CN1774272A - Substrate for tissue regeneration, material for transplantation, and processes for producing these - Google Patents
Substrate for tissue regeneration, material for transplantation, and processes for producing these Download PDFInfo
- Publication number
- CN1774272A CN1774272A CNA018202632A CN01820263A CN1774272A CN 1774272 A CN1774272 A CN 1774272A CN A018202632 A CNA018202632 A CN A018202632A CN 01820263 A CN01820263 A CN 01820263A CN 1774272 A CN1774272 A CN 1774272A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- mentioned
- tissue regeneration
- linking
- cross
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 230000017423 tissue regeneration Effects 0.000 title claims abstract description 76
- 239000000758 substrate Substances 0.000 title claims abstract description 75
- 239000000463 material Substances 0.000 title claims description 75
- 238000002054 transplantation Methods 0.000 title claims description 23
- 238000000034 method Methods 0.000 title claims description 15
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 135
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 131
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 105
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 21
- 239000007864 aqueous solution Substances 0.000 claims description 65
- 238000004132 cross linking Methods 0.000 claims description 58
- 238000004108 freeze drying Methods 0.000 claims description 47
- 230000021164 cell adhesion Effects 0.000 claims description 33
- 238000002360 preparation method Methods 0.000 claims description 28
- 238000003475 lamination Methods 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 24
- 241001597008 Nomeidae Species 0.000 claims description 23
- 102000008186 Collagen Human genes 0.000 claims description 21
- 108010035532 Collagen Proteins 0.000 claims description 21
- 229920001436 collagen Polymers 0.000 claims description 21
- 210000002950 fibroblast Anatomy 0.000 claims description 9
- 108010010803 Gelatin Proteins 0.000 claims description 7
- 229920000159 gelatin Polymers 0.000 claims description 7
- 239000008273 gelatin Substances 0.000 claims description 7
- 235000019322 gelatine Nutrition 0.000 claims description 7
- 235000011852 gelatine desserts Nutrition 0.000 claims description 7
- 239000012141 concentrate Substances 0.000 claims description 6
- 239000004744 fabric Substances 0.000 claims description 6
- 239000004745 nonwoven fabric Substances 0.000 claims description 6
- 102000009123 Fibrin Human genes 0.000 claims description 3
- 108010073385 Fibrin Proteins 0.000 claims description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 claims description 3
- 235000010443 alginic acid Nutrition 0.000 claims description 3
- 229920000615 alginic acid Polymers 0.000 claims description 3
- 229960001126 alginic acid Drugs 0.000 claims description 3
- 239000000783 alginic acid Substances 0.000 claims description 3
- 150000004781 alginic acids Chemical class 0.000 claims description 3
- 229960005188 collagen Drugs 0.000 claims description 3
- 229950003499 fibrin Drugs 0.000 claims description 3
- 229940014259 gelatin Drugs 0.000 claims description 3
- 210000001789 adipocyte Anatomy 0.000 claims description 2
- 210000001612 chondrocyte Anatomy 0.000 claims description 2
- 210000003725 endotheliocyte Anatomy 0.000 claims description 2
- 210000002919 epithelial cell Anatomy 0.000 claims description 2
- 210000003494 hepatocyte Anatomy 0.000 claims description 2
- 210000004153 islets of langerhan Anatomy 0.000 claims description 2
- 210000002510 keratinocyte Anatomy 0.000 claims description 2
- 230000002107 myocardial effect Effects 0.000 claims description 2
- 210000000963 osteoblast Anatomy 0.000 claims description 2
- 239000000049 pigment Substances 0.000 claims description 2
- 238000003825 pressing Methods 0.000 claims description 2
- 239000002344 surface layer Substances 0.000 claims description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 2
- 239000004593 Epoxy Substances 0.000 abstract description 7
- 150000001875 compounds Chemical class 0.000 abstract 2
- 238000007710 freezing Methods 0.000 description 24
- 230000008014 freezing Effects 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 208000027418 Wounds and injury Diseases 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 206010052428 Wound Diseases 0.000 description 18
- 239000007788 liquid Substances 0.000 description 17
- 239000000515 collagen sponge Substances 0.000 description 16
- -1 siloxanes Chemical class 0.000 description 14
- 239000012153 distilled water Substances 0.000 description 13
- 239000011734 sodium Substances 0.000 description 13
- 230000001954 sterilising effect Effects 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000008961 swelling Effects 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000005253 cladding Methods 0.000 description 6
- 238000012856 packing Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241001421714 Olynthus Species 0.000 description 4
- 230000006978 adaptation Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000008602 contraction Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 4
- 229940099552 hyaluronan Drugs 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000000465 moulding Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 4
- IVIDDMGBRCPGLJ-UHFFFAOYSA-N 2,3-bis(oxiran-2-ylmethoxy)propan-1-ol Chemical compound C1OC1COC(CO)COCC1CO1 IVIDDMGBRCPGLJ-UHFFFAOYSA-N 0.000 description 3
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 3
- 208000028990 Skin injury Diseases 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000037313 granulation tissue formation Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229920006264 polyurethane film Polymers 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000007142 ring opening reaction Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- WCDDVEOXEIYWFB-VXORFPGASA-N (2s,3s,4r,5r,6r)-3-[(2s,3r,5s,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O)[C@H](O)[C@H]1O WCDDVEOXEIYWFB-VXORFPGASA-N 0.000 description 1
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 229940014041 hyaluronate Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 238000005907 ketalization reaction Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
Images
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
- A61L27/50—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
- A61L27/40—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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
Abstract
A 1% aqueous hyaluronic acid solution containing a water-soluble epoxy compound as a crosslinking agent is heated and concentrated to obtain an intermolecularly crosslinked hyaluronic acid. Subsequently, this crosslinked compound is freeze-dried under vacuum to obtain a hyaluronic acid sponge. The sponge is impregnated with an atherocollagen solution and then freeze-dried under vacuum. Thus, a substrate for tissue regeneration is obtained.
Description
Technical field
The present invention relates to can be at the extensive tissue regeneration substrate that utilizes of medical field, material for transplantation and preparation method thereof.
Background technology
In recent years, cultured cell once more the regenerating tissues of constructing tissue learn and to be subjected to attracting attention.Learn according to this regenerating tissues, on the tissue regeneration substrate that the high various materials of biocompatible form, keep various cells, cultivate, obtain material for transplantation.
For example, for the cultivation skin that has embedded fibroblast etc., the known collagen sponge that can utilize is as tissue regeneration substrate (spy opens flat 4-332561 communique), and also prompting can utilize hyaluronic acid to replace collagen (spy opens flat 11-319068 communique) recently.
Hyaluronic acid is a kind of mucopolysaccharide with disaccharide repetitive structure, mainly is present in the connective tissues such as joint fluid, vitreous body of eye, umbilical cord or skin corium etc. of animal.In addition, the order of magnitude of molecular weight be hundreds thousand of～millions of, have and very a large amount of bonded character of water, this water-retaining property with the low frictional properties in for example joint and dermis of skin tissue is relevant.In addition, when the connective tissue of damaged was repaired, hyaluronic generation came to life for the moment, promoted to organize the cell when constructing again to move.Therefore, we can say that hyaluronic acid is as the good performance of wound cladding material performance.
But, though hyaluronic acid because the property of water-bearing is very high, when utilizing as tissue regeneration substrate, exists the adhesiveness of cell low, the problem of external (in vitro) cell culture difficulty as the good performance of wound cladding material performance.
Problem of the present invention is to address the above problem, and it is a kind of with the tissue regeneration substrate of hyaluronic acid as main body that its purpose is to provide, and this tissue regeneration substrate is suitable for the tissue regeneration after external cell culture and the transplanting.Another purpose is to provide the preparation method of this tissue regeneration substrate.And another purpose is to provide material for transplantation that utilizes this tissue regeneration substrate and preparation method thereof.
Disclosure of the Invention
In order to solve above-mentioned problem, the inventor has carried out concentrated research, and the result has finished following the 1st～4th invention.
The 1st of the present invention is characterised in that to possess the hyaluronic acid sponge based on hyaluronic acid and/or its derivant, and the cell adhesion portion that obtains of the sponge of pressing the macromolecular material that derives from organism to constitute at least one surface layer of above-mentioned hyaluronic acid sponge.The cell adhesion portion of this tissue regeneration substrate is because lamination derives from the sponge that the macromolecular material of organism constitutes, thereby the cell that embeds in the tissue regeneration substrate can adhere to well.In addition, owing to possess hyaluronic acid sponge, thereby promote that wound healing ability such as cell proliferation is also good based on hyaluronic acid and/or its derivant.Therefore, adopt this tissue regeneration substrate, can carry out the tissue regeneration after cell in vitro is cultivated and transplanted well.In addition, during with the high density inoculating cell, the visible bigger contraction of former collagen sponge, and tissue regeneration substrate of the present invention almost be can not see this contraction.This tissue regeneration substrate can embed cell and use as material for transplantation, and the wound cladding material that also can be used as the wound coverage face in addition uses.
Here, as hyaluronic derivant, except that hyaluronic acid slaines such as hyaluronate sodium and potassium hyaluronate, can also exemplify material that etherificates such as hyaluronic hydroxyl or carboxyl, esterification, amidatioon, acetalation, ketalization obtain etc., wherein preferably clear matter acid sodium.In addition, as hyaluronic acid sponge, the material (cross-linked-hyaluronic acid sponge) of preferred intermolecular cross-linking.In addition,, can exemplify collagen, gelatin, fibrin, alginic acid etc., the wherein preferred few atherocollagen of collagen, particularly antigenicity, the material of collagen, the preferred intermolecular cross-linking of gelatin in addition as the macromolecular material that derives from organism.
This tissue regeneration substrate preferably at the boundary vicinity of hyaluronic acid sponge and cell adhesion portion, forms the macromolecular material that derives from organism and is entrained in the interior state of hyaluronic acid sponge.At this moment, even the swelling rate of hyaluronic acid sponge and cell adhesion portion there are differences, do not worry that both can peel off when moisture yet.
This tissue regeneration substrate, fabric, non-woven fabrics or fabric that preferably clear matter acid sponge is used as supporter support.At this moment, by supporter, the intensity of tissue regeneration substrate increases, and when promptly using pincet to pick up, does not also worry damagedly, and operability is good.Here, as the material of supporter, so long as the material of hyaluronic acid sponge effect is reinforced in performance, not special the qualification can not exemplify synthesized polymer materials such as nylon, polyester, siloxanes, perhaps natural macromolecular materials such as thin,tough silk, cotton, fiber crops etc.
The 2nd of the present invention is the preparation method of this tissue regeneration substrate, it is characterized in that, comprise the steps: (1) by will being added with cross-linking agent hyaluronic acid and/or the aqueous solution of its derivant concentrate, obtain the cross-linking step of the intermolecular cross-linking thing of hyaluronic acid and/or its derivant, (2) pass through the vacuum lyophilization of above-mentioned intermolecular cross-linking thing, obtain the spongeization step of hyaluronic acid sponge, and (3) are by making after one side absorbs the aqueous solution of the macromolecular material derive from organism at least of above-mentioned hyaluronic acid sponge, vacuum lyophilization forms the lamination step of above-mentioned cell adhesion portion.According to this preparation method, by intermolecular cross-linking thing vacuum lyophilization with hyaluronic acid and/or its derivant, after making hyaluronic acid sponge, make it at least simultaneously absorb the aqueous solution of the macromolecular material that derives from organism, and then carry out vacuum lyophilization.Adopt this preparation method, can prepare the 1st of the present invention's tissue regeneration substrate with comparalive ease.
Here, in cross-linking step,, can exemplify soluble epoxide chemical compound or glutaraldehyde etc., wherein preferred water dissolubility epoxide as cross-linking agent.As this soluble epoxide chemical compound, can exemplify Ethylene glycol diglycidyl ether, glycerin diglycidyl ether, T 55 etc.When using the soluble epoxide chemical compound as cross-linking agent, its consumption is preferably about 1/2～1/10 ratio by weight with respect to hyaluronic acid and/or its derivant, and preferred especially 1/5～1/10.
In this cross-linking step, use the aqueous solution of hyaluronic acid and/or its derivant, the concentration of hyaluronic acid and/or its derivant depends on the kind of the hyaluronic acid of use and/or its derivant and molecular weight etc. in this aqueous solution, is about 0.5～1.5 weight %.In addition, as water as solvent, the ion exchange water of preferred pH5～6.
In this cross-linking step, the hyaluronic acid by will being added with cross-linking agent and/or the aqueous solution of its derivant concentrate, and carry out the intermolecular cross-linking reaction of hyaluronic acid and/or its derivant, and preferred heating is carried out.In the occasion of using the soluble epoxide chemical compound as cross-linking agent, preferably about 30～60 ℃ of the temperature when concentrating, more preferably from about 40～60 ℃, preferred about 50 ℃ especially.If heating above under 60 ℃ the high temperature, then mixed liquor can produce bubble, and the sponge structure uniformity of the hyaluronic acid sponge that obtains sometimes is insufficient.On the other hand, be lower than under 30 ℃ the temperature, intermolecular cross-linking reaction speed diminishes, and the intermolecular cross-linking thing that obtains desired concn sometimes needs the long period.In addition, the intermolecular cross-linking reaction of hyaluronic acid and/or its derivant preferably carries out at neutral region (pH5～8) or acidic region (pH3～5).In addition,, compare with the occasion of carrying out at neutral region if carry out at acidic region, the trend that has concentration time to shorten, thereby preferred.
In this cross-linking step, preferably the concentration of aqueous solution in for example hyaluronic acid and/or its derivant reaches 1～10 weight %, and preferred 2～5 weight % in the moment of preferred especially about 5 weight %, stop concentrating.Fully do not carry out spissated occasion, owing to can not fully suppress the swelling of the hyaluronic acid sponge that obtains after the following spongeization step, thereby not preferred.That is to say, if hyaluronic acid sponge has higher swelling, for example impregnated in the occasion in the fluid medium etc. when cell culture, necessary above swelling can take place, it is not firm that the result becomes, and shape also becomes greatly, therefore is difficult to operate.Relative therewith, if control spissated terminal point as mentioned above, owing to can fully suppress the swelling of hyaluronic acid sponge, necessary above swelling can not take place in the occasion that therefore impregnated in the fluid medium etc., it is not firm that the result can not become, and perhaps shape becomes excessive, easily operation.In addition, be completed into membranaceously up to concentrated solution,, can not form hyaluronic acid sponge even carry out the spongeization step by vacuum lyophilization if carry out cross-linking step, therefore be necessary concentrated solution form membranaceous before the spissated terminal point of setting.
Then, in the spongeization step, by the intermolecular cross-linking thing vacuum lyophilization after the cross-linking step is obtained hyaluronic acid sponge, in order not make it to form the crystal of bigger ice, temperature conditions during vacuum lyophilization is-85 ℃～-30 ℃ approximately, preferred-85 ℃～-50 ℃ approximately, more preferably from about-85 ℃, vacuum condition preferred 30 * 10
-3～100 * 10
-3Mmbar (3～10Pa), more preferably 30 * 10
-3～50 * 10
-3Mmbar (3～5Pa).
In this spongeization step, preferably concentrate the intermolecular cross-linking thing that obtains to not carrying out in the cross-linking step fully, carry out the freeze-thaw operation more than at least 1 time after, vacuum lyophilization obtains hyaluronic acid sponge.Do not carry out freezing, as to thaw operation occasion before the vacuum lyophilization, be formed on the sponge that is difficult to keep shape when moisture sometimes, and before vacuum lyophilization, carry out freezing, thaw the operation occasion, though think that the result probably can promote the intermolecular interaction that hydrogen bond is such, obtained the sponge that is easy to keep shape when moisture.Operation freezing before vacuum lyophilization like this, that thaw also can be carried out several, if consider the simplification of preparation section, preferably carries out 1 time.In addition, refrigerated temperature conditions is-85 ℃～-30 ℃ approximately in freezing before the vacuum lyophilization, the operation of thawing, and preferred-85 ℃～-50 ℃ approximately, more preferably from about-85 ℃, the temperature conditions that thaws in addition is about 20 ℃～30 ℃, is preferably near about 30 ℃.Wherein, also temperature can be divided into a plurality of stages risings when thawing thaws.At this moment, should note not cross-linking agent between the saboteur.In addition, in cross-linking step, under suitable concentrated condition, carrying out fully concentrating the intermolecular cross-linking thing that obtains,, therefore not necessarily must carry out the preceding freeze/thaw operation of vacuum lyophilization owing to have full intensity.
Before carrying out following lamination step, the water-washing step of the hyaluronic acid sponge for preparing in the cleansing sponge step is set preferably.In this water-washing step, also can in slurry, add the deactivator that makes unreacted cross-linking agent inactivation.For example, use the occasion of soluble epoxide chemical compound,, preferably have the function that makes epoxy ring-opening and the material harmless, for example can use glycine organism as deactivator as cross-linking agent.
Then, in lamination step, make after one side absorbs the aqueous solution of the macromolecular material derive from organism at least of hyaluronic acid sponge, carry out vacuum lyophilization, thereby form cell adhesion portion,, can exemplify collagen, gelatin, fibrin, alginic acid etc. as mentioned above as the macromolecular material that derives from organism, the wherein preferred few atherocollagen of collagen, particularly antigenicity.In addition, aqueous solution as the macromolecular material that derives from organism, can use the aqueous solution of 0.2～1.0 weight %, the aqueous solution of preferred 0.2～0.5 weight %, the more preferably aqueous solution of 0.5 weight %, the macromolecular material and the hyaluronic weight ratio that derive from organism in addition can be 1: 2～10, preferred 1: 2～4, and preferred especially 1: 4.In addition, the condition of vacuum lyophilization is identical with vacuum lyophilization condition in the spongeization step.
In addition, in this lamination step, owing to make the one side at least of hyaluronic acid sponge absorb the aqueous solution of the macromolecular material that derives from organism, thereby, form the macromolecular material derive from organism and be doped to state in the hyaluronic acid sponge at the hyaluronic acid sponge of the tissue regeneration substrate that obtains and the boundary vicinity of cell adhesion portion.Therefore, even the swelling rate of hyaluronic acid sponge and cell adhesion portion there are differences, when making tissue regeneration substrate moisture, do not worry that both can peel off yet.
In this lamination step, also can offer a plurality of holes in the one side of hyaluronic acid sponge, make its one side of having opened the hole absorb the aqueous solution of the macromolecular material that derives from organism after, vacuum lyophilization forms above-mentioned cell adhesion portion.At this moment, the aqueous solution of macromolecular material that derives from organism also enters in most the holes except in the porous of soaking into hyaluronic acid sponge and having, therefore and hyaluronic acid sponge between can also obtain the anchor ring effect.After making the aqueous solution that soaks into the organism macromolecular material in each hole or the porous like this, carry out vacuum lyophilization, can further improve the hyaluronic acid sponge of tissue regeneration substrate and the adaptation of cell adhesion portion.In addition, said here " hole " except that general hole such as circle hole or square hole, also comprises for example breaking joint or the big hole of concavo-convex geometric ratio plane contact area.
In this lamination step, when using the conduct of collagen or gelatin to derive from the macromolecular material of organism, irradiation ultraviolet radiation lamp after the preferred lamination step makes collagen or gelatin partly carry out intermolecular cross-linking, prevent when moisture that collagen or gelatin flow out, sterilize with ethylene oxide gas etc. after more preferably.In addition, when not carrying out above-mentioned water-washing step,, preferably in the aqueous solution of the macromolecular material that derives from organism, add deactivator in advance in order to make unreacted cross-linking agent inactivation.As deactivator, same as described above, for example use the occasion of soluble epoxide chemical compound as cross-linking agent, preferred glycine etc. has the function that makes epoxy ring-opening and the material harmless to organism.
In addition, also can adopt following spongeization lamination step to replace above-mentioned spongeization step and lamination step.That is to say, after also can adopting the intermolecular cross-linking thing that makes after the cross-linking step and the aqueous solution of the macromolecular material that derives from organism contacting, carry out vacuum lyophilization, form the step of above-mentioned hyaluronic acid sponge and cell adhesion portion.At this moment, because vacuum lyophilization is carried out 1 time and got final product, preparation process is simplified.
In this spongeization lamination step, because the intermolecular cross-linking thing before the spongeization is contacted with the aqueous solution of the macromolecular material that derives from organism, therefore the aqueous solution that derives from the macromolecular material of organism is difficult to the preceding intermolecular cross-linking thing of saturated spongeization, and the adaptation of the hyaluronic acid sponge of the tissue regeneration substrate that finally obtains and cell adhesion portion is probably insufficient.Therefore, most holes are set on the above-below direction of the intermolecular cross-linking thing of hyaluronic acid and/or its derivant preferably, increase the contact area with the aqueous solution of the macromolecular material that derives from organism, obtain the effect of anchor ring.After the preferred aqueous solution that makes the organism macromolecular material like this soaks into each hole, carry out vacuum lyophilization, thereby improve the hyaluronic acid sponge of tissue regeneration substrate and the adaptation of cell adhesion portion.In addition, said here " hole " except that general hole such as circle hole or square hole, also comprises for example breaking joint or the big hole of concavo-convex geometric ratio plane contact area.
Adopt this spongeization lamination step, when making the tissue regeneration substrate of the supported body support of hyaluronic acid sponge, for example carry out in the following order.That is, in advance supporter is applied bottom surface, in this container, pack into and added the hyaluronic acid of cross-linking agent and/or the aqueous solution of its derivant at container.Then,, concentrate, make the intermolecular cross-linking thing of hyaluronic acid and/or its derivant this aqueous solution heating.Then, most holes are set on the above-below direction of intermolecular cross-linking thing in this container, add the aqueous solution of the macromolecular material that derives from organism, make it to contact, carry out vacuum lyophilization with the intermolecular cross-linking thing.In addition, also can before vacuum lyophilization, (for example be provided with before the hole or after the hole is set) as required the intermolecular cross-linking thing is carried out freeze-thaw operation.By carrying out this operation, the intensity that can be improved, the sponge that is easy to keep shape when moisture.
The 3rd of the present invention is material for transplantation, it is characterized in that, possesses the cell of the cell adhesion portion maintenance of above-mentioned tissue regeneration substrate and this tissue regeneration substrate.Therefore this material for transplantation can easily carry out cell culture external because cell remains on cell adhesion portion, and adaptation is good, and promotes cell to move the wound healing ability that produces with moistening effect by hyaluronic acid, and tissue can suitably be regenerated.Here, the cell that cell adhesion portion keeps can be any cell, and for example fibroblast, keratinocyte, pigment produce cell, vascular endothelial cell, endotheliocyte, epithelial cell, chondrocyte, osteoblast, sarcoplast, adipose cell, hepatocyte, neurocyte, myocardial cell, islet cells etc.When for example using the fibroblastic material for transplantation kept deriving from other people, this material for transplantation is transplanted to the skin trauma position, covers with the film (for example polyurethane film) that contains antibacterial, the reuse binder covers.Like this, come from the various cytokines promotion healings of other people fibroblast generation by origin.
The 4th of the present invention is the preparation method of this material for transplantation, it is characterized in that, make above-mentioned tissue regeneration substrate after, cell is embedded the cell adhesion portion of this tissue regeneration substrate.According to this preparation method, can make the 3rd of the present invention's material for transplantation with comparalive ease.Here,, for example comprise, make cell take in the method that keeps in the hole simultaneously by tissue regeneration substrate be impregnated in the culture fluid as the method that cell is embedded cell adhesion portion.
Description of drawings
Fig. 1 is the key diagram of production order of the tissue regeneration substrate of expression embodiment 1.
Fig. 2 is the key diagram of production order of the tissue regeneration substrate of expression embodiment 2.
Fig. 3 is the key diagram of production order of the tissue regeneration substrate of expression embodiment 3.
Fig. 4 is the key diagram of production order of the tissue regeneration substrate of expression embodiment 4.
The preferred forms of invention
The preferred embodiments of the present invention below are described.The present invention is not subjected to any qualification of following embodiment.In addition, below short of special explanation, " % " expression weight %.
(embodiment 1)
(1) making of tissue regeneration substrate (with reference to Fig. 1)
The making of (1-1) hyaluronic acid sponge
10g is dissolved among the distilled water 1L with hyaluronate sodium (molecular weight about 2,000,000), prepares 1% hyaluronic acid aqueous solution (pH6).Hyaluronic dissolving is stirred with mechanical agitator, and stirs time enough.On the other hand, as the soluble epoxide chemical compound, デ Na コ-Le EX313 (glycerin diglycidyl ether, Na ガ セ changes into industry) 1g with distilled water 20ml dilution, is prepared デ Na コ-Le EX313 solution (hereinafter referred to as EX313 solution).The EX313 solution 20ml of preparation is like this added in the hyaluronic acid aqueous solution in the stirring, and restir 30 minutes makes hyaluronic acid-EX313 mixed liquor.
Hyaluronic acid-EX313 mixed liquor the 180ml that obtains is injected into floor space 180cm
2(in the pallet of 10cm * 18cm).At this moment the degree of depth of hyaluronic acid-EX313 mixed liquor is about 1cm.Hyaluronic acid-EX313 the mixed liquor that is injected in the pallet was at room temperature left standstill 2 hours, after removing the bubble in hyaluronic acid-EX313 mixed liquor, the ベ Application リ-ゼ (non-woven fabrics, Asahi Chemical Industry's industry) that cuts off according to the size of pallet is in advance placed on hyaluronic acid-EX313 mixed liquor.
Top hyaluronic acid-EX313 mixed liquor of having placed the state of non-woven fabrics is packed in the xeothermic device of air circulation type, under 50 ℃, left standstill 10 hours.Like this, the epoxy reaction of hyaluronic hydroxy (hydroxyl) or carboxyl and デ Na コ-Le EX313 generates hyaluronic intermolecular cross-linking thing, and liquid measure is concentrated into about 2/5 (that is, as hyaluronic acid concentration, being about 2.5 weight %) simultaneously.At this moment the degree of depth of concentrated solution is about 4mm.
Then, this concentrated solution is freezing down at-85 ℃.Cooling time is subjected to about the ability of cryoprobe, is about 5～6 hours, makes it freezing fully.After freezing like this, at room temperature place and once thawed in 1～1.5 hour, after thawing, freezing 5～6 hours of the cryoprobe of reuse-85 ℃, freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar, obtains having cross-linking agent (hereinafter referred to as the cross-linked-hyaluronic acid sponge) between the hyaluronan molecule of sponge structure.
The lamination of (1-2) and collagen sponge
Atherocollagen 1g is dissolved among the distilled water 500ml, is adjusted to pH4, preparation 0.2%atherocollagen aqueous solution with 1NHC1.The atherocollagen aqueous solution 90ml of preparation is injected into floor space 180cm
2Pallet in.The liquid measure of Atherocollagen aqueous solution is half of liquid measure (180ml) of original hyaluronic acid aqueous solution, and collagen and hyaluronic weight ratio are 1: 10.
In the pallet that the atherocollagen aqueous solution is housed, make ベ Application リ-ゼ be positioned at the top cross-linked-hyaluronic acid sponge impregnating made as mentioned above, placed 1 hour, make in the collagenic aqueous solution infiltration cross-linked-hyaluronic acid sponge.Then, with this hyaluronic acid sponge that in collagenic aqueous solution, floods-85 ℃ down freezing after, vacuum drying.
The material that cross-linked-hyaluronic acid sponge upper strata moulding olynthus after the against vacuum lyophilization obtains uses the Burdick lamp (254nm) of 15W to shine 30 minutes from the distance of 25cm in the collagen sponge side, carries out the intermolecular cross-linking of collagen.Then, in the sterilizing bag of packing into, under 60 ℃, carry out EOG (ethylene oxide gas) sterilization 20 hours.Like this, obtain promptly being possessed the cross-linked-hyaluronic acid sponge of crosslinked with collagen sponge (cell adhesion portion) by the tissue regeneration substrate of ベ Application リ-ゼ (supporter) support.
(2) preparation of material for transplantation
With the opposite one side of ベ Application リ-ゼ of tissue regeneration substrate, with 5 * 10
4Cell/cm
2Density inoculation swim behind the cultivation rabbit fibroblast in the improved Eagle minimum essential medium of the Dulbecoo that contains 10% hyclone (FBS) (DMEM+10%FBS, Gibcoo corporate system), at CO
25%, cultivated 7 days in 37 ℃ the calorstat, make cultivation corium as material for transplantation.Remove culture medium, in its freezing preservation liquid (DMEM+20%FBS that contains 10%DMSO) of packing into, in-152 ℃ refrigerator-freezer, preserve.
(3) transplant experiment
The cultivation corium (material for transplantation) that will be kept in use in the refrigerator-freezer thaws, and removes freezing preservation liquid, with Hanks liquid 30ml washing 2 times, is used for zoopery.Describe the circle of diameter 7cm at the back of rabbit, excise all skin layers, form the skin injury wound.Be suitable for above-mentioned cultivation corium at this skin injury site of injury, sew up the periphery of wound face, be suitable for polyurethane film system wound cladding material thereon, put Sterilizing pad again, sew up the periphery of wound, add press fit with the retractility binder.As a result, observe significantly reducing of good granulation tissue formation and wound area.
(embodiment 2)
(1) making of tissue regeneration substrate (with reference to Fig. 2)
The making of (1-1) hyaluronic acid sponge
Similarly to Example 1, preparation hyaluronic acid-EX313 mixed liquor is injected into floor space 180cm with the hyaluronic acid-EX 313 mixed liquor 180ml that obtain
2(in the pallet of 10cm * 18cm).In pallet, put the ベ Application リ-ゼ (as mentioned above) that cuts off according to its size in advance, add a small amount of distilled water, make it moisture, and make it to stick to tray bottom surface.The degree of depth of injecting the hyaluronic acid-EX313 mixed liquor of pallet is about 1cm.
Hyaluronic acid-EX313 the mixed liquor of this state is packed in the xeothermic device of air circulation type, under 50 ℃, left standstill 15 hours.Like this, the epoxy reaction of hyaluronic hydroxy (hydroxyl) or carboxyl and デ Na コ-Le EX313 generates hyaluronic intermolecular cross-linking thing, and liquid measure is concentrated into about 1/5 (that is, as hyaluronic acid concentration, being about 5 weight %) simultaneously.At this moment the degree of depth of concentrated solution is about 2～3mm.
Then, this concentrated solution is freezing down at-85 ℃.Cooling time is subjected to about the ability of cryoprobe, is about 5～6 hours, makes it freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar, obtains having cross-linking agent (hereinafter referred to as the cross-linked-hyaluronic acid sponge) between the hyaluronan molecule of sponge structure.Then, in order to remove unreacted cross-linking agent, in the container that the 15L ion exchange water is housed,, wash with water cross-linked-hyaluronic acid sponge impregnating 1 day.
The lamination of (1-2) and collagen sponge
Atherocollagen 2.5g is dissolved among the distilled water 500ml, is adjusted to pH3.2, preparation 0.5%atherocollagen aqueous solution with 1N HCl.Cross-linked-hyaluronic acid sponge after the washing is placed floor space 180cm
2Pallet on, make non-woven fabrics down, inject the atherocollagen aqueous solution 90ml of preparation thereon.The liquid measure of Atherocollagen aqueous solution is half of liquid measure (180ml) of original hyaluronic acid aqueous solution, and collagen and hyaluronic weight ratio are 1: 4.
Around the cross-linked-hyaluronic acid sponge some contractions are arranged, and produce the slit between the pallet side.Be immersed in this slit by a therocollagen aqueous solution, collagen be set, make it to surround hyaluronic acid sponge around, cross-linked-hyaluronic acid sponge and collagen sponge separates when suppressing swelling.
Inject the atherocollagen aqueous solution through behind the diel, the hyaluronic acid sponge that will contain the atherocollagen aqueous solution is-85 ℃ of following vacuum lyophilizations.The material that cross-linked-hyaluronic acid sponge upper strata moulding olynthus after the against vacuum lyophilization obtains uses the Burdick lamp (254nm) of 15W to shine 30 minutes from the distance of 25cm in the collagen sponge side, carries out the intermolecular cross-linking of collagen.Then, in the sterilizing bag of packing into, under 60 ℃, carry out EOG sterilization 20 hours.Like this, obtain promptly being possessed the cross-linked-hyaluronic acid sponge of crosslinked with collagen sponge (cell adhesion portion) by the tissue regeneration substrate of ベ Application リ-ゼ (supporter) support.
(2) preparation of material for transplantation
With the 180cm for preparing according to the method described above
2Tissue regeneration substrate be cut into 90cm
2Size.With the tissue regeneration substrate that the cuts off 180cm that packs into
2Pallet in, inject DMEM+10%FBS 100ml, regulate pH.Regulate (pH7.4) behind the pH, once that culture fluid is discarded, on tissue regeneration substrate with 5 * 10
5Cell/cm
2Density inoculation human fibroblasts suspension 5ml.After the inoculation, under 37 ℃, leave standstill a night, human fibroblasts is fixed on the tissue regeneration substrate.Then, add above-mentioned DMEM+10%FBS culture medium 100ml, at 37 ℃, 5%CO
2Under cultivated for 1 week.The cultivation corium (material for transplantation) that makes so not only has full intensity, and the contraction of 2～3mm degree is only arranged on every side, so operability is good, does not worry damaged.
(embodiment 3)
(1) making of tissue regeneration substrate (with reference to Fig. 3)
The making of (1-1) hyaluronic acid sponge
20g is dissolved among the distilled water 2L with hyaluronate sodium (molecular weight about 2,000,000), prepare 1% hyaluronic acid aqueous solution (pH6) after, be adjusted to pH3.5 with 1N HCl.Hyaluronic dissolving is stirred with mechanical agitator, and stirs time enough.On the other hand, as the soluble epoxide chemical compound, デ Na コ-Le EX810 (glycerin diglycidyl ether, Na ガ セ changes into industry) 4g with distilled water 40ml dilution, is prepared デ Na コ-Le EX810 solution (hereinafter referred to as EX810 solution).The EX810 solution (40ml) of preparation is like this added in the hyaluronic acid aqueous solution in the stirring, and restir 30 minutes makes hyaluronic acid-EX810 mixed liquor.
Hyaluronic acid-EX810 mixed liquor the 50g that obtains is injected into floor space 110cm
2(in the pallet of 10cm * 11cm).In pallet, put the ベ Application リ-ゼ (as mentioned above) that cuts off according to its size in advance, add a small amount of distilled water, make it moisture, and make it to stick to tray bottom surface.The degree of depth of injecting the hyaluronic acid-EX810 mixed liquor of pallet is about 5mm.
Hyaluronic acid-EX810 the mixed liquor of this state is packed in the air circulation type heater, under 50 ℃, left standstill 5 hours.Like this, the epoxy reaction of hyaluronic hydroxy (hydroxyl) or carboxyl and デ Na コ-Le EX810 generates hyaluronic intermolecular cross-linking thing, and liquid measure is concentrated into about 1/2 (that is, as hyaluronic acid concentration, being about 2 weight %) simultaneously.At this moment the degree of depth of concentrated solution is about 2～3mm.
Then, this concentrated solution is freezing down at-85 ℃.Cooling time is subjected to about the ability of cryoprobe, is about 5～6 hours, makes it freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar.Then, in order to remove unreacted cross-linking agent, in the container that the 1L distilled water is housed,, wash with water one night of cross-linked-hyaluronic acid sponge impregnating.
After the washing, the cryoprobe of reuse-85 ℃ is freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar, obtains having cross-linking agent (hereinafter referred to as the cross-linked-hyaluronic acid sponge) between the hyaluronan molecule of sponge structure.
The lamination of (1-2) and collagen sponge
Before with the collagen sponge lamination, use the sword mountain that the cross-linked-hyaluronic acid sponge is bored a hole.At this moment the hole is spaced apart about 4mm.
Atherocollagen 8g is dissolved among the distilled water 1.6L, is adjusted to pH3.5, preparation 0.5%atherocollagen aqueous solution with 1N HCl.The atherocollagen aqueous solution 40g of preparation is injected into floor space 110cm
2Pallet in.The liquid measure of Atherocollagen aqueous solution is liquid measure (50g) about 4/5 of original hyaluronic acid aqueous solution, and collagen and hyaluronic weight ratio are 2: 5.
Make the cross-linked-hyaluronic acid sponge of making as mentioned above float over the pallet that has injected the athorocollagon aqueous solution, make ベ Application リ-ゼ be positioned at the top, leave standstill a night, collagenic aqueous solution is infiltrated in the cross-linked-hyaluronic acid sponge.Then, with the hyaluronic acid sponge that infiltrated collagenic aqueous solution-85 ℃ freezing fully down after, carry out vacuum lyophilization.
The intermolecular cross-linking of collagen is carried out from the distance irradiation of 20cm 30 minutes in the both sides of the material that the cross-linked-hyaluronic acid sponge upper strata moulding olynthus after the vacuum lyophilization is obtained, the Burdick lamp (254nm) of using 15W respectively.Then, in the sterilizing bag of packing into, under 60 ℃, carry out EOG (ethylene oxide gas) sterilization 20 hours.Like this, obtain promptly being possessed the cross-linked-hyaluronic acid sponge of crosslinked with collagen sponge (cell adhesion portion) by the tissue regeneration substrate of ベ Application リ-ゼ (supporter) support.
(2) preparation of material for transplantation
The tissue regeneration substrate of preparation is according to the method described above packed in the pallet, inject DMEM+10%FBS 50ml, regulate pH.Regulate (pH7.4) behind the pH, once that culture fluid is discarded, on tissue regeneration substrate with 1 * 10
5Cell/cm
2Density inoculation human fibroblasts suspension 5ml.After the inoculation, under 37 ℃, leave standstill a night, human fibroblasts is fixed on the tissue regeneration substrate.Then, add above-mentioned DMEM+10%FBS culture medium 50ml, at 37 ℃, 5%CO
2Under cultivated for 1 week.The cultivation corium (material for transplantation) that makes is like this removed culture medium, in its freezing preservation liquid (DMEM+20%FBS that contains 10%DMSO) of packing into, preserves in-152 ℃ refrigerator-freezer.
(3) transplant experiment
The cultivation corium (material for transplantation) that will be kept in use in the refrigerator-freezer thaws, and removes freezing preservation liquid, with Lactated Ringer'S Solution 30～50ml washing 3 times, is used for the human clinical.Wound face carries out disinfection after removing residual slough, uses the normal saline thorough washing.Be suitable for above-mentioned cultivation corium at this skin injury site of injury, around the sutured cultivation corium.Be suitable for thereon and soaked into the gauze that contains antibiotic ointment, put sterile gauze again, sew up the periphery of wound, add press fit with the retractility binder.As a result, observe significantly reducing of good granulation tissue formation and wound area, grow epithelium around the wound.
(embodiment 4)
(1) making of tissue regeneration substrate (with reference to Fig. 4)
The making of (1-1) hyaluronic acid sponge
2g is dissolved among the distilled water 200ml with hyaluronate sodium (molecular weight about 2,000,000), prepare 1% hyaluronic acid aqueous solution (pH6) after, be adjusted to pH3.5 with 1N HCl.Hyaluronic dissolving is stirred with mechanical agitator, and stirs time enough.On the other hand, as the soluble epoxide chemical compound, デ Na コ-Le EX8100.2g with distilled water 2ml dilution, is prepared デ Na コ-Le EX810 solution (hereinafter referred to as EX810 solution).The EX810 solution (2ml) of preparation is like this added in the hyaluronic acid aqueous solution in the stirring, and restir 30 minutes makes hyaluronic acid-EX810 mixed liquor.
Hyaluronic acid-EX810 mixed liquor the 4.8g that obtains is injected in the dish of floor space 35mm.The degree of depth of the hyaluronic acid of injection dish-EX810 mixed liquor is about 5mm.
Hyaluronic acid-EX810 the mixed liquor of this state is packed in the air circulation type heater, under 50 ℃, left standstill 5 hours.Like this, the epoxy reaction of hyaluronic hydroxy (hydroxyl) or carboxyl and デ Na コ-Le EX810 generates hyaluronic intermolecular cross-linking thing, and liquid measure is concentrated into about 1/2 (that is, as hyaluronic acid concentration, being about 2 weight %) simultaneously.At this moment the degree of depth of concentrated solution is about 2～3mm.
Then, this concentrated solution is freezing down at-85 ℃.Cooling time is subjected to about the ability of cryoprobe, is about 5～6 hours, makes it freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar.Then,, in the container that the 1L distilled water is housed, flood a night, wash with water in order to remove unreacted cross-linking agent.After the washing, the cryoprobe of reuse-85 ℃ is freezing fully.Then, with 30 * 10
-3～50 * 10
-3(3～5Pa) carry out vacuum lyophilization handles mmbar, obtains having cross-linking agent (hereinafter referred to as the cross-linked-hyaluronic acid sponge) between the hyaluronan molecule of sponge structure.
The lamination of (1-2) and collagen sponge
Before with the collagen sponge lamination, use the sword mountain that the cross-linked-hyaluronic acid sponge is bored a hole.At this moment the hole is spaced apart about 4mm.
On the other hand, (KOKENCELLGEN I-PC: Koken Co., Ltd.'s system) 3.8g is injected in the dish of φ 35mm with 0.5% collagenic aqueous solution.The liquid measure of collagenic aqueous solution is liquid measure (4.8g) about 4/5 of original hyaluronic acid aqueous solution, and collagen and hyaluronic weight ratio are about 2: 5.
One of the cross-linked-hyaluronic acid sponge middle punch made is faced down, float over the dish that has injected collagenic aqueous solution, leave standstill a night, collagenic aqueous solution is infiltrated in the cross-linked-hyaluronic acid sponge.Then, with the hyaluronic acid sponge that infiltrated collagenic aqueous solution-85 ℃ freezing fully down after, vacuum lyophilization.
To the material both sides that the cross-linked-hyaluronic acid sponge upper strata moulding olynthus after the vacuum lyophilization obtains, use the Burdick lamp (254nm) of 15W to shine 30 minutes respectively from the distance of 20cm, carry out the intermolecular cross-linking of collagen.Then, in the sterilizing bag of packing into, under 60 ℃, carry out EOG sterilization 20 hours.Like this, obtain tissue regeneration substrate, promptly possess the cross-linked-hyaluronic acid sponge of crosslinked with collagen sponge (cell adhesion portion).For this tissue regeneration substrate, do not gain in strength, but can easily operate with pincet etc. by ベ Application リ-ゼ.
The tissue regeneration substrate of present embodiment and embodiment 1～3 are same, except can be used as the utilization of cultivation corium, also can be used as the wound cladding material and use.During as the wound cladding material, owing to have bioabsorbable, thereby do not need to remove, expectation is pressed close to wound face by making the collagen side, the cell that can obtain collagen keeps effect, be applicable to wound after, the hyaluronic acid stripping is to wound face, performance cell migration can obtain promoting the effect of curing.
In addition, as the example different with embodiment 2, also can be behind the intermolecular cross-linking reaction of hyaluronic acid-EX313 mixed liquor, do not carry out vacuum lyophilization, on the above-below direction of cross-linking reaction thing, punch, after making its face that has the hole and the atherocollagen aqueous solution contacting, vacuum lyophilization forms collagen sponge when forming hyaluronic acid sponge.At this moment, can simplify preparation process, two kinds of sponges can obtain the anchor ring effect by the hole that exists, therefore driving fit well.
Industrial applicibility
The present invention is the tissue regeneration substrate take hyaluronic acid as main body, is suitable for external cell Therefore regeneration after cultivating and transplanting can be widely used in medical field.
Claims (17)
1. tissue regeneration substrate, it is characterized in that, possess hyaluronic acid sponge based on hyaluronic acid and/or its derivant, and the cell adhesion portion that obtains of the sponge of pressing the macromolecular material that derives from organism to constitute at least one surface layer of above-mentioned hyaluronic acid sponge.
2. tissue regeneration substrate as claimed in claim 1 is characterized in that, the above-mentioned macromolecular material that derives from organism is collagen, gelatin, fibrin or alginic acid.
3. tissue regeneration substrate as claimed in claim 1 or 2 is characterized in that, above-mentioned hyaluronic acid sponge and above-mentioned cell adhesion portion have all carried out intermolecular cross-linking.
4. as any described tissue regeneration substrate in the claim 1～3, it is characterized in that the boundary vicinity of above-mentioned hyaluronic acid sponge and above-mentioned cell adhesion portion is the macromolecular material that derives from organism and is entrained in state in the hyaluronic acid sponge.
5. as any described tissue regeneration substrate in the claim 1～4, it is characterized in that fabric, non-woven fabrics or fabric that above-mentioned hyaluronic acid sponge is used as supporter support.
6. prepare the method for any described tissue regeneration substrate in the claim 1～4, it is characterized in that, comprise the steps:
(1) aqueous solution of the hyaluronic acid by will being added with cross-linking agent and/or its derivant concentrates, and obtains the cross-linking step of the intermolecular cross-linking thing of hyaluronic acid and/or its derivant,
(2) by with the vacuum lyophilization of above-mentioned intermolecular cross-linking thing, obtain the spongeization step of hyaluronic acid sponge, and
(3) by by after one side absorbs the aqueous solution of the macromolecular material derive from organism at least of above-mentioned hyaluronic acid sponge, vacuum lyophilization forms the lamination step of above-mentioned cell adhesion portion.
7. prepare the method for tissue regeneration substrate as claimed in claim 5, it is characterized in that, comprise the steps:
(1) aqueous solution of the hyaluronic acid by will being added with cross-linking agent and/or its derivant with fabric, non-woven fabrics or fabric state of contact under concentrate, obtain the cross-linking step of the intermolecular cross-linking thing of hyaluronic acid and/or its derivant,
(2) by with the vacuum lyophilization of above-mentioned intermolecular cross-linking thing, obtain the spongeization step of hyaluronic acid sponge, and
(3) by by after one side absorbs the aqueous solution of the macromolecular material derive from organism at least of above-mentioned hyaluronic acid sponge, vacuum lyophilization forms the lamination step of above-mentioned cell adhesion portion.
8. as claim 6 or the 7 described methods that prepare tissue regeneration substrate, it is characterized in that, in above-mentioned cross-linking step, reach the moment of 1～10 weight % as spissated terminal point with the concentration of hyaluronic acid and/or its derivant.
9. as any described method for preparing tissue regeneration substrate in the claim 6～8, it is characterized in that, in above-mentioned spongeization step, to above-mentioned intermolecular cross-linking thing carry out freeze-thaw operation more than at least 1 time after, vacuum lyophilization obtains hyaluronic acid sponge.
10. as any described method for preparing tissue regeneration substrate in the claim 6～9, it is characterized in that, in above-mentioned lamination step, open most holes in the one side of above-mentioned hyaluronic acid sponge, after absorbing the aqueous solution of the macromolecular material derive from organism by its one side of having opened the hole, vacuum lyophilization forms above-mentioned cell adhesion portion.
11. as any described method for preparing tissue regeneration substrate in the claim 6～8, it is characterized in that, comprise that following spongeization lamination step is to replace above-mentioned spongeization step and above-mentioned lamination step, promptly, make intermolecular cross-linking thing after the above-mentioned cross-linking step with after the aqueous solution of the macromolecular material that derives from organism contacts, carry out vacuum lyophilization, the above-mentioned cell adhesion portion that forms above-mentioned hyaluronic acid sponge and constitute by this macromolecular material.
12. the method for preparing tissue regeneration substrate as claimed in claim 11 is characterized in that, in above-mentioned spongeization lamination step, has following step:
The step in most holes is set on the above-below direction of the intermolecular cross-linking thing after the above-mentioned cross-linking step; Make it to contact, make this aqueous solution soak into the step in above-mentioned hole with the aqueous solution of the macromolecular material that derives from organism; Carry out vacuum lyophilization, form above-mentioned hyaluronic acid sponge and the step of the above-mentioned cell adhesion portion that constitutes by this macromolecular material.
13. as claim 11 or the 12 described methods that prepare tissue regeneration substrate, it is characterized in that, in above-mentioned spongeization lamination step, to the intermolecular cross-linking thing after the above-mentioned cross-linking step carry out freeze-thaw operation more than at least 1 time after, vacuum lyophilization.
14., it is characterized in that also having and make above-mentioned cell adhesion portion carry out the adhesion portion cross-linking step of intermolecular cross-linking as any described method for preparing tissue regeneration substrate in the claim 6～13.
15. material for transplantation, it is characterized in that possessing any described tissue regeneration substrate in the claim 1～5 or cell that the cell adhesion portion of the tissue regeneration substrate that makes according to any described preparation method in the claim 6～14 and above-mentioned tissue regeneration substrate keeps.
16. material for transplantation as claimed in claim 15, wherein, above-mentioned cell is fibroblast, keratinocyte, pigment generation cell, vascular endothelial cell, endotheliocyte, epithelial cell, chondrocyte, osteoblast, sarcoplast, adipose cell, hepatocyte, neurocyte, myocardial cell, islet cells.
17. prepare the method for claim 15 or 16 described material for transplantation, it is characterized in that, after making tissue regeneration substrate according to the preparation method of any described tissue regeneration substrate in the claim 6～14, cell is embedded the cell adhesion portion of this tissue regeneration substrate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP373116/2000 | 2000-12-07 | ||
| JP2000373116 | 2000-12-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1774272A true CN1774272A (en) | 2006-05-17 |
Family
ID=18842557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA018202632A Pending CN1774272A (en) | 2000-12-07 | 2001-12-07 | Substrate for tissue regeneration, material for transplantation, and processes for producing these |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP4273450B2 (en) |
| KR (1) | KR20030061378A (en) |
| CN (1) | CN1774272A (en) |
| AU (1) | AU2002221089A1 (en) |
| WO (1) | WO2002045767A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912633A (en) * | 2010-08-03 | 2010-12-15 | 孙伟庆 | Hyaluronic acid sponge and preparation method thereof |
| CN101724164B (en) * | 2008-10-31 | 2011-12-14 | 科妍生物科技股份有限公司 | Method for preparing cross-linked hyaluronic acid |
| CN102558600A (en) * | 2011-12-01 | 2012-07-11 | 上海白衣缘生物工程有限公司 | Cross-linked hyaluronan sponge and preparation method for same |
| CN106110367A (en) * | 2016-07-29 | 2016-11-16 | 北京化工大学常州先进材料研究院 | Based on natural polymer MULTILAYER COMPOSITE medical dressing and preparation method thereof |
| CN109998775A (en) * | 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 | A kind of compressed tampon dressing and the preparation method and application thereof |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6358531A (en) * | 1986-08-29 | 1988-03-14 | Hitachi Ltd | Detector for display position |
| WO2004050102A2 (en) * | 2002-06-27 | 2004-06-17 | Roberto Beretta | Methods for preparing a solid-fibrin web |
| WO2004104187A1 (en) * | 2003-05-23 | 2004-12-02 | M D Bio Inc | Fibroblast and method of culturing the same |
| PT2097116E (en) | 2006-12-22 | 2012-12-06 | Medidom Lab | In situ system for intra-articular chondral and osseous tissue repair |
| EP2606828B1 (en) * | 2011-12-20 | 2018-04-11 | Angioclinic AG | Hyaluronic acid and its use for treating venous insufficiency and varicose veins |
| KR101240518B1 (en) * | 2012-03-26 | 2013-03-11 | 주식회사 제네웰 | Raw materials for transplantation using biocompatible polymers |
| EP2910255A1 (en) * | 2014-02-19 | 2015-08-26 | MedSkin Solutions Dr. Suwelack AG | Methods for the production of biopolymers with defined average molecular weight |
| US20210085828A1 (en) * | 2018-03-22 | 2021-03-25 | Queen Mary University Of London | Implantable cell dressing for treatment of disease |
| KR102113778B1 (en) * | 2018-09-27 | 2020-05-21 | 한국화학연구원 | Myocardial-like structures prepared using porous supports method for evaluating drug toxicity using the same |
| CN114681654B (en) * | 2020-12-31 | 2023-04-18 | 广州迈普再生医学科技股份有限公司 | Absorbable sponge dressing with wound repair function and preparation method thereof |
| CN115671365B (en) * | 2022-11-04 | 2024-02-13 | 浙江诸暨聚源生物技术有限公司 | Crosslinked recombinant collagen sponge and preparation method and application thereof |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0403650B1 (en) * | 1988-03-09 | 1994-05-25 | Terumo Kabushiki Kaisha | Medical material permitting cells to enter thereinto and artificial skin |
| JPH04332561A (en) * | 1991-05-09 | 1992-11-19 | Koken Co Ltd | Matrix for culture skin |
| IT1263144B (en) * | 1993-02-04 | 1996-08-01 | Lanfranco Callegaro | PHARMACEUTICAL COMPOSITIONS INCLUDING SPONGY MATERIAL CONSTITUTED FROM FOREIGN DERIVATIVES OF HYALURONIC ACID IN ASSOCIATION WITH OTHER PHARMACOLOGICALLY ACTIVE SUBSTANCES |
| JPH06292716A (en) * | 1993-04-09 | 1994-10-21 | Shimizu Yoshihiko | Medical material |
| JPH11322807A (en) * | 1998-05-11 | 1999-11-26 | Mitsubishi Chemical Corp | Production of crosslinked hyaluronic acid sponge |
| JPH11319066A (en) * | 1998-05-11 | 1999-11-24 | Mitsubishi Chemical Corp | Wound coating material |
| DE60018814T2 (en) * | 1999-01-21 | 2006-04-06 | Nipro Corp. | Sewable membrane for adhesion prevention |
| JP2000237294A (en) * | 1999-02-18 | 2000-09-05 | Denki Kagaku Kogyo Kk | Medical material containing hyaluronic acid gel |
| JP2001212224A (en) * | 2000-02-04 | 2001-08-07 | Toyobo Co Ltd | Wound coating material |
-
2001
- 2001-12-07 CN CNA018202632A patent/CN1774272A/en active Pending
- 2001-12-07 JP JP2002547548A patent/JP4273450B2/en not_active Expired - Fee Related
- 2001-12-07 AU AU2002221089A patent/AU2002221089A1/en not_active Abandoned
- 2001-12-07 WO PCT/JP2001/010751 patent/WO2002045767A1/en active Application Filing
- 2001-12-07 KR KR10-2003-7005483A patent/KR20030061378A/en not_active Application Discontinuation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724164B (en) * | 2008-10-31 | 2011-12-14 | 科妍生物科技股份有限公司 | Method for preparing cross-linked hyaluronic acid |
| CN101912633A (en) * | 2010-08-03 | 2010-12-15 | 孙伟庆 | Hyaluronic acid sponge and preparation method thereof |
| CN102558600A (en) * | 2011-12-01 | 2012-07-11 | 上海白衣缘生物工程有限公司 | Cross-linked hyaluronan sponge and preparation method for same |
| CN106110367A (en) * | 2016-07-29 | 2016-11-16 | 北京化工大学常州先进材料研究院 | Based on natural polymer MULTILAYER COMPOSITE medical dressing and preparation method thereof |
| CN109998775A (en) * | 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 | A kind of compressed tampon dressing and the preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2002045767A1 (en) | 2004-04-08 |
| AU2002221089A1 (en) | 2002-06-18 |
| JP4273450B2 (en) | 2009-06-03 |
| KR20030061378A (en) | 2003-07-18 |
| WO2002045767A1 (en) | 2002-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1774272A (en) | Substrate for tissue regeneration, material for transplantation, and processes for producing these | |
| CN104144715B (en) | The manufacture method of collagen structure body and collagen structure body | |
| CN102430155B (en) | Cellular silk fibroin porous scaffold, and preparation method thereof | |
| CN1583836A (en) | Preparation of porous sericin sponge material | |
| CN102188747A (en) | Compound tissue engineering scaffold containing PLGA (poly(lactic-co-glycolic acid) strengthening net, and preparation method and application thereof | |
| CN103536954A (en) | Crosslinked alginate-bacterial cellulose sponge and preparation method thereof | |
| CN101537207B (en) | Preparation method of tissue engineering xenoskin | |
| CN105013013A (en) | Preparation method of skin ulcer repairing matrix | |
| CN105664260A (en) | Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin | |
| CN107149702A (en) | A kind of preparation of poly-dopamine modified lithium porous support | |
| CN107596438B (en) | A kind of natural polysaccharide self-healing hydrogel of injectable and the preparation method and application thereof | |
| CN101264337B (en) | Preparation of collagen base biological medical material | |
| CN102652841A (en) | Method for preparing sulfated silk fibroin vascular tissue engineering scaffold by using electrostatic spinning | |
| CN101856516B (en) | Preparation of collagen-chitosan-laser micropore dermal matrix composite membranes | |
| CN105126171A (en) | Gel biological material having shape memory function and preparation method of gel biological material | |
| CN103143058B (en) | Preparation of composite hydrogel tissue engineer cartilage repairing scaffold with biological activity | |
| CN109701078B (en) | Biological sponge based on acellular dermal matrix and preparation method thereof | |
| CN101496915B (en) | Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof | |
| CN101874903A (en) | Method for preparing collagen artificial skin | |
| CN101005865A (en) | Process for producing collagen sponge, process for producing artificial skin, artificial skin and cell tissue culture substrate | |
| CN111001041B (en) | Anti-inflammatory and antibacterial composite skin scaffold material and preparation method thereof | |
| CN100402097C (en) | Skin wound repairing agar/collagen dressing and its prepn and application | |
| CN105176095A (en) | Collagen-based organic silicon rubber porous composite membrane and preparation method thereof | |
| CN101204592A (en) | Process for fabricating engineering esophagus imitating biochemistry tissue | |
| CN114225118A (en) | Injectable artificial dermis for promoting wound healing and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |