CN101954124B - 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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- CN101954124B CN101954124B CN201010271010.0A CN201010271010A CN101954124B CN 101954124 B CN101954124 B CN 101954124B CN 201010271010 A CN201010271010 A CN 201010271010A CN 101954124 B CN101954124 B CN 101954124B
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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 using surface modified amniotic membrane rear as basement membrane, compound blood plasma is substrate, build the organization engineering skin (being viable skin substitute) containing basement membrane through In vitro culture, be specially adapted to skin injury reparation and the cicatrix shaping of burn, wound.
Background technology
At present, build dermis scaffold with Biodegradable materials such as polylactic acid, polyglycolic acid, collagen, hyaluronic acids, build on this basis viable skin substitute, Preliminary Applications is in reparation and the cicatrix shaping etc. of degree of depth skin injury wound surface.But the Graftskin building thus on the one hand host material extracts difficulty, complex manufacturing technology, somewhat expensive, 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, easily come off breakage or form vesicle, and affect the growth of normal epidermis structure, form.And acellular corium is taken from cadaver skin, originate limited, expensive, limit its clinical application.
Human amnion tissue is the translucent tissue containing adhesion, without blood vessel, nerve, lymphatic vessel, thickness is 0.02-0.5mm, form and the skin basement membrane of its basement membrane are closely similar, and mainly form (Zhao Min, Lu Jing by laminin，LN and IV Collagen Type VI, Zhang Qi, Deng .HGF, bFGF, ColIV, the LN expression in preservation 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, draw materials conveniently, and it is as a kind of biological dressing early for the covering of burn wound, and the inflammation that not only can reduce wound surface promotes epithelization but also can suppress the formation of cicatrix.At present existing commercial amniotic membrane product is as the lyophilization bioamnion of Jiangxi RuiJi Biotechnology Co., Ltd.
There is no bibliographical information both at home and abroad using amniotic membrane as basement membrane, compound blood plasma is substrate, builds the organization engineering skin containing basement membrane through In vitro culture.
Summary of the invention
It is a kind of taking surface modified amniotic membrane as basement membrane that the object of the invention is to provide,, the Graftskin taking autologous/allosome blood plasma as substrate, and construction method.Material source of the present invention is extensive, and preparation method is simple, with low cost, in can transplanting in vivo, keeps complete basement membrane and hemi desmosome, accelerates and promote the formation of epidermal structure form.
Of the present invention taking surface modified amniotic membrane as basement membrane, the construction method of the organization engineering skin containing basement membrane that blood plasma is 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 build organization engineering skin. Chinese Clinical rehabilitation, 2004,8 (8): 1439-1441.).
B) surface modified amniotic membrane:
Remove the amniotic membrane after chorion, after normal saline repeatedly soaks, cleans, 37 DEG C of digestion of EDTA solution in 0.02% 2 hours, residual cell debris and chorion tissue are thoroughly removed in sonic oscillation washing, are placed in containing the normal saline of 1000U/ml gentamycin, 2.5 μ g/ml amphotericin Bs and soak 20 to 50 minutes.
C) build organization engineering skin;
By B) amniotic membrane prepared of step with containing fibroblastic blood plasma matrix composite, then by epithelial cells with 1-5 × 10
5individual/cm
2density be inoculated in amniotic membrane epidermis side, In vitro culture 2-3 week.
Construction method concrete steps of the present invention are as follows:
1, separate, cultivate people's epithelial cells, fibroblast
The utilization unnecessary skin histology after postoperative discarded prepuce tissues or anaplasty of peritomizing, adopts respectively enzyme digestion and tissue block adherent method to separate, cultivate epithelial cells and fibroblast.Be prepared into respectively single cell suspension, by 2-3 × 10
5individual/ml cell density is inoculated in culture bottle, is placed in 37 DEG C 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 cell reaches when 70%-80% merges.
2, surface modified amniotic membrane
Under aseptic condition, peel off fresh amnion and (take from hepatitis virus antibody, syphilis antibody and HIV be the negative puerpera's that cuts open the belly placenta tissue all), blunt separation chorion, repeatedly soak through normal saline, after cleaning, in 0.02% EDTA solution, 37 DEG C digest 2 hours, put into subsequently high frequency ultrasound washer sonic oscillation washing 2 hours, thoroughly remove residual cell debris and chorion tissue, then through distilled water repeatedly after soaking and washing, be placed in containing 1000U/ml gentamycin, in the normal saline of 2.5 μ g/ml amphotericin Bs, soak 30min and prepare surface modified amniotic membrane.
3, build organization engineering skin
First the amniotic membrane basal surface of preparation is upwards laid on culture plate, treats that its bone dry is adherent.Get the centrifugal blood plasma that obtains after patient or allosome whole blood, remove cell debris through microfilter, fibroblast being added to compound concentration in the blood plasma of preparation is 5 × 10
5individual/ml, adds in the above-mentioned culture dish that is covered with amniotic membrane after fully mixing.The calcium chloride solution that adds subsequently 1M with the volume ratio of 1: 40, promotes its polymerization, after it aggregates into gel, adds the 1 × DMEM solution containing 20% hyclone, in In vitro culture 3-5 days.Subsequently epithelial cells is pressed to 1-5 × 10
5individual/cm
2density be inoculated on amniotic membrane, after cell fusion, carry out liquid-vapor interface cultivate 2-3 week, within every 2～3 days, change liquid once, form taking amniotic membrane as basement membrane, the viable skin substitute that plasmagel is substrate.
The present invention also provides the organization engineering skin building according to said method.
The present invention also provides the application as wound repair graft materials according to the organization engineering skin of said method structure.
The present invention is taking amniotic membrane as basement membrane, the viable skin substitute that plasmagel is substrate, and main uses is to repair full thickness dermal wounds, for serious patients with major full-thickness burn provides Pi Yuan.On the other hand, also can be applicable to skin injury wound surface and the cicatrix shapings such as chronic degree of depth skin ulcer, avulsion injury of skin.Viable skin substitute method prepared by the present invention is simple, draw materials conveniently, with low cost, in addition in In vitro culture, find the more not traditional organization engineering skin containing amniotic membrane, epidermal growth is more rapid, configuration is convergence normal skin more, possesses perfect basement membrane, and hemi desmosome is grown more ripe firm simultaneously.Show through animal (nude mice) transplantation experiments, viable skin substitute is implanted to full thickness dermal wounds, 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 amniotic membrane surface modified rear as basement membrane, and compound is substrate containing fibroblastic plasmagel, and surface grafting epithelial cells, builds the viable skin substitute containing basement membrane through In vitro culture.Manufacture method of the present invention is simple, and material source is extensive, with low cost, has got rid of the xenogenesis impact such as infection, repulsion that may cause of originating simultaneously.
Detailed description of the invention
Now in conjunction with the embodiments, the invention will be further described, but enforcement of the present invention is not limited in this.
Embodiment 1. preparations are containing the viable skin substitute of basement membrane
1, separate, cultivate people's epithelial cells, fibroblast
The utilization postoperative discarded prepuce tissues of peritomizing, adopts enzyme digestion to separate, cultivate epithelial cells, fibroblast.Be prepared into respectively single cell suspension, by 2-3 × 10
5individual/ml cell density is inoculated in culture bottle, be placed in 37 DEG C 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 cell reaches when 70 %-80% merge.
2, surface modified amniotic membrane
Under aseptic condition, peel off fresh amnion and (take from hepatitis virus antibody, syphilis antibody and HIV be the negative puerpera's that cuts open the belly placenta tissue all), blunt separation chorion, repeatedly soak through normal saline, after cleaning, (Gibco in 0.02% EDTA solution, the U.S.), 37 DEG C digest 2 hours, put into subsequently high frequency (59KHz) ultrasonic cleaner (SK8200H, section leads, Shanghai), sonic oscillation washing 2 hours, thoroughly remove residual cell debris and chorion tissue, then through distilled water repeatedly after soaking and washing, be placed in containing 1000U/ml gentamycin, in the normal saline of 2.5 μ g/ml amphotericin Bs, soak 30min for subsequent use.
3, build organization engineering skin:
First the amniotic membrane basal surface of preparation is upwards laid on culture plate, treats that its bone dry is adherent.Get patient or allosome whole blood is placed in the anticoagulant tube containing sodium citrate anticoagulant, the centrifugal 5min of 600g, collects blood plasma, removes cell debris through the filter sterilization in 20um aperture, and fibroblast being added to compound concentration in the blood plasma of preparation is 5 × 10
5individual/ml, after fully mixing, with the volume of 1: 6 (ml) and culture plate area (cm
2) than adding in the above-mentioned culture dish that is covered with amniotic membrane containing fibroblastic blood plasma.The calcium chloride solution that adds subsequently 1M with the volume ratio of 1: 40, promotes its polymerization, after it aggregates into gel, adds the 1 × DMEM solution containing 20% hyclone, in In vitro culture 3-5 days.Subsequently epithelial cells is pressed to 1-5 × 10
5individual/cm
2density be inoculated on amniotic membrane, after cell fusion, carry out liquid-vapor interface cultivate 2-3 week, within every 2～3 days, change liquid once, form taking amniotic membrane as basement membrane, the viable skin substitute that plasmagel is substrate.
The zoografting test of embodiment 2. organization engineering skins of the present invention
Male Balb/c-nu mice (nude mice, Shanghai western pul-Bi Kai laboratory animal company limited provides), 18 ± 2 grams of body weight, 16, be divided into two groups, be respectively the A group and the traditional viable skin substitute B group that do not contain amniotic membrane of the present invention containing the organization engineering skin of amniotic membrane: build and (refer to A.L.Mazlyzama taking fibrin as substrate merely, B.S.Aminuddinb, et al.Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold.Burns, 2007, 33 (3): 355-363).
Nude mice, after ketamine intraperitoneal anesthesia, is shaved except skin of back hair, 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 embodiment 1 is transplanted in wound surface.B group: the traditional viable skin substitute that does not contain amniotic membrane is transplanted in wound surface.For preventing creating all contraction of skin and epidermis is creeped, the observation of result is transplanted in impact, adopts cage ring that wound surface and periphery skin are isolated.In Composite Skin, cover oil-sand, periodic replacement dressing is also observed wound healing situation.Within after Composite skin 2 weeks, open wound surface and observe survival rate.And draw materials, make the paraffin section of thick approximately 5 μ m, row conventional organization section HE dyeing is observed and transmission electron microscope observing.
Result shows, organization engineering skin of the present invention is survival rate no significant difference compared with traditional group.Histology HE dyeing is observed visible, the epidermal structure that organization engineering skin of the present invention is transplanted latter 4 weeks physically well develops, form is convergence normal skin more, transmission electron microscope observing is shown in to possess perfect basement membrane, hemi desmosome is grown more ripe simultaneously, and matched group has no continuous basement membrane formation and hemi desmosome developmental defect.In addition the viable skin substitute that prepared by the present invention, the multiple layer epidermis cell and the dermal substitute that form adhere to close and firm more, are difficult for departing from.
Claims (1)
1. a construction method that contains the organization engineering skin of basement membrane, is characterized in that the concrete steps of the method are as follows:
A) separate, cultivate people's epithelial cells, fibroblast:
The utilization postoperative discarded prepuce tissues of peritomizing, adopts enzyme digestion to separate, cultivates epithelial cells, fibroblast, is prepared into respectively single cell suspension, by 2-3 × 10
5individual/ml cell density is inoculated in culture bottle, is placed in 37 DEG C 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 cell reaches when 70%-80% merges;
B) surface modified amniotic membrane:
Under aseptic condition, peel off fresh amnion, blunt separation chorion, after normal saline repeatedly soaks, cleans, in 0.02% EDTA solution, 37 DEG C digest 2 hours, put into subsequently high frequency ultrasound washer sonic oscillation washing 2 hours, thoroughly remove residual cell debris and chorion tissue, then through distilled water repeatedly after soaking and washing, be placed in that to soak 30min containing the normal saline of 1000U/ml gentamycin, 2.5 μ g/ml amphotericin Bs for subsequent use;
C) build organization engineering skin:
By step B) the amniotic membrane basal surface prepared is upwards laid on culture plate, treat that its bone dry is adherent, get patient or allosome whole blood is placed in the anticoagulant tube containing sodium citrate anticoagulant, the centrifugal 5min of 600g, collect blood plasma, cell debris is removed in filter sterilization through 20um aperture, and fibroblast being added to compound concentration in the blood plasma of preparation is 5 × 10
5individual/ml, after fully mixing, with the volume ml of 1: 6 and culture plate area cm
2than adding in the above-mentioned culture dish that is covered with amniotic membrane containing fibroblastic blood plasma; Add subsequently the calcium chloride solution of 1M with the volume ratio of 1: 40, after it aggregates into gel, add the 1 × DMEM solution containing 20% hyclone, in In vitro culture 3-5 days; Subsequently epithelial cells is pressed to 1-5 × 10
5individual/cm
2density be inoculated on amniotic membrane, after cell fusion, carry out liquid-vapor interface cultivate 2-3 week, within every 2～3 days, change liquid once.
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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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| CN101954124B true CN101954124B (en) | 2014-06-04 |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102499998B (en) * | 2011-12-22 | 2013-12-11 | 中国农业科学院北京畜牧兽医研究所 | Dermis equivalent constructing method |
| CN103074295B (en) * | 2013-02-06 | 2014-10-08 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN103520773B (en) * | 2013-10-24 | 2015-07-01 | 北京积水潭医院 | Method for preparing decellularized dermis material for skin grafting |
| 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 |
| CN104189950B (en) * | 2014-09-04 | 2016-09-21 | 成都清科生物科技有限公司 | A kind of amniotic membrane biological preparation and preparation method thereof |
| GB201513461D0 (en) * | 2015-07-30 | 2015-09-16 | Ucl Business Plc | Methods and devices for the production of decellularised 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 |
| CN107320781B (en) * | 2017-07-11 | 2020-03-10 | 广州润虹医药科技股份有限公司 | Tissue engineering skin containing living cells 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 |
| 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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| JP5795166B2 (en) * | 2007-11-28 | 2015-10-14 | オルガノジェネシス インク． | Biotechnological tissue constructs and methods for production and use |
| CA2717619C (en) * | 2008-03-14 | 2016-12-13 | Cook Biotech Incorporated | Graft materials and methods for staged delivery of bioactive components |
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| CN1630715A (en) * | 2003-06-16 | 2005-06-22 | 华北制药集团新药研究开发有限责任公司 | Methods of preparing a transplantable product for treatment of skin defects |
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