CN1415384A - Method for preparing all layers of skin used by tissue engineering - Google Patents

Method for preparing all layers of skin used by tissue engineering Download PDF

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Publication number
CN1415384A
CN1415384A CN02139398A CN02139398A CN1415384A CN 1415384 A CN1415384 A CN 1415384A CN 02139398 A CN02139398 A CN 02139398A CN 02139398 A CN02139398 A CN 02139398A CN 1415384 A CN1415384 A CN 1415384A
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China
Prior art keywords
preparation
collagen
fibroblast
skin
holostrome skin
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CN02139398A
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Chinese (zh)
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金岩
刘源
吕红兵
赵宇
聂鑫
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STOMATOLOGICAL COLLEGE OF 4TH MILITARY SURGEON UNIV CPLA
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STOMATOLOGICAL COLLEGE OF 4TH MILITARY SURGEON UNIV CPLA
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Priority to CN02139398A priority Critical patent/CN1415384A/en
Publication of CN1415384A publication Critical patent/CN1415384A/en
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Abstract

A process for preparing the complete skin by tissue engineering for treating burn, ulcer, wound, etc. includes preparing collegen gel, preventing shrinkage of collagen gel, culturing the complete skin, separating epiderm from dermis and digestion with collegenase.

Description

The preparation method of organizational project holostrome skin
Technical field
The present invention relates to a kind ofly prepare the method for holostrome skin, belong to the preparation field that contains living cells and cytostromatic holostrome artificial skin that to transplant with the organizational project approach.
Background technology
Skin is to cover and the vital tissue organ of protecting body surface.Owing to reasons such as inflammation, ulcer, wound, burn, tumor post-operation and congenital malformation often cause the damaged of skin with unusual.For the autodermic value of moving of the many at present employings of this tissue defect, this method not only can cause the new wound defective of skin donor site, and often is subjected to the restriction for the skin source.In addition, graft skin frequent and that transplantation site is contiguous exists the difference on color and luster, quality and the function.Ideal skin substitute products should be the corium and the epidermal area that are lacked can be repaired simultaneously, because not only cutaneous function of these two kinds of compositions and profile, and these two kinds of compositions have interactional mechanism, promotion differentiation each other.Organizational project holostrome skin comprises that two kinds of cell component are epithelial cells and fibroblast, and wherein the support of skin corium can be made up by collagen gel.
February 6 calendar year 2001, people such as Ri Wujinjin applied for relevant patent (application number is 01107099.4), and this invention comprises the preparation of various solution, the preparation of collagen gel and the dimensional culture of cell.This artificial skin elasticity and pliability are fair, can be sheared, and remain in some defectives: though 1 have more sophisticated epidermal area and skin corium, but the In vitro culture overlong time needs about 20 days altogether, has prolonged the time that patient waits for operation; 2, in undeclared this invention preparation artificial skin whether basement membrane is arranged, whether the histology views and admires not mentioned among the result most important connected mode between the epithelial cells a---appearance of desmosome, the undeclared compound artificial skin of cultivating has the ultrastructure similar with natural skin, therefore can influence the intensity and the effect of artificial skin; 3, the source of cell in the not clear and definite artificial skin; 4, still residual in the artificial skin of this invention preparation have a large amount of porose gelatinised matrix, and this will influence the growth of cell, the interaction between the cell, cause infection easily after this artificial skin transplanting.
The patent (patent No. 91109937.9) of mark-Essenberg (Autok International Company) application of announcing the prior art patent documentation that another one is relevant with the present invention---February 28 calendar year 2001, this invention is to inoculate fibroblast on the one side of the collagen sponge membrane of porous, crosslinkedization, cultivates this fibroblast and it is spread all over be grown on the collagen sponge membrane; Form one deck atresia collagen at the another side of porous collagen, then at the keratinocyte of this face inoculated and cultured.But, 1, this invention do not set up the dimensional culture environment, fibroblast can not be grown in the used collagen sponge membrane of this invention, and just in its superficial growth, thereby, do not form real dermal tissue; 2, this invention is gone up the inoculated and cultured keratinocyte at the another side (acellular atresia collagen face) of porous collagen, can not make the keratinocyte and the fibroblast in the skin corium of growth that mutual relation takes place, thereby can't form basement membrane structure important in the skin, make epidermal area and skin corium very easily peel off, repairing effect is transplanted in influence; 3, organ culture's method (promptly using gas-liquid face cultural method and specific culture fluid) is not used in this invention, do not make keratinocyte (being epithelial cells or epidermis cell) answer stratification (maturation process of epidermis), thereby the epidermal area that is obtained is a kind of monolayer membrane structure, do not meet real epidermal structure, can not form the due basal layer of epidermal area, prickle cell layer, granular layer and cuticular layer.
Content of the present invention
The technical problem to be solved in the present invention is in order to overcome the deficiency described in the background technology, and provide that a kind of shrinkage factor is low, incubation time short, obvious immunologic rejection do not take place, the preparation method of the organizational project holostrome skin more similar to the natural skin structure.
Realize technical solution of the present invention::
Prepare collagen solution: under aseptic condition, composite collagen is dissolved in 0.1% acetic acid reuse ultraviolet radiation collagen solution;
Prevent that collagen gel from shrinking: aseptic nylon membrane is inserted cultivate in the plate hole, soak to put with 1.1 described composite collagen solution and make its curing in the incubator, again nylon membrane is immersed the sodium ascorbyl phosphate buffer (PBS that contains glutaraldehyde, place down together), wash with this solution flushing with the fibroblast culture fluid, place minimum must the cultivation in the culture fluid (DMEM, down together) afterwards;
The dimensional culture of cell: aseptic nylon membrane is inserted dropping collagen solution curing in the culture dish, again the nylon membrane immersion is contained among the PBS of 1% glutaraldehyde, get the back and wash, place the fibroblast culture fluid to leave standstill then with culture fluid; Again nylon membrane is placed in the culture dish, the preparation collagen solution, (FBS, down together) uses among the NaOH and mixing with adding DMEM and hyclone behind the ultraviolet radiation, puts into incubator behind the adding fibroblast suspension and solidifies, and adds the cultivation of fibroblast culture fluid then;
Corium, epidermis separate back collagenase digesting dermal tissue.
The good effect that the present invention produced is:
By the prepared organizational project holostrome skin of the present invention, except having certain elasticity, toughness, outside can the certain frictional force of opposing, the most important thing is that this invention has also that shrinkage factor is low, incubation time is lacked, obvious immunological rejection does not take place, characteristics such as more similar to the structure of natural skin.
Collagen gel provides a timbering material for fibroblast to the growth of three-dimensional, and makes formed dermal tissue have certain thickness, has satisfied the needs in the clinical practice in the future.In addition, the epithelial cells on fibroblast in the collagen gel and collagen gel surface can directly contact, and accelerates and promote the maturation process of two kinds of cells, has shortened the incubation time of whole organization work holostrome skin.But in the process of cultivating, collagen gel is easy to shrink.And the nylon membrane method for pretreating that the present invention adopts has significantly reduced the degree that collagen gel shrinks, and the organizational project holostrome skin of formation is more conducive to operation technique clinically.
Epithelial cells of the present invention and fibroblast source are very extensive, can turn out the organizational project holostrome skin of required clinically different sizes in advance, at any time satisfy different patients' needs, the patient need not to wait for the incubation of organizational project holostrome skin, therefore can shorten patient's hospital stays greatly.
Form and Ultrastructural observation result to organizational project holostrome skin show that the structure of the biological activity Graftskin of cultivation has highly consistent double-layer structure with natural skin: epidermal area and skin corium.Contain basal layer, spinous layer, granular layer and horny layer in the epidermal area.There is the intercellular bridge between the acantholysis cell, complete basement membrane is arranged between epidermal area and the skin corium, and the prominent appearance of epithelial peg successive, that be uneven in length is arranged.There has not been the collagen residue that adds when cultivating in this skin histology.Among the transmission electron microscope observing result, in the epidermal area of biological activity skin, connect with desmosome between the acantholysis cell, and the laminated body, Tonofibrils and the fat that have in the natural skin to be had such as drip at ultrastructure.The existence of these structures can be resisted external world's friction of certain program, makes the combination between epidermis and corium tightr, is very necessary for its various functions of bringing into play similar natural skin, but also helps surgical operation.
Carried out the zoografting test of (between SD rat and the Wistar rat) between the allogeneic rat by the holostrome skin of the present invention's preparation, the result shows, organizational project holostrome skin growth healing after the transplanting is good, organizational project holostrome skin after the healing does not have obvious boundary with natural skin on every side, the structure of epidermal area and skin corium is tightr, the keratinization of epidermal area is more obvious, epithelial peg dash forward showed increased, elongation, no tangible cell infiltration in the skin corium, have a large amount of new vesselses to grow in the skin corium, it is regular, thick that the structure of collagen becomes.
The specific embodiment
The preparation of organizational project holostrome skin of the present invention can illustrate by following example.
Embodiment 1:
1,1 years old to 30 years old male's circumcision postoperative skin histology washes with the 1% 0.1M sodium ascorbyl phosphate buffer (MPBS, down together) that contains green grass or young crops/streptomycin, prunes unnecessary fat and muscular tissue.The skin histology bar is put into sterile tube, and add neutral protease (Dispase, down with) 4U/ml, epidermis and corium are separated in the digestion back under 37 ℃, the condition of 90min.
2, the acquisition of epithelial cells
With epithelial tissue pancreatin (the 0.25%)/EDTA (0.02-0.1%) (2: 1) that obtains, 37 ℃, digested 8 minutes, 100 orders or 200 mesh filter screens filter, and the epithelial cells of results is inoculated into culture bottle amplification culture.
3, fibroblastic acquisition
With 625U/ml collagenase digesting dermal tissue, behind 37 ℃ of digestion 120min, 100 or 200 mesh filter screens filter, and the fibroblast of results is inserted culture bottle, amplification culture.
4, prepare collagen solution
Under aseptic condition, collagen is dissolved in 0.1% acetic acid, concentration is 10mg/ml, this process needs to prepare before 2 days, under 4 ℃ of conditions to prevent the collagen solution degraded.Before using required collagen solution is injected culture dish, place on ice, uviol lamp is irradiation 30min down.
5, prevent the method that collagen gel shrinks
Aseptic nylon membrane inserted in the culture dish and with the bottom driving fit, drip collagen solution on the film, soak nylon membrane after, remove unnecessary collagen solution.37 ℃ solidify 30min.The nylon membrane immersion is contained among the PBS of 1% glutaraldehyde, place 1h for 4 ℃.Wash 4 times with PBS, wash 2 times with the fibroblast culture fluid, 20min at least places 4 ℃ of fresh fibroblast culture fluid 8-15h afterwards at every turn.
6, the dimensional culture of organizational project holostrome skin
1., nylon membrane is placed on the bottom of culture hole (or culture dish), prepare collagen solution, add 10 * DMEM, the FBS (hyclone) of 1/10 volume, regulate pH value to 7.2-7.4, add the fibrocyte suspension, fully mix (avoiding bubble) with the NaOH of 1M.Culture plate is put into incubator solidify about 30min, add the fibroblast culture fluid then, cultivated 3 days.
2., collect epithelial cells, with 3 * 10 6The concentration of/ml is implanted and is contained fibroblastic collagen gel surface, leaves standstill 1.5-2h, adds the epidermis cell culture fluid, cultivates 2 days.
3., when epithelial cell growth is good, the collagen gel that will have nylon membrane takes out and to be placed on the rustless steel grid, and the rustless steel grid is put into bigger culture dish.After 3-4 days, remove nylon membrane, be and cultivate successful organizational project holostrome skin, during every day change liquid 1 time.
Embodiment 2:
Get the discarded skin behind the excision,, prune unnecessary fat and muscular tissue with the 1% PBS flushing that contains the 0.1M of green grass or young crops/streptomycin.The skin histology bar is put into sterile tube, and add Dispase 4U/ml, epidermis and dermal tissue are separated in the digestion back under 37 ℃, the condition of 90min.

Claims (7)

1, a kind of preparation method of organizational project holostrome skin comprises that the epidermis with raw material separates with corium, the dimensional culture of cell, it is characterized in that:
1.1 preparation collagen solution: under aseptic condition, composite collagen is dissolved in 0.1% the acetic acid reuse ultraviolet radiation collagen solution;
1.2 preventing collagen gel shrinks: aseptic nylon membrane is inserted in the cultivation plate hole, soak to put with 1.1 described composite collagen solution and make its curing in the incubator, again nylon membrane is immersed the sodium ascorbyl phosphate buffer (PBS that contains glutaraldehyde, place down together), and wash with the flushing of this solution with the fibroblast culture fluid, place minimum must the cultivation in the culture fluid (DMEM, down together) afterwards;
1.3 the dimensional culture of cell: aseptic nylon membrane is inserted dropping collagen solution curing in the culture dish, again the nylon membrane immersion is contained among the PBS of 1% glutaraldehyde, wash with the fibroblast culture fluid after treating, place the fibroblast culture fluid to leave standstill then; Again nylon membrane is placed in the culture dish, the preparation collagen solution, (FBS, down together) uses among the NaOH and mixing with adding DMEM and hyclone behind the ultraviolet radiation, puts into incubator behind the adding fibroblast suspension and solidifies, and adds the cultivation of fibroblast culture fluid then;
1.4 corium, epidermis separate back collagenase digesting dermal tissue.
2, the preparation method of holostrome skin according to claim 1 is characterized in that the compound cultivation of holostrome skin is with dense multitudinous 1-1.5 * 10 5Fibroblast adds in the ready collagen gel, with 0.5 * 10/milliliter 6The epidermis cell of/milliliter is implanted and is contained fibroblastic collagen gel surface.
3, the preparation method of holostrome skin as claimed in claim 1 is characterized in that said ultraviolet irradiation time is 30-60min.
4, the preparation method of holostrome skin according to claim 1 is characterized in that the concentration that composite collagen is dissolved in 0.1% acetic acid is 4-10mg/ml.
5, the preparation method of holostrome skin according to claim 1 is characterized in that said incubator is CO 2Incubator, solidification temperature are 37 ℃.
6, the preparation method of holostrome skin according to claim 1 is characterized in that the operative temperature before all and collagen gel solidify is carried out at 0-6 ℃.
7, the preparation method of holostrome skin according to claim 1 is characterized in that said collagenase digesting dermal tissue is that collagenase with 625U/ml soaks dermal tissue.
CN02139398A 2002-09-02 2002-09-02 Method for preparing all layers of skin used by tissue engineering Pending CN1415384A (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100462059C (en) * 2005-05-25 2009-02-18 中国人民解放军第四军医大学口腔医院 Method for preparing artificial skin used for reparing skin defect
WO2011094963A1 (en) * 2010-02-05 2011-08-11 中国人民解放军第四军医大学 Appendage-carrying tissue engineering skin producing method
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
CN102949752A (en) * 2011-08-22 2013-03-06 中国人民解放军总医院第一附属医院 Construction method for tissue engineering skin containing complete skin appendage
CN106237312A (en) * 2016-07-28 2016-12-21 广州赛莱拉干细胞科技股份有限公司 One is dispelled scar compositions and dressing
CN107019816A (en) * 2017-03-30 2017-08-08 贵州省人民医院 A kind of compound method for going epidermis dermis to build organization engineering skin of amniotic epithelial cells
CN107349474A (en) * 2017-06-18 2017-11-17 广东博溪生物科技有限公司 A kind of artificial dermis construction method of anti-shrinkage
CN110302432A (en) * 2019-06-14 2019-10-08 华南理工大学 A kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100462059C (en) * 2005-05-25 2009-02-18 中国人民解放军第四军医大学口腔医院 Method for preparing artificial skin used for reparing skin defect
WO2011094963A1 (en) * 2010-02-05 2011-08-11 中国人民解放军第四军医大学 Appendage-carrying tissue engineering skin producing method
CN102949752A (en) * 2011-08-22 2013-03-06 中国人民解放军总医院第一附属医院 Construction method for tissue engineering skin containing complete skin appendage
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
CN106237312A (en) * 2016-07-28 2016-12-21 广州赛莱拉干细胞科技股份有限公司 One is dispelled scar compositions and dressing
CN107019816A (en) * 2017-03-30 2017-08-08 贵州省人民医院 A kind of compound method for going epidermis dermis to build organization engineering skin of amniotic epithelial cells
CN107349474A (en) * 2017-06-18 2017-11-17 广东博溪生物科技有限公司 A kind of artificial dermis construction method of anti-shrinkage
CN110302432A (en) * 2019-06-14 2019-10-08 华南理工大学 A kind of preparation method of the full thickness skin tissue engineering bracket with graded pore structure
CN110302432B (en) * 2019-06-14 2020-06-19 华南理工大学 Preparation method of full-layer skin tissue engineering scaffold with gradient pore structure

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