CN1210005C - Tissue engineering skin containing blood vessel structure and its construction method - Google Patents
Tissue engineering skin containing blood vessel structure and its construction method Download PDFInfo
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Abstract
The present invention relates to tissue engineering skin containing a blood vessel structure and a construction method thereof, which is characterized in that the tissue engineering skin contains skin flbroblasts, keratinocytes and vascular endothelial cells; the construction method of the tissue engineering skin comprises separation and cultivation methods for the skin flbroblasts, the keratinocytes and the vascular endothelial cells, and a method for constructing artificial skin containing a blood vessel structure. The construction method has the characteristics that keratinocytes, fibroblasts, and vein vascular endothelial cells of human's navels are cultivated in vitro, and then, the keratinocytes, the fibroblasts and the vein vascular endothelial cells are mixed according to a proportion in vitro by a tissue engineering method to construct artificial skin with a blood vessel. The transplantation survival rate and the anti-infection capacity of the tissue engineering skin can be enhanced, and the application range of the tissue engineering skin is further enlarged.
Description
Technical field
The invention belongs in the biomedical engineering and make up the artificial organ technical field, relate to a kind of organization engineering skin and construction method thereof that contains blood vessel structure with Method of Tissue Engineering.
Background technology
In recent years, along with the development of tissue engineering technique, the report that research and development have reparation, keep or improve the biological substitution thing of damaged tissue function increases gradually, and the research report of artificial skin substitute is also quite a lot.The present focus that the development and the exploitation of organization engineering skin have been become the skin injury transplantation treatment.
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 this tissue defect, the in the past employing from body flap or autodermic transplanting more, these methods not only exist the wound defective that causes skin donor site new, and usually exist for the limited influence in skin source.Simultaneously, graft skin frequent and that transplantation site is contiguous exists the difference on color and luster, quality and the function.
Method of Tissue Engineering is exactly by obtaining the minute quantity skin histology from body or allosome, in the sterilization of external (in the laboratory) process, digestion, separation, cultivation, when obtaining enough cell quantities, it is reassembled into skin histology, be used for the reparation of patient skin wound.The epithelial culture technique that Rheinwald in 1975 and Green propose has solved the difficult problem of the external amplification of going down to posterity of epithelial cell, makes the artificial skin graft of In vitro culture become possibility.But clinical practice finds that it has bigger contraction, and fragility is big and thin, and unsuitable clinical manipulation wears no resistance after the transplanting, easily blisters.For solving clinically the requirement of thickness, toughness and operability, needing increase holder below skin graft: such as the fibrin carrier, and acellular human dermis's layer support; The collagen sponge carrier of synthetic etc.Have the scholar that epithelial cell is seeded on the collagem membrane, cell fusion is transplanted after becoming monolayer, and opposite with standard practice, epidermis cell turns to wound surface, and the surface is a collagem membrane.Aspect artificial dermis, designed a kind of dermal replacement by people such as Burke and Yannas the earliest, purpose is to be allowed to condition to serve as support on the wound surface, is convenient to the regeneration of skin corium.Be divided into two-layerly on the structure, internal layer constitutes porous support by the collagen fiber and the chondroitin sulfate in cattle tendon source, and skin is a pellosil then, plays the epidermis effect, can be used for full thick-layer skin injury.Outer field pellosil is generally being transplanted 2～3 all backs by substituting from the body skin graft.Representative products has Integra and Dermagraft series.Dermagraft-TC product internal layer constitutes timbering material by nylon yarn, and pellosil is also adopted on the surface.DermagraftTM then uses degradation material as dermis scaffold, and its composition is the polymer of polyglycolic acid.
Ideal skin substitutes should be the corium and the epidermal area that are lacked can be repaired simultaneously, Here it is so-called holostrome composite skin.This holostrome skin histology is not yielding after plantation, is easy to healing.Composite skin comprises two kinds of cell component, promptly is positioned at the epidermis cell and the fibroblast that is positioned at skin corium on top layer.There is the scholar to use the acellular dermal of heterogenous animal or cadaver skin to make up Graftskin, but do not see the report of clinical practice as yet as support inoculation fibroblast.Structure Graftskins such as Michel do not use any timbering material, only form the fibrous membrane that contains extracellular matrix with fibroblast under ascorbic effect, and its some ultrastructure is similar to normal structure; Inoculate epithelial cells then thereon, divide a word with a hyphen at the end of a line, breed, answer stratification formative tissue engineering Graftskin.
Although the tissue engineering method is obtaining huge progress aspect the development of skin, portioned product has been applied to clinical, but or the tissue engineering product of developing imperfection still, on the method maturation, on the lead time and the first-class many-side of workinprocess cost all have defective.The organization engineering skin substitute of being reported mostly and normal group be woven with than big-difference, do not have the compositions such as hair follicle, blood vessel, sweat gland and melanocyte of normal skin as previously mentioned.
Summary of the invention
At above-mentioned prior art situation, the objective of the invention is to: a kind of organization engineering skin and construction method thereof that contains blood vessel structure is provided, and make every effort to make this organization engineering skin not yielding after plantation, be easy to healing, the In vitro culture time is short, the heterogenous skin immunologic rejection is little, construction cost is cheap.
Now the technology of the present invention solution is described below:
The organization engineering skin that contains blood vessel structure proposed by the invention is characterised in that: it is to be made of dermal fibroblast, epithelial cells and vascular endothelial cell and type i collagen; Described dermal fibroblast, epithelial cells are to take from same neonate prepuce tissues, with digestion method separation, amplification culture; Described vascular endothelial cell is to take from same neonatal umbilical vein vascular endothelial cell, with digestion method separation, amplification culture; With above-mentioned three kinds of cells is seed cell, makes up with Method of Tissue Engineering to form.Described dermal fibroblast density is 0.5 * 10
5～1 * 10
7/ ml; Used type i collagen concentration is 1～15%; Dermal fibroblast and vascular endothelial cell are pressed 1: 1～10: 1 mixed; The density of described epithelial cells is 1 * 10
5～5 * 10
6/ ml.
The present invention contains the construction method of the organization engineering skin of blood vessel structure, comprises the cultivation of dermal fibroblast, epithelial cells and vascular endothelial cell and the structure of organization engineering skin, it is characterized in that:
(1) In vitro culture of dermal fibroblast, epithelial cells and vascular endothelial cell may further comprise the steps:
1) gets the neonate prepuce tissues, separate epidermis and corium with digestion method; Get same umbilical cord venous vascular endothelial cell, separate obtaining vascular endothelial cell with digestion method;
2) isolated epidermis is obtained single cell suspension after with trypsinization, with single cell suspension with epithelial cells culture fluid inoculated and cultured; Isolated corium is obtained the single cell suspension of dermal fibroblast after with collagenase digesting, and reuse contains the DMEM culture fluid inoculated and cultured of hyclone; Isolated vascular endothelial cell is centrifugal, cleaning are used the culture fluid re-suspended cell, inoculated and cultured;
3) with above-mentioned each cell amplification culture;
4) preparation of vascular endothelial cell culture fluid: commercial culture medium M199 is 80%, and calf serum is 20%; Other contains commercial basic fibroblast growth factor bFGF is 5～20ng/ml, and commercial heparin is 60～90 μ g/ml.
(2) structure of organization engineering skin may further comprise the steps:
1) getting above-mentioned growth conditions good cell, is 0.5 * 10 with density
5～1 * 10
7The dermal fibroblast of/ml and vascular endothelial cell be by 1: 1～10: 1 mixed, and then be that 1～15% type i collagen mixes with concentration;
2) 37 ℃ hatch solidify after, add culture fluid and cultivate;
3) at above-mentioned cell surface with 1 * 10
5～5 * 10
6The density inoculation epithelial cells of/ml continues to cultivate;
4) collagen of compound cell is moved to the gas-liquid face and carry out the organ culture, cultivated 7～21 days, promptly obtain organization engineering skin, preserve standby.
Description of drawings
HE dyeing photo after Fig. 1 makes up for the present invention contains the capillary structure organization engineering skin.
SABC VIII factor antibody photo after Fig. 2 makes up for the present invention contains the capillary structure organization engineering skin.
Fig. 3 is an animal implant tests textured of the present invention photo as a result.
Fig. 4 is an animal implant tests textured SABC of the present invention photo as a result.
Fig. 1 and Fig. 2 explanation contain a large amount of capillary structures in the organization engineering skin skin corium, the tube wall cell is flat, monolayer alignment, and tube chamber is bigger, and this cell of Showed by immune group result VIII factor antibody reacting positive shows that this cell is a vascular endothelial cell.
Fig. 3 and Fig. 4 explanation, the skin injury healing of the nude mice of being made skin graft is good, smooth surface, color and luster is normal.Showed by immune group result, the skin corium of skin contains a large amount of microvessel structures after healing, and VIII factor antibody reacting positive shows that organization engineering skin healing of the present invention is good.
The specific embodiment
Now the inventive method and implementation result are described further in conjunction with experiment.
1, the cultivation of vascular endothelial cell
Get neonatal umbilical cord, be placed among the sterile phosphate buffer PBS, preserve transportation for 4 ℃, cultivate beginning in 1 hour as far as possible, otherwise cell viability will descend greatly; Umbilical cord is placed in the sterile petri dish, washes repeatedly with the removal dirt with aseptic PBS buffer, and flushing vein inner chamber, to remove blood stains; With the tight ligation of umbilical vein one end, pour into Digestive system a37 ℃ of digestion 10 minutes with stitching thread therein earlier, reuse Digestive system b digestion 5 minutes; Cut off the umbilical cord of ligation from an end, liquid to going out, is washed intravascular space with PBS then repeatedly, collect centrifugal, wash 2 times; With vascular endothelial cell culture fluid re-suspended cell, inoculated and cultured.Described Digestive system a is 625U/ml collagenase/mixed liquor of 1: 1 of 0.1% edta edta; B is 0.25% pancreatin/0.1%EDTA1: 1 mixed liquor.
Cell is identified: cell is oval type paving stone sample under the inverted microscope; Immunohistochemical staining shows the VIII factor positive; The visible W-P corpusculum of transmission electron microscope.
2, the cultivation of dermal fibroblast and epithelial cells
Get prepuce tissues behind the circumcision by surgical excision on newborn, in 75% ethanol, soaked 1 minute before handling; With prepuce tissues, clean again, remove subcutaneous tissue, will organize branch to be cut to the strip of 2mm * 10mm, use the protease digestion liquid of 1.2U/ml to digest 24 hours for 4 ℃ with cutting tweezer with being added with antibiotic PBS; Epidermis is separated with corium; Collect epidermis,, blow and beat repeatedly to obtain angle protein single cell suspension with 0.25% trypsinization 10 minutes, the angle protein is unicellular with epithelial cells culture fluid K-SFM inoculation, continue to cultivate to obtain purified epithelial cells; Collect corium, usefulness contained the DMEM culture fluid inoculated and cultured of 10% hyclone to obtain purified dermal fibroblast to usefulness 325U/ml collagenase digesting to obtain the corium single cell suspension in 4 hours; When primary cell covers with 70%-80%, go down to posterity amplification culture with the 2.5/L trypsinization.
3, the structure that contains the capillary structure organization engineering skin
Get the growth conditions good cell, with ratio be 1: 1 dermal fibroblast and vascular endothelial cell each with 1.5 * 10
6The density of/ml is mixed with the type i collagen that extracts from calf-skin, 37 ℃ hatch solidify after, add culture fluid cultivation 4 days; On its surface with 1 * 10
7/ ml density inoculation epithelial cells continues to cultivate 3 days, the collagen of compound above-mentioned cell is moved to the gas-liquid face carry out the organ culture, cultivates and 1-2 week promptly obtains organization engineering skin, preserves standby.HE dyeing (Fig. 1) and SABC VIII factor antibody (Fig. 2) result show, in the organization engineering skin skin corium, contain a large amount of capillary structures, the tube wall cell is flat, monolayer alignment, tube chamber is bigger, this cell of Showed by immune group result VIII factor antibody reacting positive shows that this cell is a vascular endothelial cell.
4, animal implant tests textured
2% pentobarbital sodium lumbar injection, 30～50mg/kg anesthesia cuts the holostrome skin at Balb/c nude mice back and makes circular wound surface, and diameter 1.5cm is for skin transplantation.Get the above-mentioned organization engineering skin that contains capillary structure, flap coverage is used suturing with thread management, pressure dressing.The matched group organization engineering skin used for reparing skin defect that does not contain vascular endothelial cell.Get specimen after 6 weeks, 4% paraformaldehyde is fixed, and it is transparent to dewater, paraffin embedding, and section dewaxes to water behind the roasting sheet, row immunohistochemical staining (VIII factor antibody).The result: when drawing materials, visible nude mice skin injury healing is good, smooth surface, color and luster normal (Fig. 3).Showed by immune group result (Fig. 4), the skin corium of skin contains a large amount of microvessel structures after healing, and VIII factor antibody reacting positive shows that the organization engineering skin repairing effect is good.
Claims (5)
1, a kind of organization engineering skin that contains blood vessel structure constitutes type i collagen, it is characterized in that: it constitutes dermal fibroblast, epithelial cells and vascular endothelial cell in addition; Dermal fibroblast density is 0.5 * 10
5/～1 * 10
7/ ml; Used type i collagen concentration is 1～15%; Dermal fibroblast and vascular endothelial cell are pressed 1: 1～10: 1 mixed; The density of epithelial cells is 1 * 10
5～5 * 10
6/ ml.
2, organization engineering skin according to claim 1 is characterized in that: described dermal fibroblast, epithelial cells are to take from same neonate prepuce tissues, with digestion method separation, amplification culture; Described vascular endothelial cell is taken from same neonatal umbilical vein vascular endothelial cell, with digestion method separation, amplification culture; With above-mentioned three kinds of cells is seed cell, makes up with Method of Tissue Engineering to form.
3, a kind of construction method that contains the organization engineering skin of blood vessel structure according to claim 1 comprises the cultivation of dermal fibroblast, epithelial cells and vascular endothelial cell and the structure of organization engineering skin, it is characterized in that:
(1) In vitro culture of dermal fibroblast, epithelial cells and vascular endothelial cell may further comprise the steps:
1) gets the neonate prepuce tissues, separate epidermis and corium with digestion method; Get same umbilical cord venous vascular endothelial cell, separate obtaining vascular endothelial cell with digestion method;
2) isolated epidermis is obtained single cell suspension after with trypsinization, with single cell suspension with epithelial cells culture fluid inoculated and cultured; Isolated corium is obtained the single cell suspension of dermal fibroblast after with collagenase digesting, and reuse contains the DMEM culture fluid inoculated and cultured of hyclone; Isolated vascular endothelial cell is centrifugal, cleaning are used the culture fluid re-suspended cell, inoculated and cultured;
3) with above-mentioned each cell amplification culture;
(2) structure of organization engineering skin may further comprise the steps:
1) getting the cell of above-mentioned amplification culture, is 0.5 * 10 with density
5～1 * 10
7The dermal fibroblast of/ml and vascular endothelial cell be by 1: 1～10: 1 mixed, and then be that 1～15% type i collagen mixes with concentration;
2) 37 ℃ hatch solidify after, add culture fluid and cultivate;
3) on above-mentioned cultured cell surface with 1 * 10
5～5 * 10
6/ ml inoculates epithelial cells, continues to cultivate;
4) collagen of compound cell is moved to the gas-liquid face and carried out the organ culture 7～21 days, promptly obtain organization engineering skin, preserve standby.
4, according to the construction method of the described skin of claim 3, it is characterized in that: the digestion step when vascular endothelial cell separates is that with a liquid digestion 10 minutes, reuse b liquid digested 5 minutes earlier; Wherein a liquid is 1: 1 the mixed liquor of ethylenediaminetetraacetic acid of 625U/ml collagenase and 0.1%; B liquid is 0.25% pancreatin and 1: 1 mixed liquor of 0.1% ethylenediaminetetraacetic acid.
According to the construction method of the described skin of claim 3, it is characterized in that 5, the proportioning of vascular endothelial cell culture fluid is: culture medium M199 is 80%, and calf serum is 20%; Other contains commercial basic fibroblast growth factor bFGF is 5～20ng/ml, and heparin is 6090 μ g/ml.
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|---|---|---|---|
| CN 03134540 CN1210005C (en) | 2003-09-02 | 2003-09-02 | Tissue engineering skin containing blood vessel structure and its construction method |
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| CN1210005C true CN1210005C (en) | 2005-07-13 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101474426B (en) * | 2009-01-14 | 2012-09-05 | 首都医科大学宣武医院 | Vascular matrix for removing cells in vascular tissue and preparation method thereof |
| CN108452381A (en) * | 2018-05-14 | 2018-08-28 | 太原理工大学 | A kind of organization engineering skin and preparation method thereof with layered structure |
| CN109529123B (en) * | 2018-11-08 | 2021-02-19 | 中国人民解放军第四军医大学 | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof |
| CN109550080A (en) * | 2019-01-24 | 2019-04-02 | 中国人民解放军陆军特色医学中心 | A kind of artificial bilayer's skin and preparation method thereof |
| EP3875124A1 (en) * | 2020-03-02 | 2021-09-08 | Urgo Recherche Innovation Et Developpement | Method for obtaining a pre-vascularised dermo-epidermic tissue |
| CN114949357B (en) * | 2022-05-20 | 2023-08-01 | 上海市第十人民医院 | Penis decellularized scaffold and preparation method and application thereof |
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