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WO2002045767A1 - Substrat de regeneration de tissus, materiel de transplantation, et procedes de production de ces elements - Google Patents

Substrat de regeneration de tissus, materiel de transplantation, et procedes de production de ces elements Download PDF

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Publication number
WO2002045767A1
WO2002045767A1 PCT/JP2001/010751 JP0110751W WO0245767A1 WO 2002045767 A1 WO2002045767 A1 WO 2002045767A1 JP 0110751 W JP0110751 W JP 0110751W WO 0245767 A1 WO0245767 A1 WO 0245767A1
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WO
WIPO (PCT)
Prior art keywords
hyaluronic acid
tissue regeneration
sponge
substrate
producing
Prior art date
Application number
PCT/JP2001/010751
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English (en)
Japanese (ja)
Inventor
Yoshimitsu Kuroyanagi
Original Assignee
Japan Tissue Engineering Co.,Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Tissue Engineering Co.,Ltd filed Critical Japan Tissue Engineering Co.,Ltd
Priority to AU2002221089A priority Critical patent/AU2002221089A1/en
Priority to JP2002547548A priority patent/JP4273450B2/ja
Priority to KR10-2003-7005483A priority patent/KR20030061378A/ko
Publication of WO2002045767A1 publication Critical patent/WO2002045767A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides

Definitions

  • the present invention relates to a tissue regeneration base material, a transplant material, and a method for producing the same, which can be widely used in the medical field.
  • transplantation materials are obtained by holding and culturing various cells on a tissue regeneration substrate formed of various materials having high biocompatibility.
  • collagen sponge can be used as a substrate for tissue regeneration in cultured skin into which fibroblasts or the like are incorporated (Japanese Patent Laid-Open No. 4-33255). It has also been suggested that hyaluronic acid can be used (Japanese Unexamined Patent Publication No. 11-316906)
  • Hyaluronic acid is a type of mucopolysaccharide having a repeating disaccharide structure, and is a substance mainly present in the joint fluid of animals, the vitreous eye, connective tissues such as the umbilical cord and the dermis.
  • the molecular weight is on the order of hundreds of thousands to millions, and has a property of binding to a very large amount of water, which is related to, for example, low friction of joints and water retention of skin dermal tissue.
  • production of hyaluronic acid is momentarily activated, facilitating cell migration in tissue remodeling. From these facts, it can be said that hyaluronic acid exhibits excellent ability as a wound dressing.
  • hyaluronic acid exhibits excellent performance as a wound dressing, it has very high water content, and therefore has low cell adhesion and is in vitro (in vitro) for use as a tissue regeneration substrate.
  • cell culture was difficult.
  • An object of the present invention is to solve the above-mentioned problems, and it is a substrate for tissue regeneration mainly composed of hyaluronic acid, which is suitable for cell culture in vitro and tissue regeneration after transplantation. The purpose is to provide what you have. It is another object to provide a method for producing the tissue regeneration substrate. It is another object of the present invention to provide a transplant material using the tissue regeneration substrate and a method for producing the same. Disclosure of the invention
  • a first aspect of the present invention is to provide a hyaluronic acid sponge mainly composed of hyaluronic acid and / or a derivative thereof, and a cell adhesion portion in which a sponge made of a biological material derived from a living body is laminated on at least one surface of the hyaluronic acid sponge. It is featured. Since the cell adhesion portion of the tissue regeneration substrate is formed by laminating sponges made of a biological material derived from a living body, cells incorporated in the tissue regeneration substrate adhere well. In addition, since it has a hyaluronic acid sponge mainly composed of hyaluronic acid and / or a derivative thereof, it has excellent wound healing ability such as promotion of cell proliferation.
  • the substrate for tissue regeneration cell culture in vitro and tissue regeneration after transplantation can be favorably performed.
  • the conventional collagen sponge shows large shrinkage
  • the substrate for tissue regeneration of the present invention hardly shows such shrinkage.
  • the substrate may be used as a material for transplantation by incorporating cells, but may also be used as a wound dressing for covering a wound surface.
  • examples of the derivatives of hyaluronic acid include hyaluronic acid metal salts such as sodium hyaluronate and potassium hyaluronate, as well as etherification and esterification of hydroxyl groups and carboxyl groups of hyaluronic acid. Examples include amidated, acetalized, and ketalized ones, and among them, sodium hyaluronate is preferable.
  • hyaluronic acid sponge an intermolecularly crosslinked one (crosslinked hyaluronic acid sponge) is preferable.
  • examples of the polymer material derived from a living body include collagen, gelatin, fibrin, and alginic acid. Among these, collagen, particularly atelocollagen having a low antigenicity, is preferable, and collagen and gelatin are intermolecularly crosslinked. Is preferred.
  • the tissue regeneration base material is in a state in which a bio-derived polymer material enters the hyaluronic acid sponge near the boundary between the hyaluronic acid sponge and the cell adhesion part. In this case, even if there is a difference in the swelling ratio between the hyaluronic acid sponge and the cell adhesion part, there is no risk that the two will be peeled off when wet.
  • the hyaluronic acid sponge is supported by a woven fabric, a nonwoven fabric, or a knitted fabric as a support.
  • the strength of the tissue regeneration base material is increased by the support, and there is no possibility that the tissue regeneration base material will be damaged when picked up with tweezers and lifted, and handling will be improved.
  • the material of the support is not particularly limited as long as it plays a role of reinforcing the hyaluronic acid sponge.
  • synthetic polymer materials such as nylon, polyester, silicone, silk, cotton, hemp, etc. Natural polymer materials and the like.
  • a second aspect of the present invention is a method for producing such a tissue regeneration base material, wherein ( ⁇ ) Condensing an aqueous solution of hyaluronic acid and / or a derivative thereof to which a cross-linking agent has been added to obtain an intermolecular cross-linked product of rihyalonic acid and / or a derivative thereof; a cross-linking step; (2) vacuuming the inter-molecular cross-linked product; A sponge step of obtaining a hyaluronic acid sponge by freeze-drying; and (3) the cell adhesion by vacuum freeze-drying after absorbing an aqueous solution of a biological material derived from a living body from at least one surface of the hyaluronic acid sponge.
  • a laminating step for forming a portion.
  • an intermolecular cross-linked product of hyaluronic acid and / or a derivative thereof is freeze-dried in vacuum to form a hyaluronic acid sponge, and after absorbing an aqueous solution of a polymer material derived from a living body from at least one surface thereof, the vacuum is applied again. Freeze dry.
  • the first tissue regeneration substrate of the present invention can be produced relatively easily.
  • examples of the cross-linking agent include a water-soluble epoxy compound and daltaraldehyde, and among them, a water-soluble epoxy compound is preferable.
  • examples of the water-soluble epoxy compound include ethylene glycol diglycidyl ether, glycerol diglycidyl ether, and glycerol triglycidyl ether.
  • the amount of the water-soluble epoxy compound to be used is preferably approximately 1 Z 2 to 10 in terms of a weight ratio with respect to hyaluronic acid and Z or a derivative thereof. / 10 is preferred.
  • an aqueous solution of hyaluronic acid and / or its derivative is used, and the concentration of hyaluronic acid and / or its derivative in this aqueous solution also depends on the type and molecular weight of the hyaluronic acid and / or its derivative used. However, it is approximately 0.5 to 1.5% by weight.
  • the water used as the solvent ion-exchanged water having a pH of 5 to 6 is preferable.
  • the aqueous solution of hyaluronic acid and / or a derivative thereof to which a crosslinking agent has been added is concentrated to form hyaluronic acid and / or a derivative thereof.
  • An intermolecular cross-linking reaction of the derivative is carried out, but is preferably carried out by heating.
  • the concentration temperature is preferably about 30 to 60 ° C, more preferably about 40 to 60 ° C, and particularly about 50 ° C. Preferred. If the mixture is heated at a high temperature exceeding 60 ° C, bubbles may be generated in the mixed solution, and the sponge structure of the obtained hyaluronic acid sponge may not be uniform enough.
  • the intermolecular crosslinking reaction of hyaluronic acid and / or a derivative thereof is preferably performed in a neutral region (pH 5 to 8) or an acidic region (pH 3 to 5). Further, it is preferable to perform the treatment in an acidic region since the concentration time tends to be shorter than that in the case of performing the treatment in a neutral region.
  • the concentration is terminated when the concentration of the aqueous solution of hyaluronic acid and Z or a derivative thereof reaches 1 to 10% by weight, preferably 2 to 5% by weight, and particularly preferably about 5% by weight. Is preferred. If the concentration is not performed sufficiently, it is not preferable because the swelling property of the hyaluronic acid sponge obtained after the next sponging step cannot be sufficiently suppressed. That is, if the hyaluronic acid sponge has a high swelling property, it will swell more than necessary when it is immersed in a liquid medium during cell culture, for example. Becomes difficult.
  • the end point of concentration is controlled as described above, so that the swelling property of the hyaluronic acid sponge is sufficiently suppressed, so that it does not swell unnecessarily when immersed in a liquid medium, etc. Handling becomes easy because it does not become too large or too large. If the cross-linking step is performed until the concentrate becomes completely film-shaped, even if the sponge step by vacuum freeze-drying is performed, a sponge of hyaluronic acid will not be formed. It is necessary to set the concentration end point You.
  • the intermolecular cross-linked product after the cross-linking step is freeze-dried in a vacuum to obtain a hyaluronic acid sponge.
  • the temperature conditions during the freeze-drying of the vacuum are such that large ice crystals are not formed.
  • the intermolecular cross-linked product obtained without sufficient concentration in the cross-linking step is subjected to at least one freeze-thawing operation, followed by freeze-drying in a vacuum to obtain hyaluronic acid. It is preferable to obtain a sponge. If the operation of freezing and thawing before vacuum freeze-drying is not performed, the sponge may become difficult to retain its shape when wet, whereas the operation of freezing and thawing before vacuum freeze-drying is performed Although this is probably due to the promotion of intermolecular interactions such as hydrogen bonding, a sponge that easily retains its shape when hydrated is obtained.
  • the operation of freezing and thawing before vacuum freeze-drying may be performed multiple times, but is preferably performed once in consideration of simplification of the manufacturing process.
  • the freezing temperature conditions in the operation of freezing and thawing before vacuum freeze-drying are about 85 ° C to 130 ° C, preferably about 85 ° C to 50 ° C, and more preferably about 50 ° C.
  • the thawing temperature is about 20 ° C. to 30 ° C., preferably about 30 ° C.
  • the temperature may be divided into multiple stages and heated for thawing. In this case, care should be taken not to break the intermolecular crosslinked product.
  • the cross-linked product obtained by performing sufficient concentration under appropriate concentration conditions in the cross-linking step has sufficient strength, so the freeze-thaw operation before vacuum freeze-drying is always performed No need.
  • a water washing step for washing the hyaluronic acid sponge produced in the sponge forming step before proceeding to the next laminating step.
  • an inactivating agent for inactivating the unreacted crosslinking agent may be added to the washing water.
  • a deactivator that has a function of opening the epoxy group and does not harm the living body is preferable. It is.
  • a cell adhesion part is formed by absorbing an aqueous solution of a biologically-derived polymer material from at least one surface of the hyaluronic acid sponge and then freeze-drying the same, but as a biologically-derived polymer material.
  • a biologically-derived polymer material As described above, collagen, gelatin, fibrin, alginic acid, and the like can be mentioned. Among them, collagen, particularly atelocollagen with low antigenicity, is preferred.
  • the aqueous solution of a polymer material derived from a living body may be an aqueous solution of 0.2 to 1.0% by weight, preferably an aqueous solution of 0.2 to 0.5% by weight, more preferably 0.5% by weight.
  • an aqueous solution of about the same degree may be used, and the weight ratio of the polymer material derived from the living body to the hyaluronic acid is about 1: 2 to 10, preferably 1: 2 to 4, and particularly preferably about 1: 4. I just need.
  • the conditions for freeze-drying in vacuum are the same as those for freeze-drying in the sponge forming step.
  • the hyaluronic acid sponge of the obtained tissue regeneration base material and the cell adhesion portion are absorbed. In the vicinity of the boundary, a biologically-derived polymer material has entered a hyaluronic acid sponge. For this reason, even if there is a difference in the swelling ratio between the hyaluronic acid sponge and the cell adhesion portion, there is no risk that the two will peel off when the tissue regeneration substrate is impregnated with water.
  • the cell adhesion portion may be formed by absorbing an aqueous solution of a polymer material derived from a living body from one side of the perforated hole, followed by freeze-drying in a vacuum.
  • the aqueous solution of the polymer material derived from a living body not only permeates the pores of the hyaluronic acid sponge but also enters a plurality of holes, so that an effect of anchoring with the hyaluronic acid sponge can be obtained.
  • hole J here refers to not only general holes such as round holes and square holes, but also holes such as cuts and irregularities that have a larger contact area than a flat surface. included.
  • the collagen or gelatin is partially intermolecularly cross-linked by irradiating an ultraviolet lamp after the laminating step, so that the collagen or gelatin can be mixed when water is contained. It is preferable to prevent the gelatin from flowing out, and it is further preferable that the gelatin be subsequently sterilized with an ethylene oxide gas or the like.
  • an inactivating agent to the aqueous solution of the polymer material derived from a living body in order to inactivate the unreacted crosslinking agent.
  • the deactivator for example, when a water-soluble epoxy compound is used as a cross-linking agent, a deactivator that has a function of opening an epoxy group such as glycine and does not harm the living body is used. preferable.
  • the following sponge forming / laminating step may be adopted. That is, a step of forming a cell adhesion portion together with the hyaluronic acid sponge by bringing the intermolecularly crosslinked product after the crosslinking step into contact with an aqueous solution of a polymer material derived from a living body and then freeze-drying the same may be employed. . In this case, vacuum freeze-drying only needs to be performed once. Can simplify the manufacturing process.
  • an aqueous solution of a bio-derived polymer material is brought into contact with the intermolecular cross-linked product before sponging, so that the aqueous solution of the bio-derived polymer material permeates into the inter-molecular cross-linked product before sponging.
  • the adhesiveness between the hyaluronic acid sponge and the cell adhesion part in the finally obtained tissue regeneration substrate may not be sufficient. Therefore, a plurality of holes are provided in the vertical direction of the intermolecular bridge of hyaluronic acid and / or its derivative to increase the contact area with the aqueous solution of the biological material derived from a living body to obtain the effect of anchoring. Is preferred.
  • hole refers to a general hole such as a round hole or square hole, or a hole whose contact area is larger than a flat surface such as a cut or unevenness. Is also included.
  • a support is spread on the bottom surface of the container in advance, and an aqueous solution of hyaluronic acid and / or a derivative thereof to which a crosslinking agent has been added is put into the container. Thereafter, the aqueous solution is heated and concentrated to produce an intermolecularly crosslinked product of rihyaluronic acid and / or a derivative thereof.
  • a third aspect of the present invention is a transplantation material, comprising the above-described tissue regeneration base material and cells held in a cell adhesion portion of the tissue regeneration base material.
  • This transplant material allows cells to be cultured easily in vitro because the cells are held in close contact with the cell adhesion area, and the tissue healing ability is enhanced by promoting hyaluronic acid cell migration and moisturizing effect. Is played properly.
  • any cells may be retained in the cell adhesion region, such as fibroblasts, keratinocytes, pigment-producing cells, vascular endothelial cells, endothelial cells, epithelial cells, chondrocytes, and bone. Examples include blast cells, myoblasts, fat cells, hepatocytes, nerve cells, cardiomyocytes, islet cells of Langerhans and the like.
  • a fourth aspect of the present invention is a method for producing such a transplant material, which comprises the above-described tissue regeneration method. After preparing the substrate, cells are incorporated into the cell adhesion portion of the tissue regeneration substrate. According to this production method, the third transplant material of the present invention can be produced relatively easily.
  • a method of incorporating cells into the cell adhesion portion for example, there is a method of immersing a substrate for tissue regeneration in a culture solution and retaining the cells by taking the cells into pores.
  • FIG. 1 is an explanatory diagram showing a procedure for producing a tissue regeneration substrate of Example 1
  • FIG. 2 is an explanatory diagram showing a procedure for producing a tissue regeneration substrate of Example 2
  • FIG. 3 is a tissue of Example 3.
  • FIG. 4 is an explanatory view showing a procedure for producing a substrate for regeneration
  • FIG. 4 is an explanatory view showing a procedure for producing a substrate for tissue regeneration in Example 4.
  • the mixed solution of hyaluronic acid-EX 3 13 with the non-woven fabric mounted thereon was placed in an air circulation type dry heater and allowed to stand at 50 ° C. for 10 hours. This allows Hyaluron
  • the hydroxyl group (hydryl xyl group) or carboxyl group of the acid reacts with the epoxy group of Denacol EX 3 13 to form an intermolecular cross-linked product of hyaluronic acid, and the liquid volume becomes about 2/5 (that is, the hyaluronic acid concentration). To about 2.5% by weight). At this time, the depth of the concentrate was about 4 mm.
  • this concentrate was frozen at 185 ° C.
  • the freezing time depends on the capacity of the freezer, but is about 5 to 6 hours, and it was completely frozen. After this freezing, it was left at room temperature for 1 to 1.5 hours to thaw it once, and after thawing, it was completely frozen again at -85 ° C for about 5 to 6 hours.
  • 3 0 X 1 0 - 3 by performing the vacuum freeze dried at ⁇ 5 0 X 1 0- 3 mm bar (3 ⁇ 5 P a), hyaluronic acid intermolecular cross-linked product having a sponge structure (hereinafter, Crosslinked hyaluronic acid sponge) was obtained.
  • atelocollagen 1 g was dissolved in 500 ml of distilled water and adjusted to pH 4 with 1 NHCI to prepare an aqueous 0.2% atelocollagen solution.
  • the prepared aqueous atelocollagen solution was injected into a tray with a bottom area of 180 cm 2 at 90 ml.
  • the aqueous solution of atelocollagen is half the volume of the original aqueous solution of hyaluronic acid (180 ml), and the weight ratio of collagen to hyaluronic acid is 1:10.
  • the crosslinked hyaluronic acid sponge prepared as described above was immersed in a tray containing an aqueous solution of atellogen collagen so that the benlyse was positioned above, and allowed to stand for 1 hour to allow the aqueous solution of collagen to soak into the crosslinked hyaluronic acid sponge. Then, the hyaluronic acid sponge immersed in the collagen aqueous solution was frozen at 185 ° C. and dried in vacuum.
  • a collagen sponge is laminated on a crosslinked hyaluronic acid sponge after vacuum freeze-drying, and a 15 W ultraviolet lamp (2 (54 nm) for 30 minutes from a distance of 25 cm to carry out intermolecular cross-linking of collagen. Then, they were packed in a sterilization bag and sterilized by EOG (ethylene oxide gas) at 60 ° C for 20 hours.
  • EOG ethylene oxide gas
  • DMEM + 10% FBS, Gibco Dulbecco's Modified Eagle Minimum Essential Medium
  • FBS fetal calf serum
  • the cultured dermis (material for transplantation) stored in the freezer was thawed at the time of use to remove the cryopreservation solution, washed twice with 30 ml of Hanks' solution, and used for animal experiments. ⁇ A 7 cm diameter circle was drawn on the back of the heron, and the skin on all layers was excised to create a skin defect wound.
  • the above cultured dermis is applied to this skin defect wound, the periphery of the wound surface is sutured, a wound dressing made of polyurethane film is applied thereon, and a sterilizing pad is further placed thereon, and the periphery of the wound is sutured and stretched. It was compressed and fixed with a sexual bandage. As a result, good granulation tissue formation and remarkable reduction in wound area were observed. (Example 2)
  • Example 2 In the same manner as in Example 1, a mixture of hyaluronic acid and EX 3 13 was prepared, 180 ml of the obtained hyaluronic acid-EX 3 13 mixture was poured into a tray having a bottom area of 180 cm 2 (10 cm ⁇ 18 cm). The tray was pre-mounted with a bemliese (see above) cut to fit its size, and a small amount of distilled water was added to make it wet and adhered to the bottom of the tray. The depth of the mixed solution of hyaluronic acid and EX3 13 injected into the tray was about 1 cm.
  • the mixed solution of hyaluronic acid and EX313 in this state was put into an air circulation type dry heater and allowed to stand at 50 ° C for 15 hours.
  • the hydroxyl group (hydroxyl xyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX 313 to form an intermolecular cross-linked product of hyaluronic acid, and the liquid volume is reduced to about ⁇ / It was concentrated to 5 (that is, about 5% by weight as hyaluronic acid concentration). At this time, the depth of the concentrate was approximately 2-3 mm. Next, the concentrate was frozen at -85 ° C.
  • the freezing time depends on the capacity of the freezer, but is about 5 to 6 hours, and it was completely frozen. Then, 3 0
  • hyaluronic acid intermolecular cross-linked product having a sponge structure hereinafter , Crosslinked hyaluronic acid sponge.
  • the cross-linked hyaluronic acid sponge was immersed in a container containing 15 L of ion-exchanged water for one day, and washed with water.
  • atelocollagen 2.5 g was dissolved in 500 ml of distilled water and adjusted to pH 3.2 with 1N HCl to prepare a 0.5% atelocollagen aqueous solution.
  • the washed cross-linked hyaluronic acid sponge was placed on a tray having a bottom area of 180 cm 2 with the nonwoven fabric facing down, and 90 ml of the prepared aqueous atelocollagen solution was poured thereon.
  • the atelocollagen aqueous solution is half the volume of the original hyaluronic acid aqueous solution (180 ml).
  • the weight ratio of lonic acid is 1: 4.
  • the periphery of the cross-linked hyaluronic acid sponge was slightly shrunk, and a gap was formed between the sponge and the side surface of the tray.
  • the infiltration of the aqueous atelocollagen solution into these gaps arranged the collagen so as to wrap around the hyaluronic acid sponge, thereby suppressing the separation of the crosslinked hyaluronic acid sponge and collagen sponge during swelling.
  • the hyaluronic acid sponge containing the aqueous atelocollagen solution was freeze-dried in vacuum at 185 ° C.
  • the collagen sponge is layered on the crosslinked hyaluronic acid sponge after vacuum freeze-drying, and irradiated on the collagen sponge side from a distance of 25 cm for 30 minutes using a 15 W ultraviolet lamp (254 nm).
  • a 15 W ultraviolet lamp (254 nm) As a result, intermolecular crosslinking of collagen was performed.
  • a tissue regeneration substrate that is, a crosslinked hyaluronic acid sponge provided with a cross-linked collagen sponge (cell adhesion portion) and supported by benlyse (support) was obtained.
  • the 180 cm 2 tissue regeneration substrate produced by the above method was cut to a size of 90 cm 2 .
  • the cut substrate for tissue regeneration was placed in a tray of 18 O cm 2 , and DMEM + 10% FBS was injected at 100 ml to adjust the pH.
  • the culture solution is once discarded, and 5 ml of the human fibroblast suspension is used as a tissue regeneration substrate at a density of 5 ⁇ 10 5 cells / m 2 . Seeded. After the seeding, the cells were allowed to stand at 37 ° C, and the human fibroblasts were fixed on a substrate for tissue regeneration.
  • the cultured dermis (material for transplantation) prepared in this way has not only a sufficient strength but also a contraction of only 2 to 3 mm around the perimeter. It was good and there was no risk of loss.
  • the resulting hyaluronic acid - base area the EX 8 1 0 mixture was 5 0 g injected into Bok Leh 1 1 0 cm 2 (1 0 cm X 1 1 cm).
  • the tray was pre-loaded with a bemliese (see above) cut to fit its size, and a small amount of distilled water was added to make it wet and adhered to the bottom of the tray.
  • the depth of the mixture of hyaluronic acid and EX810 injected into the tray was about 5 mm.
  • the mixed solution of hyaluronic acid-EX810 in this state was put into an air circulation heater, and allowed to stand at 50 ° C for 5 hours.
  • the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX810 to form an intermolecular cross-linked product of hyaluronic acid, and the liquid volume is reduced to about 1/2 (that is, (Hyaluronic acid concentration of about 2% by weight).
  • the depth of the concentrate at this time was about 2-3 mm, Next, the concentrate was frozen at -85 ° C. The freezing time depends on the capacity of the freezer, but is about 5 to 6 hours, and it was completely frozen.
  • a crosslinked hyaluronic acid sponge Prior to lamination with a collagen sponge, a crosslinked hyaluronic acid sponge was perforated using Kenzan. The hole spacing at this time was approximately 4 mm.
  • atelocollagen 8 g was dissolved in 1.6 L of distilled water and adjusted to pH 3.5 with 1N HCl to prepare a 0.5% atelocollagen aqueous solution.
  • 40 g of the prepared aqueous atelocollagen solution was poured into a tray having a bottom area of 110 cm 2 .
  • the aqueous atelocollagen solution is about 4/5 of the original hyaluronic acid aqueous solution (50 g), and the weight ratio of collagen to hyaluronic acid is 2: 5.
  • the crosslinked hyaluronic acid sponge prepared as described above was floated so that the benlyse was positioned above the tray into which the aqueous solution of atellogen was injected, and left standing for 1 minute to allow the aqueous solution of collagen to soak into the crosslinked hyaluronic acid sponge. . Then, the hyaluronic acid sponge impregnated with the collagen aqueous solution was completely frozen at -85 ° C, and then freeze-dried in vacuum.
  • the substrate for tissue regeneration prepared by the above method was placed in a tray, and DMEM + 10% FBS was injected at 50 ml to adjust the pH. After adjusting the cultivation 1-1 ( ⁇ 1 7.4), the culture solution was discarded once, and 5 ml of the human fibroblast suspension was used as a tissue regeneration substrate at 1 ⁇ 10 5 cells / cm 2. Seeded at a density of After seeding, the cells were allowed to stand at 37 ° C., and the human fibroblasts were fixed on a substrate for tissue regeneration. Thereafter, the DMEM + 1 0% FBS medium 5 0 m I were added thereto to carry out a 1-week incubation at 3 7 ° C, 5% C 0 2 below.
  • the medium was removed, and this was placed in a cryopreservation solution (DMEM containing 10% DMSO + 20% FBS) and placed in a freezer at 115 ° C. saved.
  • DMEM containing 10% DMSO + 20% FBS
  • the cultured dermis (transplant material) stored in the freezer was thawed at the time of use to remove the frozen fiber i preservation solution, washed three times with 30 to 50 ml of Ringer's lactate solution, and applied to humans.
  • the wound surface was disinfected after removing the remaining necrotic tissue, and was thoroughly washed with physiological saline.
  • the cultured dermis was applied to the skin defect wound, and the periphery of the cultured dermis was fixed by suture.
  • a gauze impregnated with an ointment containing an antibiotic was applied thereon, and sterile gauze was placed on the gauze.
  • the area around the wound was sutured, and the wound was compressed and fixed with an elastic bandage. As a result, good granulation tissue formation, a remarkable decrease in wound area, and epithelialization around the wound were observed. (Example 4)
  • the mixed solution of hyaluronic acid and EX810 in this state was put into an air circulation heater, and allowed to stand at 50 ° C for 5 hours.
  • the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX810 to form an intermolecular cross-linked product of hyaluronic acid, and the liquid volume is reduced to about 1/2 (that is, (Hyaluronic acid concentration of about 2% by weight).
  • the depth of the concentrate at this time was about 2 to 3 mm.
  • the concentrate was frozen at 185 ° C. The freezing time depends on the capacity of the freezer, but is about 5 to 6 hours, and it was completely frozen.
  • a crosslinked hyaluronic acid sponge Prior to lamination with a collagen sponge, a crosslinked hyaluronic acid sponge was perforated using Kenzan. The hole spacing at this time was approximately 4 mm.
  • a 0.5% collagen aqueous solution (K0KENCELLGENI-PC: manufactured by Koken Co., Ltd.) was injected into a ⁇ 35 mm dish (3.8 g).
  • the aqueous collagen solution is about 45 liquid volumes of the original aqueous hyaluronic acid solution (4.8 g), and the weight ratio of collagen to hyaluronic acid is approximately 2: 5.
  • the collagen sponge was laminated on the cross-linked hyaluronic acid sponge after vacuum freeze-drying, and both sides were irradiated for 30 minutes from a distance of 20 cm using a 15 W ultraviolet lamp (254 nm) on both sides, respectively. Intermolecular cross-linking of collagen was performed. Then, they were packed in a sterilization bag, and sterilized at £ 00 for 20 hours at 60 °.
  • a crosslinked hyaluronic acid sponge provided with a tissue regeneration substrate, that is, a crosslinked collagen sponge (cell adhesion portion) was obtained.
  • the substrate for tissue regeneration was not added with strength by Bemliese, but could be easily handled by tweezers or the like.
  • the substrate for tissue regeneration according to the present example can be used as a cultured dermis as well as a wound dressing material as in Examples 1 to 3.
  • a wound dressing material As in Examples 1 to 3.
  • it is bioabsorbable and therefore does not need to be removed.By applying the collagen side to the wound surface, the cell retention effect of ricollagen can be obtained. Later, it is expected that hyaluronic acid will elute to the wound surface, exhibit cell migration, and obtain a healing promoting effect.
  • Example 2 after the intermolecular cross-linking reaction of the hyaluronic acid-EX3 13 mixture, holes were formed in the vertical direction of the cross-linked product without vacuum freeze-drying, and the surface on which the holes were formed Hyaluronic acid sponge can be formed at the same time as collagen sponge by freeze-drying in vacuum after contacting with water and atelocollagen aqueous solution.
  • the manufacturing process is simplified, and the two sponges are well adhered to each other because the effect of the anchoring is obtained by the presence of the holes.
  • the present invention is a substrate for tissue regeneration mainly composed of hyaluronic acid, and is suitable for cell culture in vitro and tissue regeneration after transplantation, and thus can be widely used in the medical field.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Composite Materials (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne une solution aqueuse d'acide hyaluronique contenant un composé époxyde soluble dans l'eau en tant qu'agent de réticulation. Cette solution est chauffée et concentrée pour donner un acide hyaluronique réticulé par pontage intermoléculaire. Le composé réticulé est ensuite lyophilisé sous vide pour produire une éponge d'acide hyaluronique. L'éponge est imprégnée d'une solution athérocollagène, puis lyophilisée sous vide pour donner un substrat servant à la régénération de tissus.
PCT/JP2001/010751 2000-12-07 2001-12-07 Substrat de regeneration de tissus, materiel de transplantation, et procedes de production de ces elements WO2002045767A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2002221089A AU2002221089A1 (en) 2000-12-07 2001-12-07 Substrate for tissue regeneration, material for transplantation, and processes for producing these
JP2002547548A JP4273450B2 (ja) 2000-12-07 2001-12-07 組織再生用基材、移植用材料及びそれらの製法
KR10-2003-7005483A KR20030061378A (ko) 2000-12-07 2001-12-07 조직 재생용 기초재, 이식용 재료 및 이들 제조방법

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JP2000-373116 2000-12-07
JP2000373116 2000-12-07

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WO2002045767A1 true WO2002045767A1 (fr) 2002-06-13

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Cited By (11)

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JPS6358531A (ja) * 1986-08-29 1988-03-14 Hitachi Ltd 表示位置検出装置
WO2004103427A1 (fr) * 2003-05-23 2004-12-02 M D Bio Inc Materiau de remplissage et procede d'utilisation
JP2006514018A (ja) * 2002-06-27 2006-04-27 ベレッタ ロベルト 液体成分を分離する方法及び装置
EP2606828A1 (fr) * 2011-12-20 2013-06-26 Angioclinic AG Acide hyaluronique et son utilisation pour traiter les insuffisances veineuses et veines variqueuses
CN103360633A (zh) * 2012-03-26 2013-10-23 株式会社杰内沃 包含生物相容性聚合物的植入材料
US9592125B2 (en) 2006-12-22 2017-03-14 Laboratoire Medidom S.A. In situ system for intra-articular chondral and osseous tissue repair
JP2017508040A (ja) * 2014-02-19 2017-03-23 メドスキン ソリューションズ ドクター スベラック アクチェンゲゼルシャフト 限定された（ｄｅｆｉｎｅｄ）平均分子量を持つ生体高分子の生産方法
KR20200035736A (ko) * 2018-09-27 2020-04-06 한국화학연구원 다공성 지지체를 이용하여 제조된 심근 유사 구조체 및 이를 이용한 약물 독성 평가 방법
JP2021518136A (ja) * 2018-03-22 2021-08-02 クイーン メアリー ユニバーシティ オブ ロンドン 疾患を治療するためのインプラント型セルドレッシング
CN114681654A (zh) * 2020-12-31 2022-07-01 广州迈普再生医学科技股份有限公司 具有创面修复功能的可吸收海绵敷料及其制备方法
CN115671365A (zh) * 2022-11-04 2023-02-03 浙江诸暨聚源生物技术有限公司 交联重组胶原蛋白海绵及其制备方法和应用

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CN101724164B (zh) * 2008-10-31 2011-12-14 科妍生物科技股份有限公司 交联透明质酸的制造方法
CN101912633A (zh) * 2010-08-03 2010-12-15 孙伟庆 一种透明质酸海绵及其制备方法
CN102558600A (zh) * 2011-12-01 2012-07-11 上海白衣缘生物工程有限公司 交联透明质酸海绵及其制备方法
CN106110367A (zh) * 2016-07-29 2016-11-16 北京化工大学常州先进材料研究院 基于天然高分子多层复合医用敷料及其制备方法
CN109998775A (zh) * 2019-03-21 2019-07-12 中国人民解放军军事科学院军事医学研究院 一种压缩止血敷料及其制备方法与应用

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EP0403650A1 (fr) * 1988-03-09 1990-12-27 Terumo Kabushiki Kaisha Substance medicale dans laquelle les cellules peuvent penetrer et peau artificielle
JPH04332561A (ja) * 1991-05-09 1992-11-19 Koken Co Ltd 培養皮膚用マトリックス
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JPH06292716A (ja) * 1993-04-09 1994-10-21 Shimizu Yoshihiko 医用材料
JPH11319066A (ja) * 1998-05-11 1999-11-24 Mitsubishi Chemical Corp 創傷被覆材
JPH11322807A (ja) * 1998-05-11 1999-11-26 Mitsubishi Chemical Corp 架橋ヒアルロン酸スポンジの製造方法
EP1022031A1 (fr) * 1999-01-21 2000-07-26 Nissho Corporation Membrane suturable pour la prévention d'adhérences
JP2000237294A (ja) * 1999-02-18 2000-09-05 Denki Kagaku Kogyo Kk ヒアルロン酸ゲルを含有する医用材料
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EP0403650A1 (fr) * 1988-03-09 1990-12-27 Terumo Kabushiki Kaisha Substance medicale dans laquelle les cellules peuvent penetrer et peau artificielle
JPH04332561A (ja) * 1991-05-09 1992-11-19 Koken Co Ltd 培養皮膚用マトリックス
WO1994017840A1 (fr) * 1993-02-04 1994-08-18 Fidia Advanced Biopolymers S.R.L. Compositions pharmaceutiques comprenant une substance spongieuse composee de derives ester d'acide hyaluronique en combinaison avec d'autres substances pharmacologiquement actives
JPH06292716A (ja) * 1993-04-09 1994-10-21 Shimizu Yoshihiko 医用材料
JPH11319066A (ja) * 1998-05-11 1999-11-24 Mitsubishi Chemical Corp 創傷被覆材
JPH11322807A (ja) * 1998-05-11 1999-11-26 Mitsubishi Chemical Corp 架橋ヒアルロン酸スポンジの製造方法
EP1022031A1 (fr) * 1999-01-21 2000-07-26 Nissho Corporation Membrane suturable pour la prévention d'adhérences
JP2000237294A (ja) * 1999-02-18 2000-09-05 Denki Kagaku Kogyo Kk ヒアルロン酸ゲルを含有する医用材料
JP2001212224A (ja) * 2000-02-04 2001-08-07 Toyobo Co Ltd 創傷被覆材

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6358531A (ja) * 1986-08-29 1988-03-14 Hitachi Ltd 表示位置検出装置
JP2006514018A (ja) * 2002-06-27 2006-04-27 ベレッタ ロベルト 液体成分を分離する方法及び装置
JP2012006937A (ja) * 2002-06-27 2012-01-12 Roberto Beretta 液体成分を分離する方法及び装置
JP4875299B2 (ja) * 2002-06-27 2012-02-15 ベレッタ ロベルト 液体成分を分離する方法及び装置
WO2004103427A1 (fr) * 2003-05-23 2004-12-02 M D Bio Inc Materiau de remplissage et procede d'utilisation
WO2004104187A1 (fr) * 2003-05-23 2004-12-02 M D Bio Inc Fibroblaste et sa methode de culture
US9592125B2 (en) 2006-12-22 2017-03-14 Laboratoire Medidom S.A. In situ system for intra-articular chondral and osseous tissue repair
EP2606828A1 (fr) * 2011-12-20 2013-06-26 Angioclinic AG Acide hyaluronique et son utilisation pour traiter les insuffisances veineuses et veines variqueuses
CN103360633A (zh) * 2012-03-26 2013-10-23 株式会社杰内沃 包含生物相容性聚合物的植入材料
JP2017508040A (ja) * 2014-02-19 2017-03-23 メドスキン ソリューションズ ドクター スベラック アクチェンゲゼルシャフト 限定された（ｄｅｆｉｎｅｄ）平均分子量を持つ生体高分子の生産方法
JP2018076340A (ja) * 2014-02-19 2018-05-17 メドスキン ソリューションズ ドクター スベラック アクチェンゲゼルシャフト 限定された（ｄｅｆｉｎｅｄ）平均分子量を持つ生体高分子の生産方法
JP2021518136A (ja) * 2018-03-22 2021-08-02 クイーン メアリー ユニバーシティ オブ ロンドン 疾患を治療するためのインプラント型セルドレッシング
JP7519298B2 (ja) 2018-03-22 2024-07-19 クイーン メアリー ユニバーシティ オブ ロンドン 疾患を治療するためのインプラント型セルドレッシング
US12171911B2 (en) 2018-03-22 2024-12-24 Queen Mary University Of London Implantable cell dressing for treatment of disease
KR20200035736A (ko) * 2018-09-27 2020-04-06 한국화학연구원 다공성 지지체를 이용하여 제조된 심근 유사 구조체 및 이를 이용한 약물 독성 평가 방법
KR102113778B1 (ko) 2018-09-27 2020-05-21 한국화학연구원 다공성 지지체를 이용하여 제조된 심근 유사 구조체 및 이를 이용한 약물 독성 평가 방법
CN114681654A (zh) * 2020-12-31 2022-07-01 广州迈普再生医学科技股份有限公司 具有创面修复功能的可吸收海绵敷料及其制备方法
CN115671365A (zh) * 2022-11-04 2023-02-03 浙江诸暨聚源生物技术有限公司 交联重组胶原蛋白海绵及其制备方法和应用
CN115671365B (zh) * 2022-11-04 2024-02-13 浙江诸暨聚源生物技术有限公司 交联重组胶原蛋白海绵及其制备方法和应用

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JPWO2002045767A1 (ja) 2004-04-08
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JP4273450B2 (ja) 2009-06-03
AU2002221089A1 (en) 2002-06-18

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