CARTILAGE TISSUE PRODUCING METHOD AND CARTILAGE TISSUE

Abstract

The present invention aims to provide a method for producing a cartilage tissue, which enables production of a cartilage tissue having an appropriate thickness, form, and mechanical strength, and a cartilage tissue produced by the method for producing a cartilage tissue. Provided is a method for producing a cartilage tissue including a step of seeding a collagenase-treated cartilage tissue piece in the form of a block 50 to 1,000 m on a side onto a porous substrate composed of a bioabsorbable material.

Claims

1. A method for producing a cartilage tissue comprising a step of seeding a collagenase-treated cartilage tissue piece in the form of a block 50 to 1,000 m on a side onto a porous substrate composed of a bioabsorbable material.

2. The method for producing a cartilage tissue according to claim 1, wherein the cartilage tissue piece is in the form of a block 100 to 800 m on a side.

3. The method for producing a cartilage tissue according to claim 1, wherein the porous substrate is a nonwoven fabric having an average pore size of 20 to 50 m.

4. The method for producing a cartilage tissue according to claim 1, wherein the bioabsorbable material constituting the porous substrate is polyglycolide.

5. The method for producing a cartilage tissue according to claim 1, wherein the porous substrate is fixed to a mold composed of a bioabsorbable material to combine and integrate with the mold.

6. The method for producing a cartilage tissue according to claim 5, wherein the bioabsorbable material constituting the mold is polycaprolactone.

7. A cartilage tissue comprising a porous substrate composed of a bioabsorbable material and a collagenase-treated cartilage tissue piece in the form of a block 50 to 1,000 m on a side seeded on the porous substrate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0067] FIG. 1 shows a schematic view explaining how to measure the pore size distribution of a porous substrate by the bubble point method.

[0068] FIG. 2 shows a schematic view explaining how to estimate the pore size distribution of a porous substrate based on data obtained by the bubble point method.

[0069] FIG. 3 shows schematic views illustrating exemplary embodiments of a porous substrate combined and integrated with a mold used in the method for producing a cartilage tissue of the present invention.

[0070] FIG. 4 shows schematic views illustrating exemplary embodiments of a porous substrate combined and integrated with a mold used in the method for producing a cartilage tissue of the present invention.

[0071] FIG. 5 shows a schematic view illustrating an exemplary microslicer that can be used as a fine processing device.

[0072] FIG. 6 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Example 1.

[0073] FIG. 7 shows safranin-staining images (a) and Verhoeff's staining images (b) of cross-sections of nonwoven fabrics onto which respective collagenase-treated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Example 2.

[0074] FIG. 8 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which untreated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Comparative Example 1.

[0075] FIG. 9 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Example 3.

[0076] FIG. 10 shows safranin-staining images (a) and Verhoeff's staining images (b) of cross-sections of nonwoven fabrics onto which respective collagenase-treated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Example 4.

[0077] FIG. 11 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which untreated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation as in Comparative Example 2.

[0078] FIG. 12 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 100 m on a side subjected to collagenase treatment for 15 minutes have been seeded, taken 20 weeks after transplantation as in Example 5.

[0079] FIG. 13 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 200 m on a side subjected to collagenase treatment for 15 minutes have been seeded, taken 20 weeks after transplantation as in Example 5.

[0080] FIG. 14 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 400 m on a side subjected to collagenase treatment for 60 minutes have been seeded, taken 20 weeks after transplantation as in Example 5.

DESCRIPTION OF EMBODIMENTS

[0081] Embodiments of the present invention are described in more detail with reference to, but not limited to, Examples.

EXAMPLE 1

(1) Preparation of Nonwoven Fabric

[0082] A downy nonwoven fabric having an average fiber diameter of approximately 16 m and a thickness of approximately 0.5 mm was obtained by the following method: Polyglycolide having a weight average molecular weight of 250,000 was used as a bioabsorbable material and spun into filaments, and then made into a nonwoven fabric by needle punching.

[0083] The obtained layer into which cells penetrate was allowed to absorb a wetting liquid of a fluorine solvent (product name: Profil), and then set in Prometer 3G, the product of BEL Japan, Inc., in such a manner that the layer as a test piece was in a circular shape with a diameter of 25 mm. Air pressure was applied from the underside of the layer into which cells penetrate and the minimum pressure (bubble point) at which generation of air bubbles was observed on the membrane surface was measured. Based on the bubble point thus obtained, a graph showing the pore size distribution of the nonwoven fabric was obtained. The average pore size was 28 m as calculated from the graph.

(2) Preparation and Seeding of Cartilage Tissue Pieces

[0084] The skin, connective tissues, and perichondrium were removed from human auricular cartilage obtained by excision for the treatment of prominent ear to give a human auricular cartilage having a length of 5 mm, a width of 5 mm, and a thickness of 1 mm. The human auricular cartilage was sliced with a fine processing device into cartilage tissue pieces approximately 800 m on a side.

[0085] The obtained cartilage tissue pieces were subjected to a collagenase treatment, which included shaking in a collagenase solution having a concentration of 0.3% for 60 minutes under the conditions of a temperature of 37 C. and a revolution of 160 rpm.

[0086] The collagenase-treated cartilage tissue pieces were suspended in 50 L phosphate buffer and evenly seeded onto the nonwoven fabric using a pipette. The cartilage tissue pieces were seeded in such a manner that the area of the cartilage tissue pieces seeded was 5 mm.sup.2 with respect to 1 cm.sup.2 of the area of the cartilage tissues to be obtained.

(3) Transplantation and Evaluation of Cartilage Tissue Regeneration

[0087] The nonwoven fabric onto which the cartilage tissue pieces thus obtained had been seeded was transplanted into 4 to 6-week-old athymic mice (an average body weight of 28 g, male). After general anesthesia, an incision was made in the posterior region of neck and the nonwoven fabric were transplanted under the skin. Ten weeks after the transplantation, the mice were sacrificed and respective samples were collected.

[0088] FIG. 6 shows a safranin-staining image (a) and Verhoeff's staining image (b) of cross-sections of the nonwoven fabrics onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation. According to FIG. 6(a), it was conformed that proteoglycans was produced and regenerated cartilages were induced. In FIG. 6(b), the area stained black indicates transplanted cartilage pieces and the area stained slightly dark indicates regenarated cartilage. Induction of regenerated cartilages was confirmed also from the observation that the area stained black occupied only a small area. Further, polyglycolide constituting the nonwoven fabrics was completely degraded.

EXAMPLE 2

[0089] Seeding of the cartilage tissue pieces onto nonwoven fabrics, transplantation, and evaluation of cartilage tissue regeneration were performed in the same manner as in Example 1, except that the conditions for collagenase treatment performed on cartilage tissue pieces were set to (1) 15 minutes, (2) 30 minutes, (3) 45 minutes, and (4) 60 minutes.

[0090] FIG. 7 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of cross-sections of the nonwoven fabrics onto which respective collagenase-treated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation.

COMPARATIVE EXAMPLE 1

[0091] Seeding of the cartilage tissue pieces onto nonwoven fabrics, transplantation, and evaluation of cartilage tissue regeneration were performed in the same manner as in Example 1, except that cartilage tissue pieces were not subjected to collagenase treatment.

[0092] FIG. 8 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of cross-sections of nonwoven fabrics onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation. According to FIG. 8, it was confirmed that production of proteoglycans was poor and regenerated cartilage was hardly induced in Comparative Example 1. Though residual cartilage tissue pieces were observed, an absorption image was observed.

EXAMPLE 3

(1) Preparation of Nonwoven Fabric

[0093] A downy nonwoven fabric having an average fiber diameter of approximately 16 m and a thickness of approximately 0.5 mm was obtained by the following method: Polyglycolide having a weight average molecular weight of 250,000 was used as a bioabsorbable material and spun into filaments, and then made into a nonwoven fabric by needle punching.

[0094] The obtained layer into which cells penetrate was allowed to absorb a wetting liquid of a fluorine solvent (product name: Profil), and then set in Porometer 3G, the product of BEL Japan, Inc., in such a manner that the layer as a test piece was in a circular shape with a diameter of 25 mm. Air pressure was applied from the underside of the layer into which cells penetrate and the minimum pressure (bubble point) at which generation of air bubbles was observed on the membrane surface was measured. Based on the bubble point thus obtained, a graph showing the pore size distribution of the nonwoven fabric was obtained. The average pore size was 28 m as calculated from the graph.

(2) Preparation of Small-Pore-Size Nonwoven Fabric

[0095] A small-pore-size nonwoven fabric having an average fiber diameter of approximately 2 m and a thickness of approximately 50 m was obtained by the following method: Polyglycolide having a weight average molecular weight of 250,000 was used as a bioabsorbable material, and made into a nonwoven fabric by melt blowing.

[0096] The average pore size of the obtained small-pore-size nonwoven fabric was calculated by the bubble point method, and was 12 m.

(3) Lamination of Nonwoven Fabric and Small-Pore-Size Nonwoven Fabric

[0097] The small-pore-size nonwoven fabric having a thickness of 50 m and the nonwoven fabric having a thickness of 0.5 mm were stacked on each other and stitched at the four corners with a 5-0 nylon thread. Thus, a laminate was obtained.

(4) Preparation and Seeding of Cartilage Tissue Pieces

[0098] The skin, connective tissues, and perichondrium were removed from human auricular cartilage obtained by excision for the treatment of prominent ear to give a human auricular cartilage having a length of 5 mm, a width of 5 mm, and a thickness of 1 mm. The human auricular cartilage was sliced with a fine processing device into cartilage tissue pieces approximately 800 m on a side.

[0099] The cartilage tissue pieces thus obtained were subjected to a collagenase treatment, which included shaking in a collagenase solution having a concentration of 0.3% for 60 minutes under the conditions of a temperature of 37 C. and a revolution of 160 rpm.

[0100] The collagenase-treated cartilage tissue pieces were suspended in 50 L phosphate buffer and evenly seeded onto the nonwoven fabric side of the laminate using a pipette. The cartilage tissue pieces were seeded in such a manner that the area of the cartilage tissue pieces to be seeded is 5 mm.sup.2 with respect to 1 cm.sup.2 of the area of the cartilage tissues to be obtained.

(5) Transplantation and Evaluation of Cartilage Tissue Regeneration

[0101] The nonwoven fabric onto which the cartilage tissue pieces thus obtained had been seeded was transplanted into 4 to 6-week-old athymic mice (an average body weight of 28 g, male). After general anesthesia, an incision was made in the posterior region of neck and the cartilage tissues were transplanted under the skin. Ten weeks after transplantation, the mice were sacrificed and respective samples were collected.

[0102] FIG. 9 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation. According to FIG. 9(a), it was confirmed that a large amount of proteoglycan was produced and regenerated cartilages were more induced than Example 1. Induction of regenerated cartilages was confirmed also from the observation that the area stained black occupied only a small area in FIG. 9(b). Further, polyglycolide constituting the nonwoven fabric was completely degraded.

EXAMPLE 4

[0103] Seeding of the cartilage tissue pieces onto a nonwoven fabric, transplantation, evaluation of cartilage tissue regeneration were performed in the same manner as in Example 3, except that the conditions for collagenase treatment performed on cartilage tissue pieces were set to (1) 15 minutes, (2) 30 minutes, (3) 45 minutes, and (4) 60 minutes.

[0104] FIG. 10 shows safranin-staining images (a) and Verhoeff's staining images (b) of cross-sections of the nonwoven fabrics onto which respective collagenase-treated cartilage tissue pieces have been seeded, taken 10 weeks after transplantation.

COMPARATIVE EXAMPLE 2

[0105] Seeding of the cartilage tissue pieces onto a nonwoven fabric, transplantation, and evaluation of cartilage tissue regeneration were performed in the same manner as in Example 3, except that cartilage tissue pieces were not subjected to collagenase treatment.

[0106] FIG. 11 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces have been seeded, taken 10 weeks after transplantation. According to FIG. 11, it was confirmed that production of proteoglycan was poor and regenerated cartilages were hardly induced in Comparative Example 2. Though residual cartilage tissue pieces were observed, an absorption image was observed.

EXAMPLE 5

(1) Preparation and Seeding of Cartilage Tissue Pieces

[0107] The skin, connective tissues, and perichondrium were removed from an ear obtained by excision from a canine to give a canine auricular cartilage. The canine auricular cartilage was sliced with a fine processing device into cartilage tissue pieces in the form of a block 100 m on a side, a block 200 m on a side, and a block 400 m on a side.

[0108] The obtained cartilage tissue pieces of respective sizes were subjected to a collagenase treatment, which included shaking in a collagenase solution having a concentration of 0.3% for 0 minutes, 15 minutes, and 60 minutes under the conditions of a temperature of 37 C. and a revolution of 160 rpm.

[0109] The collagenase-treated cartilage tissue pieces of respective sizes were each suspended in 50 L phosphate buffer, thereby preparing suspensions. The suspensions were evenly seeded onto the nonwoven fabric side of laminates prepared by the same method as in Example 3 using a pipette. The cartilage tissue pieces were seeded in such a manner that the area of the cartilage tissue pieces seeded was 5 mm.sup.2 with respect to 1 cm.sup.2 of the area of the cartilage tissues to be obtained.

(2) Transplantation and Evaluation of Cartilage Tissue Regeneration

[0110] The nonwoven fabrics onto which the cartilage tissue pieces were seeded were transplanted to the canine from which one ear was excised in the above step (1). After general anesthesia, an incision was made on the head and the cartilage tissues were transplanted under the skin. Twenty weeks after transplantation, the canine was sacrificed and respective samples were collected.

[0111] Cross-sectional slices of the nonwoven fabrics 20 weeks after transplantation were prepared, and were subjected to safranin staining and Verhoeff's staining. FIG. 12 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 100 m on a side subjected to collagenase treatment for 15 minutes have been seeded, taken 20 weeks after transplantation. FIG. 13 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 200 m on a side subjected to collagenase treatment for 15 minutes have been seeded, taken 20 weeks after transplantation. FIG. 14 shows a safranin-staining image (a) and a Verhoeff's staining image (b) of a cross-section of a nonwoven fabric onto which cartilage tissue pieces in the form of a block 400 m on a side subjected to collagenase treatment for 60 minutes have been seeded, taken 20 weeks after transplantation.

[0112] In safranin staining, cartilage tissues are stained red. In FIGS. 12 to 14, many cartilage tissue parts stained red can be observed, which confirms regeneration of cartilage tissues. Image processing of the safranin-stained image enables calculation of the area of regenerated cartilage tissue parts (area of the cartilage tissue parts stained red). This method clarified that in the case of cartilage tissue pieces in the form of a block 100 m on a side and a block 200 m on a side, collagenase treatment for 15 minutes allowed especially favorable cartilage regeneration. It also clarified that in the case of cartilage tissue pieces in the form of a block 400 m on a side, collagenase treatment for 60 minutes allowed especially favorable cartilage regeneration.

[0113] The results show that the optimum collagenase treatment conditions are different according to the size (length of one side) of the cartilage tissue pieces.

EXAMPLE 6

(1) Preparation and Seeding of Cartilage Tissue Pieces

[0114] The skin, connective tissues, and perichondrium were removed from an ear obtained by excision from a canine to give a canine auricular cartilage tissue. The obtained canine auricular cartilage was cut into blocks of 1 cm1 cm (1 cm.sup.2), and then further sliced with a fine processing device into cartilage tissue pieces in the form of a block 400 m on a side.

[0115] The obtained cartilage tissue pieces were subjected to a collagenase treatment, which included shaking in a collagenase solution having a concentration of 0.3% for 15 minutes under the conditions of a temperature of 37 C. and a revolution of 160 rpm.

[0116] The collagenase-treated cartilage tissue pieces were suspended in 50 L phosphate buffer, thereby preparing a suspension. The whole suspension was evenly seeded using a pipette onto the nonwoven fabric side of a laminate prepared by the same method as in Example 3 and cut to a size of 2 cm2 cm (4 cm.sup.2).

(2) Transplantation and Evaluation of Cartilage Tissue Regeneration

[0117] The nonwoven fabric onto which the cartilage tissue pieces had been seeded was transplanted to the canine from which one ear was excised in the above step (1). After general anesthesia, an incision was made on the head and the cartilage tissues were transplanted under the skin. Twenty weeks after transplantation, the canine was sacrificed and the nonwoven fabric was collected.

[0118] A cross-sectional slice of the nonwoven fabric 20 weeks after transplantation was prepared, and subjected to safranin staining. Formation of cartilage tissue parts stained red was observed on the entire surface of the nonwoven fabric.

[0119] This result shows that larger cartilage tissues can be regenerated with only a small amount of cartilage tissue pieces.

INDUSTRIAL APPLICABILITY

[0120] The present invention can provide a method for producing a cartilage tissue which enables production of a cartilage tissue having an appropriate thickness, form, and mechanical strength, and also provide a cartilage tissue produced by the method for producing a cartilage tissue.

REFERENCE SIGNS LIST

[0121] 1, 1 porous substrate combined and integrated with mold [0122] 11 rectangular porous substrate [0123] 12 mold [0124] 2, 2 porous substrate combined and integrated with mold [0125] 21 porous substrate [0126] 22 mold shaped in the form of external ear