TISSUE REGENERATION SUBSTRATE AND METHOD FOR PRODUCING TISSUE REGENERATION SUBSTRATE

Abstract

The present invention aims to provide a tissue regeneration substrate excellent in penetrability to cells as well as capable of effectively preventing cell leakage from the tissue regeneration substrate to accelerate tissue regeneration; and a method of producing the tissue regeneration substrate. The present invention relates to a tissue regeneration substrate including: a nonwoven fabric made of a bioabsorbable material, the tissue regeneration substrate having a laminated structure in which a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm and a layer containing a nonwoven fabric having an average pore size of 5 to 20 μm are integrated.

Claims

1. A tissue regeneration substrate comprising: a nonwoven fabric made of a bioabsorbable material, the tissue regeneration substrate having a laminated structure in which a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm and a layer containing a nonwoven fabric having an average pore size of 5 to 20 μm are integrated.

2. The tissue regeneration substrate according to claim 1, which has a laminated structure in which the following three layers are integrated in the given order: a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm; a layer containing a nonwoven fabric having an average pore size of 5 to 20 μm; and a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm.

3. The tissue regeneration substrate according to claim 1, wherein the nonwoven fabric having an average pore size of 20 to 50 μm has an average fiber diameter of 10 to 50 μm and the nonwoven fabric having an average pore size of 5 to 20 μm has an average fiber diameter of 0.7 to 7 μm.

4. The tissue regeneration substrate according to claim 1, wherein the layer containing the nonwoven fabric having an average pore size of 20 to 50 μm has a thickness of 300 μm to 2.0 mm.

5. The tissue regeneration substrate according to claim 1, wherein the layer containing the nonwoven fabric having an average pore size of 5 to 20 μm has a thickness of 10 to 150 μm.

6. The tissue regeneration substrate according to claim 1, wherein the bioabsorbable material is polyglycolide.

7. The tissue regeneration substrate according to claim 6, wherein the polyglycolide has a weight average molecular weight of 30000 to 400000.

8. A method of producing the tissue regeneration substrate according to claim 1, comprising the steps of: preparing a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm; discharging threads made of a bioabsorbable material on the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm by melt blowing to form a layer containing a nonwoven fabric having an average pore size of 5 to 20 μm to produce a stack; and needle-punching the stack to integrate the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm and the layer containing a nonwoven fabric having an average pore size of 5 to 20 μm.

9. A method of producing the tissue regeneration substrate according to claim 1, comprising the steps of: preparing a layer containing a nonwoven fabric having an average pore size of 20 to 50 μm; preparing a layer containing a nonwoven fabric having an average pore size of 5 to 20 μm; stacking the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm and the layer containing a nonwoven fabric having an average pore size of 5 to 20 μm to produce a stack; and needle-punching the stack to integrate the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm and the layer containing a nonwoven fabric having an average pore size of 5 to 20 μm.

10. The method of producing a tissue regeneration substrate according to claim 8, further comprising, after the step of preparing the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm, fluffing a surface of the layer containing a nonwoven fabric having an average pore size of 20 to 50 μm on which the layer containing a nonwoven fabric having an average pore size of 5 to 20 μm is to be stacked.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 is a schematic view illustrating a method for determining the pore size distribution of a nonwoven fabric by a bubble point method.

[0074] FIG. 2 is a schematic view illustrating a method for estimating the pore size distribution of a nonwoven fabric from data obtained by a bubble point method.

[0075] FIG. 3 is a schematic view illustrating an evaluation method used in examples and comparative examples.

DESCRIPTION OF EMBODIMENTS

[0076] The embodiments of the present invention are described in more detail below with reference to examples. The present invention, however, should not be limited to the example.

Example 1

(1) Preparation of Cell Penetration Layer

[0077] Polyglycolide having a weight average molecular weight of 250000 was used as a bioabsorbable material. A fabric made of yarn spun from the polyglycolide was needle-punched to form a nonwoven fabric. In this manner, a cell penetration layer having an average fiber diameter of about 16 μm and a thickness of about 1.5 mm was obtained.

[0078] A fluorine-containing solvent (trade name: Porofil (registered trade mark)) as a wetting agent was absorbed into the obtained cell penetration layer. The layer was then placed on Porometer 3G produced by BEL Japan, Inc. such that the specimen had a circular shape with a diameter of 25 mm. Then, the air pressure was applied from the back side of the cell penetration layer to determine the minimum pressure (bubble point) at which formation of a bubble was observed at the membrane surface. A graph of the pore size distribution of the cell penetration layer was obtained based on the bubble point. The average pore size calculated from the graph was 28 μm.

(2) Stacking of Cell Leakage Prevention Layer

[0079] The obtained cell penetration layer was placed on the conveyer at a position in front of the fiber trapping points for melt blowing. While moving the conveyer, threads made of polyglycolide were discharged onto the cell penetration layer by melt blowing, thus a cell leakage prevention layer was stacked on the cell penetration layer.

[0080] The melt blowing was performed using polyglycolide having a weight average molecular weight of 250000 as a raw material at a polymer discharge rate of 0.2 kg/h and an air velocity around the outlets of 11 m/sec. The speed of the conveyor was set such that the cell leakage prevention layer obtained by the melt blowing had a density of 10 g/m.sup.2.

[0081] Separately, a cell leakage prevention layer alone was produced under the same conditions. The average pore size of this cell leakage prevention layer was 12 μm as calculated by the bubble point method.

(3) Integration of Cell Penetration Layer and Cell Leakage Prevention Layer

[0082] The obtained stack was needle-punched such that the needles entered from the cell leakage prevention layer-side of the stack, whereby the cell penetration layer and the cell leakage prevention layer were integrated.

Example 2

[0083] A tissue regeneration substrate was obtained in the same manner as in Example 1 except that in the preparation of the cell penetration layer the cell penetration layer obtained by needle punching was hot-pressed to achieve a smooth surface.

Example 3

[0084] A tissue regeneration substrate was obtained in the same manner as in Example 1 except that in the preparation of the cell penetration layer the cell penetration layer obtained by needle punching was hot-pressed to achieve a smooth surface and then fluffed by brushing with a brass brush, and that the cell leakage prevention layer was stacked on the fluffed side.

Example 4

(1) Preparation of Cell Penetration Layer

[0085] Polyglycolide having a weight average molecular weight of 250000 was used as a bioabsorbable material. A fabric made of yarn spun from the polyglycolide was needle-punched to form a nonwoven fabric. In this manner, two cell penetration layers were obtained: one having an average fiber diameter of about 16 μm and a thickness of about 1.5 mm, and the other having an average fiber diameter of about 16 μm and a thickness of about 0.5 mm.

[0086] Each obtained cell penetration layer had average pore size of 28 μm as calculated by the bubble point method.

(2) Preparation of Cell Leakage Prevention Layer

[0087] A cell leakage prevention layer having average fiber diameter of about 2 μm and a thickness of about 50 μm was obtained by melt blowing using polyglycolide having a weight average molecular weight of 250000 as a bioabsorbable material. The cell leakage prevention layer had an average pore size of 12 μm as calculated by the bubble point method.

(3) Integration of Cell Penetration Layer and Cell Leakage Prevention Layer

[0088] The cell penetration layer having a thickness of about 1.5 mm, the cell leakage prevention layer having a thickness of about 50 μm, and the cell penetration layer having a thickness of about 0.5 mm were stacked in the given order to produce a three-layer stack.

[0089] The three-layer stack was needle-punched from the side of the cell penetration layer having a thickness of about 0.5 mm to integrate the three layers. Thus, a tissue regeneration substrate was obtained.

Comparative Example 1

[0090] Polyglycolide having a weight average molecular weight of 250000 was used as a bioabsorbable material. A fabric made of yarn spun from the polyglycolide was needle-punched to form a nonwoven fabric having an average fiber diameter of about 16 μm and a thickness of about 1.5 mm. This nonwoven fabric was used as a tissue regeneration substrate. The obtained nonwoven fabric had an average pore size of 28 μm as determined by the bubble point method.

Comparative Example 2

[0091] A nonwoven fabric having an average fiber diameter of about 2 μm and a thickness of about 50 μm was obtained by melt blowing using polyglycolide having a weight average molecular weight of 250000 as a bioabsorbable material. This nonwoven fabric was used as a tissue regeneration substrate. The obtained nonwoven fabric had an average pore size of 12 μm as determined by the bubble point method.

Comparative Example 3

[0092] A tissue regeneration substrate was obtained in the same manner as in Example 1 except that the integration treatment by needle punching was not performed.

Comparative Example 4

[0093] A tissue regeneration substrate was obtained in the same manner as in Example 2 except that the integration treatment by needle punching was not performed.

Comparative Example 5

[0094] A tissue regeneration substrate was obtained in the same manner as in Example 3 except that the integration treatment by needle punching was not performed.

(Evaluation)

[0095] The tissue regeneration substrates obtained in the examples and the comparative examples were evaluated by the following methods.

[0096] Table 1 shows the results.

(1) Evaluation of Particle Trapping Rate Polystyrene particles having an average particle size of 20 μm were dispersed into an aqueous solution of ethanol (water/ethanol (volume ratio)=80/20) to prepare a particle dispersion.

[0097] The tissue regeneration substrate was cut into a square shape (20 mm×20 mm) and fixed onto metal mesh.

[0098] The particle dispersion (2 mL) was slowly added dropwise from the cell penetration layer-side (in Example 4, the about 1.5 mm-thick cell penetration layer-side) of the tissue regeneration substrate, so that the dispersion permeated the tissue regeneration substrate. The absorbance of the particle dispersion at a wavelength of 230 nm was measured before and after the permeation, and the particle concentration (pcs/mL) was calculated based on a calibration curve obtained by measuring the absorbance of a polystyrene latex particle dispersion of a known.

[0099] concentration. The number of particles in the particle dispersion before and after the permeation was determined from the obtained particle concentration before and after the permeation and the volume of the dispersion calculated from the weight of the dispersion before and after the permeation. The particle trapping rate (%) was calculated by the following formula.

Particle trapping rate (%)=(Number of particles in particle dispersion before permeation−Number of particles in particle dispersion after permeation)/Number of particles in particle dispersion before permeation

(2) Evaluation of Cell Retention

[0100] Cells were seeded on the tissue regeneration substrates in accordance with the culturing method illustrated in FIG. 3. The growth of the cells was evaluated.

[0101] Specifically, a sponge body 5 was placed on a petri dish 4. A culture medium 6 was poured into the petri dish such that the surface of the sponge body 5 was impregnated with the medium (the culture medium was thus supplied only from the sponge body 5). The tissue regeneration substrates 7 produced in the examples and the comparative examples were each placed on the corresponding sponge body 5. In the examples and Comparative Examples 3 to 5, the tissue regeneration substrate was placed with the cell leakage prevention layer downward so that it contacted the sponge body 5. In Example 4, the tissue regeneration substrate was placed with the cell penetration layer having a thickness of about 0.5 mm downward such that it contacted the sponge body 5.

[0102] On the tissue regeneration substrate 7 arranged in this manner, fibroblasts were seeded to achieve a seed density of 1×10.sup.5 pcs/cm.sup.2. The fibroblasts were then cultured for seven days. The culture medium 6 was changed every day. After the seven-day culture, the number of cells contained in the tissue regeneration substrate 7 was counted by MTT assay. The number of cells in the tissue regeneration substrates of the examples and comparative examples was expressed relative to the number of cells in the tissue regeneration substrate of Comparative Example 1 taken as 100. Table 1 shows the results.

(3) Evaluation of Integrity (Qualitative Evaluation)

[0103] The integrity of the cell regeneration substrate was evaluated according to the following criteria.

[0104] oo (Excellent): Folding the tissue regeneration substrate did not cause separation of the cell penetration layer and the cell leakage prevention layer from each other, and an attempt to manually separate the cell penetration layer and the cell leakage prevention layer from each other caused breaking of the cell penetration layer or the cell leakage prevention layer before separation.

[0105] o (Good): Folding the tissue regeneration substrate did not cause separation of the cell penetration layer and the cell leakage prevention layer from each other, and the cell penetration layer and the cell leakage prevention layer were not easily manually separated from each other.

[0106] Δ (acceptable): Folding the tissue regeneration substrate caused partial separation of the cell penetration layer and the cell leakage prevention layer from each other, and the cell penetration layer and the cell leakage prevention layer were easily manually separated from each other.

[0107] x (Poor): Folding the tissue regeneration substrate caused the entire separation of the cell penetration layer and the cell leakage prevention layer from each other, and the cell penetration layer and the cell leakage prevention layer were easily manually separated from each other.

(4) Evaluation of Integrity (Quantitative Evaluation)

[0108] The integrity of the cell regeneration substrate was evaluated by measuring the delamination strength in accordance with Determination of delamination strength specified in JIS L 1021-90.

[0109] Specifically, the obtained tissue regeneration substrate was cut into a strip of 20 mm wide×50 mm long, and the cell penetration layer and the cell leakage prevention layer were manually separated from each other by 25 mm in the length direction in advance to form grip portions. The strip was used as a sample. The grip portion of each layer of the obtained sample was held with chucks (gripping distance 20 mm) and pulled with an autograph (“AGS-J” produced by SHIMADZU Corporation, load cell 50 N) at a pulling speed of 100 mm/min to determine the delamination strength. JIS L 1021-9 specifies that the sample size is 50 mm wide×200 mm long, and the length of the portions to be separated in advance (length of the grip portions) is 50 mm. Yet, in view of the fact that the cell regeneration substrate is for implantation in tissue or an organ, the sample was prepared as described above. JIS L 1021-9 also specifies that the sample should be allowed to stand in a constant temperature and humidity chamber (20° C., 65%) for 24 hours. Yet, since the sample is a substrate made of a degradable polymer, this procedure was omitted and the measurement was performed immediately after the preparation of the sample.

[0110] For the cell regeneration substrate of Example 4, the sample was prepared by separating the layers of the 20 mm×50 mm strip from each other by 25 mm in the vertical direction, and the cell penetration layer having a thickness of about 1.5 mm and the cell leakage prevention layer having a thickness of about 50 μm were held with chucks. The delamination strength between these two layers was measured by the tensile test under the above conditions.

TABLE-US-00001 TABLE 1 Evaluation Evaluation of cell of particle retention Evaluation of integrity trapping (number of Qualitative Delamination rate (%) cells) evaluation strength (N) Exmaple 1 0.922 141 ∘∘ 0.42 Exmaple 2 0.818 128 ∘ 0.33 Exmaple 3 0.932 135 ∘∘ 0.36 Exmaple 4 0.947 145 ∘∘ Not evaluated (cell leakage prevention layer broke during delamination test) Comparative 0.840 100 — — Exmaple 1 Comparative 0.983 54 — — Exmaple 2 Comparative 0.962 138 Δ 0.02 Exmaple 3 Comparative 0.955 129 x 0.02 Exmaple 4 Comparative 0.971 130 Δ 0.06 Exmaple 5

INDUSTRIAL APPLICABILITY

[0111] The present invention provides a tissue regeneration substrate excellent in penetrability to cells as well as capable of effectively preventing cell leakage from the tissue regeneration substrate to accelerate tissue regeneration; and a method of producing the tissue regeneration substrate.

REFERENCE SIGNS LIST

[0112] 4 petri dish [0113] 5 sponge body [0114] 6 culture medium [0115] 7 tissue regeneration substrate