CN1212161C - Compound artificial skin of double side implanted two kinds of cell and its construction method - Google Patents

Abstract

The present invention relates to compound artificial skin of double side implanted two kinds of cells and a construction method of the compound artificial skin. An ox collagen film with epidermal growth factors is used as a bracket, fibroblast and epidermal cells are implanted on both sides, and the growing fibroblast is positioned in the middle; the density is respectively larger than 10<6>/cm<2>. The artificial skin of has the advantages of good physical properties, large intensity, good flexibility, strong anti-infection capability, no antigenicity, convenient transplantation operation and good combination with surrounding skin after a transplantation operation is finished.

Description

Bilateral is implanted the compound artificial skin and the construction method thereof of two kinds of cells

Technical field

The present invention relates to a kind of artificial skin, particularly bilateral and be implanted to fibrocyte and epidermis cell compound artificial skin, belong to the medical skin wound-surface cover.

The invention still further relates to the construction method of this compound artificial skin.

Background of invention

Artificial skin is used for the transplantation treatment of skin injury clinically, can promote wound healing, reduce cicatrization, protect from infection, especially large area skin disappearance patient is owing to lack transplantable autologous skin, have only by use wound-surface cover solve the early stage of skin disappearance and late period problem.

At present, abroad the skin wound covering in clinical practice has: epidermic graft, dermal transplantation thing (as allosome corium, the dead corium that removes epidermis, synthetic nethike embrane, acellular collagen sponge etc.), and the composite skin graft, as collagen gel Graftskin etc.

Compound artificial skin is a kind of double-layered artificial skin of being made up of epidermal area and skin corium, and epidermal area is differentiated to form by epidermis cell, and skin corium is inoculated in the collagen stroma by fibroblast and forms.In recent years, artificial skin makes constant progress aspect preparation, announced an artificial Composite Skin of utilizing cattle I, III Collagen Type VI to make up as U.S. patent of invention US 5 282 859, its weak point is to handle through glutaraldehyde, remaining cytotoxicity, and insufficient strength, immunogenicity is stronger, simple I, the III Collagen Type VI of adopting lacks the introducing of cytokine as support; Specially permit in disclosed JP 97 173 362 patent documentations in Japan, it is to utilize chitin etc. to be substrate, cultivates the cell in mammal skin source, and artificial skin is produced in lyophilizing then, its shortcoming is to utilize in the ammonia and the making collagen gel, be not suitable for cell culture, the cultured cell time is short, and the cultivation skin of formation has than big difference with natural skin on organizational structure, collagen gel opposing collagen degradation ability, skin fragility is big, operating difficulties, collagen gel production process complexity.

Summary of the invention

Purpose of the present invention, just for the shortcoming that overcomes above-mentioned prior art with not enough, and provide a kind of contain epidermal growth factor be implanted to the compound artificial skin of fibrocyte and epidermis cell with active bovine collagen skin tissue engineering scaffold both sides, thereby improved the performance of artificial skin, promoted wound healing.

The present invention also provides the construction method of this compound artificial skin.

The objective of the invention is to realize by following technical proposal:

Bilateral is implanted the compound artificial skin of two kinds of cells, it is characterized in that it is is skin tissue engineering scaffold with the bovine collagen film that contains the hEGF, and being implanted to fibrocyte density in a side of support is＞10 ⁶Individual/cm ², side implantation epidermis cell density is＞10 in addition ⁶Individual/cm ², inner fibroblast density for growth is＞10 ⁶Individual/cm ²

The construction method of described compound artificial skin is undertaken by following step:

1). contain the structure of the skin tissue engineering scaffold of epidermal growth factor:

A), abortive calfskin or cattle heel string are lost hair or feathers according to a conventional method, clean, freezing, shave, dewater with acetone, the reuse defat with petroleum ether, vacuum is taken out the petroleum ether in abortive calfskin or the section of cattle heel string, add gastric enzyme or ficoin or carase or chymase or bromelain, addition is by 1～5% of dried abortive calfskin or dried cattle heel string weight, at 20～40 ℃, under pH=2～8 conditions, mechanical vibration 8～12 hours, covalent bond between the degraded tissue fibers, through centrifugalize, abandon supernatant get precipitation with distilled water wash after, the 0.5M acetum that adds 50～150 times of above-mentioned siccative weight again, 0～20 ℃ of temperature, vibrate 24～32 hours, add sodium chloride again to above-mentioned solution, sodium chloride concentration reaches 2M, through centrifugalize, abandoning supernatant gets the precipitation bag filter of packing into and dialyses 3～4 times, collect dialysis back collagen solution, add dried abortive calfskin or dried cattle heel string weight 1～3% ficoin or carase or chymase or bromelain, at 30～40 ℃, under pH=2～8 conditions, antigenic determinant is removed in mechanical vibration 4～8 hours; With the acetum dissolving of 0.05～0.5M, obtain the no antigen collagen solution of solubility in acid 0.5～4mg/ml;

B), again the collagen solution of (a) item is poured in the model, and under-10～-70 ℃ of temperature freezing 4～24 hours, lyophilization is 24～48 hours again, obtain the uniform collagen sponge membrane in aperture, collagen sponge membrane is placed the glycerol solution of 0.5～10% concentration, take out behind the adsorption swelling, vacuum drying is 18～32 hours under 105～130 ℃ of temperature;

C), heparin is dissolved in 0.005～1M acetum, heparin and acetum amount ratio are W/V0.1～100mg/ml, filter with 100 mesh filter screens, obtain the heparin acetum;

D), epidermal growth factor is dissolved in the tri-distilled water, the amount ratio of epidermal growth factor and tri-distilled water is W/V0.1～100mg/ml, obtains the epidermal growth factor aqueous solution;

E), at last with heparin acetum of (c) item and (d) the epidermal growth factor aqueous solution mixing in 1: 1 by volume～50: 1 of item, vibration mixes gets mixed liquor and mixes with volume ratio 0.1～5% with 5% glycerol solution after 48 hours, soak into the collagen sponge membrane of (b) item with mixed liquor, after the moistening, freezing 4～10 hours at-10～-70 ℃, vacuum drying again must contain the collagen sponge membrane of epidermal growth factor

2). bilateral is implanted the structure of two kinds of cell compound artificial skins:

With 1) item contains the skin tissue engineering scaffold of epidermal growth factor, through Co ⁶⁰After the gamma-rays sterilization, with containing 1 * 10 ⁵After the D-Hanks liquid flushing of μ g/l penicillin and 100mg/l streptomycin, add the DMEM culture fluid that contains 10% hyclone again and soaked 24 hours, change culture fluid, the fibroblast of cultivating going down to posterity is with 3 * 10 ⁵Individual/cm ²Density is inoculated in support one side, at 37 ℃, 5%CO ₂After liquid level cultivated for two weeks under the condition, cultivated 1 day with the MCDB153 culture fluid that contains epithelical cell growth factor 10 μ g/ml, Medulla Bovis seu Bubali hypophysis extract 200mg/l, hydrocortisone 0.5mg/l, insulin 5mg/l, transferrins 10mg/l, counter-rotating support 180 degree, with epidermis cell with 3 * 10 ⁵Individual/cm ²Density is inoculated in the support opposite side, changes culture fluid every day, cultivates 10 days, supports with stainless steel mesh, and cultivates 14 days with gas-liquid interface, promptly is built into the compound artificial skin that bilateral is implanted to fiber and epidermis cell.

Compound artificial skin of the present invention and structure thereof are that employing abortive calfskin or cattle heel string are that base material is through pretreatment, dehydration, after the defat, carry out single treatment with enzyme, with the covalent bond between the degraded tissue fibers, and then through dialysis, carrying out the secondary enzyme handles, can remove antigenic determinant, make with acetate dissolution that to contain the no antigen collagem membrane be support, through molding, freezing, dry, the mixed liquor of reuse heparin and epidermal growth factor soaks into collagem membrane, constitute the skin tissue engineering scaffold that contains epidermal growth factor, be implanted to fibrocyte and epidermis cell in the support both sides, and inner fibroblast for growth.This artificial skin has good physical features, and anti-infection ability is strong, and transplant operation is simple, and can fine fusion one with surrounding skin, by zoopery, proves that it is close with the normal skin tissue structure, promotes the artificial skin of wound healing again.

Owing to take technique scheme, make the technology of the present invention compared with the prior art have following advantage and effect:

A), artificial skin of the present invention has good physical characteristic, good springiness, pliability be good, can resist certain pulling force, and can shear as natural skin, sew up, size and shape can become with wound surface, transplant operation is simple;

B), the shrinkage factor of substrate collagen membrane support is low, anti-infection ability is high, can prevent the invasion and attack of microorganism, and can resist the wound infection by microorganisms, it is few to transplant the back dressing change frequency, postoperative care is simple;

C), artificial skin of the present invention, through histological examination, dermal matrix is a cellular, the aperture is slightly larger than fibroblast, fibroblast grows in collagem membrane substrate evenly, and the fibroblast of growth is arranged in the centre, its density all＞10 ⁶Individual/cm ²

D), artificial skin of the present invention made animal implant tests textured with nude mice, the result shows that artificial skin after the transplanting and surrounding skin merge one, and a large amount of vascularization is arranged in the dermal substitute, collagen is arranged gradually rule, epidermal differentiation is good.

Description of drawings

Fig. 1 is a compound artificial skin histological structure fibroblast inoculation side of the present invention (* 400 times of H.E dyeing).

Fig. 2 is a compound artificial skin histological structure epidermis cell inoculation side of the present invention (* 1000 times of H.E dyeing).

Specific embodiments

Embodiment 1

Abortive calfskin is lost hair or feathers according to a conventional method, remove fascia, fat and blood stains, clean, freezing, be cut into thick 0.5mm thin slice, with acetone dehydration three times, with defat with petroleum ether three times, evacuation is removed petroleum ether again, the abortive calfskin of making dewatering and defatting is a raw material, get the 10g raw material, add in the reactor with distilled water washing back, adding is by the aqueous solution of 0.1g gastric enzyme and the preparation of 100g water, carrying out an enzyme handles, at 20 ℃, under the pH=2 condition, mechanical vibration 24 hours, covalent bond between the degraded tissue fibers, through centrifugalize, abandon supernatant, get precipitation, with distilled water washing three times, add the 0.5M aqueous acetic acid of 500g, under 0 ℃ of temperature, mechanical vibration are after 32 hours, add sodium chloride again, make that sodium chloride concentration reaches 2M in the solution, precipitation fully after, centrifugalize, abandon supernatant, getting the precipitation bag filter of packing into dialyses three times, collect dialysis back collagen solution, add in the reactor, and add the aqueous solution of 0.3g bromelain and the preparation of 100g water, carry out the secondary enzyme and handle, at 30 ℃, under the pH=6 condition, antigenic determinant is removed in mechanical vibration 8 hours, and enzyme-deactivating is handled according to a conventional method again, through centrifugalize, abandon supernatant, aqueous acetic acid 100g with 0.05M dissolves, and records with weight-loss method and contains 0.4mg/ml no antigen collagen solution, and pour in the pre-cooling mould of diameter 100mm, under-10 ℃ of temperature, freezing 24 hours, vacuum drying was 24 hours again, and obtaining the aperture homogeneous thickness is 0.1mm collagen sponge membrane 0.15g, insert then in the 7.5ml glycerin solution of 0.5% concentration and take out behind the adsorption swelling, under 150 ℃ of temperature, vacuum drying 32 hours must be done collagen sponge membrane, taking heparin is in the 0.005M aqueous acetic acid, and making heparin content is 0.1mg/ml.To agree skin growth factor again is dissolved in the tri-distilled water, making epidermal growth factor content is 0.01 μ g/ml, getting above-mentioned heparin aqueous acetic acid 100ml mixes with epidermal growth factor aqueous solution 100ml, after the mechanical vibration 48 hours, get above-mentioned mixed liquor 0.1ml and 5% concentration glycerin solution 100ml, mix once more, and soak into dried collagen sponge net, after the moistening under-70 ℃ of temperature freezing 4 hours, vacuum drying again, the skin tissue engineering scaffold that contains epidermal growth factor that obtains making up.Get 1cm ²Support is used Co ⁶⁰After the gamma-rays sterilization, with containing 1 * 10 ⁵After the flushing of the D-Hanks liquid of μ g/l penicillin and 100mg streptomycin, add the DMEM culture fluid that contains 10% hyclone again and soaked 24 hours, change culture fluid, with the fibroblast of going down to posterity with 3 * 10 ⁵Individual/cm ²Density is inoculated in support one side, at 37 ℃, 5%CO ₂After liquid level cultivated for two weeks under the condition, cultivated one day with the MCDB153 culture fluid that contains epidermal growth factor 10 μ g/ml, Medulla Bovis seu Bubali hypophysis extract 200mg/l, hydrocortisone 0.5mg/l, insulin 5mg/l, transferrins 10mg/l, counter-rotating support 180 degree, with epidermis cell with 3 * 10 ⁵Individual/cm ²Density is inoculated in the support opposite side, changes culture fluid every day, cultivates 10 days, supports with stainless steel mesh, and cultivates 14 days with the gas-liquid face, promptly makes up 1cm ²Bilateral is implanted to the compound artificial skin of fibrocyte and epidermis cell.By shown in Figure 1,1-support, the fibroblast of 2-fibroblast inoculation side, the epidermis cell of 3-epidermis cell inoculation side, the fibroblast of 4-growth inside.

Embodiment 2

The cattle heel string is carried out dewatering and defatting by the method for embodiment 1, evacuation is removed petroleum ether, the cattle heel string of making dewatering and defatting is a raw material, get raw material 10g, add in the reaction vessel with distilled water washing back, adding is carried out an enzyme processing by the aqueous solution of 0.3g chymase and the preparation of 100g water, at 30 ℃, under the pH=6 condition, mechanical vibration 16 hours, covalent bond between the degraded tissue fibers, through centrifugalize, abandon supernatant, get precipitation, with distilled water washing four times, add 1000g 0.5M aqueous acetic acid, under 10 ℃ of temperature, mechanical vibration 28 hours, add sodium chloride again, make that sodium chloride concentration reaches 2M in the solution, after precipitation is complete, through centrifugalize, abandon supernatant, get precipitation, the bag filter of packing into is dialysed four times, and after the collection dialysis, collagen solution adds in the reactor, and the aqueous solution that adds 0.2g carase and the preparation of 100g water carries out the processing of secondary enzyme, at 35 ℃, under the pH=7 condition, the antigenicity determinant is removed in mechanical vibration 6 hours, enzyme-deactivating is handled according to a conventional method again, through centrifugalize, abandon supernatant, with the aqueous acetic acid 100g dissolving of 0.1M, record with weight-loss method and to contain 2mg/ml no antigen collagen solution, and pour in the diameter 100mm pre-cooling mould, under-40 ℃ of temperature, freezing 10 hours, vacuum drying is 36 hours again, obtaining the aperture uniform thickness is 0.25mm collagen sponge membrane 0.3g, and inserts the 30ml glycerin solution of 5% concentration, takes out behind the adsorption swelling.Under 120 ℃ of temperature, vacuum drying 20 hours obtains dried collagen sponge membrane, and taking heparin is dissolved in the acetum of 0.1M, and making heparin content is 5mg/ml, epidermal growth factor is dissolved in the tri-distilled water again, and making epidermal growth factor content is 0.01g/ml.Getting above-mentioned heparin aqueous acetic acid 10ml and epidermal growth factor aqueous solution 200ml mixes, after the mechanical vibration 48 hours, get above-mentioned mixed liquor 1ml, mix once more with 5% concentration glycerin solution 100ml, and soak into dried collagen sponge membrane, soak into the back under-40 ℃ of temperature, freezing 6 hours, vacuum drying again, the skin tissue engineering scaffold that contains skin factor that obtains making up.Get 1cm ²Support is used Co ⁶⁰After the gamma-rays sterilization, with containing 1 * 10 ⁵After the flushing of the D-Hanks liquid of μ g/l penicillin and 100mg/l streptomycin, add the DMEM culture fluid that contains 10% hyclone again and soaked 24 hours, change culture fluid, with the fibroblast of going down to posterity with 3 * 10 ⁵Individual/cm ²Density is inoculated in support one side, at 37 ℃, 5%CO ₂After liquid level cultivated for two weeks under the condition, cultivated 10 days with the MCDB153 culture fluid that contains epidermal growth factor 10g/ml, Medulla Bovis seu Bubali hypophysis extract 200mg/l, hydrocortisone 0.5mg/l, insulin 5mg/l, transferrins 10mg/l, counter-rotating support 180 degree, with epidermis cell with 3 * 10 ⁵Individual/cm ²Density is inoculated in the support opposite side, changes culture fluid every day, cultivates one day, supports with stainless steel mesh, and cultivates 14 days with the gas-liquid face, promptly makes up 1cm ²Bilateral is implanted to the compound artificial skin of fibrocyte and epidermis cell.

Embodiment 3

Cattle heel string with the dewatering and defatting of embodiment 2 is a raw material, gets raw material 10g, adds in the reaction vessel with distilled water washing back, and the aqueous solution that adds by 0.5g bromelain and the preparation of 100g water carries out an enzyme processing.At 40 ℃, under the pH=8 condition, mechanical vibration 8 hours, covalent bond between the degraded tissue fibers, through centrifugalize, abandon supernatant, get precipitation, with distilled water washing four times, add in the 0.5M aqueous acetic acid of 1500g, under 20 ℃ of temperature, after the mechanical vibration 24 hours, add sodium chloride again, make that sodium chloride concentration reaches 2M in the solution, after precipitation is complete, centrifugalize, abandon supernatant, get precipitation, the bag filter of packing into is dialysed three times, after collecting dialysis, collagen solution adds in the reactor, and the aqueous solution that adds 0.1g chymase and the preparation of 100g water carries out the processing of secondary enzyme, at 40 ℃, under the pH=8 condition, mechanical vibration 4 hours, remove the antigenicity determinant, enzyme-deactivating is handled according to a conventional method again, through centrifugalize, abandon supernatant, get precipitation, use the aqueous acetic acid 100g dissolving of 0.5M, record with weight-loss method and contain 4mg/ml no antigen collagen solution, and pour in the diameter 50mm pre-cooling mould, under-70 ℃ of temperature, freezing 4 hours, vacuum drying was 48 hours again, obtaining the aperture uniform thickness is 0.5mm collagen sponge membrane 0.3g, insert the 45ml glycerin solution of 10% concentration, take out behind the adsorption swelling, under 130 ℃ of temperature, vacuum drying 18 hours, obtain dried collagen sponge membrane, taking heparin is dissolved in the aqueous acetic acid of 1M, and making heparin content is 10mg/ml, again epidermal growth factor is dissolved in the tri-distilled water, making epidermal growth factor content is 0.1 μ g/ml, gets above-mentioned heparin aqueous acetic acid 50ml and mixes with epidermal growth factor aqueous solution 150ml, and mechanical vibration are after 48 hours, get mixed liquor 5ml and 5% concentration glycerin solution 100ml, mix once more, and soak into dried collagen sponge membrane, after the moistening under-10 ℃ of temperature freezing 10 hours, vacuum drying again, the skin tissue engineering scaffold that contains epidermal growth factor that obtains making up.Get 1cm ²Support is used Co ⁶⁰After the gamma-rays sterilization, with containing 1 * 10 ⁵After the flushing of the D-Hanks liquid of μ g/l penicillin and 100mg/lm streptomycin, add the DMEM culture fluid that contains 10% hyclone again and soaked 24 hours, change culture fluid, with the fibroblast of going down to posterity with 3 * 10 ⁵Individual/cm ²Density is inoculated in support one side, at 37 ℃, 5%CO ₂After liquid level cultivated for two weeks under the condition, cultivated one day with the MCDB153 culture fluid that contains epidermal growth factor 10 μ g/ml, Medulla Bovis seu Bubali hypophysis extract 200mg/l, hydrocortisone 0.5mg/l, insulin 5mg/l, transferrins 10mg/l, counter-rotating support 180 degree, with epidermis cell with 3 * 10 ⁵Individual/cm ²Density is inoculated in the support opposite side, changes culture fluid every day, cultivates 10 days, supports with stainless steel mesh, and cultivates 14 days with the gas-liquid face, promptly makes up 1cm ²Bilateral is implanted to the compound artificial skin of fibrocyte and epidermis cell.

Claims (1)

1. a bilateral is implanted the construction method of the compound artificial skin of two kinds of cells, it is characterized in that it is undertaken by following step:

1). contain the structure of the skin tissue engineering scaffold of epidermal growth factor:

A), abortive calfskin or cattle heel string are lost hair or feathers according to a conventional method, clean, freezing, shave, dewater with acetone, the reuse defat with petroleum ether, vacuum is taken out the petroleum ether in abortive calfskin or the section of cattle heel string, add gastric enzyme or ficoin or carase or chymase or bromelain, addition is by 1～5% of dried abortive calfskin or dried cattle heel string weight, at 20～40 ℃, under pH=2～8 conditions, mechanical vibration 8～12 hours, covalent bond between the degraded tissue fibers, through centrifugalize, abandon supernatant get precipitation with distilled water wash after, the 0.5M acetum that adds 50～150 times of above-mentioned siccative weight again, 0～20 ℃ of temperature, vibrate 24～32 hours, add sodium chloride again to above-mentioned solution, sodium chloride concentration reaches 2M, through centrifugalize, abandoning supernatant gets the precipitation bag filter of packing into and dialyses 3～4 times, collect dialysis back collagen solution, add dried abortive calfskin or dried cattle heel string weight 1～3% ficoin or carase or chymase or bromelain, at 30～40 ℃, under pH=2～8 conditions, antigenic determinant is removed in mechanical vibration 4～8 hours; With the acetum dissolving of 0.05～0.5M, obtain the no antigen collagen solution of solubility in acid 0.5～4mg/ml;

B), again the collagen solution of (a) item is poured in the model, and under-10～-70 ℃ of temperature freezing 4～24 hours, lyophilization is 24～48 hours again, obtain the uniform collagen sponge membrane in aperture, collagen sponge membrane is placed the glycerol solution of 0.5～10% concentration, take out behind the adsorption swelling, vacuum drying is 18～32 hours under 105～130 ℃ of temperature;

C), heparin is dissolved in 0.005～1M acetum, heparin and acetum amount ratio are W/V0.1～100mg/ml, filter with 100 mesh filter screens, obtain the heparin acetum;

D), epidermal growth factor is dissolved in the tri-distilled water, the amount ratio of epidermal growth factor and tri-distilled water is W/V0.1～100mg/ml, obtains the epidermal growth factor aqueous solution;

E), at last with heparin acetum of (c) item and (d) the epidermal growth factor aqueous solution mixing in 1: 1 by volume～50: 1 of item, vibration mixes gets mixed liquor and mixes with volume ratio 0.1～5% with 5% glycerol solution after 48 hours, soak into the collagen sponge membrane of (b) item with mixed liquor, after the moistening, freezing 4～10 hours at-10～-70 ℃, vacuum drying again must contain the collagen sponge membrane of epidermal growth factor

2). bilateral is implanted the structure of two kinds of cell compound artificial skins:

With 1) item contains the skin tissue engineering scaffold of epidermal growth factor, through Co ⁶⁰After the gamma-rays sterilization, with containing 1 * 10 ⁵After the D-Hanks liquid flushing of μ g/l penicillin and 100mg/l streptomycin, add the DMEM culture fluid that contains 10% hyclone again and soaked 24 hours, change culture fluid, the fibroblast of cultivating going down to posterity is with 3 * 10 ⁵Individual/cm ²Density is inoculated in support one side, at 37 ℃, 5%CO ₂After liquid level cultivated for two weeks under the condition, cultivated 1 day with the MCDB153 culture fluid that contains epithelical cell growth factor 10 μ g/ml, Medulla Bovis seu Bubali hypophysis extract 200mg/l, hydrocortisone 0.5mg/l, insulin 5mg/l, transferrins 10mg/l, counter-rotating support 180 degree, with epidermis cell with 3 * 10 ⁵Individual/cm ²Density is inoculated in the support opposite side, changes culture fluid every day, cultivates 10 days, supports with stainless steel mesh, and cultivates 14 days with gas-liquid interface, promptly is built into the compound artificial skin that bilateral is implanted to fiber and epidermis cell.

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