JP3521226B2 - Crosslinked composite biomaterial - Google Patents

Description

Detailed Description of the Invention TECHNICAL FIELD OF THE INVENTION

The present invention reproduces and reconstructs a covering material for skin wounds such as skin ulcers and burns, and sheet-like or tube-like living tissues / organs such as skin tissues, ligaments, ligaments, blood vessels, trachea and intestines. The present invention relates to a bioabsorbable porous crosslinked biocomposite material used for

[Prior art]

Conventionally, when a living tissue or an organ is lost or damaged due to an accident or a disease, methods such as organ transplantation and artificial organ surrogate are known as treatment methods. However, the method of organ transplantation requires a large number of donors, and there are problems such as insufficient function in the case of artificial products. In recent years, as a method for solving such a problem, a treatment method using a biotissue engineering technique has been attracting attention, and its research has been actively conducted.

In this biological tissue engineering method, in order to regenerate and reconstruct biological tissues / organs, scaffolds to which biological cells are attached and three-dimensional porous substrate as a support for the formed biological tissues. The material is extremely important. Further, such a base material is required to have various conditions such as porosity, biocompatibility and bioabsorbability.

So far, polylactic acid (PLA), which is a bioabsorbable polymer, has been used as a base material for reconstructing the skin.
And polyglycolic acid (PGA) and lactic acid and polyglycolic acid copolymer (PLGA) prepared porous body and mesh body,
Prepared with collagen, which is a natural polymer derived from the living body
Three-dimensional porous materials are known.

However, although the above-mentioned bioabsorbable synthetic polymer mesh is excellent in mechanical strength, it is hydrophobic, and most of the cells pass through the gap due to the large gaps and cracks. It was extremely difficult to seed the cells on the mesh because it did not rest on the surface. On the other hand, a bio-derived natural polymer porous material, such as collagen sponge, is hydrophilic and has very excellent interaction with cells, and it is easy to seed cells, but mechanical strength is weak, In addition, it is difficult to handle clinically because it is soft and easily twisted.

[0006]

The present invention has been made under such circumstances and has excellent bioabsorbability and biocompatibility, excellent cell dissemination properties, and clinical handling properties. In addition, it has sufficient mechanical strength and is a novel cross-linking material that is useful for the regeneration and reconstruction of skin wounds such as skin ulcers and burns, skin tissues, ligaments, ligaments, and biological tissues and organs such as the trachea and intestines. It is an object to provide a composite biomaterial and an advantageous method for producing the crosslinked composite biomaterial.

[0007]

Means for Solving the Problems The present inventors have found that biological complex
After many years of studying materials, bioabsorbable synthesis
Enhance the polymer mesh body and the natural polymer porous body of biological origin
A composite material in which molecular porous materials are cross-linked by a specific means
We found that body materials are effective against the above problems,
It came to complete the Ming. That is, according to the present invention,
The following inventions are provided. (1) Living body formed from woven fabric, woven fabric or non-woven fabric
Absorbable synthetic polymer mesh body dissolved in natural polymer derived from living body
After treatment with the liquid, freeze-dry the resulting composite biomaterial
Living body obtained by treatment with a gaseous chemical cross-linking agent
Absorbable synthetic polymer mesh and bio-derived natural polymer
A crosslinked composite biomaterial composed of a porous body. (2) Bioabsorbable synthetic polymer is polylactic acid, polyglycol
Being a copolymer of lactic acid or lactic acid and glycolic acid
The crosslinked composite biomaterial according to (1) above, which is characterized in that (3) Natural polymers derived from living organisms are collagen and gelatin
A small number of selected from fibronectin, fibronectin, and laminin
The above (1) or at least one type
The crosslinked composite biomaterial according to (2). (4) Crosslinked composite biomaterial is a sheet or tube
Any of the above (1) to (3), characterized in that it is a substance
A cross-linked composite biomaterial according to item 1. (5) Biological absorption formed from woven fabric, woven fabric or non-woven fabric
A natural polymer solution derived from a biological material
And then freeze-dry the resulting composite biomaterial.
The above-mentioned characterized by being treated with a chemical cross-linking agent
Production of the crosslinked composite biomaterial according to any one of (1) to (4)
Build method.

The bioabsorbable synthetic polymer mesh used in the present invention can be formed from a woven fabric, a woven fabric, a non-woven fabric, or the like. The size of the mesh takes into consideration the intended use of the crosslinked composite biomaterial, the mechanical strength and elasticity of the crosslinked composite biomaterial, and the breaking strength of the natural polymer porous body of biological origin provided on the mesh or between the meshes. However, as the size of the mesh increases, the pore density of the natural polymer porous body derived from the living body per mesh unit becomes higher, the seed cells can be increased, and the seeding becomes easier. Therefore, in the present invention, the mechanical strength and elasticity of the crosslinked composite biomaterial, and as large as possible within the limit of the rupture strength of the natural polymer porous body of biological origin provided between the meshes. It is desirable to set it.

Examples of the bioabsorbable polymer forming the mesh body include polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, polyesters such as polymalic acid and poly-ε-caprolactone, cellulose, polyalginic acid and the like. Examples thereof include polysaccharides. The bioabsorbable polymer preferably used in the present invention is polylactic acid,
It is a polyglycolic acid or a copolymer of lactic acid and glycolic acid.

The natural polymer derived from a living body used in the present invention is derived from a living body, and any polymer having biocompatibility can be used, but it is selected from collagen, gelatin, fibronectin, and laminin. One or more kinds of collagen, particularly collagen, are preferably used. Collagen includes types I, II, III, and IV, and any of these can be used in the present invention. The pores of the biogenic natural polymer porous body are 1 to 300 μm, preferably about 20 to 100 μm. The thickness of the biogenic natural polymer porous body may be appropriately determined depending on the usage mode of the biocomposite material, but is usually 0.2 to 5 mm, preferably 0.5 to 2 mm.

The cross-linked biocomposite material of the present invention is synthesized by cross-linking the bioabsorbable synthetic polymer mesh body and the biopolymer-derived natural polymer porous body. This cross-linked composite biomaterial can be made into various structures, for example, if it is made into a sheet, it can be used as a covering material for skin wounds such as skin ulcers and burns, as a substitute tissue material for skin tissue, ligaments, ligaments. When used as a tubular material, it can be used as a substitute tissue material for blood vessels, trachea and intestines.

FIG. 1 shows a typical example of a sheet-like material, and FIG. 1 (a) shows a natural polymer porous body derived from a living body provided above and below a bioabsorbable synthetic polymer mesh body. Yes, (b) is a bioabsorbable synthetic polymer mesh body on which a biogenic natural polymer porous body is provided. The total thickness of these sheet materials is usually 0.2 to 5 mm, preferably 0.5 to 1 mm. The porosity is usually 90%
That is all. FIG. 2 shows a typical example of a tubular material, and FIG. 2 (a) shows a natural polymer porous body derived from a living body provided inside a bioabsorbable synthetic polymer mesh tube body, (B) is a bioabsorbable synthetic polymer mesh tube body provided with a biopolymer-derived natural polymer porous body both inside and outside, and (c) is a bioabsorbable synthetic polymer mesh tube body. A natural polymer porous body derived from a living body is provided outside the body. The outer diameter of these tubular materials is usually 1 mm to 5 cm, preferably 5 mm
~ 2 cm. The wall thickness is usually 0.2 to 5 mm, preferably 1 to 2 mm. The porosity of the thick part is usually 90% or more.

The crosslinked composite biomaterial of the present invention can be produced by various methods, but the most preferable method is
(1) A bioabsorbable synthetic polymer mesh is treated with a biogenic natural polymer porous solution, (2) freeze-dried, and (3) the resulting biocomposite material is then treated with a gaseous chemical crosslinker. It is a method of processing.

In step (1), it is desirable to use the natural polymer derived from a living body as an aqueous solution. PH of aqueous solution
May be prepared within a pH range that does not affect the mesh of the bioabsorbable synthetic polymer and that the natural polymer derived from the living body dissolves in water, and is usually 2 to 8. When using collagen, the pH of the normal aqueous solution is set to 3. Further, the aqueous solution concentration of the natural polymer derived from the living body is used in a concentration range of a range in which it can be made porous by freeze-drying. As a natural polymer, for example, when collagen is used, the concentration is 0.05 wt% or more. It is desirable to use.

Next, in the step (1), the bioabsorbable synthetic polymer mesh body is treated with the aqueous solution of natural polymer derived from the living body. Although there are various treatment methods, the dipping method and the coating method are preferably adopted. The immersion method is
It is effective when the concentration and viscosity of the biologically derived natural polymer aqueous solution are low. Specifically, it is performed by immersing the bioabsorbable synthetic polymer mesh body in a low concentration aqueous solution of the biologically derived natural polymer. . The coating method is effective when the concentration and viscosity of the aqueous solution of the natural polymer derived from the living body are high and the dipping method cannot be applied. It is carried out by applying to a molecular mesh body.

The composite in which the bioabsorbable synthetic polymer mesh is impregnated with and adhered to the natural polymer derived from the living body is then subjected to (2) freeze-drying. Freeze-drying is a method in which the above-mentioned composite material is frozen and freeze-dried under reduced pressure in a vacuum, but by such a step, a natural polymer derived from a living body is made porous, and a bioabsorbable synthetic polymer mesh body is obtained. A composite material with a natural polymer porous body of biological origin is formed.

As a freeze-drying method adopted in the present invention, a conventionally known method can be applied as it is. Freezing temperature is usually -2
It is 0 ° C or lower. The freeze-drying pressure may be set under a reduced pressure condition where frozen water becomes a gas, and is usually 0.2 Tor.
It is prepared under reduced pressure of about r.

In the above manufacturing process, the thickness of the biogenic natural polymer porous body is determined by the depth of the aqueous solution of the biogenic natural polymer in which the mesh body of the bioabsorbable synthetic polymer is immersed, and the thickness. It can be appropriately prepared by changing the thickness of the natural polymer derived from the living body.

In the above manufacturing process, the positional relationship between the bioabsorbable synthetic polymer mesh body and the biogenic natural polymer porous body in the composite material can be freely adjusted before freezing.

For example, in order to manufacture a sheet-like composite biomaterial as shown in FIG. 1 (a), a bioabsorbable synthetic polymer mesh is placed at the center of an aqueous solution of a natural polymer derived from a living body. When frozen and freeze-dried, a sheet-shaped composite biomaterial is formed in which a mesh body of bioabsorbable synthetic polymer is sandwiched in a natural polymer porous body of biological origin. Further, as shown in FIG. 1 (b), when a bioabsorbable synthetic polymer mesh body is frozen on the upper or lower surface of a natural polymer aqueous solution of biological origin and freeze-dried, one side of the bioabsorbable synthetic polymer mesh is frozen. A sheet-shaped composite biomaterial is formed in which the other surface of the body is a polymeric porous body of biological origin. In addition, a tubular composite biomaterial as shown in FIGS. 2A, 2B, and 2C can also be manufactured by a method substantially similar to the above.

Molded by freeze-drying in the above (2),
The composite biomaterial is then subjected to the crosslinking step (3).
In this step, the natural polymer porous body of biological origin that constitutes the composite biomaterial is crosslinked with a gaseous crosslinking agent to solidify the porous body of natural polymer of biological origin and bond with the synthetic polymer mesh body. It is necessary to increase the force and to provide sufficient elasticity and strength to stabilize the desired porous structure of the crosslinked composite biomaterial.

Generally, as the cross-linking method, a physical cross-linking method such as photo-crosslinking or thermal cross-linking by ultraviolet irradiation treatment, a chemical cross-linking method using a solution-like cross-linking agent or a gaseous cross-linking agent, etc. are known. However, in the present invention, the method using a gaseous crosslinking agent is most desirable.

That is, in the physical cross-linking method such as photo-crosslinking or thermal cross-linking by ultraviolet irradiation treatment, the degree of cross-linking is limited in the cross-linking step, and further, the bioabsorbable synthetic polymer constituting the composite biomaterial is This is because alteration or decomposition may occur, and in the method of using a solution-like crosslinking agent even in the chemical crosslinking method, the natural polymer may be dissolved in the crosslinking process. In addition, in order to prevent the dissolution of the cross-linking agent solution of the natural polymer, even if a method of performing photo-crosslinking or thermal cross-linking is adopted prior to the cross-linking agent solution method, as described above, It is not desirable because it causes decomposition and deterioration.

On the other hand, the method using a gaseous cross-linking agent overcomes all of the problems described above, and the natural polymer is cross-linked in a desired form without decomposition or alteration. It is possible to obtain a crosslinked composite biomaterial that is dimensioned and the binding force with the synthetic polymer mesh body is enhanced, and that has sufficient strength and elasticity sufficient for the purpose. Such a fact is a novel finding first discovered by the present inventors through many years of research, and is a matter that has not been known at all in the past.

As the cross-linking agent used in the present invention, any conventionally known one can be used. Examples of such a cross-linking agent include aldehydes such as glutaraldehyde, formaldehyde, and paraformaldehyde, and ethylene propylene diglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, sorbitol polyglycidyl ether, and ethylene glycol diglycidyl ether. Such as glycidyl ethers, hexamethylene diisocyanate, α-tolidine isocyanate, tolylene diisocyanate, naphthylene 1,5-diisocyanate, 4,4-diphenylmethane diisocyanate, triphenylmethane-4,4,4-triisocyanate Examples include isocyanates and calcium gluconate. Crosslinking agents preferably used in the present invention are aldehydes such as glutaraldehyde, formaldehyde, paraformaldehyde, especially glutaraldehyde.

In the cross-linking of the present invention, as described above, the above-mentioned cross-linking agent is used in a gas state. Specifically, when a natural polymer porous body derived from a living body is cross-linked, cross-linking is performed for a certain period of time under an atmosphere of a steam of a cross-linking agent saturated with an aqueous solution of a cross-linking agent having a constant concentration at a constant temperature. The crosslinking temperature may be selected within a range in which the bioabsorbable synthetic polymer mesh does not dissolve and vapor of the crosslinking agent can be formed, and is usually 20 ° C to 50 ° C.
Is set to. The cross-linking time depends on the type of cross-linking agent and the cross-linking temperature, but it is sufficient that it does not impair the hydrophilicity and bio-absorption of the natural polymer porous material of biological origin and that it does not dissolve during use and application. It is desirable to set it in a range where various cross-linking immobilization is performed. When the cross-linking time becomes short, the cross-link immobilization becomes insufficient, and when applying the biomaterial, the natural polymer porous body derived from the living body may be dissolved, and the longer the cross-linking time, the more the cross-linking proceeds, If the cross-linking time is too long, the hydrophilicity of the biomaterial sponge derived from the living body will change, and the bioabsorbability will also slow down, which is not preferable.

The crosslinked composite biomaterial of the present invention obtained as described above has a structure in which a bioabsorbable synthetic polymer mesh body and a natural polymer porous body of biological origin are judiciously crosslinked and complexed. It is a thing. The bioabsorbable synthetic polymer mesh functions as a mechanical skeleton, imparts advantageous shape controllability, excellent mechanical strength and ease of handling to the composite biomaterial, and is a natural polymer porous material derived from a living body. The plastids provide the composite biomaterial with properties such as specific interaction with cells and enhanced water wettability. Therefore, the crosslinked composite biomaterial of the present invention is excellent in biocompatibility and mechanical strength, and thus is extremely useful as a base material for regenerating and reconstructing a biological tissue / organ.

[0028]

EXAMPLES The present invention will now be described in more detail with reference to examples.

Example 1 0.5 wt% bovine I containing a copolymer (PLGA) mesh of lactic acid and glycolic acid, which is a bioabsorbable polymer having mechanical strength,
Type atelocollagen acidic aqueous solution (pH = 3.0)
It was frozen at 80 ° C for 12 hours. Next, this frozen product was lyophilized under vacuum reduced pressure (0.2 Torr) for 24 hours to produce a sheet-shaped uncrosslinked composite biomaterial of PLGA mesh and collagen sponge. The obtained uncrosslinked composite biomaterial was 25 wt% at 37 ° C.
After cross-linking with glutaraldehyde vapor saturated with the glutaraldehyde aqueous solution for 4 hours, it was washed 10 times with phosphate buffer. Furthermore, it was immersed in a 0.1 M glycine aqueous solution for 4 hours, washed 10 times with a phosphate buffer solution, washed 3 times with distilled water, and frozen at -80 ° C for 12 hours. This is vacuum reduced (0.2 T
After freeze-drying for 24 hours with orr), a sheet-like crosslinked composite biomaterial of the PLGA mesh body of the present invention and a collagen sponge was obtained. This material was coated with gold and their structure was observed by scanning electron microscopy (SEM). The result is shown in Figure 3.
Shown in.

Example 2 The sheet-like crosslinked composite biomaterial of PLGA mesh and collagen sponge obtained in Example 1 was cut into a size of 5.0 × 5.0 × 20.0 mm to prepare a sample. This sample is 2-
A static tensile test was performed in a wet state by immersing in [4- (2-hydroxyethyl) -1-piperazyl] ethanesulfone (HEPES) buffer (pH = 7.4). The results are shown in Table 1.

[0031]

[Table 1]

From Table 1, the crosslinked composite biomaterial of the PLGA mesh body of the present invention and the collagen sponge exhibits higher tensile strength when wet with the HEPES buffer solution, as compared with the biomaterial consisting of the collagen sponge alone. , PLGA mesh shows almost the same tensile strength.

Example 3 The crosslinked composite biomaterial of PLGA mesh and collagen sponge obtained in Example 1 was sterilized with ethylene oxide gas and used for culturing normal human dermal fibroblasts. Normal human skin fibroblasts NHDF purchased from Kurabo Industries were treated with 2% fetal bovine serum and 10
Medium 106S (culture medium, containing ng / mL human recombinant epidermal growth factor and 1 μg / mL hydrocortisone and 3 ng / mL human recombinant basic fibroblast growth factor and 10 μg / mL heparin
(Purchased from Kurabo) and subcultured cells
Peeling was carried out by incubating with 0.025% trypsin / EDTA, HEPES solution for 5 minutes. Removed NHDF cells from Me
Suspended in medium 106S culture medium (1.0 x 106 cells / mL), seeded this cell suspension on a composite sheet material of PLGA mesh and collagen sponge, and placed in Medium 106S culture medium 37
Culturing was performed in a 5% CO2 atmosphere at ℃. The medium was changed every two days. Cultured NHDF cells were washed 3 times with PBS (-), 0.25w
The cells were fixed with a t% glutaraldehyde / PBS (-) solution at room temperature for 1 hour, and washed with PBS (-) three times. After washing three times with distilled water, each was immersed in 50%, 60%, 70%, 80%, 90%, 95% ethanol aqueous solution and absolute ethanol for 10 minutes. It was dried under vacuum, coated with gold, and the morphology of the cells was observed by an electrophotographic microscope (SEM). The result is shown in FIG. From FIG. 4, it was found that the normal human dermal fibroblasts NHDF adhered well to the composite sheet material of PLGA mesh and collagen sponge, spread, and after culturing for 2 weeks, became almost layered.

[0034]

(1) In the crosslinked composite biomaterial according to claims 1 to 4 of the present invention, a bioabsorbable synthetic polymer mesh body and a natural polymer porous body derived from a living body are judiciously crosslinked and complexed. It has a structure. The bioabsorbable synthetic polymer mesh functions as a mechanical skeleton, imparts advantageous shape controllability, excellent mechanical strength and ease of handling to the composite biomaterial, and is a natural polymer porous material derived from a living body. The plastids provide the composite biomaterial with properties such as specific interaction with cells and enhanced water wettability. Therefore, the crosslinked composite biomaterials according to claims 1 to 4 of the present invention are excellent in biocompatibility and mechanical strength, and thus are extremely useful as a base material for regenerating and reconstructing biological tissues / organs. Is. (2) The sheet-shaped crosslinked composite biomaterial according to claim 5 of the present invention can be used as a covering material for skin wounds such as skin ulcers and burns, as a substitute tissue material for skin tissues, ligaments and ligaments, and in a tubular form. The crosslinked composite biomaterial can be used as a substitute tissue material for the trachea and intestine. (3) Further, according to the method for producing a crosslinked composite biomaterial using a gaseous crosslinker according to claim 6 of the present invention, a natural polymer derived from a living body can be formed in a desired form without decomposition or deterioration. A crosslinked composite biomaterial having biocompatibility and sufficient mechanical strength and elasticity can be industrially obtained advantageously by being crosslinked and being three-dimensionalized and the binding force with the synthetic polymer mesh body being enhanced.

[Brief description of drawings]

1A and 1B are schematic cross-sectional views of a typical sheet-like crosslinked composite biomaterial according to the present invention.

2 (a), (b) and (c) are schematic cross-sectional views of a typical tubular cross-linked composite biomaterial according to the present invention.

FIG. 3 (a) is a horizontal electron micrograph of the sheet-like crosslinked composite biomaterial according to Example 1 of the present invention, (b).
3A is an electron micrograph in the longitudinal direction of the sheet-like crosslinked composite biomaterial according to Example 1 of the present invention.

4 (a) is an electron micrograph of skin fibroblasts after culturing on the sheet-like crosslinked composite biomaterial according to Example 1 of the present invention for 5 days, (b) is Example 1 of the present invention. 2 is an electron micrograph of skin fibroblasts after culturing for 2 weeks on the sheet-like crosslinked composite biomaterial according to the present invention.

Continuation of the front page (56) Reference JP-A-7-116242 (JP, A) JP-A-3-23864 (JP, A) JP-A-2000-143291 (JP, A) International Publication 99/063908 (WO, A1) ) Guoping Chen et al. , Chemistry Letters, 1999, NO. 7, 561-562 Guoping Chen et al. , Advanced Materials, 2000, vol. 12, no. 6,455-457 (58) Fields surveyed (Int.Cl. ⁷ , DB name) A61L 15/00-15/64 A61L 27/00-27/60 CAPLUS (STN) MEDLINE (STN) JISST File (JOIS)

Claims (5)

(57) [Claims] 1. Formed from woven, woven or non-woven fabrics
Bio-absorbable synthetic polymer mesh
A composite organism that is formed by freeze-drying after treatment with a molecular solution
Obtained by treating the material with a chemical crosslinker in gaseous form
Bio-absorbable synthetic polymer mesh and bio-derived natural
A crosslinked composite biomaterial composed of a molecular porous material. 2. A bioabsorbable synthetic polymer is polylactic acid or polylactic acid.
Glycolic acid or a copolymer of lactic acid and glycolic acid
The crosslinked composite biomaterial according to claim 1, wherein
Fee. 3. A natural polymer derived from a living body is collagen,
Selected from latin, fibronectin, and laminin
It is at least 1 sort (s), Claim 1 or
2. The crosslinked composite biomaterial according to item 2. 4. A crosslinked composite biomaterial, which is a sheet-like material or a chisels.
4. A tube-shaped material according to any one of claims 1 to 3.
A cross-linked composite biomaterial according to item 1. 5. Raw material formed from woven, woven or non-woven fabrics
Bioabsorbable synthetic polymer mesh is a natural polymer of biological origin
Composite biomaterial produced by freeze-drying after treatment with solution
Claims, characterized in that is treated with a gaseous chemical cross-linking agent.
Item 5. A method for producing the crosslinked composite biomaterial according to any one of Items 1 to 4.
Law.