed by sirius red staining, and shown as the integrated interface% of total interface area.

Figure 25 depicts alignment of collagen fiber orientation of DMS with collagen fiber orientation in a meniscus defect, according to embodiments herein. Collecting tissue in a defined orientation, (a) vertically piercing cylindrical AVAS slices; (b) horizontally piercing cylindrical AVAS slices; (c) vertically puncturing cylindrical VAS slices; and (d) horizontally puncturing cylindrical VAS sections.

Fig. 26 depicts collagenase digestion of DMS according to embodiments herein. In one embodiment, collagenase digestion promotes cell migration and infiltration.

Fig. 27 depicts PDGF-conjugated DMS inducing cell migration and proliferation according to embodiments herein.

Fig. 28 depicts PDGF-conjugated DMS inducing cell migration and proliferation according to embodiments herein.

Detailed Description

All references, publications, and patents cited herein are incorporated by reference in their entirety as if fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Hornyak et al, Introduction to Nanoscience and nanotechnology, CRC Press (2008); singleton et al, Dictionary of Microbiology and molecular Biology, third edition, J.Wiley & Sons (New York, NY 2001); march, Advanced Organic Chemistry Reactions, mechanics and Structure seventh edition, j.wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual fourth edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide those skilled in the art with a general guidance for many of the terms used in this application. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein which can be used in the practice of the present invention. Indeed, the invention is in no way limited to the methods and materials described.

Meniscal tears are the most common injuries to the knee joint. Meniscal tears, especially the most common form occurring in the medial third, do not usually heal spontaneously and are a major risk factor for developing knee osteoarthritis.

As described herein, and in accordance with various embodiments herein, the inventors have developed a novel chemotactic acellular meniscal graft for integrated meniscal healing. The inventors have characterized a Decellularized Meniscal Scaffold (DMS) for host cell infiltration; the effect of PDGF coating with DMS on cell recruitment and meniscus repair was examined in vitro; and PDGF coated DMS was tested for efficacy in meniscal integrated healing using an animal model.

In one embodiment, the inventors have shown that only chemotactic growth factors can be applied to the scaffold without any exogenous cells, and that endogenous cells migrating to the damaged area are expected to undergo the healing process. In one embodiment, the inventors have found that PDGF has a strong chemotactic effect on progenitor cells. In one embodiment, the inventors have shown that administration of PDGF to an acellular meniscus as a scaffold can cure meniscal damage through endogenous cell migration.

In various embodiments, the inventors have shown that heparin-conjugated decellularized meniscal scaffolds bind and slowly release PDGF-BB for at least two weeks. In another embodiment, the inventors have shown that insertion of a PDGF-treated scaffold at a defect in an avascular meniscus causes PDGFR β expression to increase and cells to migrate into the defect region. In another embodiment, safranin O and sirius red staining shows tissue integration between the scaffold and the injured explant. In another embodiment, the extension performance of the lesion explants treated with the PDGF coated stent is significantly better than without the PDGF coated stent.

In one embodiment, the scaffold disclosed herein comprises decellularized meniscal tissue, wherein the scaffold is covalently conjugated to heparin and a growth factor. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor β), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines, such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the scaffold further comprises stem cells. In one embodiment, the scaffold further comprises meniscal cells. In one embodiment, the decellularized meniscal tissue comprises collagen fibers, and wherein the orientation of the collagen fibers matches the orientation of the meniscal defect. In one embodiment, the decellularized meniscal tissue comprises pores. In one embodiment, the holes are formed in the decellularized meniscal tissue by collagenase digestion, mechanical puncture, and/or application of a laser. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 10 days after administration, or at least 20 days after administration, or at least 30 days after administration. In one embodiment, the scaffold releases growth factors at substantially first order kinetics over a period of at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, or 30 days after administration. In one embodiment, the scaffold has a tensile strength at least twice that of a similar decellularized meniscal tissue not covalently conjugated to heparin and growth factors. In one embodiment, the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated to heparin and growth factors. In one embodiment, the tensile modulus of the scaffold is greater than 0.6 young's modulus (MPa). In one embodiment, the growth factor is from 10ng/mL scaffold to 1mg/mL scaffold. In one embodiment, the growth factor is from 1ng/mL to 1 μ g/mL, or from 1 μ g/mL to 500 μ g/mL, or from 500 μ g/mL to 1mg/mL, or from 1mg/mL to 10 mg/mL. In one embodiment, the decellularized meniscal tissue is substantially sheet-like. In one embodiment, the decellularized meniscal tissue has a three-dimensional form. In one embodiment, the stent is in the form of a medical dressing. In one embodiment, the decellularized meniscal tissue is derived from a mammal. In one embodiment, the decellularized meniscal tissue is derived from a human. In one embodiment, the rack is in sterile condition and packaged in a sterile container.

In one embodiment, the growth factor conjugated to DMS may be PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the growth factor is administered at a dose of 10ng/ml to 1 mg/ml. In one embodiment, the source of the scaffold may be meniscal tissue. The meniscus may be derived from a human or other mammal. In one embodiment, a three-dimensional form of DMS may be prepared to fill in larger meniscal defects. The decellularization process and heparin/PDGF conjugation is similar to that described herein with respect to the DMS tablet. In one embodiment, PDGF-conjugated scaffolds can also be used to attach stem cells or meniscal cells (either native or modified by pre-culture in growth factor or viral gene transfer) for implantation into meniscal defects. In one embodiment, the heparin/PDFG conjugated DMS is inserted into the meniscal defect during arthroscopic surgery. The DMS is secured by using glue, sutures, staples or anchors. In one embodiment, the scaffold may be additionally modified to facilitate treatment of tissue damage. For example, in one embodiment, to promote cell migration, the collagen fiber orientation of the DMS is matched to the collagen fiber orientation of the meniscal defect. This is achieved by cutting the DMS horizontally from the meniscus and inserting the heparin/PDGF conjugated DMS in the same orientation. In another embodiment, to promote cell migration and infiltration, pores are formed in the dense collagen fiber network of the DMS by using collagenase digestion, mechanical puncture, or application of a laser.

In one embodiment, disclosed herein is a method of repairing and/or treating a tissue injury in a subject in need thereof, comprising: providing a scaffold comprising decellularized meniscal tissue; and repairing and/or treating the tissue damage by implanting a scaffold into the tear, wherein the scaffold is covalently conjugated to heparin and a growth factor. In one embodiment, the tissue injury is a tear in the tissue. In one embodiment, the tissue is meniscal tissue. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the PDGF is PDGF-AA, PDGF-BB and/or PDGF-AB. In one embodiment, the scaffold recruits a new cell population to initiate repair in the avascular or vascular region of the meniscal tissue. In one embodiment, the scaffold is optimized to achieve efficient cell infiltration and migration from the host cells to the scaffold. In one embodiment, the decellularized scaffold is implanted at the meniscal tear by arthroscopic surgery. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 10 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 20 days after administration. In one embodiment, the scaffold releases the growth factor at substantially first order kinetics over a period of at least 30 days after administration. In one embodiment, the scaffold has a tensile strength at least twice that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF. In one embodiment, the scaffold has a tensile strength at least three times that of a similar acellular meniscal tissue not covalently conjugated with heparin and PDGF. In one embodiment, the tensile modulus of the scaffold is greater than 0.6 young's modulus (MPa). In one embodiment, the PDGF is 10ng/mL scaffold to 1mg/mL scaffold. In one embodiment, the method of repairing and/or treating a tear in a tissue further comprises a second treatment regimen. In one embodiment, the second treatment regimen comprises a non-surgical treatment, such as rest, ice therapy, compression, elevation, and/or physical treatment. In one embodiment, the second treatment protocol comprises a surgical treatment, such as a surgical repair, a meniscal resection, and/or a total meniscectomy. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a horse.

In one embodiment, the inventors have developed a novel scaffold that can be inserted at the site of a damaged meniscal lesion to promote integrated tissue healing. In particular, in various embodiments, methods are disclosed for preparing human decellularized menisci (with appropriate collagen orientation); methods of heparin and PDGF-BB conjugation of decellularized menisci; and methods of inserting a decellularized meniscal scaffold into a torn meniscus. Bovine meniscal explants were used to generate meniscal tears. Insertion of a PDGF-BB conjugated meniscal scaffold results in migration of cells to the scaffold, creating a new collagen extracellular matrix, thereby remedying the defect and improving biomechanical properties. In one embodiment, a decellularized meniscus can be inserted into a meniscus tear during arthroscopy to promote healing of meniscus damage and prevent chronic knee pain and dysfunction.

In one embodiment, disclosed herein is an apparatus comprising: an acellular scaffold covalently conjugated to heparin and a growth factor, wherein the device is used to repair tissue. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the decellularized scaffold is a biological decellularized scaffold. In one embodiment, the decellularized scaffold is derived from decellularized natural meniscal tissue. In one embodiment, the decellularized scaffold has biological and mechanical properties similar to those of a natural meniscus. In one embodiment, the growth factor recruits a new population of cells to initiate repair of an avascular or vascular area. In one embodiment, the decellularized scaffold is optimized to achieve efficient cellular infiltration and migration from the host cells to the scaffold. In one embodiment, the device enables slow release of growth factors. In one embodiment, the slow release is sustained for a period of up to 30 days.

In one embodiment, disclosed herein is a method of inducing cell migration comprising: providing a decellularized meniscal scaffold to immobilize one or more growth factors; and induce cell migration to the decellularized meniscal scaffold. In one embodiment, the one or more growth factors is PDGF. In one embodiment, heparin is used for fixation. In one embodiment, the decellularized meniscal scaffold is implanted directly into the subject. In one embodiment, the subject is a human. In one embodiment, the subject is a horse.

In various embodiments, the present disclosure provides heparin-conjugated DMS that exhibits strong immobilization of PDGF-BB such that PDGF-BB is slowly released. PDGF-BB coated DMS promotes migration of endogenous meniscal cells into the defect area and the scaffold. A new matrix is formed that bridges the space between the natural meniscus and the scaffold, which is associated with improved biomechanical properties. PDGF-BB coated DMS is a promising approach for integrated healing of meniscal tears.

The present disclosure also relates to kits comprising the scaffolds. The kit may be used to practice the method of the present invention for repairing and/or treating a tear in a tissue. The kit is a collection of materials or components comprising at least one scaffold of the invention. Thus, in some embodiments, the kit comprises a sterile container comprising a scaffold covalently conjugated to heparin and a growth factor; and instructions for using the kit. In one embodiment, the growth factor is selected from PDGF (platelet derived growth factor), TGF (transforming growth factor beta), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), FGF (fibroblast growth factor), and other chemokines such as CCL20, CXCL3, CXCL6, CCL3, CCL3L 1. In one embodiment, the growth factor is Platelet Derived Growth Factor (PDGF). In one embodiment, the kit further comprises means for delivering the scaffold to the damaged meniscus. In one embodiment, the delivery means is a medical glue, a medical suture, a medical staple and/or a medical anchor. In one embodiment, the scaffold is a biological decellularized scaffold. In one embodiment, the scaffold is derived from decellularized natural meniscal tissue. In one embodiment, the decellularized scaffold recruits a new population of cells to initiate repair of the avascular region. In one embodiment, heparin conjugation enables slow release of growth factors. In one embodiment, the slow release is sustained for a period of up to 30 days.

The exact nature of the components configured in the kit of the invention depends on their intended purpose. For example, some embodiments are configured for the purpose of treating and/or healing tears in tissue. In one embodiment, the kit is specifically configured for the purpose of treating a mammalian subject. In another embodiment, the kit is specifically configured for the purpose of treating a human subject. In other embodiments, the kit is configured for veterinary use, treating subjects such as, but not limited to, farm animals, livestock, and laboratory animals.

Instructions for use may be included in the kit. "instructions for use" generally include tangible forms of expression that describe techniques employed in using the components of the kit to achieve a desired result, such as treating, repairing, and/or healing tissue. Optionally, the kit also comprises other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring instruments, dressing materials, or other useful items readily recognized by one skilled in the art.

The materials or components assembled in the kit can be provided to the practitioner in any convenient and suitable manner that maintains their operability and usefulness. For example, the components may be in dissolved, dehydrated or lyophilized form; they may be provided at room temperature, refrigerated or frozen temperatures. The components are typically contained in a suitable packaging material. As used herein, the phrase "packaging material" refers to one or more than one physical structure for containing the contents of a kit, such as the compositions of the present invention and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contamination-free environment. The packaging materials used in the kit are those commonly used in the pharmaceutical or biomedical field. As used herein, the term "package" refers to a suitable solid substrate or material, such as glass, plastic, paper, foil, etc., capable of holding the various kit components. Thus, for example, the package may be a glass vial for containing an appropriate amount of a composition of the invention comprising a decellularized scaffold coated with a chemotactic growth factor. The packaging material typically has an external label which indicates the contents and/or purpose of the kit and/or its component parts.

Embodiments of the present disclosure are further described in the following examples. These examples are merely illustrative and do not in any way limit the scope of the invention as claimed.