CN101361990A - Double layer artificial skin and preparation method thereof - Google Patents
Double layer artificial skin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a double-layer artificial skin and a preparation method thereof, wherein, cell-free membranous biological derivative material is used as a surface layer, and a fibroblast, extracellular matrix synthesized and secreted by the fibroblast and a cell growth factor are compounded in the interior of biological support material to form a dermis, and then the surface layer and the dermis are combined to form the double-layer artificial skin; a compact surface layer structure can effectively reduce the loss of water, electrolytes and protein from surface of wound, avoid the invading and the reproduction of bacteria to the impaired surface of wound as well as the infection of the surface of wound, and be beneficial to epitheliosis and epithelial growth; the dermis can directly repair the surface of wound, promote the ingrowth of cells around the surface of wound and the angiogenesis, induce the differentiation from stem cells to skin cells and quicken wound healing; the artificial skin has the advantages of being capable of promoting the regeneration of skin, improving the elasticity, the flexibility and the mechanical abrasion resistance of skin after the surface of wound is healed, reducing excess scar tissues, controlling the contracture, having excellent biocompatibility, increasing the success rate of transplant and improving the quality of healing; the double-layer artificial skin has wide material resources and simple production method, and is applicable to the clinical treatment of skin defect caused by inflammation, ulcer, thermal burns, iatrogenicity and the like.
Description
Technical field
The invention belongs to the technical field of biological materials of organizational project, be specifically related to a kind of double-layered artificial skin and preparation method thereof.
Background technology
Skin is the vital tissue organ that covers and protect body surface.Owing to reasons such as inflammation, ulcer, wound, burn, tumor post-operation and congenital malformation often cause the damaged and unusual of skin.For the at present methods that adopt auto-skin grafting of this tissue defect, this not only causes the new wound defective of skin donor site, and often is subjected to the restriction for the skin source more.
Along with the development of tissue engineering technique, the preparation artificial skin has become the focus of skin injury treatment.Ideal tissue engineering skin can imitate the structure of natural skin, has the compound skin of epidermal area and skin corium.The skin corium composition wherein and the composition of dermis of skin are approaching, are beneficial to growing into of hypodermal cell; The epidermal area covering plays the isolation external environment, prevents water evaporates, avoids bacterial invasion, and more compound somatomedin or medicine can play the purpose that promotes wound healing, treatment dermatosis simultaneously.
Commercial at present double-layered artificial skin has Integra (production of Integra LifeSciences company), artificial skin for double-deck substrate, the corium part is made the support with certain hole size by the collagen protein and the chondroitin sulfate of cattle tendon, epidermal area then is made up of pellosil, can control evapotranspiring of wound aqueous vapor, at wound healing process, skin corium can combine with wound, the pellosil of epidermal area can be removed in skin corium healing back, further do auto-skin grafting again, U.S. FDA was checked and approved Integra and has been used in the burn and scald treatment in 1996; Its deficiency mainly is, because there is nondegradable pellosil on its surface, is difficult for observing the early stage infection conditions of wound surface, is unfavorable for creeping and the sealing of wound surface of edge of wound epidermis cell, and the later stage is taken pellosil off also needs second operation.Apligraf (production of Organogenesis company) also is double-deck artificial skin, and skin corium is to be planted in the bovine collagen albumen by human dermal fibroblast, and epidermal area is to be made of human epidermal cell, attached to forming epidermal area on the corium; Its deficiency is that mainly epidermal area is to be made of the higher allosome epidermis cell of immunogenicity, has increased the immunological rejection of product, makes complex manufacturing, production time length, cost increase simultaneously.
Summary of the invention
Purpose of the present invention just provides a kind of double-layered artificial skin and preparation method thereof, has that manufacturing cycle is short, cost is low, method is easy, the advantage of easy operating; Prepared artificial skin has hemostasis, prevent that body fluid runs off, protects wound surface and promotes the function of wound healing; excellent biological compatibility is arranged; can be used for used for reparing skin defect, control traumatic infection, the difficult more wound surface of property of treatment, also have the advantage that shape, size and thickness can prepare according to demand simultaneously.
The double-layered artificial skin that the present invention proposes, include fibroblast, it is characterized in that: be by taking off the membranaceous bio-derived material of cell as the top layer, extracellular matrix and cell growth factor by fibroblast and synthesis secretion thereof are compound in the inner skin corium, both chimeric formations of forming of biologic bracket material; Described to take off the membranaceous bio-derived material of cell be natural biological tissue after taking off cell and handling formed membranaceous bio-medical material, comprise take off cell submucous layer of small intestine, acellular dermal matrix, take off the cell fascia, take off the cell submucous layer of bladder, human acellular amniotic membrane, take off cell dural any; Described fibroblast is the fibroblast from body or allosome source, or can be to the stem cell of fibroblast differentiation, comprise mesenchymal stem cells MSCs, fat mesenchymal stem cell, hematopoietic stem cell, skin progenitor cell, mescenchymal stem cell, muscle stem cell, any of liver stem cells, neural stem cell; Described biologic bracket material is to provide three-dimensional backing material for cell growth, comprises any or several mixing of Fibrinogen, fibrin, alginate, collagen protein, hyaluronic acid, chondroitin sulfate, chitosan, hydrogel, gelatin, polylactic acid, polyglycolic acid.
The preparation method of double-layered artificial skin of the present invention includes fibroblastic obtain, the take off preparation of cell biological derived material and the structure of double-layered artificial skin, and concrete steps are as follows:
Step 1). fibroblastic obtaining: fibroblast is from tissue, and cell culture processes routinely carries out In vitro culture, and the extensive amplification of cell can adopt existent method to carry out;
Step 2). take off the preparation of cell biological derived material: will take from dermal layer of the skin of the same race or xenogeneic or fascia or submucous layer of small intestine or amniotic membrane or dural any membranaceous biomaterial, place and contain peracetic acid and alcoholic acid aqueous solution soaking disinfection was not less than 1 hour, clean with phosphate buffer (PBS solution); If (adopt thicker biomaterial, as dermal layer of the skin, need place-80 ℃ more than freezing half an hour this moment, guarantee that the material internal and external temperature reaches consistent, take out the back and thaw naturally that so multigelation is 2～5 times, make the cell disintegrate of breaking fully, clean) with PBS solution; The NaOH solution soaking that is placed on 0.1～1M is carried out defat, is taken off the cell processing; Embathe the neutrality to pH with PBS solution, it is inserted in the mixed solution that contains DNA enzyme and alpha-galactosidase again, 37 ℃ of environment are handled more than 25 minutes down, remove residual DNA and α-ga1 native antigen composition, reduce immunogenicity, clean with PBS solution; For it is combined with timbering material firmly, adopt mechanical means evenly drilling on its material, aperture 0.1～1mm, spacing 0.5～5mm, taking off the cell biological derived material and foraminously take off cell biological derived material stack and compress it is combined closely atresia, become bilayer behind the dry sterilization and take off the cell biological derived material, standby;
Wherein, described peracetic acid and alcoholic acid volume final concentration are respectively 0.1～0.5% and 2～10%; The final concentration of described DNA enzyme and alpha-galactosidase is respectively 30～50U/ml and 10～25U/ml;
Step 3). the preparation of double-layered artificial skin culture fluid, its component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM 67.5%, commercial culture fluid F12 22.5%, hyclone 5～15%, basic fibroblast growth factor 2～100ng/ml, epithelical cell growth factor 2～100ng/ml, insulin 1～50ng/ml, hydrocortisone 10～500ng/ml, adenine 0.2～0.25mM, transferrins 1～10 μ g/ml;
Step 4). the structure of double-layered artificial skin: the gel solution of preparation biologic bracket material; After the bilayer of preparation taken off the cell biological derived material and soak into gel solution, place precuring under 37 ℃ of environment; The fibroblast of In vitro culture is pressed 10
4～10
6The concentration of individual/ml is mixed in the gel solution, again by 0.1～1ml/cm
2The bilayer that is added drop-wise to precuring takes off on porose of cell biological derived material, places 5%CO
2Solidify under 37 ℃ of conditions in the environment; Insert again cultivate 2～3 days in the bilayer skin culture fluid after, be placed on and continue on the cultivation support of cultivating in the vessel to cultivate 5～8 days, change liquid every day between culture period, double-layered artificial skin is cultivated and is finished; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment.
Wherein, the gel solution of described biologic bracket material can be by following preparation: under 4 ℃ of conditions, press mass ratio with collagen 7～10: chitosan 0.5～1: hyaluronic acid 2～3: chondroitin sulfate 0.5～1 mixes, with the acetic acid of 0.1～0.5M it being mixed with concentration is 6～20mg/ml solution, behind the ultraviolet radiation, the hyclone by its volume adding 10% adds the DMEM culture medium again and makes its final concentration reach 10mg/ml under the ice bath, regulate pH value to 7.2～7.4, make gel solution.Also can adopt the formulated of other gel solutions.
The double-layered artificial skin that the present invention is prepared has top layer and skin corium double-decker, includes the living cells from human body, can survive at wound surface after being applied to wound surface, can participate in the reparation of wound surface directly; Taking off the cell biological derived material forms fine and close surface structure and can reduce moisture, electrolyte and protein losing by wound surface effectively, stop invasion, the breeding of antibacterial to impaired wound surface, prevent traumatic infection, induce of the migration of the epidermis cell in wound week, help the propagation and the growth of epithelium to wound surface; Skin corium and dermis of skin composition are approaching, the extracellular matrix and the various kinds of cell somatomedin that include human body cell and synthesis secretion thereof, direct wound repairing, excellent biological compatibility can promote to create the generation, induced dry-cell of the growing into of pericyte, the blood vessel differentiation to Skin Cell, can obviously promote the healing of wound surface.
Double-layered artificial skin of the present invention has certain elasticity and toughness, and big or small thickness can be controlled, and is with short production cycle, does not have obvious immunological rejection, has excellent biological compatibility.Its effect in clinical practice has: as skin large defect patient's the wound surface covering and the covering of skin donor site; Repair malnutrition and infective wound surface; Treat the chronic difficulty ulcer wound surface of healing; The covering of postoperative wound surface and prevention infection; Promote growing into of wound surface granulation tissue, cell, promote the regeneration of damaged skin.
The double-layered artificial skin of the present invention's preparation, compare with present existing skin substitutes product, have the following advantages: can promote the regeneration of wound surface skin, skin elasticity, pliability and mechanical endurance behind the enhancing wound healing, reduce scar hyperplasia, the control contracture has excellent biological compatibility, can improve the artificial skin transplanting succeed rate, and improve healing quality; Surface structure and skin corium are entrenched togather, in conjunction with tight; Material source is extensive, production technology is simple; The treatment of the skin injury that can directly apply to prepared double-layered artificial skin inflammation, ulcer, burn and scald, reason such as iatrogenic cause is carried out personalized treatment at various disease; The shape size and the thickness of skin can prepare according to real needs.
The specific embodiment
Below in conjunction with example technical solution of the present invention is described in further detail.
Embodiment 1:
Step 1). fibroblastic obtaining: get prepuce tissues after the neonate excision, subcutaneous fat and Musclar layer were removed with phosphate buffer (PBS) flushing in the sterilization back, and skin is cut into strip, with 4U/ml neutral protease liquid digestion 2 hours; Remove epidermal area, dermal tissue is shredded, use the collagenase liquid of 625U/ml to digest 2.5 hours, results are cultivated by fibroblast; Former fibroblast of being commissioned to train foster is used cell factory or method amplification culture such as revolving bottle culture systems or bioreactor.
Step 2). take off the preparation of cell submucous layer of small intestine: the fresh pig jejunum is clean with distilled water flushing, be cut into required fragment, place and contained 0.2% peracetic acid and 10% alcoholic acid aqueous solution soaking disinfection 1 hour, with the rinsing of PBS liquid; Mechanical curettage mucous membrane of small intestine, flesh layer and serous coat, clean with the PBS rinsing, obtain tela submucosa; Again it is inserted in the NaOH solution of 1M, under 4 ± 2 ℃ of conditions, soaked 10 minutes, use the rinsing of PBS liquid to pH neutrality; It is inserted in the mixed solution that contains 40U/ml DNA enzyme and 10U/ml alpha-galactosidase soaked 1 hour, clean with the rinsing of PBS liquid; The cell submucous layer of small intestine is taken off in acquisition; Use void formers taking off evenly drilling on the cell submucous layer of small intestine, the aperture is 0.2mm, spacing 1mm, respectively get identical size stack, compress it is combined closely with the foraminous cell submucous layer of small intestine that takes off that takes off cell submucous layer of small intestine and atresia, dry back ethylene oxide sterilizing forms bilayer and takes off the cell submucous layer of small intestine;
Step 3). the preparation of bilayer skin culture fluid, each component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM 67.5%, commercial culture fluid F12 22.5%, hyclone 10%, basic fibroblast growth factor 10ng/ml, epithelical cell growth factor 5ng/ml, insulin 5ng/ml, hydrocortisone 20ng/ml, adenine 0.25mM, transferrins 5 μ g/ml;
Step 4). the structure of double-layered artificial skin: under 4 ℃ of conditions, press mass ratio with collagen 10: chitosan 1: hyaluronic acid 2: chondroitin sulfate 0.5 mixes, acetic acid with 0.1M is mixed with the solution that concentration is 10mg/ml with it, ultraviolet radiation under the ice bath, hyclone by its volume adding 10%, adding final concentration again is the DMEM culture medium of 10mg/ml, and regulating pH value is 7.2～7.4, becomes gel solution; The bilayer of preparation is taken off the cell submucous layer of small intestine and soaks into gel solution, precuring 30 minutes under 37 ℃ of environment in culture dish; The fibroblast of In vitro culture is pressed 10
6The concentration of individual/ml is mixed in the above-mentioned gel solution, presses 1ml/cm again
2The bilayer that is added drop-wise to precuring takes off on porose of cell submucous layer of small intestine, in 5%CO
2Solidify after 30 minutes under 37 ℃ of conditions in the environment, insert the bilayer skin culture fluid and cultivate after 3 days, be placed on again and continue on the cultivation support of cultivating in the vessel to cultivate 5 days, during every day change liquid, double-layered artificial skin is cultivated and is finished; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment.
The double-layered artificial skin of this examples preparation comprise human fibroblasts and synthesize, excretory extracellular matrix and somatomedin, be convenient to clinical use, can participate in the reparation of wound surface directly, be suitable for the damaged repair materials of skin histology.
Embodiment 2:
Step 1). fibroblastic obtaining: reference literature Dermal matrix as a carrier forin vivo delivery of human adipose-derived stem cells.Biomaterials.2008 Apr; 29 (10): 1431-42. has proved that fat mesenchymal stem cell can be divided into fibroblast.Get depleted healthy fatty tissue in the operation, with phosphate buffer (PBS) flushing, shred fatty tissue after sterilizing, use the collagenase liquid of 625U/ml to digest 150 minutes, the fat mesenchymal stem cell of gathering in the crops is seeded to culture bottle cultivates; The method of reuse cell factory or revolving bottle culture systems or bioreactor is carried out amplification culture.
Step 2). the acellular dermal preparation: fresh pig skin is clean with distilled water flushing, and machinery is removed epidermal area and hypodermis layer, is cut into 0.4mm thickness, 5cm
2Size places and contained 0.2% (v/v) peracetic acid and the alcoholic acid aqueous solution soaking disinfection of 10% (v/v) 1 hour, uses the PBS rinsing; Place-80 ℃ of freezing half an hour again, guarantee that the material internal and external temperature reaches consistent, take out the back and thaw naturally, so multigelation is 3 times, makes the cell disintegrate of breaking fully, with the cleaning of PBS solution; It is inserted in the NaOH solution of 0.3M, under 4 ± 2 ℃ of conditions, soaked 30 minutes, use the PBS rinsing to pH neutrality; Again it is inserted in the solution that contains 30U/ml DNA enzyme and 20U/ml alpha-galactosidase and soaked 1 hour, clean with the PBS rinsing, obtain acellular dermal; Use void formers evenly drilling on acellular dermal, the aperture is 0.5mm, spacing 3mm, with the acellular dermal and the foraminous acellular dermal of atresia respectively get identical size stack, compress it combined closely, dry back ethylene oxide sterilizing forms double-deck acellular dermal material;
Step 3). the preparation of bilayer skin culture fluid, each component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM 67.5%, commercial culture fluid F12 22.5%, hyclone 10%, basic fibroblast growth factor 10ng/ml, epithelical cell growth factor 10ng/ml, insulin 5ng/ml, hydrocortisone 20ng/ml, adenine 0.15mM, transferrins 5 μ g/ml;
Step 4). the structure of double-layered artificial skin: under 4 ℃ of conditions, press mass ratio with collagen 8: chitosan 1: hyaluronic acid 3: chondroitin sulfate 1 mixes, with the acetic acid of 0.3M it being mixed with concentration is 6mg/ml solution, ultraviolet radiation under the ice bath, hyclone by its volume adding 10%, add the DMEM culture medium again and make its final concentration reach 10mg/ml, regulating pH value is 7.2～7.4, becomes gel solution; With the preparation double-deck acellular dermal soak into gel solution after, precuring under 37 ℃ of environment; The fat mesenchymal stem cell of In vitro culture is pressed 5 * 10
5The concentration of individual/ml is mixed in the gel solution, again this gel solution is pressed 1ml/cm
2Be added drop-wise on porose of double-deck acellular dermal of precuring, place 5%CO
2After solidifying under 37 ℃ of conditions of environment, add the bilayer skin culture fluid and cultivate after 2 days, be placed on again and continue on the cultivation support of cultivating in the vessel to cultivate 7 days, during every day change liquid, double-layered artificial skin is cultivated and is finished; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment.
The double-layered artificial skin of this examples preparation includes people's fat mesenchymal stem cell, can be divided into the healing that fibroblast is participated in wound surface directly at wound surface, have unrestricted, the molecular marker for increased proliferation in fat mesenchymal stem cell source simultaneously, promote the strong advantage of wound healing ability.
Claims (5)
1. double-layered artificial skin, include fibroblast, it is characterized in that, it is by taking off the membranaceous bio-derived material of cell as the top layer, extracellular matrix and cell growth factor by fibroblast and synthesis secretion thereof are compound in the inner skin corium, both chimeric formations of forming of biologic bracket material; The described membranaceous bio-derived material of cell that takes off is that natural biological tissue is through taking off the formed membranaceous bio-medical material of cell; Described fibroblast is the fibroblast from body or allosome source, or can be to the stem cell of fibroblast differentiation; Described biologic bracket material is to provide three-dimensional backing material for the cell growth.
2. the method for preparing the described double-layered artificial skin of claim 1 includes the step that cell obtained, took off the preparation of cell biological derived material, it is characterized in that concrete steps are as follows:
Step 1). fibroblastic obtaining: fibroblast is from tissue, and cultural method routinely carries out In vitro culture and extensive amplification;
Step 2). take off the preparation of cell biological derived material: adopt dermal layer of the skin, fascia, submucous layer of small intestine, amniotic membrane, dural any membranaceous biomaterial preparation; Material placed contain peracetic acid and alcoholic acid aqueous solution soaking disinfection was not less than 1 hour, after cleaning with phosphate buffer; Being placed on the NaOH solution soaking handles; Embathe neutrality with phosphate buffer to pH, it being inserted in the mixed solution that contains DNA enzyme and alpha-galactosidase handles more than 25 minutes again, evenly drilling thereon after embathing with phosphate buffer, with the taking off cell biological derived material stack and compress it is combined closely of atresia, become bilayer behind the dry sterilization and take off the cell biological derived material again with foraminous;
Step 3). the preparation of double-layered artificial skin culture fluid, its component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM67.5%, commercial culture fluid F1222.5%, hyclone 5～15%, basic fibroblast growth factor 2～100ng/ml, epithelical cell growth factor 2～100ng/ml, insulin 1～50ng/ml, hydrocortisone 10～500ng/ml, adenine 0.2～0.25mM, transferrins 1～10 μ g/ml;
Step 4). the structure of double-layered artificial skin: the gel solution of preparing biologic bracket material earlier; After the bilayer of preparation taken off the cell biological derived material and soak into gel solution, place precuring under 37 ℃ of environment; The fibroblast of In vitro culture is pressed 10
4～10
6The concentration of individual/ml is mixed in the gel solution, again by 0.1～1ml/cm
2Be added drop-wise on porose of precuring material, place 5%CO
2Solidify under 37 ℃ of conditions in the environment; Insert again cultivate 2～3 days in the double-layered artificial skin culture fluid after, be placed on and continue on the cultivation support of cultivating in the vessel to cultivate 5～8 days, change liquid every day between culture period, cultivation is finished; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment.
3. preparation method according to claim 2 is characterized in that step 2) in be 0.1～1mm in the described aperture of taking off the drilling of cell biological derived material, spacing is 0.5～5mm.
4. preparation method according to claim 2 is characterized in that, in step 2) described in peracetic acid and alcoholic acid volume final concentration be respectively 0.1～0.5% and 2～10%; The final concentration of described DNA enzyme and alpha-galactosidase is respectively 30～50U/ml and 10～25U/ml.
5. preparation method according to claim 2, it is characterized in that, in step 2) described in soaking disinfection clean after, before the NaOH solution soaking, material is placed-80 ℃ more than freezing half an hour, take out the back and thaws naturally, so multigelation is 2～5 times, with after the phosphate buffer cleaning again in the NaOH solution soaking.
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