CN100415309C - Application of selective cell-removed pork skin as skin substitute of human body and its preparation method - Google Patents
Application of selective cell-removed pork skin as skin substitute of human body and its preparation method Download PDFInfo
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- CN100415309C CN100415309C CNB031482910A CN03148291A CN100415309C CN 100415309 C CN100415309 C CN 100415309C CN B031482910 A CNB031482910 A CN B031482910A CN 03148291 A CN03148291 A CN 03148291A CN 100415309 C CN100415309 C CN 100415309C
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- 238000002360 preparation method Methods 0.000 title claims description 11
- 235000015277 pork Nutrition 0.000 title 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 241000282898 Sus scrofa Species 0.000 claims abstract description 4
- 241000282894 Sus scrofa domesticus Species 0.000 claims description 41
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 4
- 230000002459 sustained effect Effects 0.000 claims description 4
- 206010048768 Dermatosis Diseases 0.000 claims description 3
- 241000381142 Pachydermia Species 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 208000017520 skin disease Diseases 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 2
- 210000003491 skin Anatomy 0.000 abstract description 48
- 230000002500 effect on skin Effects 0.000 abstract description 9
- 239000011159 matrix material Substances 0.000 abstract description 9
- 108010002823 integra artificial skin Proteins 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 5
- 210000004207 dermis Anatomy 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 abstract description 2
- 150000003016 phosphoric acids Chemical class 0.000 abstract 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000002504 physiological saline solution Substances 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 206010052428 Wound Diseases 0.000 description 45
- 208000027418 Wounds and injury Diseases 0.000 description 45
- 210000004027 cell Anatomy 0.000 description 31
- 206010063560 Excessive granulation tissue Diseases 0.000 description 13
- 210000001126 granulation tissue Anatomy 0.000 description 13
- 230000002980 postoperative effect Effects 0.000 description 10
- 239000003921 oil Substances 0.000 description 7
- 229940099259 vaseline Drugs 0.000 description 7
- 210000000416 exudates and transudate Anatomy 0.000 description 6
- 210000004969 inflammatory cell Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000029663 wound healing Effects 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 3
- 210000002615 epidermis Anatomy 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 206010053615 Thermal burn Diseases 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010562 histological examination Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 229940012022 pentobarbital sodium 50 mg Drugs 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 108010023728 Alloderm Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 206010040844 Skin exfoliation Diseases 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000035618 desquamation Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 231100000075 skin burn Toxicity 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Abstract
The present invention discloses the new application of pigskin, namely the application of pigskin as the substitute for human skin. The present invention also discloses a producing method of pigskin as the substitute for human skin, which comprises the following steps: an about 50Kg white healthy domestic pig without dermatopathy is killed; the pigskin is depilated and cleaned; a large sheet of full-thickness skin is taken off and reversely shaved into skin pieces; the skin pieces are crosslinked with glutaraldehyde and cleaned by balanced phosphoric acid salt water; the mixture is soaked in IM physiological saline containing trypsinase, shocked by Triton-100, washed by balanced phosphoric acid salt water and placed into a refrigerator at 4 DEGC for preservation and backup. The present invention as the substitute for human skin has the functional characteristics of the two skin substitutes of Integra artificial skin and acellular dermal matrix (ADM), and thus, the pigskin produced with the present invention can be used as not only a temporary wound surface covering for a human body to protect the wound surface but also a permanent substitute for dermis to repair the wound surface. The material therefor has the advantages of wide source, easy acquisition, low price and easy production.
Description
Technical field
The present invention relates to the purposes of Corii Sus domestica, relate in particular to application as the human body skin sub.The invention still further relates to the preparation method of Corii Sus domestica as the human body skin sub.
Background technology
At present, to the skin burn wounded's treatment, especially to the extensive deep burn wounded's treatment, early stage cutting scab in time seals burn wound, is to improve one of burn wounded survival rate most critical, effective measures.Wherein best wound-surface cover is the wounded's a autologous skin.But wounded's auto-skin grafting not only can produce the new wound surface of skin donor site, and because the corium damage also can cause skin donor site cicatrization and pigmentation, the more important thing is the available skin scarcity of the large-area burns wounded self.Therefore, seek ideal permanent human body skin sub and become the orthopedic important topic of burn.
At present, the human body skin sub that can supply usefulness mainly comprises two kinds of temporary skin sub and permanent skin subs.
Temporary human body skin sub mainly comprises allosome fell and glutaric pigskin etc., and it finally can be ostracised.Wherein with the best results of allosome fell flap coverage.But allosome fell source is limited, and risk of disease transmission is arranged; Glutaric pigskin wound surface adhesiveness is relatively poor, and the occupy-place phenomenon is arranged, and is unfavorable for wound healing.
Permanent human body skin sub mainly comprises acellular dermal matrix (ADM), Integra artificial skin etc.Removing acellular dermal matrix (trade name Alloderm) is that Wainwright1995 succeeds in developing and be applied to clinical.This material be to people's allograft skin through processing such as trypsin, detergents, removed the cell component that epidermis and intradermal can be brought out immunological rejection, have that scalable induces host cell to grow into, absorbance is lower, can with characteristics such as autologous skin cograft.Shortcoming: the one, being applied to needs with other dressing covering protection behind the wound surface, and the 2nd, cost an arm and a leg; The Integra artificial skin is to be applied to clinical by Burke invention in 1996 and to obtain drugs approved by FDA.It is a kind of acellular artificial polymeric material, constitutes dermis scaffold by the I type bovine collagen and the 6-chondroitin sulfate of glutaraldehyde cross-linking.Support is the porous crack, can hold from the body fibroblast and grow into.It can be used after having burn immediately, transplants back corium composition and can degrade voluntarily, and can induce host's epidermal growth, and material plasticity is good, and shape, size and thickness are easy to change; Can see through the characteristics such as variation that silicon fiml is observed corium.Shortcoming is: transplant the back easy infection; The epithelial cells diaphragm is difficult for adhering to; The expense costliness.
Summary of the invention
One of task of the present invention is to provide selectivity to go the new purposes of cell Corii Sus domestica, promptly as the application of human body skin sub.
Another task of the present invention provides selectivity and goes the preparation method of cell Corii Sus domestica as the human body skin sub.
Selectivity of the present invention goes the preparation method of cell Corii Sus domestica as the human body skin sub, the healthy white tame pig of getting no dermatosis about 50Kg lives and kills, shave the clean skin of hair, take off the big full pachydermia of opening, its counter cutting is skin graft, clean behind the glutaraldehyde cross-linking with the washing of phosphoric acid balanced salt, be put in to contain in the tryptic IM normal saline and soak, reuse phosphoric acid balanced salt water washing behind the Triton-100 sustained oscillation, 4 ℃ of refrigerators are preserved standby.
Selectivity of the present invention goes the preparation method of cell Corii Sus domestica as the human body skin sub, the anti-thickness of cutting to skin graft of wherein said big Zhang Quanhou Corii Sus domestica is 0.3-0.4mm, with the phosphoric acid balanced salt washing 5 times after 10 minutes of 0.5% glutaraldehyde cross-linking, place it in then and contain in the 0.25% tryptic IM normal saline 37 ℃ and soaked 2 hours, put into 36 hours reuse phosphoric acid of 100 times/minute sustained oscillations of the 37 ℃ of distilled water balanced salt washing 5 times that contains 0.5%Triton-100 again.
Go application and the dermatoplastic effect thereof of cell Corii Sus domestica as the human body skin sub in order to understand selectivity of the present invention better, the result is proved below by zoopery.
1. selectivity goes the zoopery of cell Corii Sus domestica as temporary wound-surface cover
1.1 animal model
Get 96 of healthy SD rats, male and female are not limit, body weight 205-250 gram, the capable intraperitoneal anesthesia of 3% pentobarbital sodium 50mg/Kg.After hair was shaved at the back, 100 ℃ of boiled water caused the about 4cm in back * 3cmIII degree scald 12 seconds, normal saline 100ml/Kg subcutaneous injection, and excision holostrome skin is evenly smeared from the body microparticle skin on the wound surface to sarolemma.Be divided into 4 groups then at random, 24 every group, go cell Corii Sus domestica, allosome Corium Mus, glutaric pigskin and vaseline oil yarn to cover skin injury with selectivity respectively, reuse kpetrolatum gauze and aseptic dressing cover, and wrapping is fixing.Experimental mouse postoperative list cage is raised.
1.2 experimental result
(1) gross examination of skeletal muscle
Postoperative 3 days, selectivity go cell Corii Sus domestica and allosome Corium Mus group, and the covering of transplanting is complete, the surface wettability softness.Open covering, wound surface has the granulation tissue of more transudate and visible comparatively fresh.Transudate is through antibacterial culturing, except that 2 examples have the bacillus cloacae growth, and surplus no bacterial growth.Microparticle skin combines with wound surface closely; Glutaric pigskin flap coverage group except that the wound surface marginal portion have a little exposed, the covering of transplanting is complete, the surface is dry, wound surface has the granulation tissue of less transudate and visible comparatively fresh; Vaseline oil yarn group combines with wound surface closely, takes off by force from oily yarn, visible more oozing of blood, the fresh fragility of granulation tissue.
In 1 week of postoperative, selectivity goes the still more complete softness in the Corii Sus domestica surface of cell Corii Sus domestica group.Open covering, wound surface has less transudate and visible a large amount of granulation tissue, visible epithelial island on the granulation tissue; It is red that as seen the allograft skin of allosome Corium Mus group changes gradually, and wound surface still has more transudate and visible a large amount of granulation tissue, visible epithelial island on the granulation tissue; The Corii Sus domestica drying of glutaric pigskin group, part has been peeled off wound surface, and it exposes granulation tissue and desiccation down.Unstripped place its following visible granulation tissue and epithelial island.
In 2 weeks of postoperative, selectivity goes the Corii Sus domestica quality of cell Corii Sus domestica group harder, and part has been peeled off wound surface, the visible a large amount of granulation tissuies of wound surface, visible a large amount of epithelial island on the granulation tissue; The allograft skin of allosome Corium Mus group is dry gradually, still has a little transudate and visible a large amount of granulation tissue under it, visible a large amount of epithelial island on the granulation tissue; The Corii Sus domestica drying of glutaric pigskin group, and peel off wound surface substantially, still have more remaining wound surface; Vaseline oil yarn group also still has more remaining wound surface.
In 4 weeks of postoperative, the allograft skin major part of allosome Corium Mus group is peeled off wound surface, and wound surface shrinks, and residual wound surface is less; Selectivity goes the Corii Sus domestica of cell Corii Sus domestica group substantially all to peel off, and wound surface shrinks, also residual fraction wound surface, the not still visible granulation tissue of healing place; Glutaric pigskin is formed face and is shunk obviously residual more wound surface; The vaseline oil yarn is formed face and is shunk obviously, and residual wound surface is more, and the prognosis quality is the poorest.Each is organized skin graft and adheres to natural law and form respectively that the face healing rate sees Table 1, table 2.
Table 1 is respectively organized skin graft and is adhered to relatively (X ± s) of natural law
| Group | The example number | The average natural law that adheres to |
| Allosome Corium Mus group | 24 | 20.4±1.1 |
| Selectivity goes cell Corii Sus domestica group | 24 | 18.6±0.9 |
| The glutaric pigskin group | 24 | 14.7±1.0 * |
*Glutaric pigskin group and allosome Corium Mus group compare, P＜0.05, and significant difference is remarkable.
Table 2 is respectively organized healing rate relatively (%, X ± s)
*Selectivity goes cell Corii Sus domestica group, glutaric pigskin group and vaseline oil yarn group and allosome Corium Mus group relatively, P＜0.05, and significant difference is remarkable.
#Glutaric pigskin group and vaseline oil yarn group and selectivity go cell Corii Sus domestica group relatively, P＜0.05, and significant difference is remarkable.
(2) histological examination
Visible newborn little blood vessel has a large amount of inflammatory cells with the wound surface contact surface in the postoperative 3 days, the allograft skin of allosome Corium Mus group; Selectivity removes the visible a large amount of inflammatory cell infiltration of cell Corii Sus domestica group, visible fibroblast in the skin corium; The also visible more inflammatory cell infiltration of glutaric pigskin group, but go cell Corii Sus domestica group few than selectivity.
In 1 week of postoperative, selectivity removes the still visible a large amount of inflammatory cell infiltration of cell Corii Sus domestica group, and inflammatory cell tiering and boundary are comparatively obvious, and the glutaric pigskin group obviously reduces.
In 4 weeks of postoperative, selectivity goes cell Corii Sus domestica group and allosome Corium Mus to form the most of epithelization of face; Glutaric pigskin group part wound surface epithelization.
Above result shows that selectivity goes the obducent wound healing effect of cell Corii Sus domestica slightly to be worse than the obducent wound healing effect of allosome Corium Mus, but obviously is better than the effect of glutaric pigskin and vaseline oil yarn flap coverage.
2. selectivity goes the zoopery of cell Corii Sus domestica as permanent corium sub row two-step method wound repairing
2.1 animal model
Get 12 of healthy SD rats, male and female are not limit, body weight 205-250 gram, the capable intraperitoneal anesthesia of 3% pentobarbital sodium 50mg/Kg.After hair is shaved at the back, 100 ℃ of boiled water cause the about 4cm in back * 3cmIII degree scald 12 seconds, normal saline 100ml/Kg subcutaneous injection, and excision holostrome skin is to sarolemma, wound surface goes the cell Corii Sus domestica to cover back reuse kpetrolatum gauze with selectivity and aseptic dressing covers, and wrapping is fixing.Experimental mouse postoperative list cage is raised.Postoperative was changed dressings in 10 days, threw off epidermis, and visible lower floor corium changes red, and combined closely with the wound base.Get the full thick skin of 3cm * 3cm after the experimental mouse abdominal part is shaved hair, instead cut the thick skin graft into 0.2mm, transplant in back corium top, wrapping is fixing, and the abdominal part wound surface is sewed up.
2.2 experimental result
(1) gross examination of skeletal muscle
10 days visible wound surface of post-transplantation heal substantially, change red from the body skin graft, and wound surface shrinks not obvious, and elasticity is better.Post-transplantation 30 days survives well from the body skin graft, and ruddy, absent hair growth has the desquamation phenomenon.
(2) histological examination
Wound surface is transplanted selectivity and was gone behind the cell Corii Sus domestica 10 days, the visible more newborn little blood vessel of intradermal, and arrangement of collagen fibers is orderly, fibroblast, little blood vessel hyperplasia.
The above results shows that selectivity goes the cell Corii Sus domestica to can be used as corium sub row two-step method wound repairing, improves the wound healing quality.
Selectivity of the present invention goes the cell Corii Sus domestica to show as temporary wound-surface cover with as the above zoopery result of permanent corium sub row two-step method wound repairing, selectivity goes the cell Corii Sus domestica to have with the similar structure function characteristics of Integra artificial dermis, and it goes acellular dermal matrix part to approach people's dermal matrix more, has Integra artificial dermis and the advantage of going two kinds of subs of acellular dermal matrix (ADM).Both can be used as temporary wound-surface cover protection wound surface, can be used as nonvolatil corium sub wound repairing again.
Advantage of the present invention:
Material source extensively, be easy to get, cheap.
2. preparation easily.Only need to Corii Sus domestica carry out that the epidermis cell inactivation is handled, corium goes cell to handle can to make.
3. the product no antigen has Integra artificial skin and the functional characteristics of going two kinds of skin subs of acellular dermal matrix (ADM).Selectivity removes the cell Corii Sus domestica, and not only structure function is similar to the Integra artificial skin, and its dermal matrix part approaches people's dermal matrix more.Therefore, both can be used as temporary wound-surface cover protection wound surface, can be used as nonvolatil corium sub wound repairing again.
The specific embodiment
The present invention goes the preparation method of cell Corii Sus domestica as the human body skin sub, the healthy white tame pig of getting no dermatosis about 50Kg lives and kills, shave the clean skin of hair, take off the big full pachydermia of opening, with its anti-skin graft of cutting to 0.3-0.4mm, with the phosphoric acid balanced salt washing 5 times after 10 minutes of 0.5% glutaraldehyde cross-linking, place it in then and contain the interior 37 ℃ of immersions of 0.25% tryptic IM normal saline 2 hours, put into 36 hours reuse phosphoric acid of 100 times/minute sustained oscillations of the 37 ℃ of distilled water balanced salt that contains 0.5%Triton-100 again and wash 5 times, put 4 ℃ of refrigerators and preserve standby.
Claims (1)
1. selectivity goes the application of cell Corii Sus domestica in preparation human body skin substitute, wherein said selectivity goes the preparation method of cell Corii Sus domestica as follows: be object with the Corii Sus domestica, this Corii Sus domestica obtains from alive the killing of healthy white tame pig that 50kg does not have dermatosis, shave the clean skin of hair, take off the big full pachydermia of opening, its counter cutting is skin graft, the thickness of skin graft is 0.3-0.4mm, wash 5 times with the phosphoric acid balanced salt after 10 minutes with 0.5% glutaraldehyde cross-linking, place it in then and contain the interior 37 ℃ of immersions of 0.25% tryptic 1M normal saline 2 hours, put into 100 times/minute sustained oscillations of 37 ℃ of distilled water 36 hours that contain 0.5%Triton-100 again, reuse phosphoric acid balanced salt washing 5 times, 4 ℃ of refrigerators are preserved standby.
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| CNB031482910A CN100415309C (en) | 2003-07-03 | 2003-07-03 | Application of selective cell-removed pork skin as skin substitute of human body and its preparation method |
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| CN100415309C true CN100415309C (en) | 2008-09-03 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100408107C (en) * | 2003-10-17 | 2008-08-06 | 胡杰 | Biological wound dressing |
| CN100372511C (en) * | 2005-11-30 | 2008-03-05 | 烟台正海生物技术有限公司 | Acellular dermal matrix and its preparing method |
| CN100463963C (en) * | 2006-10-20 | 2009-02-25 | 扬州大学 | Freeze preservating method for domestic pig skin tissue |
| CN101843922B (en) * | 2009-03-25 | 2013-08-14 | 上海交通大学医学院附属瑞金医院 | Amino acid enriched acellular dermal matrix implant as well as preparation method and application thereof |
| CN103990179B (en) * | 2014-05-24 | 2017-08-29 | 河北爱能生物科技股份有限公司 | A kind of method for preparing xenogenesis acellular matrix and products thereof |
| CN108744044B (en) * | 2018-06-08 | 2019-03-01 | 李峰 | A kind of MEEK skin-grafting material in conjunction with allosome skin graft |
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