JPH10234844A - Base material for regenerating cartilaginous tissue and regenerating method of cartilaginous tissue using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable decomposition and absorption of a cartilaginous tissue after the elapsing of a specified period with easier formation of the tissue by forming a sponge- like molded product having a cell holding structure out of a polycondensation body of any of lactic acid, glycolic acid and caprolactone or a copolymer thereof to make it adapted to the proliferation of cartilaginous cells. SOLUTION: Material herein used is a polycondensation body of any of lactic acid, glycolic acid and caprolactone or copolymer thereof, the example, a copolymer of lactic acid and glycolic acid. This copolymer is properly treated and molded as desired in a porous form to make a sponge-like molded product for surgery. The pore diameter of the molded product is not limited if the structure thereof can hold cells, preferably cartilaginous cells. But, normally, the pore diameter is about 1mm or less, preferably about 5-100μm. The density of the product is about 0.1-10g/cm<3> , preferably about 1-5g/cm<3> . This structure can improve breaking strength, breaking by elongation and elasticity thereby easily producing a mold according to the figure of a patient. This also enables regeneration of a cartilaginous tissue in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel sponge-shaped bioabsorbable polymer comprising a polycondensate of lactic acid, glycolic acid or caprolactone or a copolymer thereof.

[0002] The present invention also relates to a novel base material serving as a good scaffold for regenerating cartilage tissue in a cartilage tissue reconstruction method, and a cartilage tissue regeneration method using the base material.

[0003] The "cartilage tissue reconstruction method" is a method of regenerating cartilage by reconstructing cartilage tissue lost by surgery, trauma, or the like by a cell culture method and transplanting it into a patient.

[0004]

2. Description of the Related Art Recent advances in cell culture technology have enabled the cultivation of many animal cells, and attempts are being made to reconstruct tissues from those cells (Cell Engineering, 14 (12) 1995). ). The most important thing in such an attempt is the preparation of a matrix that serves as a scaffold for the seeded cells to reconstruct the living tissue.

[0005] Skin is the most advanced in practical application in this field. Maruguchi et al. Prepared a foam using collagen, inoculated with fibroblasts, and then inoculated epidermal keratinocytes on the foam to produce a cultured artificial skin having an epidermal and dermal structure (Plast . Reconstr. Sur
g. 93: 537, 1994). Collagen has excellent biocompatibility,
It is also very suitable for such purposes, as it is degraded and absorbed after tissue regeneration.

In cartilage regeneration, tissue reconstruction has been attempted. Have succeeded in the reconstruction of cartilage tissue by using a collagen sponge similar to that used for the artificial skin as a scaffold and seeding it with chondrocytes (Biomaterials 17 (1996) 155-162). Also Vaca
nti et al. have attempted to regenerate cartilage tissue by seeding chondrocytes on a fiber consisting of a copolymer of glycolic acid and lactic acid, which are bioabsorbable polymers (Plast. Reconstr. Surg. . 88, 753, 1991).

However, these methods have problems in the physical properties of the base material, and it is difficult to regenerate cartilage tissue for practical use. That is, when a collagen sponge is used as a base material, collagen is very excellent as a base material for cell culture, but on the other hand, only a very flexible one can be produced, so it is difficult to produce a required shape. is there. That is, after transplantation into a living body after the necessary cultured chondrocytes have been formed in a test tube, the shape cannot be maintained in the living body without a certain strength. Even in the case where a bioabsorbable polymer is used, it is difficult to freely produce a substrate having a required structure in an attempt to produce a matrix substrate using fibers.

Therefore, there is a need for a material capable of easily forming a three-dimensional shape freely as an ideal base material of a chondrocyte matrix. For example, when forming an auricle, it is ideal that the tissue can be regenerated by forming the shape of the auricle with a matrix and then implanting the same.

[0009]

Accordingly, an object of the present invention is to provide (1) an optimal scaffold for chondrocyte proliferation,
(2) It has an appropriate strength and can easily produce the desired shape of cartilage tissue. (3) It is said that after transplantation into a living body, it is decomposed and absorbed after a desired period of time. An object of the present invention is to provide a sponge-like molded product having properties, preferably a substrate for cell seeding, more specifically, a substrate for cartilage tissue regeneration.

Another object of the present invention is to provide a cartilage tissue-regenerating substrate-chondrocyte complex which has a cartilage tissue regenerating effect in a short period of time and efficiently.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned objects, and as a result, have found that the above properties can be obtained by using a porous body made of a specific bioabsorbable polymer as a scaffold for chondrocytes. We have found that a cartilage tissue regeneration base material can be provided, and based on this knowledge, we have completed the present invention.

That is, a first aspect of the present invention is a sponge-like molding comprising a polycondensate of any of lactic acid, glycolic acid and caprolactone or a copolymer thereof, and having a cell holding structure. Things.

[0013] A second aspect of the present invention is a cartilage tissue regeneration method comprising a polycondensate of any of lactic acid, glycolic acid and caprolactone, or a porous material composed of any of these copolymers. It is a substrate. In addition, as a porous body, a sponge-like thing is more preferable. The porous body may be coated with a cell adhesion promoting substance.

[0014] A third aspect of the present invention is a cartilage tissue regeneration complex containing the above-mentioned cartilage tissue regeneration base material and chondrocytes.

Further, a fourth aspect of the present invention is a method for regenerating cartilage tissue, wherein cartilage is regenerated by sowing chondrocytes on the substrate for regenerating cartilage tissue and transplanting the same.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The molded product of the present invention is a polycondensate of lactic acid (polylactic acid), a polycondensate of glycolic acid (polyglycolic acid), and a polycondensate of caprolactone, which are synthetic bioabsorbable polymers. (Polycaprolactone), a copolymer of lactic acid and glycolic acid, a copolymer of glycolic acid and caprolactone, a copolymer of lactic acid and caprolactone, or a copolymer of lactic acid, glycolic acid and caprolactone, To make it porous by
It is a surgical molded product formed into a desired form.

Here, the porous shape means a shape having a large number of small voids (holes: pores) therein, and specific examples thereof include a sponge shape, a honeycomb shape, and the like. , Preferably in the form of a sponge.

The pore size is not particularly limited as long as it can hold cells, preferably chondrocytes.
mm or less, preferably 5 to 100 [mu] m, density of 0.1 to 10 g / cm ^3, preferably from 1 to 5 g / cm ^3.

The average molecular weight, mixed composition, polymerization ratio, polymerization method and the like of the polymer used are not particularly limited. However, when cells are seeded on the prepared molded product, the cells adhere and retain on the surface or inside of the molded product. It is preferable that the cells have the property of allowing cells to proliferate when cultured with a molded article. More preferably, a molded product obtained by molding into a porous shape,
When the cells are seeded to form cartilage, they are appropriately selected so as to have appropriate strength and elasticity as cartilage, and to be decomposed and absorbed after cartilage is regenerated.

For example, one embodiment of the molded article of the present invention is a molded article comprising the above-mentioned polycondensate or copolymer, having a tensile strength at break of 0.1 kgf / cm ² or more and an average viscosity after molding. When the molecular weight is in the range of 100,000 to 500,000 and is implanted in the body, it can be decomposed and absorbed within one year, preferably within six months.

The method for producing a sponge-like molded product having the above-mentioned properties is not particularly limited, but as an example, the viscosity average molecular weight is 100,000 to 500,000, preferably 50,000 to 500,000.
A polycondensate or copolymer solution having 300,000 is placed in a desired mold, frozen, and then freeze-dried under vacuum to obtain a sponge-like molded product in a desired form. Alternatively, the formed sponge can be formed by cutting the sponge into an appropriate shape.

More specifically, for example, the lactic acid polycondensate (polylactic acid) used in the present invention can be prepared from an optically active L-form or D-form lactic acid by a conventional method (CE Lowe.
SP2668, 162), a lactide that is a cyclic dimer of lactic acid is synthesized, and then the lactide is subjected to ring-opening polymerization. The polymerization is generally carried out at a polymerization temperature of 100 to 2 under reduced pressure or an inert gas atmosphere.
The reaction can be carried out at 50 ° C. using a tin-based, zinc-based, aluminum-based compound or the like as a catalyst.

In the present invention, a polycondensate of glycolic acid (polyglycolic acid) or a polycondensate of caprolactone (polycaprolactone) is used in place of the lactic acid polycondensate. These production methods can also be performed according to a conventional method.

Further, in the present invention, a copolymer composed of lactic acid, glycolic acid or caprolactone can be used instead of the above polycondensate.

The copolymer may be either a random copolymer or a block copolymer. These block copolymers consist of a series of chain segments of various lengths, each segment consisting of a homopolymer of monomers or a random copolymer comprising two or more copolymers. You may.

The mixing ratio of these copolymers is not particularly limited as long as they constitute a sponge molded product having the above characteristics, and the copolymer can be produced according to a conventional method.

The sponge-like molded article of the present invention is not limited in its use, but can be used for any purpose by taking advantage of its properties. Preferably, use as a cell scaffold or use as a cell seeding material is exemplified.

The form of the molded product is not particularly limited, and can be appropriately selected according to the purpose.

The cartilage tissue regeneration base material according to the present invention is one of the above-mentioned molded products. In this case, the form of the molding is processed so as to conform to the shape of the object to be reconstructed, for example, the ear or the nose, in consideration of the desired form.

The “substrate for regenerating cartilage tissue” is a scaffold on which cartilage cells can be suitably proliferated when seeded in vitro, and when cartilage cells are embedded in a living body while containing the cells. Means that the chondrocytes therein rapidly differentiate and regenerate, and the substrate itself is decomposed and absorbed. The `` substrate for regenerating cartilage tissue '' of the present invention is particularly easy to handle because it has moderate cartilage-like strength and elasticity, and also contains cartilage cells and, when transplanted into a living body, regenerates cartilage tissue. It is characterized by being performed in a short period of several weeks.

The base material for cartilage tissue regeneration of the present invention exhibiting the above-mentioned effects is one of the above-mentioned molded products, which has the strength not to lose its shape after implantation into a living body, has a certain flexibility, and decomposes in about 6 months. Those having the property of being absorbed are preferred.

The molded product and the substrate for cartilage tissue regeneration of the present invention may further have a porous surface coated with a cell adhesion promoting substance.

The cell adhesion promoting substance is not particularly limited as long as it has a property of promoting cell adhesion, and specific examples thereof include collagen, gelatin and the like.

The coating method is not particularly limited and can be carried out according to a conventional method. For convenience, as shown in Example 2 below, after preparing a porous substrate, it is immersed in a cell adhesion promoting substance. Then, a method of freeze-drying again is exemplified.

The present invention also provides a chondrocyte complex using the above-mentioned substrate for cartilage tissue regeneration.

Such a cartilage tissue regeneration substrate-chondrocyte complex comprises a matrix composed of the above-mentioned cartilage tissue regeneration substrate and containing chondrocytes, and is used as a cartilage tissue regeneration graft or cartilage tissue regeneration. Useful as a therapeutic material.

The preparation method is not particularly limited. For example, after preparing the above-mentioned ointment tissue regenerating base material into an appropriate size and shape according to the purpose of use, for example, a chondrocyte dispersion liquid is poured into the base material. at 5% CO _2/37 ℃ incubator and a method of culturing about 24 hours of culture.

The chondrocytes used preferably include those having immunogenicity close to or the same as the subject to which the complex is applied. The chondrocytes can be prepared according to a conventional method, but generally prepared by treatment with an enzyme such as collagenase.

The cell concentration of the chondrocyte dispersion when preparing the complex can be appropriately changed depending on the purpose of use, the site of use, the degree of disease, and the like.
The cell concentration per cm ³ is 1 × 10 ² or more, preferably 1 × 10 ³ to 1 × 10 ⁸ , more preferably 1 × 10 ³ to 1 × 10 ^8.
It is desirable to adjust so as to be × 10 ⁴ to 1 × 10 ⁷ .

When the composite of the present invention is used for, for example, reconstruction of cartilage of an ear, a chondrocyte dispersion is injected into a substrate for cartilage tissue regeneration molded and processed so as to be adapted to a site to which the ear is applied. This is carried out by implanting the complex of the present invention prepared by culturing for a period of time in the site of application to the ear.

Since the molded article and the cartilage tissue regeneration substrate of the present invention use a synthetic bioabsorbable polymer, they have a stable and uniform quality unlike natural polymers such as collagen. It can be provided, and can be easily sterilized to prevent the growth and contamination of bacteria.Furthermore, the molecular weight, polymerization degree, and mixed composition can be freely adjusted, so that it has desired strength and decomposition rate. It is also useful in that the base material can be easily prepared.

Further, the present invention is a method for regenerating cartilage tissue, which comprises regenerating cartilage by disseminating chondrocytes on the above-mentioned substrate for regenerating cartilage tissue.

The method of seeding chondrocytes, the chondrocytes used and the like are as described above. In addition, the regeneration of cartilage is performed by a method of embedding a cartilage tissue regeneration base material (complex for cartilage tissue regeneration) containing seeded chondrocytes in a living body, or by culturing in vitro.

The cartilage tissue that has been cultured in vitro and the cartilage tissue has been regenerated is used for reconstruction of the cartilage tissue by appropriately implanting it in the living body. The method of cartilage tissue regeneration by culturing in vitro has the advantage that cartilage tissue required in advance can be regenerated and stocked.

[0045]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples, but the present invention is not limited to these examples.

Example 1 Preparation of Foam of Lactic Acid / Caprolactone Copolymer Lactic acid / caprolactone copolymer (P (LA-CL))
(50:50) 2 g in 100 ml dioxane, 4
The mixture was stirred and dissolved at 0 ° C. This was cast on a glass mold,
Place in a 30 ° C freezer, freeze for 1 hour, then
Vacuum freeze-dried for hours. The sponge (base material 1) produced in this manner had a flexible property.

When this was observed with a scanning electron microscope, it was found to have a porous structure in which a myriad of holes were exposed on the surface, which was clearly different from structures such as woven cloth and nonwoven cloth (see FIG. 1).

Example 2 Preparation of Foam of Gelatin-Coated Lactic Acid-Caprolactone Copolymer 2 g of medical gelatin was placed in 100 ml of distilled water,
The mixture was stirred and dissolved at ℃. This was sufficiently infiltrated into the substrate 1 prepared in Example 1, frozen at -135 ° C for 1 hour, and then freeze-dried in vacuum. Observation of the thus-prepared product with a scanning electron microscope revealed a porous body as in the case of the substrate 1 (see FIG. 2). Further, as compared with the base material 1, it exhibited a styrofoam-like hardness and could be molded by a cutter.

Experimental Example 1 For each of the copolymer sponges prepared in Examples 1 and 2, the tensile strength at break in dry and wet states
The tensile elongation at break was measured. The measurement was performed by an autograph using a sample having a width of 20 mm at a tensile speed of 500 mm / min. The same experiment was performed using a collagen sponge as a comparative sample (Comparative Example).

Table 1 shows the results.

[0051]

[Table 1]

From this, the copolymer sponge of the present invention is
It can be seen that the breaking strength is significantly improved and the breaking elongation is better than that of the collagen sponge. In addition, it was found that the tensile breaking strength was improved by coating with gelatin, which is a cell adhesion promoting substance.

Example 3 (1) Collection of chondrocytes [0053] The costal cartilage of a 4-week-old Lewis male rat was aseptically collected, the attached soft tissue was removed as much as possible, and the removed cartilage was fragmented. Next, an enzyme solution [first solution: 0.1% EDTA
(Nacalai Tesque) / PBS (-) (Roman Industries), second liquid: 0.25% trypsin-EDTA (Gibco) / PBS (-) (Roman Industries), third liquid:
0.1% collagenase (manufactured by Wako Pure Chemical Industries) / PBS (+)
(Manufactured by Gibco)] was placed in a test tube, and treated at 37 ° C. for 15 minutes, 1 hour and 3 hours, respectively, to decompose the cartilage tissue. The collected cartilage dispersion was placed in a medium [D-MEM (Dulbeco's Modified Eagle Mediu
m: Gibco) + 10% FCS (FETAL BOVINE SERUM)
+ Penicillin + streptomycin) at 37 ° C., 5% CO _{2 for} about 2 weeks until confluent.
Cultured underneath. The obtained culture was subjected to 0.25% trypsin / P
The cells were treated with BS (-) for 5 minutes, centrifuged at 1000 rpm for 10 minutes, and the chondrocytes were collected.

(2) Seeding of chondrocytes The chondrocytes collected by the above method were placed on each of the base materials 1 and 2 (1 cm × 1 cm) prepared in Examples 1 and 2 by 5 × 1.
0 ⁶ was injected at a syringe at a concentration of Cells / 50 [mu] l, 3 h, 37 ℃, 5% CO ₂ After culturing under, and further cultured for 24 hours the medium was added 2 ml, cartilage tissue regeneration substrate - Cartilage A cell complex was prepared.

Experimental Example 2 Transplantation Experiment The cartilage tissue regeneration substrate-chondrocyte complex prepared in Example 3 was subcutaneously transplanted into the back of a nude mouse (KSN-nuSlc: Japan SLC).

Four weeks after transplantation, the transplant was collected and stained with hematoxylin and eosin to observe the tissue. In addition, cartilage proteoglycan was subjected to Alcian-Blue staining, Azan staining, and Aggrecan immunoantibody staining, and observed with an optical microscope. As a result of observing the tissue section 4 weeks after the transplantation, a high staining area showing the formation of a cartilage mass inside the complex was observed by Azan staining and Aggrecan immunoantibody staining (see FIGS. 3 and 4, respectively).

From the above, the porous cartilage tissue regeneration base material of the present invention has moderate strength and elasticity,
The three-dimensional shape can be freely created and the molding process can be easily performed according to the shape of the diseased part.Furthermore, cartilage tissue can be regenerated in just a few weeks by attaching chondrocytes to it and implanting it in the living body Turned out to be.

[Brief description of the drawings]

FIG. 1 is an electron micrograph instead of a drawing, in which the structure of a spongy porous body of a lactic acid / caprolactone copolymer prepared in Example 1 was observed with a scanning electron microscope.

FIG. 2: Gelatin-coated lactic acid /
3 is an electron micrograph instead of a drawing, in which the structure of a sponge-like porous body of a caprolactone copolymer is observed with a scanning electron microscope.

FIG. 3. After transplantation of the substrate-chondrocyte complex of the present invention, 4
It is a photograph substituted for the drawing which shows the cartilage mass formation state of the week by Azan staining.

FIG. 4. After transplantation of the substrate-chondrocyte complex of the present invention, 4
It is a photograph substituted for the drawing which shows the cartilage mass formation state of the week by Aggrecan immunological antibody staining.

Claims (6)

[Claims] 1. A sponge-like molded product comprising a polycondensate of any of lactic acid, glycolic acid or caprolactone or a copolymer thereof, and having a cell holding structure. 2. A cartilage tissue regeneration base material comprising a porous body composed of a polycondensate of any of lactic acid, glycolic acid and caprolactone or a copolymer thereof. 3. The substrate for cartilage tissue regeneration according to claim 2, wherein the porous body is sponge-like. 4. The substrate for cartilage tissue regeneration according to claim 2, wherein the porous body is coated with a cell adhesion promoting substance. 5. A cartilage tissue regeneration complex comprising the cartilage tissue regeneration substrate according to claim 2 and a chondrocyte. 6. A method for regenerating cartilage by disseminating cartilage by seeding a chondrocyte on the substrate for regenerating cartilage tissue according to claim 2.