JP4314421B2 - HELP - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an artificial heart valve and a method for manufacturing the same, a method for regenerating a heart valve, and a heart valve.
[0002]
[Prior art and problems]
Valves do not work normally, such as mitral stenosis, mitral regurgitation (reflux), aortic stenosis, aortic regurgitation, tricuspid regurgitation If stenosis or regurgitation occurs, the valve needs to be replaced. There are three types of valves currently used in surgery: (1) mechanical valves, (2) heterogeneous biological valves, and (3) homogeneous valves.
[0003]
The mechanical valve is excellent in durability, but it is necessary to continue to drink the anticoagulant for a lifetime. In addition, heterogeneous biological valves that use animal valves do not need to continue to drink anticoagulants, but can cause valve dysfunction in 6-10 years. On the other hand, human frozen homogenous valves provided by cadaver are generally used in Europe and the United States where long-term remote results are superior to heterogeneous biological valves and the use of cadaver tissue is advanced, but supply is not sufficient in Japan There's a problem.
[0004]
On the other hand, in recent years, attempts have been made to regenerate many tissues of a living body using a tissue culture technique. This is an attempt to regenerate own tissue by seeding and culturing tissue cells on a scaffold made of bioabsorbable polymer. Already skin (MLCooper, LF Hansbrough, RL Spiel vogel et.al .: In vivo optimization of dermal substitute to cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials, 12: 243-248, 1991) and cartilage (CAVacanti, R. Langer et.al.:Synthetic polymers seeded with chondrocytes provide a templete for new cartilage formation. Plast.Reconstr.Surg., 88: 753-759,1991).
[0005]
Attempts have also been made to regenerate heart valves using tissue culture technology, and good research results on the regeneration of leaflet structures have been reported (T. Shinoka et.al .: Tissue-engineered heart valve leaflets. Autologous valv leaflet replacement study ina lamb model. Circulation, 94 (suppl.II): II-164-II-168,1996.T.Shinokaet.al.: Tissue-engineered heart valve leaflets. Does cell origin affect outocome? Circulation, 96 (suppl II): II-102-II-107,1996).
[0006]
However, a practical bioabsorbable substrate for producing the entire heart valve from a bioabsorbable material has not been obtained.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to provide a prosthetic heart valve base material and a heart valve that do not leak blood.
[0008]
[Means for Solving the Problems]
The present invention relates to the following items 1 to 8.
Item 1. A heart valve base material comprising a bioabsorbable material having a heart valve main body and a blood leakage prevention layer covering the outer surface of the main body.
Item 2. Item 2. The artificial heart valve substrate according to Item 1, wherein the blood leakage preventing layer is a film.
Item 3. Item 2. The heart valve substrate according to Item 1, wherein the heart valve main body includes a valve leaflet inside a cylindrical base and forms a Valsalva sinus.
Item 4. A method for producing a heart valve base material, comprising a step of forming a blood leakage prevention layer by sticking a film to an outer surface of a heart valve main body.
Item 5. A method for producing a heart valve base material, wherein the heart valve body is immersed in a biodegradable absorbable polymer solution and then dried to form a film-like blood leakage prevention layer.
Item 6. A method for producing a heart valve base material, comprising a step of forming a Valsalva sinus on a cylindrical base body provided with a film-like blood leakage prevention layer on an outer surface and a step of forming a valve leaflet inside the base body.
Item 7. Item 5. A method for regenerating a heart valve, comprising seeding endothelial cells and fibroblasts simultaneously or separately on the artificial heart valve substrate according to any one of Items 1 to 3, and regenerating heart valve tissue.
Item 8. A heart valve comprising an inner surface of a heart valve body made of a bioabsorbable material, a Valsalva sinus, and a leaflet covered with a biological cell layer containing fibroblasts and endothelial cells.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the heart valve body (including the base body, Valsalva sinus and valve leaflet) constituting the heart valve base material, the blood leakage prevention layer, etc. are all made of a bioabsorbable material.
[0010]
Bioabsorbable materials include polyglycolic acid, polylactic acid (D-form, L-form, DL-form), polycaprolactone, glycolic acid-lactic acid (D-form, L-form, DL-form) copolymer, glycolic acid-caprolactone co Synthetic bioabsorbable polymers such as polymers, lactic acid (D-form, L-form, DL-form) -caprolactone copolymer, poly (p-dioxanone) and natural polymers such as collagen, modified collagen, gelatin, chitin and chitosan Etc.
[0011]
The base of the heart valve base material of the present invention is made of a foam, a film, a nonwoven fabric, or the like made of a bioabsorbable material. When strength is required, a woven fabric, a knitted fabric, or a nonwoven fabric also made of a bioabsorbable polymer. It is also possible to reinforce with a reinforcing material such as.
[0012]
A film made of a bioabsorbable polymer is formed on the outer surface of the substrate for the purpose of preventing blood leakage in the acute phase.
[0013]
Examples of the method for producing the heart valve base material include the following methods.
(1) Production of base having Valsalva sinus and blood leakage prevention layer A bioabsorbable material film is placed on the outside mold for producing a base having Valsalva sinus structure and used as a reinforcing material on the inside as needed. A base material with Valsalva sinus and blood leakage prevention layer is prepared by fitting woven fabric, knitted fabric, non-woven fabric, etc. made of functional polymer, inserting a mold from the inside, pouring a bioabsorbable polymer solution into the gap, and freeze-drying after freezing it can. The substrate having Valsalva sinus and a film-like blood leakage prevention layer is made of a porous foam, and is reinforced with a reinforcing material as necessary.
(2) Manufacture of valve leaflets (inner valves) A cylindrical woven fabric or knitted fabric, or a flat woven fabric or knitted fabric is wound around a Teflon test tube in a cylindrical shape, and is made into a cylindrical shape by fusing or stitching. This is put in an outer mold, a bioabsorbable polymer solution serving as a base material is poured into the gap, and after freeze-freeze, freeze-dried. The end of one side of the cylindrical base material taken out is folded so that the inside overlaps (from two directions in the case of two cusps and from three directions in the case of three cusps) to obtain a leaflet (FIG. 1).
(3) Compositing A leaflet is inserted around the Valsalva sinus of the base fabricated as described above, and the non-folded part of the leaflet and the edge of the Valsalva sinus are sutured with a bioabsorbable suture. The produced heart valve base material is sterilized with ethylene oxide gas and subjected to the following experiment.
(4) Cell culture and seeding Living cells (endothelial cells and fibroblasts, etc.) are collected from the femoral artery, mixed and cultured, then seeded on an artificial heart valve, the inner surface of the artificial heart valve, Valsalva sinus and valve leaflet Endothelial cells to cover. The inner surface of the artificial heart valve, the Valsalva sinus, and the leaflet need only be substantially covered with endothelial cells and fibroblasts, but are more preferably completely covered.
(5) Transplantation The heart valve thus produced can be used for transplantation not only for humans and adults of animals but also for infants and children.
[0014]
The base of the heart valve substrate of the present invention is preferably a foam. The pore diameter of the foam is preferably such that the cells adhere appropriately and proliferate, and at the same time blood does not leak when transplanted as a heart valve. The pore diameter is usually 1 mm or less, preferably 5 to 100 μm. The thickness of the substrate is determined from the absorption period or the ease of suturing, and is usually 5 mm or less, preferably 500 μm to 2 mm.
[0015]
A preferable material for the blood leakage prevention layer is bioabsorbable and flexible, particularly polylactic acid-caprolactone (P (CL / LA)).
[0016]
The preferable thickness of the blood leakage preventing layer is preferably a thickness that does not impair flexibility and can prevent blood leakage, and is specifically 1 μm to 5 mm, preferably 5 μm to 1 mm, more preferably Examples are 10 μm to 100 μm. As a preferred method for manufacturing the blood leakage prevention layer, it is preferable that the prevention layer is integrally formed when the base material is produced, for example, it is preferably integrated with the heart valve body at the time of completion. It is.
[0017]
Examples of the method for producing the foam include the following methods.
(1) Freeze-drying method A polymer solution used as a base material is frozen in a mold and then freeze-dried. Foams having various pore sizes can be obtained depending on the freezing temperature and the polymer concentration.
(2) Elution method A polymer solution containing a water-soluble substance as a base material is mixed. After drying, the water-soluble substance is washed away with water. A foam having a diameter corresponding to the water-soluble substance particles is obtained. In this example, sucrose can be used appropriately.
[0018]
The reinforcing material needs to be stronger than the foam as the base material. It can be selected from fibrous, non-woven, film, and the like.
[0019]
The reinforcing material is preferably integrated with the foam, and the position can be any of the inner surface, the center, and the outer surface. However, since the inner surface of the foam is involved in adhesion to vascular endothelial cells, the center or the outer surface is Although preferred, the inner surface is also possible.
[0020]
Examples of the cells to be seeded include endothelial cells, smooth muscle cells, and fibroblasts. These two or three types of mixed cultured cells can be exemplified, and tissue culture is performed using the mixed cultured cells. Preferably, mixed cultured cells containing endothelial cells and fibroblasts and further containing smooth muscle cells as optional constituents can be preferably used.
[0021]
The culture conditions and seeding method of the cells used are shown below.
A. Cell isolation, cell culture, increase in cell number Vascular tissue collected under complete cleanliness is immersed in a cell culture solution and washed with a phosphorylated saline in a clean bench. Next, the tissue is cut according to a simple explant technique using a scalpel on a Petri dish. About 1-2 mm ² large tissue pieces are evenly distributed on the dish, and after about 20 minutes, the culture solution is added after the tissue is firmly adhered to the lower surface of the dish. The culture solution was supplemented with Dulbecco's Modified Eagles Media (DMEM) with 10% fetal bovine serum and 1% antibiotic solution (L-glutamine 29.2mg / ml, penicillin G 1000u / ml, streptomycin sulfate 10,000μg / ml). Use things. The vessel wall cells begin to migrate from the tissue onto the dish after 5-7 days, and a mixed cell colony forms around the explant tissue piece after another week. Two to three weeks later, the mixed cells form a confluent state on the dish. Immediately, Passage was performed with 0.25% trypsin, and cultivation on a 75 cm ² culture flask was started. When this flask became confluent, about 2 million cells were obtained. Cell culture is performed in an environment of 5% CO ₂ and 95% O _{2 and} the culture is continued until 10 × 10 ⁶ cells are obtained. Although the culture medium is changed every 4-5 days, the doubling time of the cells is about 48 hours according to the result of the preliminary experiment. The number of cells in progress is calculated according to the classic exclusion method using trypan blue.
B. Cell isolation and endothelial cell purification When mixed cells reach confluence and a certain number of cells are obtained, according to the following procedure, endothelial cells are selected and separated from the mixed cells using FACS. Biomedical Technologies' Dil-acetylated LDL (fluorescent dye marker) (hereinafter Dil-Ac-LDL) is added to the mixed cell culture solution at a concentration of 1 μg / ml, and incubation is performed for 24 hours. This marker is taken into cells through the scavenger pathway unique to endothelial cells and macrophages. After 24 hours, tripsinize is performed to prepare a mixed cell suspension, which is sorted using a cell sorter (FACS machine: manufactured by Bectin Dickenson). Cells are sorted for Dil-Ac-LDL positive and negative based on their size and fluorescence. After separation, these are cultured separately and continued until there are 2 million endothelial cells.
C. Tissue building The first step in building tissue is in vitro cell seeding. Specifically, about 1 million cells / cm ² of Dil-Ac-LDL-negative fibroblasts are seeded on a biodegradable heart valve base material.
[0022]
Immediately after seeding the concentrated cell suspension onto the polymer, leave it in the clean bench on the culture dish for 30-60 minutes, and then add about 50 ml of the culture solution. The culture medium is basically changed every day, and 7 days later, one day before surgical transplantation, further seeding with a cell suspension of endothelial cells (about 2 million cells) is performed, and this work aims to make a single layer of endothelial cells. .
[0023]
A. above. ~ C. Exemplifies cell collection, culture, and seeding methods during heart valve production.
[0024]
[Example 1]
(1) Production of substrate having Valsalva sinus and blood leakage prevention layer Film made of lactic acid-caprolactone copolymer (molar ratio 50:50) on outer cylinder mold having Valsalva sinus 1 having a diameter of 20 mm (thickness about 150 μm) After insertion, a cylindrical non-woven fabric made of polyglycolic acid was inserted inside. After fitting the inner mold from the inside, a dioxane solution (5%) of a copolymer of lactic acid-caprolactone (molar ratio 50:50) was poured into the gap, frozen at −30 ° C. and then freeze-dried at 20 ° C. for 24 hours. . The substrate taken out after drying had a foam structure, a fibrous reinforcing material was incorporated in the core, and a film-like blood leakage prevention layer was formed on the outer surface (FIG. 2: cross-sectional photograph 1).
(2) Preparation of valve leaflets Cylindrical polyglycolic acid woven fabric was fitted to a Teflon test tube having a diameter of 18 mm. This is put into a cylindrical mold with a diameter of 20 mm, a dioxane solution (5%) of a copolymer of lactic acid-caprolactone (molar ratio 50:50) is poured into the gap, frozen at -30 ° C. and then frozen at 20 ° C. for 24 hours. Dried. The valve leaflet taken out had a foam structure and had a structure in which a fibrous reinforcing material was incorporated in the core material (FIG. 3: sectional photograph 2). In the case of producing the tricuspid valve 4 shown in FIG. 1, the end is folded inward from three sides, stitched at the center, and then heat-set at 100 ° C. for 3 hours under vacuum. The suture was cut after the heat setting was completed.
[0025]
When combining, a sheet-like base 2 may be used as shown in FIG. 6, and the Valsalva sinus 1 and the tricuspid valve 4 may be stitched together as described above to form a cylindrical base.
(3) The compound leaflet is inserted into the cylindrical base 2, the edge of the Valsalva sinus 1 is integrally sutured with a polyglycolic acid suture, and the other end is integrally sutured in a cylindrical shape to thereby connect the valve 6. The heart valve substrate 3 of the present invention having the present invention was obtained (FIGS. 4 and 5).
(4) Cell culture
A. Cell Isolation, Cell Culture, Increase in Cell Number From the 20-day-old Dover lamb, about 2 cm of the femoral artery was collected under general anesthesia while preserving the deep femoral artery. The tissue collected under complete cleanliness was immersed in a cell culture medium and washed with a phosphorylated saline in a clean bench. Next, the tissue was cut according to a simple explant technique using a scalpel on a Petri dish. About 1-2 mm ² large tissue pieces were evenly distributed on the dish, and after about 20 minutes, the culture solution was added after the tissue was firmly adhered to the lower surface of the dish. At this time, care was taken so that the tissue pieces did not peel off from the dish.
[0026]
The culture solution used was Dulbecco's Modified Eagles Media supplemented with 10% fetal bovine serum and 1% antibiotic solution (L-glutamine 29.2mg / ml, penicillin G 1000u / ml and Streptomycin sulfate 10,000μg / ml). .
[0027]
Sheep vascular wall cells began to migrate from tissue to the dish after 5-7 days, and a further week later, mixed cell colonies formed around the explant tissue pieces. After a further 2-3 weeks, the mixed cells became confluent on the dish. Immediately, Passage was performed with 0.25% trypsin, and culture on a 75 cm ² culture flask was started, but when this flask was almost confluent, about 2 million cells were obtained. Cell culture was performed in an environment of 5% CO 2 and 95% O 2 and continued until 10 × 10 ⁶ cells were obtained. The culture medium was changed every 4-5 days. The cell doublling time was about 48 hours.
B. Cell isolation and purification of endothelial cells When the mixed cells reached confluence and a certain number of cells were obtained, the endothelial cells were selected and separated from the mixed cells using FACS according to the following procedure. Biomedical Technologies' Dil-acethylated LDL (fluorescent marker) (hereinafter D-Ac-LDL) was added to the mixed cell culture at a concentration of 1 μg / ml and incubated for 24 hours. This marker is taken into cells through the scavenger pathway unique to endothelial cells and macrophages. After 24 hours, tripsinize was performed to prepare a mixed cell suspension, which was sorted using a cell sorter (FACS machine: Bectin Dickenson, Mountainview, California). Cells are sorted for Dil-Ac-LDL positive and negative based on cell size and fluorescence. Endothelial cells were positive and about 5-8% of the mixed culture was recognized. After separation, these were cultured separately and continued until there were 2 million endothelial cells. In addition, calculation of the number of cells in progress followed the classic exclusion method with trypan blue.
C. About 20 million Dil-Ac-LDL negative myofibroblasts were seeded on the inner surface of the leaflet tissue construction substrate and the leaflet substrate. Immediately after seeding the concentrated cell suspension onto the polymer, it was left in a clean bench on a culture dish for 30-60 minutes, after which approximately 50 ml of culture was added. The culture medium is basically changed daily, and 7 days later, one day before transplantation into the animals, further seeding is performed with a suspension of endothelial cells (approximately 2 million cells). As a result, a heart valve was obtained in which the inner surface of the base body of the heart valve body, Valsalva sinus and valve leaflet were covered with fibroblasts and endothelial cells.
D. Animal experiment When the heart valve prepared in C. above was replaced with the heart valve of a puppy, there was no blood leakage immediately after the replacement, and after that, good patency was obtained without the use of an anticoagulant, It was confirmed that it functions sufficiently as a heart valve.
[0028]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the artificial heart valve which replaces a mechanical valve, a heterogeneous biological valve, and a homogeneous valve can be provided. Further, since the whole is composed of a bioabsorbable polymer, it disappears after tissue regeneration and does not remain in the body as a foreign substance. Growth can be expected especially in children.
[0029]
Furthermore, blood leakage could be prevented immediately after replacement due to the presence of the blood leakage prevention layer.
[Brief description of the drawings]
FIG. 1 shows a tricuspid valve.
FIG. 2 is a drawing-substituting photograph showing a cross section of a heart valve substrate.
FIG. 3 is a drawing-substituting photograph showing a cross-section of a heart valve substrate.
FIG. 4 shows a cross-sectional view of the prosthetic heart valve of the present invention.
FIG. 5 shows a plan view of the prosthetic heart valve of the present invention.
FIG. 6 shows a view in which a tricuspid valve 4 is integrally stitched to the Valsalva sinus 1 of a sheet-like base body 2
1 Valsalva Sinus 2 Heart Valve Body 3 Heart Valve Base Material 4 Tricuspid Valve 5 Top 6 Valve 7 Blood Leakage Prevention Layer

Claims (6)

A heart valve substrate made of a bioabsorbable material having a heart valve body and a blood leakage prevention layer covering the outer surface of the body ,
The heart valve body is composed of a foam made of a polylactic acid-caprolactone copolymer, and
A heart valve base material, wherein the blood leakage prevention layer is composed of a film made of a polylactic acid-caprolactone copolymer . The heart valve base material according to claim 1, wherein the heart valve main body includes a valve leaflet inside a cylindrical base body and forms a Valsalva sinus. A method for producing a heart valve base material, characterized in that a blood leakage prevention layer is formed by sticking a film on the outer surface of a heart valve body ,
The heart valve body is composed of a foam made of a polylactic acid-caprolactone copolymer, and
A method for producing a heart valve substrate, wherein the blood leakage prevention layer is composed of a film comprising a polylactic acid-caprolactone copolymer . A method for producing a heart valve substrate according to claim 2,
A method for producing a heart valve base material, comprising a step of forming a Valsalva sinus on a cylindrical base body provided with a film-like blood leakage prevention layer on an outer surface and a step of forming a valve leaflet inside the base body. A method for producing a cell-containing heart valve , which comprises seeding endothelial cells and fibroblasts on the artificial heart valve substrate according to claim 1 or 2 simultaneously or separately in vitro to form a heart valve tissue. The heart valve which covers the inner surface of the heart valve main body of the heart valve base material of Claim 2 , Valsalva sinus, and a leaflet with the biological cell layer containing a fibroblast and an endothelial cell.

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