CN101954124A - Tissue engineered skin with basilar membrane and construction method thereof - Google Patents
Tissue engineered skin with basilar membrane and construction method thereof Download PDFInfo
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- CN101954124A CN101954124A CN2010102710100A CN201010271010A CN101954124A CN 101954124 A CN101954124 A CN 101954124A CN 2010102710100 A CN2010102710100 A CN 2010102710100A CN 201010271010 A CN201010271010 A CN 201010271010A CN 101954124 A CN101954124 A CN 101954124A
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- amniotic membrane
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Abstract
The invention relates to the technical fields of tissue engineering and medical wound repair. At present, a living skin substitute constructed by using the materials of polylactic acid, polyglycolic acid, collagen, hyaluronic acid, and the like as a dermic bracket has the defects that on one hand, host materials are difficult to extract, the living skin substitute has complicated manufacturing technology and is expensive in cost and difficult to widely popularize and apply clinically; and on the other hand, the living skin substitute does not have a skin basilar membrane structure so that healed skin does not resist pressure and wear, and the living skin has unfirm adhesion to the epidermis and is easy to shed and break or form water blisters so that the structural and morphological development of the normal epidermis is influenced; and allogeneic acellular dermis is taken from cadaver skin and is limited in sources and expensive in cost, and thus clinical application is limited. The invention aims at providing a skin substitute which uses surface-finished and modified amnion as the basilar membrane and blood plasma as stroma, which has the advantages of wide material sources, low cost and simple preparation method. An animal experiment proves that the complete basilar membrane and hemidesmosomes can be retained in in-vivo transplantation, and the formation of an epidermal structural form is accelerated and promoted.
Description
Technical field
The present invention relates to organizational project and medical science wound repair technical field, be specifically related to a kind of that amniotic membrane is surface modified back as basement membrane, compound blood plasma is substrate, make up the organization engineering skin (being the viable skin substitute) contain basement membrane through In vitro culture, be specially adapted to burn, the skin injury reparation and the cicatrix shaping of wound.
Background technology
At present, make up dermis scaffold with Biodegradable materials such as polylactic acid, polyglycolic acid, collagen, hyaluronic acids, make up the viable skin substitute on this basis, Preliminary Applications is in the reparation of degree of depth skin injury wound surface and cicatrix shaping etc.But the host material extraction is difficult on the one hand for the Graftskin that makes up thus, complex manufacturing technology, the expense costliness, be difficult in clinical wide popularization and application, it does not possess the basement membrane structure of skin on the other hand, causes healing skin not withstand voltage and wear-resisting, and epidermis adheres to not firm, come off easily breakage or form vesicle, and influence the growth of normal epidermis structure, form.And the allosome acellular dermal is taken from cadaver skin, and it is limited to originate, and costs an arm and a leg, and has limited its clinical application.
Human amnion tissue is the translucent tissue that contains the simple epithelium cell, no blood vessel, nerve, lymphatic vessel, thickness is 0.02-0.5mm, the form and the skin basement membrane of its basement membrane are closely similar, and mainly form (Zhao Min, Lu Jing by laminin and IV Collagen Type VI, Zhang Qi, Deng .HGF, bFGF, ColIV, the LN expression in preserving amniotic membrane. ophthalmology research, 2006,24 (5): 514-517.).This stranger's amniotic membrane is the discarded tissue of medical treatment, and wide material sources are drawn materials conveniently, and it early is used for the covering of burn wound as a kind of biological dressing, and the inflammation that not only can reduce wound surface promotes epithelization but also can suppress the formation of cicatrix.The lyophilization bioamnion of at present existing commercial amniotic membrane product such as Jiangxi RuiJi Biotechnology Co., Ltd.
Still do not have both at home and abroad bibliographical information with amniotic membrane as basement membrane, compound blood plasma is substrate, makes up the organization engineering skin contain basement membrane through In vitro culture.
Summary of the invention
It is basement membrane with surface modified amniotic membrane that the object of the invention is to provide a kind of,, being the Graftskin of substrate from body/allosome blood plasma, and construction method.Material source of the present invention is extensive, and preparation method is simple, and is with low cost, and the basement membrane and the hemi desmosome that are kept perfectly in can transplanting in vivo quicken and promote the formation of epidermal structure form.
Of the present invention is basement membrane with surface modified amniotic membrane, and blood plasma is the construction method of the organization engineering skin that contains basement membrane of substrate, comprises the steps:
A) separate, cultivate epithelial cells, fibroblast (reference: Liu Dewu, Li Guohui, Zou Ping, Liu Deming. epidermis cell, the compound acellular dermal matrix of fibroblast make up organization engineering skin. Chinese clinical rehabilitation, 2004,8 (8): 1439-1441.).
B) surface modified amniotic membrane:
Amniotic membrane after the removal chorion, after normal saline soaks repeatedly, cleans, 37 ℃ of digestion of EDTA solution in 0.02% 2 hours, residual cell debris and chorion tissue are thoroughly removed in the sonic oscillation washing, place the normal saline that contains 1000U/ml gentamycin, 2.5g/ml amphotericin B to soak 20 to 50 minutes.
C) make up organization engineering skin;
With B) amniotic membrane of step preparation with contain fibroblastic blood plasma matrix composite, again with epithelial cells with 1-5 * 10
5Individual/cm
2Density be inoculated in the amniotic membrane epidermis side, In vitro culture 2-3 week.
Construction method concrete steps of the present invention are as follows:
1, separates, cultivates people's epithelial cells, fibroblast
The utilization unnecessary skin histology after discarded prepuce tissues of postoperative or the anaplasty of peritomizing adopts enzyme digestion and tissue block adherent method to separate, cultivate epithelial cells and fibroblast respectively.Be prepared into single cell suspension respectively, press 2-3 * 10
5Individual/ml cell density is inoculated in the culture bottle, places 37 ℃ of incubators, and epithelial cells is cultivated with serum-free medium, and fibroblast is cultivated with perfect form DMEM culture fluid, goes down to posterity, increases when 70%-80% merges when cell reaches.
2, surface modified amniotic membrane
Peel off fresh amnion under the aseptic condition and (take from hepatitis virus antibody, the puerpera's that cuts open the belly that syphilis antibody and HIV are all negative placenta tissue), passivity is separated chorion, soak repeatedly through normal saline, after the cleaning, in 0.02% EDTA solution, 37 ℃ digested 2 hours, put into high frequency ultrasound washer sonic oscillation washing 2 hours subsequently, thoroughly remove residual cell debris and chorion tissue,, place to contain the 1000U/ml gentamycin repeatedly after the soaking and washing through distilled water then, prepare surface modified amniotic membrane 2.5g/ml soak 30min in the normal saline of amphotericin B.
3, make up organization engineering skin
At first the amniotic membrane basal surface with preparation upwards is laid on the culture plate, treats that its bone dry is adherent.Get the centrifugal blood plasma that obtains behind patient or the allosome whole blood, remove cell debris, fibroblast is added compound concentration is 5 * 10 in the blood plasma of preparation through microfilter
5Individual/ml, fully add behind the mixing in the above-mentioned culture dish that is covered with amniotic membrane.Add the calcium chloride solution of 1M subsequently with 1: 40 volume ratio, promote its polymerization, treat that it aggregates into gel after, add the 1 * DMEM solution that contains 20% hyclone, in In vitro culture 3-5 days.Subsequently epithelial cells is pressed 1-5 * 10
5Individual/cm
2Density be inoculated on the amniotic membrane, treat cell fusion after, carry out liquid-vapor interface and cultivate 2-3 week, changed liquid once in per 2～3 days, forming with the amniotic membrane is basement membrane, plasmagel is the viable skin substitute of substrate.
The present invention also provides the organization engineering skin that makes up according to said method.
The present invention also provides the application as the wound repair graft materials of the organization engineering skin that makes up according to said method.
The present invention is basement membrane with the amniotic membrane, and plasmagel is the viable skin substitute of substrate, and main uses is to repair holostrome skin injury wound surface, for serious extensive deep burn patient provides Pi Yuan.On the other hand, also can be applicable to skin injury wound surface and cicatrix shapings such as chronic degree of depth skin ulcer, avulsion injury of skin.The viable skin substitute method of the present invention's preparation is simple, draw materials conveniently, with low cost, find more not contain traditional organization engineering skin of amniotic membrane in addition in the In vitro culture, epidermal growth is more rapid, configuration is the convergence normal skin more, possesses perfect basement membrane, and hemi desmosome is grown more ripe firm simultaneously.Show that through animal (nude mice) transplantation experiments the viable skin substitute is implanted holostrome skin injury wound surface, and survival rate reaches 88%, visible complete basement membrane and well-developed epidermal area form and the hemi desmosome of newborn skin.
Therefore, the present invention carries out surface modified back as basement membrane with amniotic membrane, and compound to contain fibroblastic plasmagel be substrate, and plantation epithelial cells in surface makes up the viable skin substitute that contains basement membrane through In vitro culture.Manufacture method of the present invention is simple, and material source is extensive, and is with low cost, got rid of influences such as the infection that the xenogenesis source may cause, repulsion simultaneously.
The specific embodiment
Now in conjunction with the embodiments, the present invention is further described, but enforcement of the present invention is not limited in this.
Embodiment 1. preparations contain the viable skin substitute of basement membrane
1, separates, cultivates people's epithelial cells, fibroblast
The utilization discarded prepuce tissues of postoperative of peritomizing adopts enzyme digestion to separate, cultivate epithelial cells, fibroblast.Be prepared into single cell suspension respectively, press 2-3 * 10
5Individual/ml cell density is inoculated in the culture bottle, place 37 ℃ of incubators, epithelial cells is cultivated (DK-SFM with serum-free medium, Gibco, the U.S.), fibroblast is cultivated with perfect form DMEM culture fluid (Gibco, the U.S.), goes down to posterity, increases when 70%-80% merges when cell reaches.
2, surface modified amniotic membrane
Peel off fresh amnion under the aseptic condition and (take from hepatitis virus antibody, the puerpera's that cuts open the belly that syphilis antibody and HIV are all negative placenta tissue), passivity is separated chorion, soak repeatedly through normal saline, after the cleaning, (Gibco in 0.02% EDTA solution, the U.S.), 37 ℃ digested 2 hours, put into high frequency (59KHz) ultrasonic cleaner (SK8200H subsequently, section leads, Shanghai), residual cell debris and chorion tissue are thoroughly removed in sonic oscillation washing 2 hours,, place to contain the 1000U/ml gentamycin repeatedly after the soaking and washing through distilled water then, 2.5g/ml it is standby to soak 30min in the normal saline of amphotericin B.
3, make up organization engineering skin:
At first the amniotic membrane basal surface with preparation upwards is laid on the culture plate, treats that its bone dry is adherent.Get the patient or the allosome whole blood places the anticoagulant tube that contains sodium citrate anticoagulant, the centrifugal 5min of 600g collects blood plasma, removes cell debris through the filter sterilization in 20um aperture, fibroblast is added compound concentration is 5 * 10 in the blood plasma of preparation
5Individual/ml, fully behind the mixing, with 1: 6 volume (ml) and culture plate area (cm
2) add in the above-mentioned culture dish that is covered with amniotic membrane than containing fibroblastic blood plasma.Add the calcium chloride solution of 1M subsequently with 1: 40 volume ratio, promote its polymerization, treat that it aggregates into gel after, add the 1 * DMEM solution that contains 20% hyclone, in In vitro culture 3-5 days.Subsequently epithelial cells is pressed 1-5 * 10
5Individual/cm
2Density be inoculated on the amniotic membrane, treat cell fusion after, carry out liquid-vapor interface and cultivate 2-3 week, changed liquid once in per 2～3 days, forming with the amniotic membrane is basement membrane, plasmagel is the viable skin substitute of substrate.
The zoografting test of embodiment 2. organization engineering skins of the present invention
Male Balb/c-nu mice (nude mice, west, Shanghai pul-Bi Kai laboratory animal company limited provides), body weight 18 ± 2 grams, 16, be divided into is two groups, be respectively the present invention contain amniotic membrane organization engineering skin A group and do not contain traditional viable skin substitute B group of amniotic membrane: be merely that substrate makes up and (sees A.L.Mazlyzama for details with the fibrin, B.S.Aminuddinb, et al.Reconstruction of living bilayerhuman skin equivalent utilizing human fibrin as a scaffold.Burns, 2007,33 (3): 355-363).
Nude mice is shaved except that the skin of back hair after the ketamine intraperitoneal anesthesia, and excision spinal column inclined to one side veutro portion's holostrome skin and deep fascia reach Musclar layer.A group: the organization engineering skin through In vitro culture 2-3 week described in the embodiment 1 is transplanted in wound surface.The B group: traditional viable skin substitute that will not contain amniotic membrane is transplanted in wound surface.All skin shrinks and epidermis is creeped in order to prevent to create, and result's observation is transplanted in influence, adopts cage ring with wound surface and the isolation of peripheral skin.Cover oil-sand on the Composite Skin, periodic replacement dressing is also observed the wound healing situation.Open wound surface after the Composite skin 2 weeks and observe survival rate.And draw materials, make the paraffin section of thick about 5 μ m, row conventional organization section HE dyeing is observed and transmission electron microscope observing.
The result shows that organization engineering skin of the present invention is compared the survival rate no significant difference with traditional group. As seen histology HE dyeing observed, the epidermal structure in 4 weeks physically well developed after organization engineering skin of the present invention was transplanted, form is the convergence normal skin more, transmission electron microscope observing sees to possess perfect basilar memebrane, hemidesmosome is grown more ripe simultaneously, and control group has no continuous basilar memebrane formation and hemidesmosome developmental defect. The in addition viable skin substitute of the present invention preparation, formed multiple layer epidermal cell adheres to more close and firm with dermal substitute, is difficult for disengaging.
Claims (4)
1. a construction method that contains the organization engineering skin of basement membrane is characterized in that this method comprises the steps:
A) separate, cultivate epithelial cells, fibroblast;
B) surface modified amniotic membrane:
Amniotic membrane after the removal chorion, after normal saline soaks repeatedly, cleans, 37 ℃ of digestion of EDTA solution in 0.02% 2 hours, residual cell debris and chorion tissue are thoroughly removed in the sonic oscillation washing, place the normal saline that contains 1000U/ml gentamycin, 2.5g/ml amphotericin B to soak 20 to 50 minutes;
C) make up organization engineering skin;
With B) amniotic membrane of step preparation with contain fibroblastic blood plasma matrix composite, again with epithelial cells with 1-5 * 10
5Individual/cm
2Density be inoculated in the amniotic membrane epidermis side, In vitro culture 2-3 week.
2. the construction method that contains the organization engineering skin of basement membrane according to claim 1 is characterized in that the concrete steps of this method are as follows:
A) separate, cultivate people's epithelial cells, fibroblast:
The utilization discarded prepuce tissues of postoperative of peritomizing adopts enzyme digestion to separate, cultivates epithelial cells, fibroblast, is prepared into single cell suspension respectively, presses 2-3 * 10
5Individual/ml cell density is inoculated in the culture bottle, places 37 ℃ of incubators, and epithelial cells is cultivated with serum-free medium, and fibroblast is cultivated with perfect form DMEM culture fluid, goes down to posterity, increases when 70%-80% merges when cell reaches;
B) surface modified amniotic membrane:
Peel off fresh amnion under the aseptic condition, passivity is separated chorion, after normal saline soaks repeatedly, cleans, in 0.02% EDTA solution, 37 ℃ digested 2 hours, put into high frequency ultrasound washer sonic oscillation washing 2 hours subsequently, thoroughly removed residual cell debris and chorion tissue, through distilled water repeatedly after the soaking and washing, it is standby to place the normal saline that contains 1000U/ml gentamycin, 2.5g/ml amphotericin B to soak 30min then;
C) make up organization engineering skin:
With step B) preparation the amniotic membrane basal surface upwards be laid on the culture plate, treat that its bone dry is adherent, get the patient or the allosome whole blood places the anticoagulant tube that contains sodium citrate anticoagulant, the centrifugal 5min of 600g, collect blood plasma, cell debris is removed in filter sterilization through the 20um aperture, is 5 * 10 with compound concentration in the blood plasma of fibroblast adding preparation
5Individual/ml, fully behind the mixing, with 1: 6 volume (ml) and culture plate area (cm
2) add in the above-mentioned culture dish that is covered with amniotic membrane than containing fibroblastic blood plasma.The calcium chloride solution that adds 1M subsequently with 1: 40 volume ratio, treat that it aggregates into gel after, add the 1 * DMEM solution that contains 20% hyclone, in In vitro culture 3-5 days; Subsequently epithelial cells is pressed 1-5 * 10
5Individual/cm
2Density be inoculated on the amniotic membrane, treat cell fusion after, carry out liquid-vapor interface and cultivate 2-3 week, changed liquid once in per 2～3 days.
3. organization engineering skin that method as claimed in claim 1 or 2 makes up.
4. the application of organization engineering skin as claimed in claim 3 in preparation wound repair graft materials.
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| CN201010271010.0A CN101954124B (en) | 2010-08-31 | 2010-08-31 | Tissue engineered skin with basilar membrane and construction method thereof |
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|---|---|---|---|
| CN201010271010.0A CN101954124B (en) | 2010-08-31 | 2010-08-31 | Tissue engineered skin with basilar membrane and construction method thereof |
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| Publication Number | Publication Date |
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| CN101954124A true CN101954124A (en) | 2011-01-26 |
| CN101954124B CN101954124B (en) | 2014-06-04 |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102499998A (en) * | 2011-12-22 | 2012-06-20 | 中国农业科学院北京畜牧兽医研究所 | Dermis equivalent constructing method |
| CN103074295A (en) * | 2013-02-06 | 2013-05-01 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN103520773A (en) * | 2013-10-24 | 2014-01-22 | 北京积水潭医院 | Method for preparing decellularized dermis material for skin grafting |
| CN103520772A (en) * | 2013-10-24 | 2014-01-22 | 北京积水潭医院 | Improved process for preparing acellular dermal material for skin transplantation |
| CN104189950A (en) * | 2014-09-04 | 2014-12-10 | 成都清科生物科技有限公司 | Amniotic biological agent and preparation method thereof |
| CN105688287A (en) * | 2016-01-26 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | Amniotic membrane patch for treating skin wound and preparation method thereof |
| CN107320781A (en) * | 2017-07-11 | 2017-11-07 | 广州润虹医药科技股份有限公司 | Organization engineering skin containing living cells and preparation method thereof |
| CN108026509A (en) * | 2015-07-30 | 2018-05-11 | Ucl商业有限公司 | Method and apparatus for producing decellularization organization bracket |
| CN108760933A (en) * | 2018-05-02 | 2018-11-06 | 梧州市食品药品检验所 | The method that LC-MS detects amphotericin B content in blood |
| CN109481737A (en) * | 2017-09-12 | 2019-03-19 | 中国人民解放军第三军医大学第附属医院 | Bionical double-deck dressing of one kind and preparation method thereof |
| CN109771697A (en) * | 2018-12-29 | 2019-05-21 | 江苏艾尔康生物医药科技有限公司 | A kind of dermal fibroblast skin graft and its construction method and application |
| CN114028618A (en) * | 2021-10-25 | 2022-02-11 | 广东普洛宇飞生物科技有限公司 | Biological material based on amniotic membrane basement membrane and preparation method and application thereof |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102499998A (en) * | 2011-12-22 | 2012-06-20 | 中国农业科学院北京畜牧兽医研究所 | Dermis equivalent constructing method |
| CN103074295A (en) * | 2013-02-06 | 2013-05-01 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN103074295B (en) * | 2013-02-06 | 2014-10-08 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN103520773A (en) * | 2013-10-24 | 2014-01-22 | 北京积水潭医院 | Method for preparing decellularized dermis material for skin grafting |
| CN103520772A (en) * | 2013-10-24 | 2014-01-22 | 北京积水潭医院 | Improved process for preparing acellular dermal material for skin transplantation |
| CN103520772B (en) * | 2013-10-24 | 2015-05-20 | 北京积水潭医院 | Improved process for preparing acellular dermal material for skin transplantation |
| CN103520773B (en) * | 2013-10-24 | 2015-07-01 | 北京积水潭医院 | Method for preparing decellularized dermis material for skin grafting |
| CN104189950A (en) * | 2014-09-04 | 2014-12-10 | 成都清科生物科技有限公司 | Amniotic biological agent and preparation method thereof |
| CN108026509A (en) * | 2015-07-30 | 2018-05-11 | Ucl商业有限公司 | Method and apparatus for producing decellularization organization bracket |
| CN108026509B (en) * | 2015-07-30 | 2021-07-06 | Ucl商业有限责任公司 | Method and apparatus for generating decellularized tissue scaffolds |
| CN105688287A (en) * | 2016-01-26 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | Amniotic membrane patch for treating skin wound and preparation method thereof |
| CN107320781A (en) * | 2017-07-11 | 2017-11-07 | 广州润虹医药科技股份有限公司 | Organization engineering skin containing living cells and preparation method thereof |
| CN109481737A (en) * | 2017-09-12 | 2019-03-19 | 中国人民解放军第三军医大学第附属医院 | Bionical double-deck dressing of one kind and preparation method thereof |
| CN109481737B (en) * | 2017-09-12 | 2021-07-06 | 中国人民解放军第三军医大学第一附属医院 | Bionic double-layer dressing and preparation method thereof |
| CN108760933A (en) * | 2018-05-02 | 2018-11-06 | 梧州市食品药品检验所 | The method that LC-MS detects amphotericin B content in blood |
| CN109771697A (en) * | 2018-12-29 | 2019-05-21 | 江苏艾尔康生物医药科技有限公司 | A kind of dermal fibroblast skin graft and its construction method and application |
| CN109771697B (en) * | 2018-12-29 | 2021-09-07 | 江苏艾尔康生物医药科技有限公司 | Dermal fibroblast skin sheet and construction method and application thereof |
| CN114028618A (en) * | 2021-10-25 | 2022-02-11 | 广东普洛宇飞生物科技有限公司 | Biological material based on amniotic membrane basement membrane and preparation method and application thereof |
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