JP2004194944A - Substrate for cartilage cultivation and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate, which offers a more convenience in handling, easier cutting/sutures to arbitrary shapes and sizes, and improves cell reproduction rate etc., for regenerating cartilage tissues by cell cultivation. <P>SOLUTION: This substrate for cartilage cultivation is made of crosslinked collagen sponge combined with a reinforcement of a biodegradable absorbence. It is manufactured in the process of pouring an aqueous solution of collagen, its mixed solution with acetic acid/ethanol, or bubble solution of those onto the reinforcement laid down, and freeze-drying them. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a culture substrate used for regenerating cartilage tissue by cell culture and a method for producing the same.
[0002]
[Prior art]
A technique of regenerating cartilage tissue lost by surgery, trauma, joint deformity, etc. by cell culture and transplanting it into the human body is being put into practical use as regenerative medicine.
That is, recent advances in cell culture technology enable the cultivation of various cells. For example, Kashiki et al. Have succeeded in seeding chondrocytes in a collagen sponge and regenerating cartilage tissue (Non-patent Document 1). reference). Vacanti et al. Attempted to regenerate cartilage tissue by seeding chondrocytes on a fiber composed of a copolymer of glycolic acid and lactic acid, which is a bioabsorbable polymer, as a base material (Non-patent Document 2). reference). It is also known to use a biodegradable polymer or a porous body thereof as a substrate for culturing chondrocytes (see Patent Document 1 and Patent Document 2). Furthermore, the present applicant has also proposed a base material for cartilage tissue regeneration using a sponge-like molded product comprising a polymer of any of lactic acid, glycolic acid or caprolactone, or a copolymer thereof, and having a cell holding structure. (See Patent Document 3).
[0003]
[Non-Patent Document 1]
Biomaterials 17 (1996) 155-162
[Non-Patent Document 2]
Plast.Reconstr.Surg.88,753,1991
[Patent Document 1]
WO90 / 2091 [Patent Document 2]
WO98 / 31345 [Patent Document 3]
Japanese Patent Laid-Open No. 10-234844
[Problems to be solved by the invention]
However, the above configuration still has room for improvement in handling, ease of cutting into an arbitrary shape and size, stitchability, cell proliferation efficiency, and the like.
[0005]
[Means for Solving the Problems]
The present invention has been made to solve the above problems, and has the following features.
Item 1. A base material for cartilage culture characterized by comprising a cross-linked collagen sponge as a constituent material.
Item 2. The substrate for cartilage culture according to Item 1, which is a collagen sponge having a pore size of Item 2.1 to 500 microns.
Item 3. Item 3. The substrate for cartilage culture according to Item 1-2, wherein the thickness is in the range of 1-20 mm.
Item 4. Item 4. The cartilage culture substrate according to any one of Items 1 to 3, which is combined with a biodegradable and absorbable reinforcing material.
Item 5. Item 5. The cartilage culture substrate according to Item 4, wherein the reinforcing material is integrated on one side or in the center.
Item 6. Item 5. The base material for cartilage culture according to Item 4, wherein the reinforcing material is configured in a mesh shape.
Item 7. Item 7. The base material for cartilage culture according to Item 6, wherein the reinforcing material is selected from knitted fabric, woven fabric, non-woven fabric, and film.
Item 8. Item 8. The cartilage culture substrate according to Item 7, wherein the reinforcing material is selected from a polymer of lactic acid, glycolic acid, caprolactone, a copolymer thereof, or a mixture of polymers.
Item 9. Item 9. The cartilage culture substrate according to Item 8, wherein the reinforcing material has been subjected to a hydrophilic treatment.
Item 10. Item 5. The cartilage culture substrate according to Item 4, wherein the cross-linked collagen sponge and the reinforcing material are bonded together with a biodegradable absorbable polymer.
Item 11. Item 11. The substrate for cartilage culture according to Item 10, wherein the biodegradable absorbable polymer used for adhesion is a copolymer of lactic acid and caprolactone.
Item 12. A method for producing a substrate for cartilage culture, which comprises pouring an aqueous solution of collagen, a mixed solution with acetic acid or ethanol, or a foamed solution thereof from the laid reinforcing material, and then freeze-drying.
Item 13. A method for producing a substrate for cartilage culture, comprising pouring a biodegradable absorbable polymer solution from a reinforcing material, placing a collagen sponge, and lyophilizing to adhere the reinforcing material and the collagen sponge. .
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is particularly characterized in that it uses moderately cross-linked collagen as a porous scaffold (sponge) and is reinforced by a reinforcing material made of a biodegradable material, thereby improving handling properties, Improvements such as ease of cutting into arbitrary shapes and sizes, stitchability, and cell growth efficiency have been made.
Hereinafter, this configuration will be described in detail.
[0007]
(Cross-linked collagen sponge)
Collagen used in the present invention can achieve the purpose regardless of its structure, whether it is derived from skin, bone, tendon, animal, human or the like, denatured collagen, fibrotic collagen, etc. For the purpose of reducing antigenicity, it is preferable to use atelocollagen in which the terminal telopeptide is enzymatically removed.
The main purpose of the cross-linking treatment is to eliminate the disadvantage that it becomes difficult to handle due to excessive swelling when it is attached to a culture solution and control of in vivo degradation. As the method, thermal crosslinking, ultraviolet crosslinking, or chemical crosslinking using glutaraldehyde, hexamethylene diisocyanate, carbodiimide, or the like can be used. The degree of cross-linking may be adjusted by appropriately selecting these conditions. However, it is preferable that the degree of crosslinking is usually adjusted in the range of 5 days to several months, preferably in the range of several weeks to 3 months. Specifically, adjustment to a moisture content range of 60% to 95% is a standard.
On the other hand, sponge formation is performed, for example, by freeze-drying a collagen aqueous solution, a mixed solution with acetic acid or ethanol, or a foamed solution thereof. At this time, the stability and uniformity of the porous material can be improved by adding chloroform to the collagen aqueous solution, adding acetic acid, or adding ethanol. Further, by appropriately changing the concentration of the solution, the degree of stirring, the solvent, the additive to the solvent, and the lyophilization conditions, the pore size is several microns to several hundred microns, specifically 1 micron to 500 microns, and the thickness is 1 to It is desired to be 20 mm. The fact that both are within this range is a preferable range in terms of chondrocyte proliferation, strength, convenience of handling, and the like. Furthermore, in this structure, it is good also as a structure which compounded with the sponge body of the lactic acid-caprolactone copolymer, for example, bonded together or piled up.
[0008]
(Reinforcing material)
Biolactic acid, lactic acid-caprolactone copolymer, polyglycolic acid, lactic acid-glycolic acid copolymer, lactic acid-ethylene glycol copolymer, polydioxanone, glycolic acid-caprolactone copolymer, glycol An acid-trimethylene carbonate copolymer can be exemplified, but in the present invention, in terms of control of degradability and flexibility, a polymer of lactic acid, glycolic acid, caprolactone and a copolymer thereof, or a mixture of these polymers It is preferable that it is more selected.
In the present invention, these polymers are spun and stretched by a known method using a knitted, woven, non-woven fabric, flat braid, etc., or extruded, cast, etc. The obtained film is used as a reinforcing material. In addition, as a method for forming the nonwoven fabric, in addition to the needle punching method, those obtained by the melt blow method can also be used.
These reinforcing materials are in the form of a mesh (mesh shape) in consideration of the nutritional supply to the cells to be cultured, and the basis weight is particularly preferably in the range of 5 to 200 g / m ² . Therefore, regarding the film, it is desirable to provide fine holes by an appropriate method. Furthermore, it is desirable to perform a hydrophilic treatment in advance for the purpose of improving the adhesion with the collagen sponge.
Examples of such hydrophilic methods include plasma treatment, glow discharge treatment, corona discharge treatment, ozone treatment, surface graft treatment, coating treatment, chemical treatment, etc., but treatment with plasma is preferable from the viewpoint of operability and effects. desirable.
[0009]
(Method of compounding)
In the present invention, examples of the composite form include a form in which a reinforcing material is integrated on one side of a collagen sponge, or a form in which both sides of a reinforcing material are sandwiched between collagen sponges. . Further, the reinforcing material may be arranged on the entire surface or a part thereof, but it is preferable to arrange the reinforcing material on the entire surface from the viewpoint of convenience of handling. Furthermore, a biodegradable absorbable polymer solution may be used as an adhesive for integration. An example of such an adhesive is a lactic acid-caprolactone copolymer solution.
The compounding method is specifically illustrated below, but is not limited thereto.
(Method A)
Put a reinforcing material in an appropriate container ⇒ pour a homogenized collagen aqueous solution or an aqueous solution containing acetic acid or ethanol ⇒ freeze-dry ⇒ cross-link treatment (two-layer structure)
(Method B)
Put a reinforcing material in an appropriate container ⇒ pour a lactic acid-caprolactone copolymer solution as an adhesive ⇒ stick a cross-linked collagen sponge on it ⇒ freeze-dry (two-layer structure with an adhesive layer)
(Method C)
Pour the homogenized collagen solution into an appropriate container ⇒ Place the reinforcing material ⇒ Pour the homogenized collagen aqueous solution or the aqueous solution with acetic acid or ethanol added ⇒ Freeze-dry and cross-link (sandwich three-layer structure)
Hereinafter, specific examples will be described.
[0010]
[Example 1]
1. Preparation of collagen sponge-Using atelocollagen (manufactured by Nitta Gelatin Co., Ltd.) as a raw material, a 3 mg / ml, pH 3.0 collagen aqueous solution is prepared.
Add 0.5 g of chloroform to 50 g of this solution, and vigorously stir for 1 minute at 6000 rpm using a homogenizer.
-Pour the resulting creamy foam into an aluminum mold (11cm x 8.5cm).
・ Leave for 1 hour in a -40 ℃ freezer and freeze.
Heat at a constant rate over 12 hours from −40 ° C. to 40 ° C. under vacuum reduced pressure (0.01 mmHg), and further dry at 40 ° C. under vacuum reduced pressure for 12 hours to obtain a collagen sponge.
2. Preparation of heat-crosslinked collagen sponge • The above-described collagen sponge is heat-dried at 105 ° C under vacuum for 24 hours to obtain a heat-crosslinked collagen sponge.
3. Preparation of Chemically Crosslinked Collagen Sponge • The above-mentioned thermally cross-linked collagen sponge is immersed in a 0.2 wt% glutaraldehyde / acetic acid solution at 5 ° C. for 24 hours for chemical crosslinking.
-Wash the resulting sponge thoroughly with ion-exchanged water.
-After washing, immerse in 15% ethanol aqueous solution.
・ Leave in a freezer at -135 ° C for 12 hours to freeze.
Heat at a constant rate from −40 ° C. to 40 ° C. under vacuum under reduced pressure (0.01 mmHg) over 12 hours, and further dry at 40 ° C. under vacuum under reduced pressure for 12 hours to obtain a chemically crosslinked complexed collagen sponge.
4). Composite with poly-L lactic acid fiber (cloth)-A reinforcing cloth is prepared from poly-L lactic acid fiber (PLLA fiber).
(Organization: 7 types: milling fabric, double-sided knitting fabric, twin knitted fabric, plain woven fabric, tengu knitted fabric, non-woven fabric)
-A 4% dioxane solution of lactic acid-caprolactone copolymer (P (LA / CL); polymerization ratio 50:50) is prepared.
-Put the PLLA cloth on a 20cm x 20cm glass mold and pour the P (LA / CL) solution there.
-Place the sponge obtained in 3 above on it, quickly transfer it to a -80 ° C freezer and freeze it.
・ Heat from -40 ° C to 40 ° C at a constant rate over 12 hours under vacuum reduced pressure (0.01mmHg), further dry at 40 ° C under vacuum reduced pressure for 6 hours, and further at 70 ° C under vacuum reduced pressure. Dry for 12 hours to obtain a complex of collagen sponge and PLLA cloth and P (LA / CL) sponge.
[0011]
[Example 2]
1. Preparation of lactic acid-caprolactone reinforcing material (cloth)-Fabricate a lactic acid-caprolactone copolymer (P (LA / CL); polymerization ratio 75:25) fiber. (Organization: 7 types: milling fabric, double-sided knitting fabric, twin knitted fabric, plain woven fabric, tengu knitted fabric, non-woven fabric)
・ Plasma treatment (oxygen, 100 W, 0.5 torr, 30 seconds) of P (LA / CL) cloth to make it hydrophilic. 2. Complexed with collagen sponge ・ Atelocollagen (Nitta gelatin) is used as a raw material to prepare a 3 mg / ml, pH 3.0 collagen aqueous solution.
Add 0.5 g of chloroform to 50 g of this solution, and vigorously stir for 1 minute at 6000 rpm using a homogenizer.
-Place the hydrophilic P (LA / CL) cloth on an aluminum mold (11cm x 8.5cm) and pour the resulting creamy collagen foam.
-Let stand for 1 hour in a -40 ° C freezer and freeze.
-Heated at a constant rate from -40 ° C to 40 ° C for 12 hours under vacuum reduced pressure (0.01mmHg), and further dried at 40 ° C under vacuum reduced pressure for 12 hours to reinforce P (LA / CL) A collagen sponge complexed with is obtained.
3. Thermal cross-linking The above composite is heat-dried at 105 ° C. for 24 hours under vacuum under reduced pressure to be thermally cross-linked.
4). Chemical cross-linking The above composite is immersed in a 0.2 wt% glutaraldehyde / acetic acid solution at 5 ° C. for 24 hours for chemical cross-linking.
-Wash the resulting sponge thoroughly with ion-exchanged water.
-After washing, immerse in 15% ethanol aqueous solution.
・ Leave in a freezer at -135 ° C for 12 hours to freeze.
-Heating at a constant rate from -40 ° C to 40 ° C under vacuum reduced pressure (0.01 mmHg) over 12 hours, and further drying at 40 ° C under vacuum reduced pressure for 12 hours to obtain a chemically crosslinked complexed collagen sponge.
[0012]
(Transplant to animals)
Rabbit bone marrow was collected, mixed with DMEM medium containing 15% fetal bovine serum, and cultured for about 1 week. Further, the cells were subcultured for about 1 week and grown to about 10 ⁶ . The cells were seeded on a composite of the milled knitted fabric obtained in Example 2 and a cross-linked collagen sponge obtained by cutting into a cylindrical shape having a diameter of 4 mm and a thickness of 3 mm. The rabbit's knee joint was exposed, a 4 mm diameter and 4 mm deep osteochondral defect was created using a drill, and a cell seeded sample was inserted into the defect. Rabbits were sacrificed 1, 2, and 4 weeks after the operation, and tissue sections of the transplanted sites were prepared. After the operation, regeneration of the cartilage tissue at the joint surface and regeneration of the bone tissue at the bone defect were observed with time.
[0013]
【The invention's effect】
In the present invention, the cross-linking of collagen and the reinforcing effect by the reinforcing material can improve handling convenience, can be cut into a free shape and size, can be added with sufficient suturing strength, and the reinforcing material can be absorbed. Since the reinforcing material is a porous body (with moderately open eyes), it has many features and effects such as nutrition permeation.
Therefore, it can be used not only for cartilage regeneration, but also as a substrate for regeneration of other organs, for example, as a substrate for cultured skin. In particular, a three-layer type having a sandwich structure can be suitably used as a cultured skin base material.

Claims (13)

A base material for cartilage culture, characterized by comprising a cross-linked collagen sponge as a constituent material. The cartilage culture substrate according to claim 1, which is a collagen sponge having a pore size of 1 to 500 microns. The substrate for cartilage culture according to claim 1 or 2, wherein the thickness is in the range of 1 to 20 mm. The base material for cartilage culture according to any one of claims 1 to 3, wherein the base material for cartilage culture is compounded with a biodegradable and absorbable reinforcing material. The base material for cartilage culture according to claim 4, wherein the reinforcing material is located on one side or the central part and integrated. The base material for cartilage culture according to claim 4, wherein the reinforcing material is configured in a mesh shape. The base material for cartilage culture according to claim 6, wherein the reinforcing material is selected from knitted fabric, woven fabric, non-woven fabric, and film. The base material for cartilage culture according to claim 7, wherein the reinforcing material is selected from a polymer of lactic acid, glycolic acid, caprolactone and a copolymer thereof, or a mixture of these polymers. The base material for cartilage culture according to claim 8, wherein the reinforcing material is hydrophilized. The base material for cartilage culture | cultivation of Claim 4 to which the cross-linked collagen sponge and the reinforcing material are adhere | attached with the biodegradable absorbable polymer. The substrate for cartilage culture according to claim 10, wherein the biodegradable absorbable polymer used for adhesion is a copolymer of lactic acid and caprolactone. A method for producing a base material for cartilage culture, which comprises pouring an aqueous solution of collagen, a mixed solution with acetic acid or ethanol, or a foaming solution thereof from the laid reinforcing material, followed by freeze-drying. A method for producing a base material for cartilage culture, comprising pouring a biodegradable absorbable polymer solution from a reinforcing material laid, placing a collagen sponge, and freeze-drying to adhere the reinforcing material and the collagen sponge. .