CN1363398A - Stratified artificial skin using chitosan or its derivative as matrix clathrum - Google Patents
Stratified artificial skin using chitosan or its derivative as matrix clathrum Download PDFInfo
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- CN1363398A CN1363398A CN 02100072 CN02100072A CN1363398A CN 1363398 A CN1363398 A CN 1363398A CN 02100072 CN02100072 CN 02100072 CN 02100072 A CN02100072 A CN 02100072A CN 1363398 A CN1363398 A CN 1363398A
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Abstract
A stratified artificial skin is disclosed, which uses porous chitosan network as matrix and is composed of epiderm layer and dermis layer. Its preparing process and its application in covering wound and repairing skin and hypoderm are also disclosed.
Description
The present invention relates to body surface wound covering and repair materials.Specifically, the present invention relates to chitin porous rack is host material, comprises the multiple layer artificial skin of epidermal area with vitality and skin corium.The invention further relates to the method for the said multiple layer artificial skin of preparation, and this multiple layer artificial skin reparation of preparation application makes damaged tissues regeneration by human or other mammalian skin tissue defect that a variety of causes causes.
In the vital movement of human body, common because of reasons such as mechanical trauma, physics, chemistry and biological damage cause the body part soft tissue, particularly skin is damaged.When corium is involved in damage (as burn), general being difficult to regenerated, and usually is to form the connective tissue cicatrix to substitute skin histology in the damaged zone.Clinically, transplanted (as using the tissue of monozygotic twins) though once can use autograft, allohisto compatibility, or (as using treated Corii Sus domestica, abortive calfskin and Corii Caprae seu Ovis etc.) method is transplanted by heteroplasm, or use natural or chemical synthetic material covering (as using polyester fiber, crosslinked polyvinyl alcohol foam lamella etc.), temporarily fill and cover impaired skin and hinder face.But said method all has unsurmountable shortcoming.Autograft is actually with wound and cures the wound, and the graft materials source is limited; Heteroplastic transplantation is difficult to overcome immunological rejection; The obducent histocompatibility of chemosynthesis is poor, is unfavorable for exudate discharge or the like.In recent years, many skin substitute products have also appearred on the market, and so-called " artificial skin ".Known these artificial skins comprise with the collagen protein to be the collagen film of feedstock production and to be the chitin film of the non-woven fibre form of raw material with hydrobiological chitin, and freeze dried Corii Sus domestica and fibrin film etc.Yet these artificial skins all can not provide desirable therapeutic, because these so-called artificial skins all are the temporary transient skin-covering agents of no vitality.No Skin Cell composition, its histocompatibility is poor, and pliability is poor, finally will come off, and needs the secondary skin transplantation, can not promote growing into of original position cell proliferation and normal cambium effectively.
As with the closely-related prior art of the present invention; here for example United States Patent (USP) 5 that can mention; 698; provide for No. 228 a kind of skin wound of the squid chitin on the collagen protein of fish skin floor that coincides that comprises more to create compositions; its advantage is to have good flexibility; and can promote the generation of lysozyme, with the protection wound.Simultaneously, said composition also helps fibroblastic adhesion and propagation.In addition, United States Patent (USP) 4,651, described for No. 725 a kind of be applicable to apply cover the skin injury wound surface, comprise the wound dressing of the binding agent that chitin non-woven fibre (fiber number is less than 1 denier, and intensity is greater than 2g/d) and macromolecular material are made.Said wound dressing has the better tissues compatibility, pliability and suitability.Therefore, the former chitin that utilizes is used for making the method for skin culture or Graftskin as tissue engineering material, all is basically to make diaphragm or non-woven fibre tabletting, inoculating or do not inoculating under the situation of body and variant cell, directly be used for applying and cover the skin wound surface.The shortcoming of these materials comprises: be difficult to make from body or variant cell grow in its surface, breed, can not be from the wound surface that is capped for cell provides nutritional support, the wound part that is capped has that too much transudate gathers, covering is easy to be decomposed and shrinkage by wound fluid, and then come off etc.
People such as Eugene Bell are (referring to Science, 211:1052-1054,1981) pass through to cultivate from isolating epidermis cell of skin histology and fibroblast in a large number, and having mixed inoculation epidermis cell in stratification ground on fibroblastic collagen gel, to obtain the skin culture.People (referring to Surgery, 103:421-431,1988) such as Steven Boyce have also reported the epidermis cell that a large amount of cultivations derive from skin, and the method for these cells is cultivated and bred to stratification on spongy collagen matrices.Wherein said spongy substrate adds a small amount of chondroitin sulfate and makes in collagen protein.Moreover, United States Patent (USP) 6,043, No. 089 discloses a kind of is substrate with the collagen material, and has inoculated Skin Cell culture of Skin Cell and preparation method thereof on this substrate.So far, in looking into domestic and foreign literature, patent, Shang Weijian is with the artificial skin of chitin or derivatives thereof as the substrate rack.
As previous work of the present invention, this seminar is former to be engaged in artificial dermis and the research of multiple layer artificial skin for many years, used mammal (comprising the people) collagen protein as the substrate rack, successfully made up have with the multiple layer artificial skin of natural skin analog structure and function (referring to Liu Jinyu etc., China's reconstruction surgical magazine, 15 (4): 235-239,2001; Ma Gang etc., Chinese journal of dermatology, 31 (2): 121-122,1998).The inventor considers all advantageous properties of chitin or derivatives thereof such as the multiple layer of chitosan conduct dermal matrix material, on the basis of above-mentioned previous work, use the porous three-dimensional structure of chitin or derivatives thereof to be the substrate rack, further prepared and had epidermis and the double-deck engineered artificial skin of corium, thereby finished the present invention.
In addition, the three dimensional matrix planar network architecture of also using the chitin or derivatives thereof has built up cartilaginous tissue, shows that this rack has good plastotype character.For being applied to make up other organizational project member, it provides clue.
1. the purpose of this invention is to provide a kind of artificial multiple layer skin, be used for the skin histology of the reconstruction of wound commitment or the impaired wound surface that heals.Be characterised in that the porous three-dimensional support that uses the chitin or derivatives thereof is as artificial skin substrate rack, the medial surface inoculation and the cultivation fibroblast near wound of this substrate rack, and in outside inoculation and the cultivation epidermal keratinocytes of this substrate rack facing to wound.Formed multiple layer artificial skin with epidermis and corium.
2. according to a preferred embodiment of the invention, the optimum thickness of wherein said substrate rack is about 1.5mm.
3. according to a preferred embodiment of the invention, wherein said chitin derivatives comprises chitosan.
4. according to a preferred embodiment of the invention, wherein said somatomedin is epidermal growth factor and fibroblast growth factor.
5. according to a preferred embodiment of the invention, wherein said substrate rack or space contain the somatomedin and the hyaluronic acid of debita spissitudo.
Fig. 1 shows the structure flow process of multiple layer artificial skin.
Fig. 2 shows the scanning electron microscope structure by the chitin substrate rack of the inventive method preparation.
Fig. 3-1 (HE * 100) and Fig. 3-2 (HE * 400) are presented at animal holostrome skin injury position respectively and implant by the Histological change 14 days time the behind the multiple layer artificial skin of the inventive method preparation.
Fig. 4-1 and Fig. 4-2 shows the laboratory animal (nude mice) that has skin of back disappearance (diameter 2.5cm) respectively, and the multiple layer artificial skin and the chitin diaphragm of not inoculating Skin Cell in contrast that prepare by the present invention change in the reparation of transplanting behind the skin injury position.
The present invention relates to for applying lid and repairing the people and defect of skin that mammal causes because of a variety of causes Multiple layer artificial skin (comprising epidermis and corium) preparation method. Be characterised in that said compound people Worker's skin is as the three dimensional matrix rack with the porous spongy chitin or derivatives thereof that obtains after the freeze-drying Material. Engineered Compound artificial skin of the present invention has good histocompatbility, enough Biomechanical strength, can be grown well and breed by epidermis and the dermal cell inoculated, and can with Surface of a wound periphery normal skin tissue fully merges. In addition, multiple layer artificial skin of the present invention has synthetic And the ability of extracellular matrix secretion (such as collagen and proteoglycan), show that this artificial skin is " skin of living " that vitality is arranged. Thereby it can be used as " real artificial skin equivalent " fully Clinically extensive use.
The same with other organizational project, comprise equally having three-dimensional structure in the skin tissue engineering research Extracellular matrix rack, respective organization cell (comprising stem cell), and regulation and control respective organization cell This three basic key element of growth factor of growth, propagation and differentiation. So far, be used for making up artificial skin Extracellular matrix or rack material be the collagen of treated biogenetic derivation mostly, there is not yet Use tegument polysaccharide or derivatives thereof such as partially deacetylated chitin as making up artificial multiple layer skin The report of the host material of skin. The former basis of being engaged in for a long time organizational engineering research of the inventor On, it is artificial at first successfully to have prepared spongy chitin or derivatives thereof (such as chitosan) Multiple layer dermal matrix rack material, and be used for the again preparation of layer artificial skin.
Chitin is to form by the 2-acetylamino-2-deoxy-D-glucose unit that β-Isosorbide-5-Nitrae glycosidic bond connects Linear polysaccharide. The ectoskeleton of insect and crustacean such as crab, lobster and river prawn etc. contains a large amount of shells Polysaccharide makes it to become nature and has this characteristic polysaccharide and be only second to cellulosic second the richest Rich biopolymer. Yet, because chitin is to many aqueous solvents commonly used or organic solvent all Be inert reaction, so both instability also was not easy to processing. For this reason, on the market warp had appearred again afterwards Cross chitin derivatives such as the chitosan of processing. Chitosan is partly or entirely to take off second The chitin of acyl group, and be by monomer β-(Isosorbide-5-Nitrae)-D-aminoglucose unit (accounting for 60%) and The polysaccharide that monomer β-(Isosorbide-5-Nitrae)-2-acetylamino-2-deoxy-D-glucose unit (accounting for 40%) forms. Take off The acetyl Chitosan and its derivatives dissolves in the organic acids such as fumaric acid, acetic acid, propionic acid, some derivative Such as D-carboxymethyl chitosan even water soluble. Because chitosan has good group Knit compatibility and biodegradability, and have bacteria growing inhibiting and promote wound healing etc. useful Biological property, so medical domain can use it for make operation suture thread, wound-surface cover, Immunologic adjuvant and medicinal slow release agent etc. Particularly in the application facet as medicinal slow release agent, for example beautiful State's patent discloses for 4,946, No. 870 and has contained glycosaminoglycan (being chitin) derivative, is used for to office Section's mucous membrane slowly discharges the drug sustained release system of medicine. United States Patent (USP) 5,422 in addition, and No. 116 also open Contain the opthalmological slow releasing composition of chitosan.
In general, the amount of the required acid of dissolving chitosan is about as much as amino group in the molecule Stoichiometric amount. In addition, for obtaining soluble product, required chitin degree of deacetylation must Must be more than 80%, namely the acetyl content of chitosan must be less than 4～4.5%. Therefore, take off The acetyl chitin is not a kind of single, clear and definite chemical entities, and is based on working condition not With and the composition that produces.
For the Matrix sponge for the preparation of artificial skin of the present invention, for example can be with homemade or from the city The deacetylation of buying on the field is dissolved in greater than 85% chitosan and is selected from formic acid, acetic acid, third In the organic acid weak solutions such as acid, tartaric acid, fumaric acid and citric acid, make concentration and be 1～5% Solution adds growth factor (such as EGF and FGF) and the hyaluronic acid of appropriate amount then. To mix The compound high-speed stirred is poured the casting molds of different sizes into after form uniform foam by certain thickness Also directly put into the freeze-drying apparatus freeze drying in the groove. After the freeze-drying, the highly basic of available debita spissitudo (as NaOH) solution soaked 2～4 hours, was washed till neutrality with distilled water again, and is namely available after the dry also sterilization In being implanted to fibrocyte and keratinocyte.
Holostrome skin is made up of epidermis, corium and subcutaneous tissue three parts.Wherein, epidermis mainly comprises and is in different propagation, keratinocyte (KC) and a spot of non-keratinocyte of differential period.Epidermal keratinocytes is the main cell that constitutes epiderm skin, and it is normally bred and breaks up normal configuration and the function that directly has influence on epidermis.Therefore, the In vitro culture of keratinocyte is a common problem in the artificial skin reconstruction technique always.Up to now, the keratinocyte cultural method of having set up comprises trophoderm culture method (Cuono et al, Plast Reconstr Surgery 80 (4): 626-635,1987), serum-free culture method (Steven et al, Surgery 103 (40): 421-431,1988) gentle surface culture method (Boyce et al, J Biomed Mater Res 22:937-957,1988).The present invention finds through the comparative experiments to several method, with neutral protease and trypsin associating digestion method separating table chrotoplast, use the NIH3T3 cell as trophoderm, and use contains blood serum medium and low inoculum density is cultivated epidermal keratinocytes, can realize the optimal separation and the culture effect of epidermal keratinocytes.What should particularly point out is, the inventor is on the basis of having carried out a large amount of preliminary experiments, designed especially except that containing hyclone and antibiotic and HEPES, also contained the DMEM culture medium of insulin, transferrins, 3, cholera toxin, hydrocortisone and the various kinds of cell somatomedin of appropriate amount.Cultivation results shows that in such cell culture medium, the keratinocyte multiplication rate obviously improves, and passage is respond well.
As the formation cell of dermal tissue, fibroblast (FB) has the function of synthetic justacrine extracellular matrix and cytokine, and it plays a significant role in tissue formation, organ generation and repair in trauma.For example, fibroblast divides a word with a hyphen at the end of a line, assembles and activate to target site by himself chemotactic factor, produces the justacrine collagen protein simultaneously forming a series of collagen tissues, and then causes tissue regeneration and repair in trauma.The isolated culture method of the skin flbroblast of having set up at present, comprises tissue mass cell culture, collagenase digestion and collagenase and neutral protease associating digestion method.The present invention selects for use collagenase to separate with neutral protease associating digestion method, and uses the DMEM culture medium that contains 15% (v/v) hyclone to cultivate fibroblast according to a conventional method.In general, cultivated 7～8 days, cell can form cell monolayer in culture bottle.
In order to prepare of the present invention is the multiple layer artificial skin of substrate rack with the chitin or derivatives thereof, can with the chitin substrate net frame cell culture fluid that as above prepares and alcoholic solution (75%) soaks and with after the irradiation under ultraviolet ray sterilization, inoculate suitable cell density successively respectively in these spongy rack both sides, as stated above separation and purification and the epidermal keratinocytes (wound surface distally) and the fibroblast (being close to the wound surface side) of going down to posterity and cultivating.Therebetween, can be behind the inoculation fibroblast, with the cell culture 4～6 days that is attached on the rack, then, again in offside surface seeding epidermal keratinocytes.The substrate rack of having inoculated two kinds of Skin Cells is being cultivated different time respectively on the culture medium liquid level He on the liquid gas interface successively.When breaking up, epidermal growth has well-bedded each layer, the gradual change of the most surperficial (horny layer) cell is flat, and skin corium have a large amount of fibroblasts and by extracellular matrix when accumulation of generations justacrine, resulting multiple layer artificial skin material can be transplanted on the skin wound of animal experimental model, so as to observing treatment and the repairing effect of multiple layer artificial skin of the present invention skin trauma.The structure flow process of the multiple layer of the present invention artificial skin as shown in Figure 1.
Observed result shows, transplant back 7 days promptly visible of the present invention be the multiple layer artificial skin of host material with the chitin and normal skin tissue's good knitting on every side, the epidermis thickening also the keratinization phenomenon occurs, and the skin corium cell number increases simultaneously, and the substrate rack is decomposed by cell gradually.After transplanting the 14th day, visible blood capillary number increases sharply, and epidermal area engages well with skin corium.Transplanted the back the 21st day, normal skin combines together substantially around graft and the wound surface, and the substrate rack disappears substantially, and replaces newborn Skin Cell and extracellular matrix.
The present invention is that to an important improvement of prior art the using-system compatibility is good, have antiinflammatory antibacterial, promote wound healing and blood capillary regeneration, and the chitin or derivatives thereof that material source is extensive, cost is relatively low replaces the collagen protein that generally uses the at present substrate as the multiple layer of preparation artificial skin.Existing result of study shows that chitin is the important polysaccharose substance that constitutes mantle, itself is easy to be organized very much lysozyme and decomposes.Therefore, chitin has more some unique advantage than collagen protein.Our experimental studies results further shows, use the substrate rack material of chitin or derivatives thereof as multiple layer artificial skin, more help inoculating other the growth of Skin Cell and propagation, generation extracellular matrix, local blood capillary generates and rebuilds, thereby has quickened the regeneration and the reparation of wound site skin histology greatly.Achievement in research of the present invention is for using the chitin host material, the multiple layer of the organizational project of preparation " work " artificial skin, and for the clinical successful experience that provides is provided for organization engineering skin the most at last, also can be for the experiment basis of using for reference for using in the future chitin to make up as the substrate rack that other tissue or organ provide.
Below by specific embodiment the present invention is described for example further, but it will be appreciated by those skilled in the art that, under the prerequisite that does not deviate from the present invention's spirit and principle, to any change and the change that some ins and outs involved in the present invention are done, all will fall into the present invention and await the reply in the claim scope.
Embodiment 1
Present embodiment uses from rat skin and separates the also epidermis and the hypodermal cell of purification, use mainly spongy body by the chitin material preparation as the substrate rack, preparing multiple layer artificial skin, and observe its good effect in the skin histology repair in trauma of nude mice receptor.
(1) histiocytic separation of rat skin and purification
Select the healthy adult rat, under anesthesia, cut the holostrome skin of about 3 * 3cm size from back part of animal.After 3 minutes, (every liter contains 4g KCl, 0.6g KH to the D-Hanks liquid of reuse dilution in 1: 10 with 75% soak with ethanol
2PO
4, 80g NaCl, 2.50g NaHCO
3With 0.8g Na
2HPO
412H
2O) wash 3 times.Skin histology is cut into 5 * 5mm fritter and with digestion under neutral protease (40g/L) room temperature 24 hours, separates epidermis and dermal tissue then.
With epidermal tissue with containing trypsin 0.25g/L) and EDTA (0.1g/L) solution, 37 ℃ of digestion were crossed 200 order mesh screens with digestion product after 5 minutes.Reuse contains penicillin (10
5After the unit/L) and the DMEM culture medium of streptomycin (100mg/L) are washed 3 times with cell, check the viability of cell with the blue staining of tongue phenol.Containing 15% (v/v) hyclone, penicillin (10 then
5Unit/L) and in the DMEM culture medium of streptomycin (100mg/L), insulin (6mg/L), transferrins (5mg/L), hydrocortisone (0.8mg/L) and epidermal growth factor (20 μ g/L) and at 6.8%CO
2Under the environment, 37 ℃ of cultured cells.
Use 37 ℃ of digestion of type i collagen enzyme (2.5g/L) after 4 hours dermal tissue, basically handle by the same quadrat method of above-mentioned processing epidermal tissue, and use contain hyclone (15%, v/v), HEPES (10mM) and antibiotic (penicillin and streptomycin, concentration is the same) culture medium, at 5%CO
2The following 37 ℃ of cultured cells of environment.
Treat that two kinds of cells all breed to enough cell densities (1～5 * 10
6Behind the cell/ml), carry out morphocytology once more according to a conventional method and identify.Frozen these seed cells in liquid nitrogen or-70 ℃ of refrigerators then.In cell freezing and the recovery process, impaired for preventing cell, must in cell freezing liquid, add dimethyl sulfoxide (DMSO) and glycerol as protective agent, and select suitable freezing and freeze thawing speed.Employed cell freezing liquid is for being added with the above-mentioned culture medium of 10% (v/v) DMSO in the present embodiment, and chilling rate is-1 ℃/minute.Cell suspension is sub-packed in finishes freezing and recovery process in the 1ml cryovial.During use, cryovial is taken out from liquid nitrogen container or cryogenic refrigerator and directly puts into 37 ℃ of water baths and vibrate, melt fully up to content.Behind the low-speed centrifugal cell, add fresh culture and continue according to a conventional method and cultivate.
The NIH3T3 cell that uses mitomycin (4mg/L) processing is inoculated keratinocyte (2.0 * 10 as trophoblastic cell on trophoblastic cell
4/ cm
2) back in 37 ℃ and 6.8%CO
2Cultivate it under the condition.Changed culture fluid every 3 days, the cell growth is linked to be cell monolayer after about 10 days.Repeatedly with containing the trypsin 0.25g/L of EDTA (0.1g/L)) after the simple digestion of solution, continue cultured cell and also go down to posterity.
In addition, the fibroblast suspension that separates and cultivate is pressed 1.0 * 10
5/ cm
2Density is inoculated in the glass culture bottle, cultivates according to the conventional method of above-mentioned cultivation keratinocyte basically and goes down to posterity.
With normal dyeing exclusive method (using tetrazolium bromide (MTT) dyestuff) or colony-forming efficiency detection method detection by quantitative cell survival rate, and definite growth curve.
(2) with chitin be the preparation of the multiple layer artificial skin substrate rack of basic host material
Purified commercial chitin (moral prosperous biotech company in Jilin produces deacetylation＞87%) is dissolved in and makes 2% (w/v) chitin solution in 1.5% acetic acid.With resulting solution fast (2000rpm) be stirred to cystose, casting mold and lyophilization in nappy then obtains the artificial skin substrate rack of the about 1.5mm of thickness.
By the morphology ultrastructure of the artificial skin substrate rack of the method for the invention preparation as shown in Figure 2.
(3) preparation of multiple layer artificial skin
At first, will through cultivation and the fibroblast of the purification that goes down to posterity by 3.0 * 10
5Cell/cm
2Density be inoculated on the side of the chitin substrate rack of preparation in the above-mentioned steps (2), cultivate 4 days (middle change culture fluid 1 time) for 37 ℃.Then, will be through the above-mentioned keratinocyte of handling with quadrat method by 4.0 * 10
5Cell/cm
2Density be inoculated in the opposite side of this substrate rack.The substrate rack of inoculating two kinds Skin Cell is placed the complex medium of above-mentioned cultivation keratinocyte, and 37 ℃ are continued to cultivate 6 days (changing culture fluid 2 times in the incubation), use gas-liquid face culture method then instead and continue to cultivate.After cultivation is finished, can will be somebody's turn to do treatment and reparation that artificial multiple layer skin graft is used for consubstantiality or allogenic animal skin and subcutaneous part soft tissue injury wound surface.
(4) application and the effect observation of multiple layer artificial skin of the present invention in repairing the nude mice skin trauma
(1.5 * 1.5cm) adult nude mice is as experimental animal model in order to cause the holostrome skin injury with the back people.After the wound 2 hours, press close to wound surface to inoculate a fibroblastic side, (1.5 * 1.5cm) are transplanted to wound site with the multiple layer artificial skin of as above preparation.After the transplanting, passed through gross examination of skeletal muscle, light microscopic and Electronic Speculum histological examination, and the repairing effect of multiple layer artificial skin of the present invention to the receptor skin trauma observed and analyzed to the infrared thermal imagery scanning technique every 3 days.The result as seen, 3 days grafts of post-transplantation and normal skin tissue's good knitting, and fit closely with wound surface.As seen transplant had a small amount of blood capillary to grow in graft from normal surrounding tissue under the mirror after 6 days, and the newborn number of capillaries in the implant matrix of growing into increases gradually.Normal skin color is very approaching on every side for the artificial skin of transplanting and its.Postoperative 9 days, as seen graft zone blood capillary rolls up, the high-visible well-bedded basal layer of epidermal area, prickle cell layer, granular layer and horny layer, and visible top layer has the keratinocyte layer to peel off to come off, simultaneously visible skin corium cell quantity and extracellular matrix significantly increase, and the substrate rack almost all is absorbed and is replaced (referring to Fig. 3-1 and 3-2) by these proliferating cells and secreted extracellular matrix thereof.Postoperative 15 days, substantially visible wound surface heal substantially, it is normal to repair district's skin color, and residual cicatrix seldom (referring to Fig. 4-1 and 4-2).
Claims (5)
1. one kind is used for that the wound commitment is rebuild or the artificial multiple layer skin of the skin histology of healing damaged, be characterised in that and wherein use chitin or derivatives thereof mandruka as the cell culture matrix scaffold, and said matrix scaffold near the inoculation of the medial surface of wound and cultivate fibroblast, simultaneously, inoculate and cultivate epidermal keratinocytes in the outside facing to wound of said substrate.
2. according to the multiple layer artificial skin of claim 1, wherein the optimum thickness of said matrix scaffold is about 1.5mm.
3. according to the multiple layer artificial skin of claim 1, wherein said chitin derivatives also comprises chitosan.
4. according to the multiple layer artificial skin of claim 1, wherein said matrix scaffold or space contain the somatomedin and the hyaluronic acid of debita spissitudo.
5. according to the multiple layer artificial skin of claim 1, wherein said somatomedin is selected from epidermal growth factor and fibroblast growth factor.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003076604A3 (en) * | 2002-03-06 | 2004-03-04 | Univ Cincinnati | Surgical device for skin therapy or testing |
| CN1297324C (en) * | 2003-07-25 | 2007-01-31 | 吕伟光 | Tissue engineering autologous complex skin and its preparation method |
| CN101967464A (en) * | 2010-09-28 | 2011-02-09 | 浙江大学 | Preparation method of porous rack mechanism matrix and application thereof |
| CN101316618B (en) * | 2005-11-14 | 2011-12-14 | 株式会社大熊 | Sustained release film formulation for healing wound comprising epidermal growth factor |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | Process for the production of patches or dressings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
| CN103764816A (en) * | 2011-05-30 | 2014-04-30 | 莱雅公司 | Reconstructed scalp model and process for screening active molecules |
| CN110841113A (en) * | 2019-11-27 | 2020-02-28 | 黑龙江紫泰科技有限公司 | Preparation method of tissue engineering skin |
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2002
- 2002-01-16 CN CN 02100072 patent/CN1363398A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003076604A3 (en) * | 2002-03-06 | 2004-03-04 | Univ Cincinnati | Surgical device for skin therapy or testing |
| US7741116B2 (en) | 2002-03-06 | 2010-06-22 | University Of Cincinnati | Surgical device for skin therapy or testing |
| EP2302035A3 (en) * | 2002-03-06 | 2011-06-01 | University Of Cincinnati | A method of producing a cultured skin device |
| US8765468B2 (en) | 2002-03-06 | 2014-07-01 | University Of Cincinnati | Surgical device for skin therapy or testing |
| US9089417B2 (en) | 2002-03-06 | 2015-07-28 | Steven T. Boyce | Surgical device for skin therapy or testing |
| CN1297324C (en) * | 2003-07-25 | 2007-01-31 | 吕伟光 | Tissue engineering autologous complex skin and its preparation method |
| CN101316618B (en) * | 2005-11-14 | 2011-12-14 | 株式会社大熊 | Sustained release film formulation for healing wound comprising epidermal growth factor |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | Process for the production of patches or dressings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
| CN103003415B (en) * | 2010-05-21 | 2017-02-08 | 罗德里戈·福西翁·索托帕雷哈 | Process for the production of patches or dressings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
| CN101967464A (en) * | 2010-09-28 | 2011-02-09 | 浙江大学 | Preparation method of porous rack mechanism matrix and application thereof |
| CN103764816A (en) * | 2011-05-30 | 2014-04-30 | 莱雅公司 | Reconstructed scalp model and process for screening active molecules |
| CN110841113A (en) * | 2019-11-27 | 2020-02-28 | 黑龙江紫泰科技有限公司 | Preparation method of tissue engineering skin |
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