CN1504241A - Tissue engineering artificial skin constructed on the rack of heterogenous or xenogenous bone matrix gelatin - Google Patents
Tissue engineering artificial skin constructed on the rack of heterogenous or xenogenous bone matrix gelatin Download PDFInfo
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- CN1504241A CN1504241A CNA021510202A CN02151020A CN1504241A CN 1504241 A CN1504241 A CN 1504241A CN A021510202 A CNA021510202 A CN A021510202A CN 02151020 A CN02151020 A CN 02151020A CN 1504241 A CN1504241 A CN 1504241A
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Abstract
The invention provides a double layer artificial skin transplant which comprises, (a) a corium layer including bone matrix gelatin, fibroblast and stromatin, (b) a cuticular layer above one primary surface of the corium layer. In the double layer artificial skin according to the present invention, continuous cuticle cells are growing on the surface of the fibroblast-bone matrix gelatin compound, the cuticle cell differentiation has been found good with appreciable stratification. The invention also provides the method for preparation and use of the artificial skin.
Description
Technical field
The present invention relates to medical science and biomedical engineering field, relating more specifically to xenogenesis or allograph bone matrix gelatin is the double-layered artificial skin that support makes up, and its production and use.
Background technology
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.
The tissue of body is made of jointly cell and extracellular matrix (extracellular matrix), and the content of extracellular matrix differs in the various tissues.Extracellular matrix is suitable for the function needs of various tissues in the physical behavior of pericellular decision tissue.Although extracellular matrix is various informative, its common characteristic is to form highly organized network structure by multiple macromolecular components.These compositions comprise collagen, non-collagen sugar albumen, elastin laminin, Dan Baijutang and aminoglycan etc.Wherein collagen is the important component in the extracellular matrix, is the rich in protein of animal body intensive amount, accounts for more than 30% of human body protein total amount, belongs to insoluble fibre shape protein, is dispersed throughout various organs and tissue in the body.The type of collagen has 12 kinds more than, and the collagen fiber of skin mainly are made of I type and III Collagen Type VI, and content less I V-type, VII Collagen Type VI are the important composition compositions of basement membrane.Normal skin accounts for more than 75% based on type i collagen, and III Collagen Type VI content is 25%.
Bone is made up of the extracellular matrix of cell and calcification.Bone matrix (bone matrix) is called for short sclerotin, i.e. the extracellular matrix of the osseous tissue of calcification comprises organic principle and inorganic constituents, and is moisture few.Organic principle comprises a large amount of collagen fiber and a small amount of substrate, and wherein collagen fiber account for 90%, and chemical constituent mainly is a type i collagen albumen.Substrate is gel, and main component is proteoglycan and complex thereof, has the effect of binder fibre.Inorganic constituents claims bone mineral again, accounts for 65% of dried bone weight, and based on calcium, P elements, the existence form of bone mineral mainly is a hydroxylapatite crystal, is fine acicular, and long 10-20nm also combines closely with it along the arrangement of collagen fibril major axis.
The fifties in last century, Kreutz etc. find allosome freeze drying bone transplanting not only can not causing host's immunological rejection the earliest, and the osteocyte creeping substitution and new bone formation (Kreutz JP that can also induce the host, HyattGW, Turner TC, et al.The preservation and clinical use of freeze dried bone.J Bone Joint Surg.1956; 33A:863).Subsequently, on the basis of freeze drying bone, with chemical method remove the inorganic calcium phosphorus composition and be prepared into decalcified bone matrix (Demineralized bone matrix, DBM).Decalcified bone matrix now has been widely used in clinical, numerous application report and has shown that the decalcification bone is having a good application prospect aspect the treatments such as bone is damaged.Though DBM has the induced osteogenesis effect, owing to, only leave the organic moiety bone matrix, cause mechanical strength to descend by demineralization, can not meet with stresses, can't substitute skeleton heavy burden function.(bonematrix gelatin is on the basis of decalcification bone BMG) to bone matrix gelatin, and the method for handling with continuous chemical is prepared from.Urist had prepared the allogeneic bone matrix gelatin first in 1973, owing in preparation process, removed most non-collagenic structure proteins and lipid, made antigenicity further reduce (Urist MR, et al.Bone morphogenesisin implants of insoluble bone gelatin, Proc Natl Acad Sci.1973; 70:3511-3515).BMG has full decalcified bone matrix gelatin, surperficial decalcified bone matrix gelatin etc., and full decalcification BMG lacks the support effect, can not repair stressed large segmental bone defect separately, though the BMG of partly decalcifying has support effect preferably, uses in conjunction with slower separately.Yet, still BMG is not used to make up the report of artificial skin.
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.
At present, skin injury reparation approach 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, seek a kind of ideal Graftskin and be always difficult problem of needing solution badly clinically for the district.
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 contain full decalcified bone matrix gelatin, fibroblast and stromatin;
(b) be positioned at epidermal area on first type surface of skin corium.
In a preference, described skin corium contains the 10-30% bone matrix gelatin, 5-15% fibroblast, 55-85% stromatin.
In another preference, described bone matrix gelatin is the bone matrix gelatin derived from pig, cattle, sheep or Canis familiaris L..
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, it is characterized in that described epidermal keratinocytes and fibroblast comprise epidermal keratinocytes and the fibroblast through genetic modification, transformation.
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 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 full decalcified bone matrix gelatin;
(b) under the condition that is fit to growth, cultivate, thereby be formed into fibrocyte-complete gelatin-compounded thing of decalcified bone matrix;
(c) keratinocyte is inoculated in the described fibroblast-complete gelatin-compounded thing of decalcified bone matrix surface;
(d) under the condition that is fit to the keratinocyte growth, cultivate, thereby be formed into fibrocyte-full decalcified bone matrix gelatin-epidermis complex.
In a preference, described full decalcified bone matrix gelatin prepares in order to the below method:
Spongy bone is made the lamellar of 1-2 millimeters thick;
The distilled water rinsing of pre-cooling;
Continue in hydrochloric acid-10% sodium chloride solution to stir, 4 ℃, 24h ± 12h;
The distilled water rinsing of pre-cooling;
Chloroform: methanol (1: 1), 25 ℃, 24h ± 12h;
The distilled water rinsing of pre-cooling;
Hydrazoic acid,sodium salt, 25 ℃, 12h ± 6h;
The distilled water rinsing of pre-cooling;
Hydrogen peroxide soaks, 30 minutes ± 15 minutes;
Phosphate buffer, 25 ℃;
Lyophilization behind the oxirane disinfection, makes full decalcified bone matrix gelatin.
In another preference, in step (a), fibroblastic inoculum concentration is 1 * 10
5-1 * 10
7Individual cell/10mg bone matrix gelatin more preferably is 5 * 10
5-5 * 10
6Individual cell/10mg bone matrix gelatin.
In another preference, in step (c), the inoculum concentration of keratinocyte is 1 * 10
4-1 * 10
7Individual cells/square cm more preferably is 1 * 10
5-5 * 10
6Individual cells/square cm,
In another preference, described 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.
Description of drawings
Fig. 1 is Histological section's photo of monolayer artificial skin of the present invention.
Fig. 2 is Histological section's photo of double-layered artificial skin of the present invention.
The specific embodiment
The inventor has improved the structure and the preparation method of double-layered artificial skin through extensive and deep research, and the xenogenesis bone matrix gelatin of the full decalcification of first Application makes up artificial dermis and double-layer tissue engineering skin as cultivating support, has made double-layered artificial skin.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.For example, be used for the reparation of refractory wound surface (diabetic foot ulcers and venous ulcer etc.) and covering of early burn wound surface and burn back cicatrix patient's reparation in late period.
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 bone matrix gelatin rack, fibroblast and excretory stromatin thereof.Should be understood that in artificial skin of the present invention, the content of bone matrix gelatin rack, fibroblast and excretory stromatin thereof is not unalterable.Because used bone matrix gelatin can be degraded along with the time in the artificial skin of the present invention, so its content descends gradually.Usually, skin corium contains the 10-30% bone matrix gelatin, 5-15% fibroblast, 55-85% stromatin.More preferably, described skin corium contains the 15-25% bone matrix gelatin, 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 the bone matrix gelatin of full decalcification.Full decalcified bone matrix gelatin method for making is as known in the art.Its main component is a type i collagen albumen.Because the collagen protein in the skin also mostly is the I type, therefore, full decalcified bone matrix gelatin is suitable as the host material of cultivation, at external structure monolayer dermal tissue and bilayer, activated artificial skin.
Be used for bone matrix gelatin of the present invention and can be of the same race or xenogeneic, also can be allochthonous or same individuality.Preferably, described bone matrix gelatin is xenogenesis or allosome.Bone matrix gelatin commonly used is from non-human mammals such as pig, cattle, sheep, Canis familiaris L.s.Particularly preferably be from pig and cattle.
The shape of bone matrix gelatin 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 full decalcified bone matrix gelatin.Fibroblastic inoculum concentration is about 1 * 10
5-1 * 10
7Individual cell/10mg bone matrix gelatin preferably is 5 * 10
5-5 * 10
6Individual cell/10mg bone matrix gelatin.In addition, with " individual cell/ml culture fluid " expression, inoculum concentration is generally 1 * 10
5-100 * 10
6Individual cell/ml culture fluid preferably is about 5 * 10
6-50 * 10
6Individual cell/ml culture fluid.
(b) under the condition that is fit to growth (as at 37 ℃, 5%CO2 is under the condition of saturated humidity) cultivates a period of time (being about 4-14 days usually), makes fibroblast proliferation, thereby is formed into fibrocyte-bone matrix gelatin complex.(annotate: form this moment is monolayer artificial dermis tissue).
(c) keratinocyte is inoculated in described fibroblast-bone matrix gelatin 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-bone matrix gelatin-epidermis complex.A kind of preferred mode is to divide two stages to cultivate in step, 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 at cell-bone matrix gelatin complex-metal rack on, 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.Another kind of optimal way is the permeable filter membrane of cushioning under cell-bone matrix gelatin 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 bone matrix gelatin 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.
Can be formed with active, monolayer or double-layer tissue engineering skin with the inventive method.The skin corium of organizational project bilayer skin is made of jointly bone matrix gelatin 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.
Major advantage of the present invention is:
(1) bone matrix gelatin 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) artificial skin that is made of jointly bone matrix gelatin and cell and excretory stromatin thereof is an 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 preparation of xenogenesis decalcification ossein sponge
In the present embodiment, prepare the xenogenesis decalcification ossein sponge of pig and cattle respectively, method is as follows:
1, gets the spongy bone of donor (pig and cattle) long bone of limbs, make the lamellar of 1-2 millimeters thick, put into following solution then and handle continuously.
2, the distilled water rinsing of pre-cooling;
3, continue in 5% hydrochloric acid-10% sodium chloride solution to stir, 4 ℃, 24h ± 12h; (effect is decalcification)
4, the distilled water rinsing of pre-cooling;
5, chloroform: methanol (1: 1), 25 ℃, 24h ± 12h; (effect is defat)
6, the distilled water rinsing of pre-cooling;
7, Hydrazoic acid,sodium salt, 25 ℃, 12h ± 6h; (effect is to remove blood constituent)
8, the distilled water rinsing of pre-cooling;
9, hydrogen peroxide soaks, 30 minutes ± 15 minutes; (effect is further to remove antigenicity)
10, phosphate buffer, 25 ℃;
11,, behind the oxirane disinfection, be stored under the room temperature standby again through lyophilization.
Embodiment 2
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 (DispaseII) 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 * 106/75cm2 density inoculated and cultured, serum-free keratinocyte culture medium (GIBRO, USA), at 37 ℃, 5%CO2 cultivates in the incubator of 100% relative humidity, and 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-0.53mMEDTA, 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 3
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 * 106/75cm2 density inoculated and cultured, contain the DMEM culture fluid of 10% hyclone, at 37 ℃, 5%CO2 cultivates in the incubator of 100% relative humidity, and 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 4
The preparation double-layered artificial skin
(1) is built into fibrocyte-bone matrix gelatin complex
To the dermal fibroblast of preparation among the embodiment 3, collect with the trypsinization method, then respectively with 2 * 10
6Individual cell/ml culture fluid, 10 * 10
6Individual cell/ml culture fluid, 50 * 10
6The concentration of individual cell/ml culture fluid is inoculated in the pig of preparation among the embodiment 1 or the bone matrix gelatin (bone matrix gelatin) of cattle with it.Then with postvaccinal bone matrix gelatin material, at 37 ± 0.5 ℃, under 5%CO2, 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-bone matrix gelatin complex (artificial dermis) (see figure 1).
(2) formation of bilayer skin
Collect the keratinocyte of preparation among the embodiment 2, then respectively with 0.5 * 10
6/ square centimeter, 5 * 10
6/ square centimeter, 50 * 10
5The density of/square centimeter is seeded in fibroblast-bone matrix gelatin composite surface that previous step makes suddenly with it.Postvaccinal complex at 37 ± 0.5 ℃, under 5%CO2, the saturated humidity condition, was cultivated 4-14 days in the keratinocyte culture medium.
After 4-14 days, the permeable filter membrane of cushioning under cell-bone matrix gelatin 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 ℃, continue cultivation 4-21 days under 5%CO2, the saturated humidity condition, thereby make the further differentiation (being keratinization) of keratinocyte.Thereby obtain to have double-deck artificial skin transplant.
| Fibroblast inoculum concentration (10 6Individual cell/ml culture fluid) | Keratinocyte inoculum concentration (10 6/ square centimeter) | Full decalcified bone matrix gelatin source | |
| Artificial skin 1 | ????2 | ????0.5 | Pig |
| Artificial skin 2 | ????10 | ????0.5 | Pig |
| Artificial skin 3 | ????50 | ????0.5 | Pig |
| Artificial skin 4 | ????2 | ????5 | Pig |
| Artificial skin 5 | ????10 | ????5 | Pig |
| Artificial skin 6 | ????50 | ????5 | Pig |
| Artificial skin 7 | ????2 | ????50 | Pig |
| Artificial skin 8 | ????10 | ????50 | Pig |
| Artificial skin 9 | ????50 | ????50 | Pig |
| Artificial skin 10 | ????2 | ????5 | Cattle |
| Artificial skin 11 | ????10 | ????5 | Cattle |
| Artificial skin 12 | ????50 | ????5 | Cattle |
Detection method
Each group is respectively at 1,2,3,4 all specimen takens after the compound cultivation, and liquid-solid fixed with 10% neutral formalin, paraffin embedding is made the 4um slab, does conventional H E dyeing.
The histology: fibroblast is a large amount of substrate of well-grown justacrine on bone matrix gelatin; The visible successive epidermis cell of the 1-2 layer well-grown on " fibroblast-bone matrix gelatin " complex in one week of inoculation epidermal keratinocytes back; Bone matrix gelatin 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.
The result
Result of the test as shown in Figure 2.In the tissue slice of artificial skin 1-12, can see successive epidermal growth in fibroblast-bone matrix gelatin 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.
In addition, the result also shows, all can be used for the present invention from the bone matrix gelatin of different plant species.
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 contain full decalcified bone matrix gelatin, fibroblast and stromatin;
(b) be positioned at epidermal area on first type surface of skin corium.
2. graft as claimed in claim 1 is characterized in that described skin corium contains the 10-30% bone matrix gelatin, 5-15% fibroblast, 55-85% stromatin.
3. graft as claimed in claim 1 is characterized in that, described bone matrix gelatin is the bone matrix gelatin derived from pig, cattle, sheep or Canis familiaris L..
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 epidermal area contains keratinocyte.
6. graft as claimed in claim 5 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 full decalcified bone matrix gelatin;
(b) under the condition that is fit to growth, cultivate, thereby be formed into fibrocyte-complete gelatin-compounded thing of decalcified bone matrix;
(c) keratinocyte is inoculated in the described fibroblast-complete gelatin-compounded thing of decalcified bone matrix surface;
(d) under the condition that is fit to the keratinocyte growth, cultivate, thereby be formed into fibrocyte-full decalcified bone matrix gelatin-epidermis complex.
9. method as claimed in claim 8 is characterized in that, described full decalcified bone matrix gelatin prepares in order to the below method:
Spongy bone is made the lamellar of 1-2 millimeters thick;
The distilled water rinsing of pre-cooling;
Continue in hydrochloric acid-10% sodium chloride solution to stir, 4 ℃, 24h ± 12h;
The distilled water rinsing of pre-cooling;
Chloroform: methanol (1: 1), 25 ℃, 24h ± 12h;
The distilled water rinsing of pre-cooling;
Hydrazoic acid,sodium salt, 25 ℃, 12h ± 6h;
The distilled water rinsing of pre-cooling;
Hydrogen peroxide soaks, 30 minutes ± 15 minutes;
Phosphate buffer, 25 ℃;
Lyophilization behind the oxirane disinfection, makes full decalcified bone matrix gelatin;
And in step (a), fibroblastic inoculum concentration is 1 * 10
5-1 * 10
7Individual cell/10mg bone matrix gelatin;
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 bone matrix gelatin;
In the step (c), the inoculum concentration of keratinocyte is 1 * 10
5-5 * 10
6Individual cells/square cm,
And described 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.
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| CN101138655B (en) * | 2007-07-20 | 2010-08-25 | 中国人民解放军第三军医大学第一附属医院 | Farrow skin prosoma organization for renovating skin wound, preparing method and use method thereof |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | 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 |
| CN101538555B (en) * | 2008-03-17 | 2013-08-21 | 莱雅公司 | Functional pigmented skin equivalent |
| CN113633827A (en) * | 2021-08-16 | 2021-11-12 | 中国人民解放军总医院第四医学中心 | Silk woven meniscus implant and preparation method thereof |
| CN114980939A (en) * | 2020-12-18 | 2022-08-30 | 爱恩斯生物科技(昆山)有限公司 | Artificial skin based on dermis containing substrate film layer and manufacturing method thereof |
| CN117582557A (en) * | 2024-01-19 | 2024-02-23 | 四川恒普科技有限公司 | Demineralized bone fiber and preparation method thereof |
-
2002
- 2002-12-04 CN CN 02151020 patent/CN1224429C/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101138655B (en) * | 2007-07-20 | 2010-08-25 | 中国人民解放军第三军医大学第一附属医院 | Farrow skin prosoma organization for renovating skin wound, preparing method and use method thereof |
| CN101538555B (en) * | 2008-03-17 | 2013-08-21 | 莱雅公司 | Functional pigmented skin equivalent |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | 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 |
| CN103003415B (en) * | 2010-05-21 | 2017-02-08 | 罗德里戈·福西翁·索托帕雷哈 | 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 |
| CN114980939A (en) * | 2020-12-18 | 2022-08-30 | 爱恩斯生物科技(昆山)有限公司 | Artificial skin based on dermis containing substrate film layer and manufacturing method thereof |
| CN113633827A (en) * | 2021-08-16 | 2021-11-12 | 中国人民解放军总医院第四医学中心 | Silk woven meniscus implant and preparation method thereof |
| CN117582557A (en) * | 2024-01-19 | 2024-02-23 | 四川恒普科技有限公司 | Demineralized bone fiber and preparation method thereof |
| CN117582557B (en) * | 2024-01-19 | 2024-03-19 | 四川恒普科技有限公司 | Demineralized bone fiber and preparation method thereof |
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