CN1193802C - Double-layered artificial skin and its prepn process - Google Patents
Double-layered artificial skin and its prepn process Download PDFInfo
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- CN1193802C CN1193802C CNB02136043XA CN02136043A CN1193802C CN 1193802 C CN1193802 C CN 1193802C CN B02136043X A CNB02136043X A CN B02136043XA CN 02136043 A CN02136043 A CN 02136043A CN 1193802 C CN1193802 C CN 1193802C
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Abstract
The present invention provides a double-layer artificial skin graft which comprises: a dermis layer and an epidermal layer, wherein the dermis layer comprises biodegradable materials, such as polyglycollic acid, etc., fibroblast and stromatin; the epidermal layer is positioned on one main surface of the dermis layer. Continuous epidermal cells grow on the surface of a fibroblast-PGA compound in the artificial skin of the present invention, and epidermal cells are favorably differentiated and have comparatively obvious layers. A section shows that the tissue engineering artificial skin has a tissue structure characteristic which is similar to normal human body skin. The present invention also provides a preparation method and a purpose of the artificial skin.
Description
Technical field
The present invention relates to medical science and biomedical engineering field, relating more specifically to the polyglycolic acid is the double-layered artificial skin that support makes up, and its production and use.
Background of invention
Skin covering directly contacts with external environment in human body whole body surface, is anatomy and physiological critical boundaries organ.Skin accounts for into 16% of body weight for humans, the about 1.2-2.2m of area
2Skin is made up of epidermis and corium, borrows subcutaneous tissue to link to each other with deep fascia, aponeurosis (aponeuroses) or the periosteum in deep.Skin originates from ectoderm and mesoderm; the complicated and eggcase of structure has important barrier action and protective effect, can prevent that extraneous stimulation from damaging intravital tissue; can stop foreign body and microorganism intrusion, and can stop body fluid to blend the absorption of material to external world outward.
Skin is the barrier that contacts with external environment as the organ of human body maximum.When causing skin injury owing to factors such as extraneous damage or diseases, its harm can be extremely slight, also can be fatal.
Before the history of skin injury reparation can be traced back to hundreds of years.Reparation approach at present commonly used has: from body skin-grafting, allograft of skin, xenogenesis skin-grafting, but because, immunologic rejection not enough and shortcoming such as spread disease for the district, seek a kind of ideal Graftskin and be always a difficult problem (Naughton GK.Skin and Epithelia.In Lanza RP who needs solution badly clinically, Langer R, Chick WL.Principles of Tissue Engineering.R.G.Landes Company and AcademicPress, Inc., 1997,769.).
Tissue engineering is the principle and the method for application project and life sciences, make up biological substitute, to recover, to keep or promote a science (the Nerem RM.Tissueengineering in the USA.Med Biol Eng Comput of impaired organ or tissue function, 1992,30 (4): CE8).The proposition of " organizational project " provides brand-new and challenging approach for artificial constructed a kind of Graftskin.
Dermagraft
It is a kind of artificial dermis of producing by Advanced Tissue Sciences company.The fibroblast that will obtain from the neonate foreskin is inoculated on biodegradable synthetic material polyglycolic acid (PGA) rack, after 14-17 days, fibroblast breeds justacrine collagen, fibronectin, proteoglycan, somatomedin etc. in a large number, forms the artificial dermis that is made of fibroblast, extracellular matrix and degradable biomaterial.Dermagraft is mainly used in reparation (Eaglstein WH, Falanga V.Tissue engineering for skin:An update.JAm Acad Dermatol, 1998,39 (6): 1007) of burn wound and chronic ulcer.Yet, Dermagraft
Be the monolayer organization's engineering skin that only has the artificial dermis layer, transplant and behind wound surface, still need first phase or the second stage of autologous transplanting split-thickness skin graft.Ideal tissue engineering skin should comprise epidermis and corium is two-layer, because epidermis and corium all are the integral body of mutual dependence for existence on function still on dissecting.
Apligraft
(being called Graftskin again) is first kind of commercial tissue engineering skin that had not only contained epidermal area but also contained skin corium, and this product of being produced by Organogenesis register of company gets permission to be used for clinical treatment venous ulcer and diabetic foot ulcers in the Canada and the U.S..Apligraft
Method for making be that the fibroblast that will obtain from the neonate foreskin is inoculated in the cattle type i collagen, 1 Zhou Houzai is at its surface seeding neonate keratinocyte.It is in blocks that cutin forms cell fusion after 4 days, covers " skin corium " under it.Whole complex is placed in gas-liquid interface subsequently to impel the further differentiation and maturation of keratinocyte.In a clinical research, Apligraft compares with the pressure therapy of traditional treatment venous ulcer, and cure rate was respectively 63% and 48.8% (P=0.012) in 6 months; The average healing was respectively 61 days and 181 days (P=0.003); Be significantly shorter than matched group (92 days/190 days for the intractable ulcer Apligraft treatment group healing time that surpasses 6 months, P=0.001) (Falanga V, Margolis D, Alvarez O, et al.Rapid healing of venous ulcers and lackof clinical rejection with an allogeneic cultured human skinequivalent.Arch Dermatol, 1998,134:293.).Yet the shortcoming of Apligraft is that the shrinkage factor of collagen gel is bigger; Use homogeneous variant cell and bovine collagen, exist the risk of unpredictable immunological rejection and latent viral infection; Fragility is big, operating difficulties.
Therefore, this area presses for new safe, the workable artificial skin succedaneum of exploitation.
Summary of the invention
Purpose of the present invention just provides a kind of new safe, artificial skin that method for making is easy.
Another object of the present invention just provides the method for making and the purposes of described artificial skin.
In a first aspect of the present invention, a kind of artificial skin transplant is provided, it comprises:
(a) skin corium, described skin corium contains pharmaceutically acceptable Biodegradable material, fibroblast and stromatin;
(b) be positioned at epidermal area on first type surface of skin corium.
In a preference, described pharmaceutically acceptable Biodegradable material is selected from: polyglycolic acid, polylactic acid, polylactic acid-glycolic acid or its mixture.
In another preference, described skin corium contains the 10-30% polyglycolic acid, 5-15% fibroblast, 55-85% stromatin.More preferably, described skin corium contains the 15-25% polyglycolic acid, 5-15% fibroblast, 60-80% stromatin.
In another preference, the thickness of described skin corium is 0.2-4mm, and the thickness of described epidermal area is 20-120um.
In another preference, described epidermal area contains keratinocyte.
In another preference, described fibroblast is selected from: dermal fibroblast, and multiple connective tissue cell, endotheliocyte and the smooth muscle cell in embryo's mesenchymal cell in period source.
In another preference, described epidermal keratinocytes and fibroblast comprise epidermal keratinocytes and the fibroblast through genetic modification, transformation.
In a second aspect of the present invention, a kind of method for preparing artificial skin transplant is provided, comprise step:
(a) fibroblast is inoculated in pharmaceutically acceptable Biodegradable material;
(b) under the condition that is fit to growth, cultivate, thereby be formed into fibrocyte-pharmaceutically acceptable biodegradable material compound;
(c) keratinocyte is inoculated in described fibroblast-pharmaceutically acceptable biodegradable material compound surface;
(d) under the condition that is fit to the keratinocyte growth, cultivate, thereby be formed into fibrocyte-pharmaceutically acceptable Biodegradable material-epidermis complex.
In a preference, described pharmaceutically acceptable Biodegradable material is selected from: polyglycolic acid, polylactic acid, polylactic acid-glycolic acid or its mixture;
In another preference, in step (a), fibroblastic inoculum concentration is 1 * 10
5-1 * 10
7Individual cell/10mg Biodegradable material (as polyglycolic acid).More preferably, fibroblastic inoculum concentration is 5 * 10
5-5 * 10
6Individual cell/10mg Biodegradable material (as polyglycolic acid).
In another preference, in step (c), the inoculum concentration of keratinocyte is 1 * 10
4-1 * 10
7Individual cells/square cm.More preferably, the inoculum concentration of keratinocyte is 1 * 10
5-5 * 10
6Individual cells/square cm.
In another preference, step (d) comprises two stages:
(i) complex that step (c) is obtained is under complete buried condition, in 37 ± 0.5 ℃, and 5%CO
2With cultivation under saturated humidity 4-14 days, thereby make keratinocyte propagation, form the keratinocyte layer;
(ii) the keratinocyte layer segment with the complex of acquisition in the step (i) is exposed in the air, continues to cultivate 4-21 days, thereby makes the keratinocyte keratinization.
Accompanying drawing is described
Fig. 1 is Histological section's photo of double-layered artificial skin of the present invention.
Fig. 2 A and 2B are the transmission electron microscope photos of double-layered artificial skin of the present invention.
The specific embodiment
The inventor passes through extensive and deep research, has improved the structure and the preparation method of double-layered artificial skin, has made first based on polyglycolic acid (Polyglycolic Acid, PGA) double-layered artificial skin of Biodegradable material such as grade.Double-layered artificial skin of the present invention has corium and epidermis double-decker, does not have and shrinks, and can directly sew up flap coverage, and alternative auto-skin grafting is used for the reparation of skin injury.
As used herein, term " epidermal area " refers to be positioned at the artificial skin surface, mainly the cellular layer that is made of keratinocyte.
As used herein, term " skin corium " refers to be positioned at the artificial skin lower face, mainly the cellular layer that is made of Biodegradable material racks such as polyglycolic acid, fibroblast and excretory stromatin thereof.Should be understood that the content of Biodegradable material rack, fibroblast and excretory stromatins thereof such as polyglycolic acid is not unalterable in artificial skin of the present invention.Because used materials such as polyglycolic acid are biodegradable in the artificial skin of the present invention, meeting is along with the time can degrade, so its content descends gradually.Usually, skin corium contains the 10-30% polyglycolic acid, 5-15% fibroblast, 55-85% stromatin.More preferably, described skin corium contains the 15-25% polyglycolic acid, 5-15% fibroblast, 60-80% stromatin.(should point out that the moisture in the artificial skin of the present invention all exists with the form of bound water basically, therefore be included in cell component and the stromatin.)
The material that can be used for the rack of artificial skin of the present invention is medically acceptable Biodegradable material, preferably is selected from: polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid (PLGA) or its mixture.More preferably be selected from polyglycolic acid (PGA).
The shape of Biodegradable material is not particularly limited, and is generally tridimensional network flat, the band space.
Can be used for fibroblast of the present invention and be not particularly limited, can be the fibroblast in any source, especially dermal fibroblast.Fibroblastic separation and cultural method all are to know in this area.
Can be used for keratinocyte of the present invention and be not particularly limited, can be the keratinocyte in any source.The separation of keratinocyte and cultural method all are to know in this area.
In preference, fibroblast should be from identical source, promptly from same mammal, more preferably from same individuality with keratinocyte.
The thickness of artificial skin of the present invention is not particularly limited.The thickness of common described skin corium is about 0.2-4mm, and the thickness of described epidermal area is about 20-120um.
Artificial skin of the present invention can prepare with following method:
(a) fibroblast is inoculated in Biodegradable material such as polyglycolic acid (as braiding shape polyglycolic acid).Fibroblastic inoculum concentration is about 1 * 10
5-1 * 10
7Individual cell/10mg polyglycolic acid preferably is about 5 * 10
5-5 * 10
6Individual cell/10mg polyglycolic acid.
(b) under the condition that is fit to growth, cultivate a period of time (being about 4-14 days usually), make fibroblast proliferation, thereby be formed into fibrocyte-polyglycolic acid complex.
(c) keratinocyte is inoculated in described fibroblast-polyglycolic acid composite surface; The inoculum concentration of keratinocyte is about 1 * 10 usually
4-1 * 10
7Individual cells/square cm preferably is about 1 * 10
5-5 * 10
6Individual cells/square cm.
(d) under the condition that is fit to the keratinocyte growth, cultivate complex a period of time (about 4-21 days), thereby be formed into fibrocyte-polyglycolic acid-epidermis complex.A kind of preferred mode is to cultivate in branch two stages, i.e. in the phase I with the complex that obtains not with condition that air contact under, promptly be immersed in the culture fluid, cultivation is 4-14 days under condition of culture, thereby keratinocyte is bred rapidly; In the second stage, the keratinocyte layer segment is exposed in the air, continues to cultivate 4-21 days, thereby keratinocyte is further broken up, be i.e. keratinization.So just formed double-deck artificial skin of the present invention.
Available various means of different is exposed to air with the keratinocyte layer segment.For example will place on the metal rack, and make the part epidermal area be exposed to air, and remainder still is positioned under the liquid level of culture fluid and gets final product at cell-PGA complex.Another kind of optimal way is the permeable filter membrane of cushioning under cell-PGA complex, makes gas-liquid interface.
Be used for culture fluid of the present invention and can select the common culture medium that is used for fibroblast and keratinocyte in this area for use.The culture fluid that a kind of preferred artificial dermis is cultivated is the DMEM culture medium that contains 10% hyclone.A kind of culture medium (keratinocyte culture medium) that preferably is used for the compound cultivation of double-layered artificial skin is: the mixed culture medium of DMEM and F12, by about 3:1 (v/v) mixed, also can add other additives during use, the about 50ug/ml of for example about 50ug/ml Niu Chuiti extract (BPE), the about 5ng/ml of recombinant human epidermal growth factor (rEGF) etc.
Can be formed with active, double-layer tissue engineering skin with the inventive method.The skin corium of organizational project bilayer skin is made of jointly PGA and fibroblast and excretory stromatin thereof, and epidermis is constantly to be bred, be differentiated to form by the keratinocyte that is seeded in the surface.The histology shows that artificial skin of the present invention has the characteristics of organizational structure similar with normal human skin, does not possess appendages and blood vessels such as hair follicle, sebaceous gland, sweat gland, does not also contain melanocyte, Langerhans cell, Merkel cell etc.
Double-layer tissue engineering artificial skin of the present invention has corium and epidermis double-decker, does not have and shrinks, and can directly sew up flap coverage, and alternative auto-skin grafting is used for the reparation of skin injury.
The invention has the advantages that:
(1) polyglycolic acid is the degradable absorbing material, has good mechanical performance, reliable biological safety and biocompatibility;
(2) have the epidermal area of keratinization, can bring into play the important barrier and the defencive function of skin;
(3) being made of jointly degradable absorbing material and cell and excretory stromatin thereof, is activated double-layered artificial skin truly.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The separation of keratinocyte and cultivation
1) draws materials
Children's's foreskin of excision is inserted and is contained in the antibiotic keratinocyte culture fluid, brings cell culture chamber into.
2) separate epidermis
Carefully cut off subcutaneus adipose tissue and part corium with shears, and be trimmed to the strip of wide about 1-2mm, with no calcium magnesium PBS rinsing twice, epidermis side is dipped in the 0.24U/ml neutral protease (Dispase II) 4 ℃ up and spends the night.
3) digestion
After 18 hours, with two ophthalmology tweezers epidermis is torn from corium, be cut into fragment, reuse 0.05% trypsin-0.53mM EDTA was 37 ℃ of digestion 15 minutes, and 10ml pancreatin inhibitor (10mg/ml) stops digestion.Filter through 150 order stainless steel filtering nets, with 1000r/min centrifugal 10 minutes, supernatant discarded added keratinocyte culture fluid 10ml, mixing, trypan blue dyeing counting, cell viability＞85%.
4) former be commissioned to train foster
Make single cell suspension behind the counting, by 3 * 10
6/ 75cm
2The density inoculated and cultured, and serum-free keratinocyte culture medium (GIBRO, USA), at 37 ℃, 5%CO
2, to cultivate in the incubator of 100% relative humidity, culture fluid changed once in 3 days.
5) cultivation of going down to posterity
When cell fusion arrives 60%-75% density, there is not calcium magnesium PBS flushing with 10ml, digested 5-10 minute for 37 ℃ through 0.05% pancreatin-0.53mM EDTA, add the 10ml pancreatin inhibitor and stop digesting, move in the centrifuge tube centrifugal 10 minutes of 1000r/min.Abandoning supernatant adds keratinocyte culture fluid 10ml, mixing, and counting is in the cultivation of going down to posterity of 1: 3 ratio.Change culture fluid 2-3 day one time, during to 60%-75% density, repeated transmission is commissioned to train foster up to cell fusion.
Embodiment 2
Dermal fibroblast separates and cultivates
1) digestion
Corium behind the epidermis of tearing is cut into fragment, and the collagenase with 0.1% filters through 150 order stainless steel filtering nets 37 ℃ of digestion 2 hours, and with 1000r/min centrifugal 5 minutes, supernatant discarded added no calcium magnesium PBS cleaning 3 times.Add the DMEM culture fluid 10ml that contains 10% hyclone, mixing, trypan blue dyeing counting.
2) former be commissioned to train foster
Make single cell suspension behind the counting, press 1-2 * 10
6/ 75cm
2The density inoculated and cultured contains the DMEM culture fluid of 10% hyclone, at 37 ℃, and 5%CO
2, to cultivate in the incubator of 100% relative humidity, culture fluid changed once in 3 days.
3) cultivation of going down to posterity
When cell fusion arrives 60%-75% density, there is not calcium magnesium PBS flushing with 10ml, through 37 ℃ of digestion of 0.25% pancreatin 5 minutes, add the DMEM culture fluid that contains 10% hyclone and stop digesting, move in the centrifuge tube centrifugal 5 minutes of 1000r/min.Abandoning supernatant adds the DMEM culture fluid 10ml contain 10% hyclone, mixing, and counting is in the cultivation of going down to posterity of 1: 3 ratio.Change culture fluid 2-3 day one time, during to 60%-75% density, repeated transmission is commissioned to train foster up to cell fusion.
Embodiment 3
The preparation artificial skin
(1) is built into fibrocyte-PGA complex
To the dermal fibroblast of preparation among the embodiment 2, collect with the trypsinization method, then respectively with 0.2 * 10
6/ 10mg polyglycolic acid, 1 * 10
6/ 10mg polyglycolic acid, 5 * 10
6The concentration of/10mmg polyglycolic acid is inoculated in netted polyglycolic acid (PGA) with it.Then with postvaccinal PGA material, at 37 ± 0.5 ℃, 5%CO
2, under the saturated humidity condition, in containing the DMEM culture fluid of 10% hyclone (culture fluid changed once in per 3 days), cultivate 1-2 week, be formed into fibrocyte-PGA complex.
(2) formation of bilayer skin
Collect the keratinocyte of preparation among the embodiment 1, then respectively with 0.5 * 10
5/ square centimeter, 5 * 10
5/ square centimeter, 50 * 10
5/ cm
2Density it is seeded in fibroblast-PGA composite surface that previous step makes suddenly.With the mixed culture medium (by about 3: 1 (v/v) mixed, be added with Niu Chuiti extract (BPE) 50ug/ml, recombinant human epidermal growth factor (rEGF) 5ng/ml) of postvaccinal complex at DMEM and F12, in 37 ± 0.5 ℃, 5%CO
2, under the saturated humidity condition, in the keratinocyte culture medium, cultivated 4-14 days.
After 4-14 days, the permeable filter membrane of cushioning under cell-PGA complex, the keratinocyte layer segment is exposed in the air, make gas-liquid interface, wherein fibroblast layer (being skin corium) and part cutin formation cellular layer (being epidermal area) still is arranged in culture fluid, and part cutin formation cellular layer (being epidermal area) is exposed to air.At 37 ± 0.5 ℃, 5%CO
2, continue under the saturated humidity condition to cultivate 4-21 days, thereby make the further differentiation (being keratinization) of keratinocyte.Thereby obtain to have double-deck artificial skin transplant.
| Fibroblast inoculum concentration (10 6/10mg PGA) | Keratinocyte inoculum concentration (10 5/ square centimeter) | |
| Artificial skin 1 | 0.2 | 0.5 |
| Artificial skin 2 | 1 | 0.5 |
| Artificial skin 3 | 5 | 0.5 |
| Artificial skin 4 | 0.2 | 5 |
| Artificial skin 5 | 1 | 5 |
| Artificial skin 6 | 5 | 5 |
| Artificial skin 7 | 0.2 | 50 |
| Artificial skin 8 | 1 | 50 |
| Artificial skin 9 | 5 | 50 |
Detection method
Each group is cut into two respectively at 1,2,3,4 all specimen takens after the compound cultivation, and one liquid-solid fixed with 10% neutral formalin, and paraffin embedding is made the 4um slab, does conventional H E dyeing.Another fritter tissue is fixing with 2% glutaraldehyde, the acid of 1% blade of a sword, and gradient ethanol dewaters step by step, after the EPON embedding, and the row transmission electron microscope observing.
The result
Fibroblast is a large amount of substrate of well-grown justacrine on PGA; Inoculation epidermal keratinocytes-visible successive the epidermis cell of the 1-2 layer well-grown on " fibroblast-PGA " complex in week back; PGA part degraded during 4 weeks, the skin lamination differentiation is more perfect, and visible basal layer, prickle cell layer and cuticular layer are with the normal skin structural similarity.Wherein, skin corium contains the 10-30% polyglycolic acid, 5-15% fibroblast, 55-85% stromatin.
The tissue slice result as shown in Figure 1.In the tissue slice of artificial skin 1-9, can see successive epidermal growth in fibroblast-PGA composite surface, and the epidermis cell well differentiated, significantly layering is arranged.Section display organization engineering artificial skin has the characteristics of organizational structure similar with normal human skin, does not possess appendages and blood vessels such as hair follicle, sebaceous gland, sweat gland, does not also contain melanocyte, Langerhans cell, Merkel cell etc.Ultrastructure shows epidermal keratinocytes and fibroblastic form of artificial skin are normal, epidermal differentiation is more complete, the multiple layer differentiation of basal cell layer, prickle cell layer and cuticular layer arranged, and collagen fiber are crisscross in the corium, visible polyglycolic acid material of not degrading fully as yet.
The ultrastructure photo is shown in Fig. 2 A and 2B.Scanning electron microscope is shown as a large amount of stromatin compositions of fibrocyte, epidermal keratinocytes tactophily justacrine on PGA.The transmission electron microscope observing organization engineering skin, 2-4 is visible epidermis cell layering well differentiated during week, as seen disperse in the endochylema, very thin keratin filament, the adjacent surface of cell has desmosome to connect, the fibroblast form is normal in the artificial dermis layer, be distributed with a large amount of collagen fiber, the still visible PGA that does not degrade fully is at the visible Discontinuous basement membrane composition of two confluent monolayer cells intersections.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. artificial skin transplant is characterized in that it comprises:
(a) skin corium, described skin corium contains pharmaceutically acceptable Biodegradable material, fibroblast and stromatin, and skin corium contains the 5-15% fibroblast, the 55-85% stromatin;
(b) be positioned at epidermal area on first type surface of skin corium, described epidermal area contains keratinocyte.
2. graft as claimed in claim 1 is characterized in that, described pharmaceutically acceptable Biodegradable material is selected from: polyglycolic acid, polylactic acid, polylactic acid-glycolic acid or its mixture.
3. graft as claimed in claim 1 is characterized in that described skin corium contains the 15-25% polyglycolic acid, 5-15% fibroblast, 60-80% stromatin.
4. graft as claimed in claim 1 is characterized in that, the thickness of described skin corium is 0.2-4mm, and the thickness of described epidermal area is 20-120um.
5. graft as claimed in claim 1 is characterized in that described skin corium contains the 10-30% polyglycolic acid.
6. graft as claimed in claim 1 is characterized in that, described epidermal keratinocytes and fibroblast comprise epidermal keratinocytes and the fibroblast through genetic modification, transformation.
7. graft as claimed in claim 1 is characterized in that, described fibroblast is selected from: dermal fibroblast, and multiple connective tissue cell, endotheliocyte and the smooth muscle cell in embryo's mesenchymal cell in period source.
8. a method for preparing artificial skin transplant is characterized in that, comprises step:
(a) fibroblast is inoculated in pharmaceutically acceptable Biodegradable material;
(b) under the condition that is fit to growth, cultivate, thereby be formed into fibrocyte-pharmaceutically acceptable biodegradable material compound;
(c) keratinocyte is inoculated in described fibroblast-pharmaceutically acceptable biodegradable material compound surface;
(d) under the condition that is fit to the keratinocyte growth, cultivate, thereby be formed into fibrocyte-pharmaceutically acceptable Biodegradable material-epidermis complex; Wherein this incubation step comprises two stages:
(i) complex that step (c) is obtained is under complete buried condition, in 37 ± 0.5 ℃, and 5%CO
2With cultivation under saturated humidity 4-14 days, thereby make keratinocyte propagation, form the keratinocyte layer;
(ii) the keratinocyte layer segment with the complex of acquisition in the step (i) is exposed in the air, continues to cultivate 4-21 days, thereby makes the keratinocyte keratinization.
9. method as claimed in claim 8 is characterized in that, described pharmaceutically acceptable Biodegradable material is selected from: polyglycolic acid, polylactic acid, polylactic acid-glycolic acid or its mixture;
And in step (a), fibroblastic inoculum concentration is 1 * 10
5-1 * 10
7Individual cell/10mg polyglycolic acid;
In step (c), the inoculum concentration of keratinocyte is 1 * 10
4-1 * 10
7Individual cells/square cm.
10. method as claimed in claim 9 is characterized in that, in the step (a), fibroblastic inoculum concentration is 5 * 10
5-5 * 10
6Individual cell/10mg polyglycolic acid;
In the step (c), the inoculum concentration of keratinocyte is 1 * 10
5-5 * 10
6Individual cells/square cm.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9259445B2 (en) | 2006-06-07 | 2016-02-16 | Universidad Tecnica Federico Santa Maria | Integrated implant system (IIS) biocompatible, biodegradable and bioactive, comprising a biocompatible sterile porous polymeric matrix and a gel, integrating in situ the tridimensional matrix structure |
| CN100522264C (en) * | 2006-09-14 | 2009-08-05 | 中国人民解放军第三军医大学第三附属医院 | Preparation method of double-layer tissue engineering skin comprising basement membrane structure |
| CN101361989B (en) * | 2008-09-03 | 2012-09-19 | 陕西瑞盛生物科技有限公司 | Double membrane tissue patching material and preparation method thereof |
| CN101361990B (en) * | 2008-09-03 | 2012-09-19 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
| PT2573167T (en) * | 2010-05-21 | 2019-12-10 | Rodrigo Focion Soto Pareja | 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 |
| US10273549B2 (en) | 2016-04-21 | 2019-04-30 | Vitrolabs Inc. | Engineered skin equivalent, method of manufacture thereof and products derived therefrom |
| CN106390205B (en) * | 2016-11-16 | 2019-08-02 | 浙江省人民医院 | A kind of preparation method of 3D printing artificial skin |
| CN106474567B (en) * | 2016-11-16 | 2019-08-02 | 浙江省人民医院 | A kind of flexibility artificial skin and preparation method |
| 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 |
| CN111035812A (en) * | 2019-12-20 | 2020-04-21 | 厦门大学附属中山医院 | Human-derived cell biological composite patch |
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