JP5939565B2 - Tissue regeneration substrate - Google Patents

Description

The present invention relates to a tissue regeneration substrate suitable for a scaffold for reinforcing a fragile tissue and promoting tissue regeneration, which is excellent in cell invasiveness and hardly causes tissue adhesion.

Recent advances in cell engineering technology have enabled the culturing of numerous animal cells, including human cells, and rapid research on so-called regenerative medicine that uses these cells to reconstruct human tissues and organs. Is going on. In regenerative medicine, the point is that cells can proliferate and differentiate to construct a three-dimensional biological tissue-like structure. A method of regenerating a tissue or an organ by invading into a base material and growing and differentiating is performed.

Further, as a method of excising a lesion generated in a living tissue under an endoscope, excision using an automatic suturing device or the like is performed. When excision is performed on fragile tissues such as the lungs, bronchi, liver and digestive tract, or tissues weakened by lesions, there is a risk of tissue tearing only by suturing, and for example, lung surgery May cause air leakage. In view of this, it has been practiced to sew the suture reinforcement material to the excision site of the living tissue.

As such a regenerative medical base material and suture reinforcing material, for example, it has been proposed to use a nonwoven fabric made of a bioabsorbable material as disclosed in Patent Document 1. When a nonwoven fabric made of a bioabsorbable material is used as a base material for regenerative medicine, it is expected that cells will invade and proliferate into the voids and that tissues will be regenerated early. In addition, when used as a reinforcing material for a fragile tissue, it is possible to prevent the tissue from tearing and to prevent the occurrence of air leakage. Furthermore, since it decomposes | disassembles after a fixed period progress and is absorbed by the biological body, it has the outstanding performance that it is not necessary to take out by reoperation.

However, when a non-woven fabric made of a conventional bioabsorbable material is transplanted, there is a problem that tissues may adhere to each other at the site of use. Another problem is that cells do not enter as much as expected and it takes time to regenerate the tissue. Furthermore, it causes chronic inflammation at the site of use, which can cause scar formation.

Japanese Patent Laid-Open No. 5-315561

An object of the present invention is to provide a tissue regeneration substrate suitable for scaffolds for reinforcing fragile tissue and promoting tissue regeneration, which is excellent in cell invasion and hardly causes tissue adhesion.

The present invention is a tissue regeneration substrate made of a nonwoven fabric made of polyglycolide having an average fiber diameter of 0.9 to 7.0 μm.
The present invention is described in detail below.

The present inventors investigated the invasion of cells and the presence or absence of tissue adhesion using nonwoven fabrics having various materials and shapes. As a result, when a non-woven fabric made of polyglycolide having an average fiber diameter of 0.9 to 7.0 μm is used, the cell invasion is remarkably compared with the case of using a non-woven fabric having another material or fiber diameter. The present invention was completed by finding that it is excellent and can suppress the occurrence of tissue adhesion.

The tissue regeneration substrate of the present invention comprises a nonwoven fabric made of polyglycolide.
As bioabsorbable materials, in addition to polyglycolide, polylactide (D, L, DL form), polycaprolactone, lactide (D, L, DL form) -ε-caprolactone copolymer, poly (p-dioxanone), etc. Many resins are known. Among these bioabsorbable materials, only when polyglycolide is used, it is possible to exert an excellent effect of being excellent in cell invasion and hardly causing tissue adhesion. For example, when polyglycolide is fibrous and immersed in physiological saline at 37 ° C., the period until the tensile strength becomes 1/2 before immersion is about 14 days. By having such degradability, the base material is gradually decomposed and absorbed when the cells proliferate and the tissue is regenerated, and a tissue regenerated to the inside of the base material is constructed. As a result, high quality regeneration is achieved. An organization is established. Furthermore, since the inflammatory cells disappear within a few days after implantation in a living body, it is considered that the excellent effect of hardly causing tissue adhesion can be exhibited. If a material that decomposes earlier than polyglycolide is used, the base material loses strength before the tissue regeneration proceeds, and cannot serve as a scaffold. If a material that is harder to decompose than polyglycolide is used, the base material remains at the site of use over a long period of time, causing adhesion or chronic inflammation. In general, it is said that polyglycolide is likely to cause early inflammation as compared with the other bioabsorbable materials. However, early inflammation may also contribute to the better progress of tissue regeneration compared to the other bioabsorbable materials, because cells can be collected locally by the inflammation mechanism. Is expensive.
In the present specification, polyglycolide means a polymer of glycolide such as polyglycolic acid, but other bioabsorbables such as lactic acid, ε-caprolactone, trimethylene carbonate, etc., as long as the effects of the present invention are not impaired. It is good also as a copolymer with a component. Moreover, it is good also as a mixture with other bioabsorbable materials, such as a polylactide, in the range which does not inhibit the effect of this invention.

The weight average molecular weight of the polyglycolide is not particularly limited, but a preferable lower limit is 30000 and a preferable upper limit is 200000. If the weight average molecular weight of the polyglycolide is less than 30000, the strength may be insufficient and a sufficient tissue reinforcing effect may not be obtained. If the weight average molecular weight exceeds 200000, the degradation rate in the living body is slowed down, causing a foreign body reaction. Sometimes. The minimum with a more preferable weight average molecular weight of the said polyglycolide is 50000, and a more preferable upper limit is 150,000.

The average fiber diameter of the nonwoven fabric constituting the tissue regeneration substrate of the present invention (hereinafter also simply referred to as “nonwoven fabric”) is 0.9 to 7.0 μm. When the average fiber diameter of the nonwoven fabric is within this range, it is possible to exhibit an excellent effect of being excellent in cell proliferation on the substrate and hardly causing tissue adhesion. When the average fiber diameter of the non-woven fabric is less than 0.9 μm, it becomes difficult for cells to invade, and even if transplanted, granulation tissue does not sufficiently infiltrate and tissue regeneration may be delayed. When the average fiber diameter of the nonwoven fabric exceeds 7.0 μm, it tends to cause tissue adhesion. In addition, many edema and inflammatory cells are observed at the transplant site. A preferable upper limit of the average fiber diameter of the nonwoven fabric is 3 μm. Among these, particularly excellent effects are exhibited when the average fiber diameter is 0.9 μm. Even if the nonwoven fabric has the same fiber diameter, the spatial rate inside the nonwoven fabric is changed by changing the three-dimensional thickness, and the spatial rate is also considered to affect the invasion property of cells.
Thus, it is not clear why the average fiber diameter of the nonwoven fabric greatly influences the invasion property and tissue adhesion property of the nonwoven fabric, but the interfiber distance varies depending on the fiber diameter, and the interfiber distance varies depending on the cell infiltration property and the layer. It is thought that this may affect the uniformity of the image.
In the present specification, the average fiber diameter of the nonwoven fabric means an average fiber diameter obtained by cutting a part of the center of the fabric, observing it with an electron microscope, measuring 10 fiber diameters at random.

The above-mentioned nonwoven fabric has a preferred basis weight of 5 g / m ² and a preferred upper limit of 50 g / m ² . If it is less than 5 g / m ² , the tissue reinforcing effect may not be sufficiently obtained, and if it exceeds 50 g / m ² , the adhesiveness to the tissue may be reduced and sufficient tissue regeneration may not be possible. The upper limit with the more preferable fabric weight of the said nonwoven fabric is 30 g / m < ² >.

The method for producing the non-woven fabric is not particularly limited. A conventionally known method such as a wet method can be used. Among these, the melt blow method is preferable.

The thickness of the tissue regeneration substrate of the present invention is not particularly limited, but a preferred lower limit is 10 μm and a preferred upper limit is 0.5 mm. If the thickness is less than 10 μm, the strength may be insufficient and cannot be used to reinforce a fragile tissue, and if it exceeds 0.5 mm, the handleability is poor. The more preferable lower limit of the thickness is 20 μm, and the more preferable upper limit is 0.3 mm.

The tissue regeneration substrate of the present invention is suitable as a scaffold for reinforcing fragile tissue and promoting tissue regeneration. In particular, it is useful as a scaffold for reinforcing fragile tissue and promoting tissue regeneration by applying it to sites where early healing is desired, such as lung leakage, bronchial stump, pancreatic fistula, bile leakage, etc. The effect is prominent when attached to sites such as few bronchial stumps, pancreatic fistulas, and bile leakage.

The tissue regeneration substrate of the present invention is pasted so as to cover fragile tissue after surgery. The tissue regeneration substrate of the present invention can maintain close contact with the tissue without using a medical adhesive such as fibrin glue. In addition, when using an automatic suturing device, by performing a suturing operation using the automatic suturing device after attaching the tissue regeneration substrate of the present invention to the tissue, air leakage from the lungs and body fluids from various tissues It is also useful in that leakage can be prevented.

ADVANTAGE OF THE INVENTION According to this invention, the tissue regeneration base material excellent in the penetration | invasion property of a cell and being hard to cause the adhesion | attachment of a tissue suitable for the reinforcement | strengthening of a weak structure | tissue and the promotion of tissue regeneration can be provided.

It is a HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polyglycolide whose average fiber diameter is 0.7 micrometer. It is a HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polyglycolide whose average fiber diameter is 0.9 micrometer. It is the HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polyglycolide whose average fiber diameter is 3.1 micrometers. It is a HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polyglycolide whose average fiber diameter is 7.0 micrometers. It is a HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polyglycolide whose average fiber diameter is 20.0 micrometers. It is a HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of polylactide with an average fiber diameter of 7.3 micrometers. It is the HE dyeing | staining image of the structure | tissue which embedded the nonwoven fabric which consists of a lactide-epsilon-caprolactone copolymer with an average fiber diameter of 3.1 micrometers.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Experimental example 1)
Polyglycolide is used as a bioabsorbable material, and a nonwoven fabric obtained by an electrospinning deposition method (average fiber diameter 0.7 μm) or a melt blow method (average fiber diameters 0.9, 3.1, 7.0 μm) is drawn. The average fiber diameter is 0.7, 0.9, 3.1, 7.0, 20.0 μm by the method or the method of making a spun tubular knitted fabric into a nonwoven fabric by the needle punch method (average fiber diameter 20 μm). A nonwoven fabric having a thickness of 0.13 to 0.30 mm was obtained.

Polylactide was used as a bioabsorbable material, and a nonwoven fabric having an average fiber diameter of 7.3 μm and a thickness of 0.093 mm was obtained by a method of stretching a nonwoven fabric obtained by a melt blow method. Further, a non-woven fabric having an average fiber diameter of 3.1 μm and a thickness of 0.148 mm was obtained by using a lactide-ε-caprolactone copolymer as a bioabsorbable material and stretching the non-woven fabric obtained by the melt blow method.

(Evaluation of organization regeneration promotion)
By a method in which a female rat having a body weight of 200 g and a 9-week-old body weight reared under the conditions of specific pathogen free was subjected to basic anesthesia by ether inhalation anesthesia, and then 5 mg of pentobarbital was diluted with saline to give 1 ml intraperitoneally. General anesthesia was applied.
After general anesthesia, for each animal, two left and right sides were placed on the head side of the back and two left and right sides on the caudal side. Each non-woven fabric cut into a size of 10 mm × 10 mm was buried in each of the four subcutaneous pockets at random.

On the third, second and third weeks after the operation, the rats were euthanized by intraperitoneal administration of an excessive amount of pentobarbital, and the buried portion was removed.
The photomicrograph images obtained by staining the obtained specimen with hematoxylin-eosin (HE staining) are shown in FIGS.
In addition, the obtained HE-stained image was observed with a microscope to evaluate granulation tissue invasion, edema formation, presence of inflammatory cells, granulation tissue layer formation, and the like. A comprehensive evaluation of regeneration promotion was performed. The results are shown in Table 1.

Tissue regeneration promotion comprehensive evaluation A: Granulation tissue has penetrated into the material, and there are few inflammatory cells.
In addition, the granulation tissue forms a layer.
○: Granulation tissue penetrates into the material. The granulation tissue forms a layer, but the layer is more uneven than よ り. There are few inflammatory cells.
Δ: The granulation tissue penetrates into the material, but the granulation tissue does not form a layer.
There are many inflammatory cells. Edema is observed.
X: Granulation tissue does not penetrate into the material.

(Evaluation of non-adhesiveness)
Wistar / ST rats (8 weeks old, female, 180 g) were subjected to basic anesthesia by ether inhalation anesthesia, and then 5 mg of pentobarbital was diluted with saline to give 1 ml of general anesthesia by intraperitoneal administration. It was over.
After general anesthesia, the abdomen was opened by a 4.5cm midline abdominal incision, and each non-woven fabric cut to a size of 1.5cm x 1.5cm was sewn with a 7-0 non-absorbing thread for vascular suture on the wall of the left abdominal wall. I wore it. Thereafter, the abdominal wall was closed by continuous stitching of peritoneum and fascia using 4-0 nylon monofilament suture and knot stitching of the skin.

One week after surgery, 8 of each group were randomly selected, rats were euthanized by intraperitoneal administration of pentobarbital overdose, necropsied, and the degree of gross adhesion was evaluated according to the following criteria: . The results are shown in Table 1.
Area of adhesion 0: No adhesion 1: Adhesion of 1-25% of treatment area 2: Adhesion of 26-50% of treatment area 3: Adhesion of 51-75% of treatment area 4: 76-100% of treatment area adhesion

Strength of adhesion 0: No adhesion 1: Easy peeling 2: Strong adhesion requiring forceful peeling 3: Strong adhesion with serous damage of blunt peeling, requiring sharp peeling of 50% or less 4: Blunt Strong adhesion that requires 51% or more sharp detachment with detachment serosal damage

Non-adhesion comprehensive evaluation A value obtained by multiplying the value of the adhesion area evaluated by the above criteria and the value of the adhesion strength was calculated and evaluated according to the following criteria.
A: The value obtained by multiplying the adhesion area value and the adhesion strength value is 0.
○: The value obtained by multiplying the adhesion area value and the adhesion strength value is less than 5 Δ: The value obtained by multiplying the adhesion area value and the adhesion strength value is 5 or more and less than 10 ×: Adhesion The value of the area value multiplied by the adhesion strength value is 10 or more

From Table 1, in the nonwoven fabric made of polyglycolide having an average fiber diameter of 20 μm, the degree of adhesion was high, and strong adhesion requiring peeling occurred. On the other hand, in the non-woven fabric made of polyglycolide having an average fiber diameter of 0.9 μm, the degree of adhesion was low, and no adhesion that required peeling was generated.

(Experimental example 2)
By the same method as in Experimental Example 1, nonwoven fabrics made of polyglycolide having average fiber diameters of 0.67 μm, 0.90 μm, 3.1 μm, 7.0 μm, and 20.6 μm were prepared.
Each obtained non-woven fabric was cut out into a circular shape having a diameter of 1.5 cm (well size of 24 well plate), immersed in ethanol and dried overnight. Each non-woven fabric after drying was placed in each well of a 24 well plate, and a ring was put on and fixed so as not to float.

A suspension of F334 rat fibroblasts at a concentration of 1.33 × 10 ⁴ cells / μL was prepared, and 750 μL per well (cell number 10 ⁴ cells / well) was seeded on each well on which the nonwoven fabric was placed. After sowing, the cells were cultured in a CO ₂ incubator for 5 days and 7 days. After completion of the culture, the nonwoven fabric was transferred to a new 24 well plate, and the number of cells was counted by the ATPassay method. Each measurement was performed at N = 3.
The results are shown in Table 2.

As shown in Table 2, the number of cells was hardly increased by culturing in the non-woven fabric having an average fiber diameter of 0.67 μm and 20.6 μm (in the non-woven fabric having an average fiber diameter of 0.67 μm, 7 days rather than after culturing for 5 days. On the other hand, the number of cells increased in the non-woven fabrics having average fiber diameters of 0.9 μm, 3.1 μm, and 7.0 μm.

ADVANTAGE OF THE INVENTION According to this invention, the tissue regeneration base material excellent in the penetration | invasion property of a cell and being hard to cause the adhesion | attachment of a tissue suitable for the reinforcement | strengthening of a weak structure | tissue and the promotion of tissue regeneration can be provided.

Claims (1)

A tissue regeneration substrate used as a scaffold for promoting weak tissue reinforcement and tissue regeneration , comprising a non-woven fabric made of polyglycolide having an average fiber diameter of 0.9 μm , and the polyglycolide has a weight average molecular weight of 30,000 to 200,000. tissue regeneration substrate which is characterized by certain.