JP2007275388A - Artificial organ to promote differentiation of cells and control their inflammation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an artificial organ with excellent biocompatibility and healing power that causes no inflammation or fibrogenesis of host cells/tissues but promotes their differentiation. <P>SOLUTION: The artificial organ is a fibrous structure of which the one or both of outer and inner surfaces are covered with polysaccharide sulfide, especially hyaluronic acid sulfide. This artificial organ should preferably have the shape of a tube or sheet with peameability or porosity degree of 0-50 ml/(cm<SP>2</SP>×min) (120 mmHg, 37°C). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an artificial organ such as an artificial blood vessel and the like, and relates to an artificial organ excellent in biocompatibility and curability that does not cause an inflammatory reaction or fibrosis to a host cell or tissue and promotes differentiation. It is.

A blood vessel anastomosis connector comprising a porous tubular body made of a biodegradable material in which at least an inner surface is treated with an antithrombotic biodegradable substance has been disclosed (for example, see Patent Document 1). An intravascular treatment material characterized by being composed of a biodegradable material containing catechins has been disclosed (for example, see Patent Document 2). There has been disclosed a prosthesis characterized in that the outer surface and / or the inner surface of a tubular prosthesis is covered with a biodegradable synthetic polymer (for example, see Patent Document 3).
JP-A-5-123391 JP 2002-085549 A JP 2004-313310 A

  Foreign body reaction is an unavoidable problem in prosthetic transplantation. A foreign body reaction occurs when an artificial organ made of an artificial object comes into contact with a cell or tissue of a living body. It can cause inflammation, encapsulation, adhesion, etc., affecting the function of the prosthesis. Especially in serious situations where the function of the prosthesis cannot be maintained, such as occlusion of the lumen due to the development of granulation from the anastomosis part of the tubular object, excessive adhesion and scarring on the flat body, and fibrosis There is much to develop. The present invention relates to an artificial organ such as an artificial blood vessel, and provides an artificial organ excellent in biocompatibility and curability that does not cause an inflammatory reaction or fibrosis in a host cell or tissue and promotes differentiation. The purpose is to do.

In order to achieve the above-mentioned object, the present invention uses various fiber structures as prosthetic devices as a result of various studies, and the outer surface or the inner surface or both are coated with sulfated polysaccharides. The present invention has been completed by finding that the above structure achieves the above object. That is, the first aspect of the present invention relates to a structure composed of fibers, the outer surface or the inner surface of which is covered with a sulfated polysaccharide. The second aspect of the present invention also relates to a structure using sulfated hyaluronic acid as a sulfated polysaccharide. Furthermore, the third aspect of the present invention uses sulfated hyaluronic acid having a degree of sulfation within a range containing 0.1 to 2.0 equivalent sulfate groups per disaccharide unit. Concerning structure. And the 4th side surface of this invention is related with the structure whose weight average molecular weight of the sulfated hyaluronic acid to be used is the range of 10,000 to 2 million. The fifth aspect of the present invention relates to a structure in which the prosthesis is tubular or sheet-like. Furthermore, the sixth aspect of the present invention relates to a structure in which the prosthesis has water permeability with a porosity of 0 to 50 ml / (cm ² · min) (120 mmHg, 37 ° C.).

  An artificial organ including an artificial blood vessel according to the present invention is a prosthetic device that can promote differentiation without causing an inflammatory reaction or fibrosis to a host cell or tissue, and is excellent in biocompatibility and curability. . Therefore, it is a highly effective and highly safe prosthesis that exhibits excellent effects of suppressing foreign body reaction and promoting healing due to contact with a living body.

  The prosthesis of the present invention is a structure composed of fibers, and is characterized in that the outer surface, the inner surface or both of them are coated with a sulfated polysaccharide. Preferably, the prosthesis of the present invention is characterized in that the sulfated polysaccharide is sulfated hyaluronic acid. Since the prosthesis of the present invention is coated with sulfated hyaluronic acid, it has excellent affinity with a living body, and it has a good healing process by suppressing inflammation, promoting cell proliferation with the living body, and affinity with tissue. Is preferable.

Porosity at 37 ° C. of the prosthesis of the present ^{invention, 0~50ml / (cm 2 · min} ) is ^{preferable, 0~10ml / (cm 2 · min} ) is more preferable, 0~5ml / (cm ² · min ) Is more preferable, and 0 to 3 ml / (cm ² · min) is particularly preferable.

  The structure made of fibers is a structure made of fibers selected from thermoplastic resin fibers and metal fibers, and is a tube made of fibers selected from thermoplastic resin fibers and metal fibers in a planar shape or in a cylindrical shape. A knitted fabric, a woven fabric, a braided fabric or a nonwoven fabric selected from thermoplastic resin fibers and metal fibers, and combinations thereof can be used. As the knitted fabric, known knitted fabrics and woven fabrics such as plain weave and twill weave can be used. As the tubular material, a knitted fabric, a woven fabric or a braid of thermoplastic resin fibers formed in a cylindrical shape, a nonwoven fabric of thermoplastic resin fibers formed in a cylindrical shape, or the like can be used. As the tubular material, a knitted fabric of thermoplastic resin fibers formed into a cylindrical shape, and a plain woven fabric of thermoplastic resin fibers formed into a cylindrical shape are particularly preferable because of excellent strength, porosity, and productivity. As the tubular material, a tubular material composed of fibers selected from thermoplastic resin fibers and metal fibers combined with monofilaments, wires, threads and the like can be used.

  As the sulfated polysaccharide, sulfated hyaluronic acid is preferable.

  The degree of sulfation of sulfated hyaluronic acid is preferably in the range containing 0.1 to 2.0 equivalent sulfate groups per hyaluronic acid unit consisting of disaccharides, and is equivalent to 0.2 to 1.6 equivalents. More preferably, 0.3 to 1.0 is more preferable, and 0.35 to 0.8 is more preferable.

  The weight average molecular weight of sulfated hyaluronic acid is preferably in the range of 10,000 to 3 million, more preferably 500,000 to 2.5 million, and even more preferably 1 million to 2 million.

  The structure made of fibers is a structure made of fibers selected from thermoplastic resin fibers and metal fibers, and is a tube made of fibers selected from thermoplastic resin fibers and metal fibers in a planar shape or in a cylindrical shape. A knitted fabric, a woven fabric, a braided fabric or a nonwoven fabric selected from thermoplastic resin fibers and metal fibers, and combinations thereof can be used. As the knitted fabric, known knitted fabrics and woven fabrics such as plain weave and twill weave can be used. As the tubular material, a knitted fabric, a woven fabric or a braid of thermoplastic resin fibers formed in a cylindrical shape, a nonwoven fabric of thermoplastic resin fibers formed in a cylindrical shape, or the like can be used. As the tubular material, a knitted fabric of thermoplastic resin fibers formed into a cylindrical shape, and a plain woven fabric of thermoplastic resin fibers formed into a cylindrical shape are particularly preferable because of excellent strength, porosity, and productivity. As the tubular material, a tubular material composed of fibers selected from thermoplastic resin fibers and metal fibers combined with monofilaments, wires, threads and the like can be used.

Porosity at 37 ° C. of the structure is preferably 0~2950ml / (cm ² · min) in the case of knitted fabric of the thermoplastic resin ^{fibers, 10~2000ml / (cm 2 · min} ) is more preferable, 20 ~ 1000 ml / (cm ² · min) is more preferable, and 50 to 500 ml / (cm ² · min) is particularly preferable. In the case of a woven fabric made of metal fibers, the porosity is very high and measurement is difficult, but considering the mechanical properties and compatibility with the living body, it is preferably 100 ml / (cm ² · min) or more, It is further preferably 500 ml / (cm ² · min) or more, more preferably 750 ml / (cm ² · min) or more, and particularly preferably 2000 ml / (cm ² · min) or more.

  The tensile strength of the structure may be any strength as long as it is practically used. For example, a tensile strength of preferably 5.0 kg to 20 kg, more preferably 7.5 kg to 20 kg, and particularly preferably 8.0 kg to 20 kg. The thickness of the structure may be any outer diameter that is practically used. For example, a structure having a thickness of 0.01 mm to 20 mm, 0.05 to 15 mm, particularly 0.1 to 10 mm can be used. The outer diameter of the tubular material may be an outer diameter used for practical use, and for example, a diameter of 2 to 45 mm, further 4 to 40 mm, particularly 5 to 40 mm can be used.

  Tubular objects with crimps such as crimping can also be used. As the crimping process, a method of processing the surface of a plain woven tubular product of thermoplastic resin fibers into an uneven shape can be used. For example, the method described in the specification of US Pat. No. 3,337,673, that is, a flat-woven tubular product of thermoplastic resin fibers is fitted on the surface of a round bar, and threads are spirally wound from above the tubular product at equal intervals. A method of forming a wrinkle by compressing and contracting in the axial direction, heating and heat setting, a method described in JP-A-1-155860, that is, a plain woven tubular product of thermoplastic resin fibers, For example, a method of fitting in a sufficiently polished screw rod, winding an appropriate yarn along the screw groove, heat-treating the state as it is, and heat setting is preferable. By having a portion to be crimped as a tubular object, it is strong against expansion and contraction and bending, and is easily adapted to an organ shape such as a blood vessel shape of a human body.

  Examples of the thermoplastic resin forming the thermoplastic resin fiber include polyolefins such as polyethylene, polypropylene, ethylene-α-olefin copolymer, polyamide, polyurethane, polyethylene terephthalate, polybutylene terephthalate, polycyclohexane terephthalate, polyethylene-2,6- Examples thereof include polyesters such as naphthalate and fluororesins such as PTFE and ETFE. More preferably, it is chemically stable and highly durable and has little tissue reaction, fluororesin such as PTFE and ePTFE, chemically stable and highly durable and has little tissue reaction, and excellent mechanical properties such as tensile strength Polyesters such as polyethylene terephthalate are preferred. Particularly preferred is a polyester such as polyethylene terephthalate having a glass transition temperature of 60 ° C. or higher because the strength of the polyester resin may be reduced by body temperature.

  As the material of the metal fiber, a shape memory effect by heat treatment or a shape memory alloy imparted with superelasticity is preferably employed, but stainless steel, tantalum, titanium, platinum, gold, tungsten, or the like may be used depending on applications. . As the shape memory alloy, Ni—Ti, Cu—Al—Ni, Cu—Zn—Al, and the like are preferably used. Further, the surface of the shape memory alloy may be coated with gold, platinum or the like by means such as plating. The thickness of the metal fiber is not particularly limited.

  As the thermoplastic resin fiber and the metal fiber, a monofilament, a bundle of two or more fibers, a twist of two or more fibers, or the like can be used. The thermoplastic resin fibers and metal fibers are 0.1 to 5 denier, more preferably 0.5 to 3 denier, especially more than 0.8 and not more than 3 denier monofilaments to several hundred, further 10 to 700, especially 10 Up to 100 bundled or twisted yarns can be used. As the thermoplastic fiber, a monofilament having an outer diameter of 1 μm to 1 mm can be used. As the metal fiber, a wire having an outer diameter of 20 μm to 1 mm can be used. Thermoplastic resin fibers and metal fibers can be a combination of monofilaments, wires, threads, etc., and the shape is circular, tubular hollow body, ribbon shape, etc. Can be used.

  The prosthesis of the present invention is preferably produced by preparing a tubular product made of fibers selected from thermoplastic resin fibers and metal fibers, and then coating the outer surface and / or inner surface of the tubular product with sulfated polysaccharide. As a coating method, a known coating method such as coating, dipping, impregnation, spraying, polymerization, or crosslinking can be used. In the prosthesis of the present invention, as a method of coating the outer surface and / or inner surface of the tubular material with a biodegradable synthetic polymer, application, immersion, impregnation, spraying, polymerization using a sulfated polysaccharide solution is possible. The solution of the sulfated polysaccharide can be performed by a known method such as cross-linking, and the solution of the sulfated polysaccharide is uniformly dissolved, partially dissolved, dispersed, Monomers, precursors, and the like can be used. When a sulfated polysaccharide is coated on a tubular material, a pore-forming component can be used. By removing the pore-forming component by a method such as elution after coating, the sulfated polysaccharide coating tends to be spongy, and a favorable coating for mechanical properties, tissue / cell invasion, etc. can be achieved.

  Examples of the method of coating the outer surface and / or inner surface of the structure with the sulfated polysaccharide include immersing the tube in a solution of the sulfated polysaccharide, and applying the solution of the sulfated polysaccharide to the tube Or spraying. At this time, it is preferable to perform the dipping process a plurality of times and to invert the top and bottom to achieve uniformity. Moreover, the application of the sulfated polysaccharide to the tubular structure fixed to the core rod, spraying, and dipping can be mentioned. At this time, it is preferable to make the core rod uniform by rotating it. A solvent that does not significantly affect the base material or that does not significantly deteriorate the solvent is preferable. As an example, when hexafluoroisopropanol or heated phenol is used for a textile base material made of polyester, the fibers are dissolved and coating is difficult.

  Hereinafter, embodiments of the present invention will be described in detail by way of examples. The present invention is not limited only to these examples.

The measuring method of the characteristic value of the structure obtained by an Example and a comparative example is shown.
(1) Water permeability: 120 mmHg of water is allowed to flow through the structure at 37 ° C., and the water permeability is calculated from the weight of the outflowed water, the surface area of the tubular material, and time.
(2) Tensile strength: The tensile strength in the weft direction of a plain woven fabric using thermoplastic resin fibers was measured. The tensile strength was measured under the conditions of a temperature of 23 ° C., a tensile speed of 10 mm / min, a sample having a length of 1 cm in the warp direction (width) and a length of 2 cm in the weft direction (length). The tensile strength is an average value of 5 measurement samples.

[Example 1]
As the thermoplastic resin fiber, a 1.0 denier polyethylene terephthalate monofilament was twisted. A plain woven fabric and a tubular product having an outer diameter of 20 mm were prepared using 50 denier thermoplastic resin fibers (307) as weft (length direction) and 50 denier thermoplastic resin fibers as warp (circumferential direction). The obtained plain-woven cloth and tubular product had a tensile strength of 9.5 kg, a wall thickness of 64 μm, and a porosity of 1700 ml / (cm ² · min). The wall thickness was 150 μm. Sulfated hyaluronic acid was used as the sulfated polysaccharide. This was synthesized as previously reported. The degree of sulfation was 0.4 (sulfate group per disaccharide unit), and the weight average molecular weight was 1,680,000. At room temperature (25 ° C.), sulfated hyaluronic acid was uniformly dissolved in distilled water to prepare a solution of 50 μg / mL. Then, in a dry nitrogen atmosphere, the tubular body was immersed in this solution, allowed to stand for 5 minutes and then lifted. After excess liquid was shaken off, it was suspended vertically and allowed to air dry. The obtained sulfated hyaluronic acid-coated tubular structure was swollen and the porosity was measured and found to be 0 ml / (cm ² · min). This wall thickness was 300 μm. The raw sulfated hyaluronic acid was coated on both the outer and inner surfaces of the tubular body.

[Example 2]
A tubular structure was prepared in the same manner as in Example 1. The tubular product was subjected to the same coating treatment with sulfated hyaluronic acid as in Example 1, and was cut open to obtain a planar structure. When the porosity of the tubular structure coated with the sulfated hyaluronic acid obtained was measured, it was 0 ml / (cm ² · min). This wall thickness was 300 μm. Sulfated hyaluronic acid was coated on both the outer and inner surfaces of the tubular body.

[Example 3]
The tubular structure of Example 1 having an outer diameter of 20 mm was coated with sulfated hyaluronic acid. The method of covering with sulfated hyaluronic acid is that the tubular material is fixed to the core rod, the sulfated hyaluronic acid is applied to the outer surface of the tubular structure rotating in the axial direction, and air-dried while rotating. A tubular structure coated with sulfated hyaluronic acid was prepared in the same manner as in Example 1. The porosity was 0 ml / (cm ² · min). This wall thickness was 250 μm. Sulfated hyaluronic acid was mainly coated only on the outer surface of the tubular body.

[Example 4]
A tubular structure having an inner diameter of 6 mm knitted with 72 nitinol alloys having a thickness of 30 μm was used. This porosity was not measurable. This wall thickness was 75 μm. Except for using this tubular structure, the porosity of the tubular structure subjected to the same coating treatment with sulfated hyaluronic acid as in Example 3 was 0 ml / (cm ² · min). This wall thickness was 350 μm. The coating with sulfated hyaluronic acid was applied to both the outer and inner surfaces.

[Example 5]
A tubular body knitted with 60 nitinol alloys having a thickness of 25 μm and having an inner diameter of 4 mm was used. This porosity was not measurable. This wall thickness was 50 μm. The same aqueous solution of sulfated hyaluronic acid as in Example 1 was applied to a Teflon (registered trademark) core rod having an outer diameter of 3.5 mm. The above tubular body was covered thereon and allowed to air dry at room temperature. The porosity of this sulfated hyaluronic acid-coated tubular structure was 0 ml / (cm ² · min). This wall thickness was 250 μm. The sulfated hyaluronic acid coating was applied only to the inner surface.

[Example 6]
A tubular structure was prepared in the same manner as in Example 5. The tubular product was subjected to the same coating treatment with sulfated hyaluronic acid as in Example 1, and was cut open to obtain a planar structure. When the porosity of the obtained planar structure coated with sulfated hyaluronic acid was measured, it was 0 ml / (cm ² · min). This wall thickness was 250 μm. Sulfated hyaluronic acid was coated only on one side of the tubular body.

[Example 7]
Cell culture was performed on the planar structure prepared in Example 2. The cells used were human heart disease patient fibroblasts (HCF) obtained from Cell Application. Using a medium obtained from Cell Application, the cells were cultured at 37 ° C. in a humidified 5% CO ₂ atmosphere. The medium was changed every other day, and secondary culture was performed when HCF reached 85-95% of saturation. The cells were seeded on the structure and allowed to adhere for 48 hours, and then a tensile load was applied at a frequency of 60 times / minute in one axial direction. In addition, as a control, culture was performed in the same system except that a structure not containing the structure of the present invention was used. After culturing for 1, 3 and 6 hours, respectively, the structure to which the cells were attached was washed with phosphate buffer (PBS), the cells were treated, and mRNA expression was determined by RT-PCR. And quantified. As a result, in the HCF cultured on the structure containing sulfated hyaluronic acid, the expression of mRNA related to the expression of collagen type I was significantly reduced as compared with the control. In the control, the expression of collagen increased as the period of loading was increased, but no expression of collagen was observed on the structure containing sulfated hyaluronic acid.

  From this result, it was considered that sulfated hyaluronic acid suppresses fibrosis of cardiomyocytes. The medical device treated with sulfated hyaluronic acid has the potential to be a prosthetic device with extremely high biocompatibility that prevents fibrosis and scarring of the living tissue that comes into contact with it, that is, does not cause a foreign body reaction. .

[Example 8]
Cell culture was performed on the planar structure prepared in Example 2. The cells used were normal human astrocytes obtained from Cambrex Bio Science. As the medium, ABM medium supplemented with 5% FCS, rhEGF and IGF obtained from Cambrex Bio Science was used. The cells were seeded on the structure and cultured for 24 hours. Then, the cell culture surface was cut straight using a sharp razor, and TetraColor ONE, which is a fluorescent dye, was added. After allowing to stand for 5 minutes, the medium was removed and further washed with the medium. This was observed with a fluorescence microscope, and it was measured how far the dye had reached from the cut portion. Thereby, the communication function (gap junction activity) between cells was evaluated. In addition to this sample, the same as in Example 2, except that the coating solution concentration of sulfated hyaluronic acid was adjusted to 10 μg / mL instead of 50 μg / mL. Further, as a control, the same operation was performed on the structure prepared in the same manner as in Example 2 except that sulfated hyaluronic acid was not included. As a result, the propagation distance between the fluorescent dye cells was significantly increased in the system coated with sulfated hyaluronic acid (FIG. 1). There was no difference due to the concentration of the sulfated hyaluronic acid treatment solution. From this result, it was considered that sulfated hyaluronic acid enhances the intercellular communication function.

  Connexin is known as a protein subunit having an important function for cell-cell communication. The expression of mRNA related to main connexins was examined in the same manner as in Example 7. As a result, as shown in FIGS. 2 (A) to (C), an increase in expression proportional to the amount of sulfated hyaluronic acid was observed for each connexin of Cx26, Cx32, and Cx43. From these results, it was shown that sulfated hyaluronic acid enhances the intercellular communication function.

  In addition, cell proliferation was evaluated by crystal red staining. As a result, no significant difference was observed between the control and the system coated with sulfated hyaluronic acid. When the cell shape was observed, no abnormality was observed in any of the systems. The results are shown in FIG.

[Example 9]
In the same experimental system as in Example 8, normal human astrocytes were cultured and observed for differentiation. Some astrocytes dedifferentiate and become neural progenitor cells, and then redifferentiate into neurons, oligodendrocytes, and astrocytes. Nestin for neural progenitor cells, Nurr-1 for neurons, OLIG1 for oligodendrocytes, and Glial filamentous acidic protein (GFPA) for astrocytes were used as expression indexes, and the degree of gene expression was evaluated in the same manner as in Examples 7 and 8. As a result, as shown in FIGS. 4 (A) to (D), promotion of differentiation proportional to the amount of sulfated hyaluronic acid was observed.

  From these results, it was found that treatment with an appropriate concentration of sulfated hyaluronic acid does not affect the proliferation of aortic smooth muscle cells and enhances the intercellular communication function. This suggests that the strength of the smooth muscle tissue can be maintained by suppressing the thickening due to blood vessel anastomosis and the like, and maintaining the patency without causing stenosis and maintaining the cell-cell communication. In other words, a medical device treated with sulfated hyaluronic acid can become a prosthetic device with extremely high biocompatibility that can suppress malfunction due to foreign body reaction while maintaining a high level of function of the living tissue to be contacted. Sex was shown.

  The present invention relates to an artificial organ such as an artificial blood vessel, and to an artificial organ excellent in biocompatibility and curability that does not cause an inflammatory reaction or fibrosis to a host cell or tissue and promotes proliferation. It is. Therefore, since a foreign body reaction due to contact with a living body is suppressed and healing is promoted, a prosthetic device having high effectiveness and high safety in clinical practice is provided.

The intracellular propagation distance (astrocyte) of the fluorescent dye from the cut portion is shown. The mRNA expression level of Cx43 is shown. The mRNA expression level of Cx26 is shown. The Cx32 mRNA expression level is shown. Shows cell growth and morphology. The state of differentiation into Nestin is shown. The state of differentiation into GFAP is shown. The state of differentiation into OLOGI is shown. The state of differentiation into Nurr-1 is shown.

Claims (6)

  A prosthesis, which is a structure composed of fibers, and whose outer surface and / or inner surface is coated with a sulfated polysaccharide.   The prosthesis according to claim 1, wherein the sulfated polysaccharide is sulfated hyaluronic acid.   The prosthetic device according to claim 2, wherein the sulfated hyaluronic acid has a degree of sulfation in a range containing 0.1 to 2.0 equivalent sulfate groups per unit of hyaluronic acid composed of disaccharides. .   The prosthesis according to any one of claims 1 to 3, wherein the weight average molecular weight of the sulfated hyaluronic acid is in the range of 10,000 to 3,000,000.   The prosthesis according to any one of claims 1 to 4, wherein the prosthesis is tubular or sheet-like. The prosthesis according to any one of claims 1 to 5, wherein the prosthesis has a water permeability of 0 to 50 ml / (cm ² · min) (120 mmHg, 37 ° C).

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