Implant for Repairing a Cartilage Defect

Abstract

An implant for repairing a cartilage defect comprising a first layer and a second layer. The first layer comprises a membrane-like structure and the second layer comprises a sponge-like structure with directional and/or interconnected pores. The first layer is facing the synovial space and the second layer is located towards bone.

Claims

1. An implant for repairing a cartilage defect comprising: a first layer comprising a stable scaffolding in the form of a membrane structure; and a second layer comprising at least one wettable material and a sponge structure comprising interconnected pores; wherein 1) the pores form hollow fiber structures orientated directionally in columns and that run substantially parallel to each other, have a substantially consistent pore size and 2) said first layer faces an opposite direction relative to said second layer and 3) wherein said first layer comprises a material having a resorption time exceeding a resorption time of said second layer.

2. The implant according to claim 1, wherein that said first layer and said second layer each comprises biocompatible materials.

3. (canceled)

4. The implant according to claim 1, wherein said first layer comprises a material which is selected from the group consisting of collagen, bioresorbable polymers, pericardium, composites and glycosaminoglycans, or mixtures of two or more of these materials.

5. The implant according to claim 1, wherein said second layer comprises a physiological component of bone selected from the group consisting of calcium phosphates, calcium sulfates, calcium fluorides, calcium oxides, hydroxyl apatites, or mixtures of two or more of these components.

6. The implant according to claim 1, wherein said at least one wettable material comprises a material which is selected from the group consisting of collagen, hyaluronic acid, alginate, chitosan, gelatine, blood borne components, processed materials and composites, or mixtures of two or more of these materials.

7. The implant according to claim 1, wherein said second layer further comprises substances which are selected from the group consisting of antiangiogenesis, morphogenic, mitogenic, and antiinflammatoric agents, and mixtures of two or more of these substances.

8. The implant according to claim 1, wherein said pore structure comprises pores of a size of about 50 μm to about 250 μm.

9. The implant according to claim 8, wherein said pore structure comprises pores of a size of about 130 μm to about 200 μm.

10. The implant according to claim 1, wherein said sponge-like structure is suitable to be seeded with cells.

11. The implant according to claim 10, wherein said cells are selected from the group consisting of chondrocytes, chondroprogenitor cells, bone-precursor cells, stem cells, cells from periosteum tissue, and cells from perichondrium tissue, or mixtures of two or more of these cell types.

12. The implant according to claim 10, in which said cells are taken from a source which is xenogenic to the patient into whom the implant is to be introduced.

13. The implant according to claim 1, wherein said first layer is having a depth of about 0.01 mm to about 0.5 mm.

14. The implant according to claim 1, wherein said second layer is having a depth of about 0.3 mm to about 3 mm.

15. A method for treating a cartilage defect comprising the steps of: a) providing an implant in a vessel, wherein the implant comprises a first and at least a second layer, wherein said first layer comprises a membrane-like structure and said second layer comprises a sponge-like structure having directional and interconnected pores of a size of about 50 μm to about 250 μm and wherein said second layer is adapted to be located at or near the cartilage defect, and wherein the first layer comprises a material having a resorption time exceeding the resorption time of the second layer; b) cutting the defect cartilage of a patient with a punching device and subsequent curettage to obtain a defect contact surface; c) cutting the implant to the same size as the defect contact surface obtained in step b; and d) introducing the implant onto the defect contact surface.

16. The method according to claim 15, further comprising a step of: seeding and cultivating cells on the implant to obtain a cell-seeded implant either prior to or after step d).

17. The method according to claim 16, wherein said cells are selected from the group consisting of chondrocytes, chondroprogenitor cells, bone-precursor cells, stem cells, cells from periosteum tissue, and cells from perichondrium tissue, or a mixture of two or more of these cell types.

18. The method according to claim 15 further comprising the step of: e) affixing the implant to the defect cartilage by fixation means which are selected from the group consisting of stitches, pins and tissue adhesives.

19. The implant according to claim 5, wherein said calcium phosphates may be a compound selected from the group consisting of tricalcium phosphate, tetracalcium triphosphate and calcium hydroxide, or mixtures of two or more of these components.

20. The implant according to claim 1, wherein said first layer and said second layer are fixed to one another.

21. A three-dimensional implant for repairing a cartilage defect, comprising; a) a first layer comprising a membrane structure comprising stable scaffolding; and b) a second layer comprising at least one wettable material and a sponge structure comprising interconnected pores, wherein cells are seeded on the sponge structure of the implant, further wherein 1) the pores form hollow fiber structures arranged as columns that run substantially parallel to each other and have a substantially consistent pore size, 2) said first layer is configured to face in one direction towards a synovial space and said second layer is configured to face in an opposed direction towards bone, 3) wherein said first layer comprises a material having a resorption time exceeding a resorption time of said second layer and 4) wherein said first layer is fixed to said second layer through lyophilisation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG. 1 shows an implant according to the invention comprising a first membrane-like structure (A) and a second sponge-like structure (A′, A″); in (B) the distribution of fluorescence labelled cells in an implant according to the invention in shown;

[0077] FIG. 2 shows expression of IL-1 (A) and collagen type II (B) of chondrocytes cultured in an implant according to the invention (“TETEC”) and in commercially available implants (“T1”, “T2”);

[0078] FIG. 3 shows the protection of the second layer during its resorption till the defect's replenishment (A) (safranine o staining) and (B) (hematoxilin/eosin staining); the second layer is marked with arrows 2 and seeded with cells, the first layer is marked with arrows 1 and functions as protective layer for cells and matrix underneath; (C) (safranine o staining) shows the reestablishment of hyaline cartilage using an implant according to the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

EXAMPLE 1

Preparation of an Implant According to the Invention

[0079] An implant according to the invention can be prepared—for example—by a method disclosed in EP 1 275 405. By using this method a sponge-like layer or protein matrix can be anchored in a membrane-like layer. Briefly, a membrane-like layer was provided comprising collagen—other suitable materials are, e.g., bioresorbable polymers such as polylactide or polyglycolic acid, collagen, pericardium, composites, glycosaminoglycanes, natural tissue sources like elastin, and mixtures of two or more of these materials. The sponge-like layer was applied thereon in form of a suspension. Alternatively it can be supplied as a dispersion or paste. The suspension was comprising collagen—other materials can be used, e.g. hyaluronic acid, alginate, chitosan, gelatine, processed materials, composites, blood born components such as fibrin, and mixtures of two or more of these materials—and was introduced into the membrane-like structure by means of pressure vacuum. Alternatively, the suspension, dispersion or paste can be introduced by centrifugation. Subsequently, the sponge-like structure of the second layer was formed by unilaterally cooling the membrane-like structure with the suspension applied thereon. The cooling process was performed by gradually lowering the temperature from room temperature to −50° C. thereby generating directional and/or interconnected pores.

[0080] The implant produced in that way was seeded with chondrocytes; alternatively it can be seeded with other cells, e.g. with chondroprogenitor cells, stem-cells, cells from periosteum tissue, and cells from perichondrium tissue or mixtures of two or more of these cell types, before introducing it into the defect site. On the other hand, the implant can be placed directly into the defect without having cells seeded onto it. In the latter case, after putting it under normal strain the implant is compressed and relaxed repeatedly whereby substances such as cells, fluids, nutrients, etc., which are present in the defect site, are taken up. Due to the hollow fiber structure of the sponge-like structure's pores cells can grow and/or migrate into the pores resulting in a homogenous three-dimensional distribution of the cells.

[0081] In FIG. 1 the layers of the implant prepared accordingly are shown. The implant comprises a membrane-like first layer (A) serving as a cover for the underlying sponge-like second layer (A′) that comprises pores orientated directional with respect to the surface in a column-like fashion. First layer and second layer are tightly connected. The pores are interconnected and comprise a directional pore size, whereby a three dimensional distribution of cells can be achieved. The upper picture of the porous layer A′ was taken using transmitted light microscopy, the lower picture of the porous layer (A″) was taken using scanning electron microscopy.

[0082] In FIG. 1, B shows the distribution of fluorescence labelled chondrocytes within the sponge-like second layer of the implant. When preparing the implant the base of the second layer (for example collagen) can comprise additional physiological components of the hyaline cartilage to enhance stable regeneration.

[0083] As can be seen in pictures A′ and A″ of FIG. 1, the pores of the sponge-like structure are directional and interconnected. In that way a homogenous distribution of the cells growing into or migrating into the implant can be achieved which is shown in picture B of FIG. 1.

EXAMPLE 2

[0084] With respect to the biocompatibility of the implants, induction of Interleukin IL-1 expression and reduction of collagen type II expression of chondrocytes seeded into different implants was assessed in vitro.

[0085] The results of these tests are shown in FIG. 2. In FIG. 2A (“IL-1 Induktion”) it is shown that IL-1 induction in chondrocytes seeded on an implant according to the invention (“TETEC”) is lower than IL-1 expression in chondrocytes seeded on commercially available implants (“T1” and “T2”). When IL-1 expression was increased hypertrophy and degeneration of chondrocytes seeded into the implants could be observed in vitro. A marker and controls “GAPDH” and “H2O” are displayed in lanes 1 to 3 respectively.

[0086] Further, expression of collagen type II, which is an essential structural protein in cartilage, was remarkably reduced in chondrocytes seeded into commercially available implants (“T1” and “T2”) in comparison to chondrocytes seeded into implants according to the invention (“TETEC”). These results are shown in FIG. 2B (“COL2A1-Expression”). In FIG. 2B, a marker and controls “GAPDH” and “H2O” are displayed in lanes 1 to 3 respectively.

EXAMPLE 3

[0087] The implant prepared as mentioned above (see Example 1) was tested in animal studies. Experiments were conducted in SCID (“severe combined immunodeficiency”) mice, into which human cells can be transplanted without rejection of these cells, since in the mentioned mice the enzyme adenosine deaminase is deficient and—as a result—T or B cells are not being developed.

[0088] It was previously shown in SCID mice that human articular chondrocytes do only produce solid hyaline cartilage when the transplanted cells express certain marker genes, the fact of which has to be proofed in molecular/biological quality assays. Chondrocytes not expressing collagen type II, BMP-2 (bone morphogenetic protein 2) and FGFR-3 (fibroblast growth factor receptor 3) any more are not able to regenerate high quality cartilage.

[0089] In test group A, human articular chondrocytes expressing relevant marker genes were seeded on an implant according to the invention (5×105 cells/cm2 carrier layer, i.e. second layer). The implant was subsequently transplanted into SCID mice. After incubation in SCID mice, high quality hyaline cartilage was consistently formed, which could be proofed by hematoxilin/eosin staining and safranine o staining. When implanting an implant without the membrane-like first layer, immigration of unspecific connective tissue cells into the sponge-like structure was observed leading to a softening of the implant.

[0090] In control group B, transplantation of human articular chondrocytes not expressing the relevant marker genes any more (5×105 cells/cm2 carrier (layer)) resulted in a clearly inferior and inhomogeneous regeneration, which was shown by hematoxilin/eosin staining and safranine o staining as well.

EXAMPLE 4

[0091] After transplantation, the healing process was checked for resorption of the implant and replenishment of the cartilage defect in animal model (mouse).

[0092] To control the healing process, dissection of the implant after a retention time of about 8 to 12 weeks in the defect cartilage and section staining was performed. The results are shown in FIG. 3. As can be seen in FIG. 3A (safranine o staining) and FIG. 3B (hematoxilin/eosin staining), the membrane-like first layer (indicated by arrows 1) protects the sponge-like second layer underneath and functions as a barrier for cells, thereby ensuring that the underlying sponge-like second layer is kept in a stable condition (indicated by arrows 2) required for replenishment of the cartilage.

[0093] After 8 to 12 weeks the transplantation site was checked for regeneration. Complete resorption of all portions of the implant could be observed, which is shown in FIG. 3C (safranine o staining). The defect site was completely recovered and replenished with hyaline cartilage.