CN101537207A - Preparation method of tissue engineering xenoskin - Google Patents
Preparation method of tissue engineering xenoskin Download PDFInfo
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- CN101537207A CN101537207A CN200910022257A CN200910022257A CN101537207A CN 101537207 A CN101537207 A CN 101537207A CN 200910022257 A CN200910022257 A CN 200910022257A CN 200910022257 A CN200910022257 A CN 200910022257A CN 101537207 A CN101537207 A CN 101537207A
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Abstract
The invention relates to a preparation method of tissue engineering xenoskin, which adopts a baby pig with rich source as a skin source to obtain the finished product through the steps of taking the skin, fixing the skin, cutting, removing hypodermal cells, modifying three-dimensional collagenic structure, changing physical characteristics and sterilizing and packaging, and the finished product can be stored for more than 2 years under the environment of 2-10 DEG C. The tissue engineering xenoskin is applied to deep burn and defect of skin, has the functional characteristics of low immunogenicity, compact adhesion on wound surface, and moisture maintenance, can be used as a protection wound surface of a provisional wound surface cover of a human body, and can also be used as a growth repair wound surface of permanently remaining corium bracket. The invention solves the defects of high cost, limit source, long fabrication time, short storage time and organism hurt by secondary skin-grafting in the prior artificial skin product. The prepared product material has wide material source, short product period, short storage time and low cost, and is widely applied to the clinic.
Description
Technical field
The invention belongs to the tissue engineering technical field of biological materials, be specifically related to a kind of preparation method of tissue engineering xenoskin.
Background technology
Serious deep burn, skin injury need solve fast the effectively problem of wound closure.Treat deep burn at present clinically and mainly use self-skin transplant, will increase new wound surface like this.Have from the insufficient problem in body skin source at the large-area burns patient, it is to cover the most frequently used effective means of extensive deep burn wound surface at present that allograft skin adds from body microparticle skin implantation technique, but the allograft skin source is more and more limited, and it is most important in extensive deep burn patient's treatment to develop a kind of Graftskin so.And the large skin defect that severe trauma causes also need be faced the problems referred to above.In order to overcome these difficult problems, carried out multiple research at the external structure artificial skin.Sophisticated artificial skin launch has abroad been arranged, as: the product of Integra, Alloderm, Epicel, Dermagraft, Apligraft etc., but, multiple cause influences such as Production Time long, holding time weak point limited owing to cost an arm and a leg, originate do not form the scale application at home.Integra is as a kind of synthetic leather material, and the wound surface that has been widely used in large-area burns covers; It is grown into and " surviving " by promoting basic capillary endothelial cell of wound and fibroblast under the protection of one deck silicon fiml, removes silicon fiml, can obtain function and outward appearance preferably behind the autologous transplanting thin skin sheet; Shortcoming is to need the treatment of secondary self-skin transplant.Alloderm obtains by the cell component of removing in the allograft skin skin corium, also be to promote basic capillary endothelial cell of wound and fibroblast to grow into and " surviving ", its effect is better, immunogenicity is low, shortcoming be the source limited, exist pathophorous potential may.
Be applied to clinical pigskin dressing on the domestic market at present and mainly contain cell-removed pig dermis matrix, fresh porcine skin, glutaric pigskin, radiated pig skin, lyophilizing Corii Sus domestica.Prove that through zoopery and clinical practice pigskin dressing has following defective: cell-removed pig dermis matrix table of absence skin structure, still need during application to repay covering from body sword pachydermia, need the treatment of secondary self-skin transplant, the patient is formed new damage; The antigenicity of fresh porcine skin is stronger, and contamination rate is higher, is used to cut behind (cutting) crust wound surface repel early, brings out infection easily, the exposed second operation that needs of wound surface; The elasticity of glutaric pigskin is relatively poor, the water penetration height, be applied to behind the wound surface easily dry, can not with wound surface tight adhesion, skin new vessels poor growth; Radiated pig skin is the same with the lyophilizing Corii Sus domestica, and poor adhesion does not have the growth effect that promotes body epidermis cell and hypodermal cell.Though these dressing can play the effect of protection wound surface, all there is shortcoming in various degree, still can not replace allograft skin and be used to cover extensive deep burn and cut (cutting) crust wound surface, can only use as provisional covering.
Therefore press for a kind of clinically for the Graftskin of transplanting.It should possess following requirement: 1) excellent biological compatibility and histocompatibility; 2) similar to the normal skin double-layer structure, good springiness; 3) promote the growth of body epidermis cell and hypodermal cell, accelerate the growth of skin new vessels; 4) there is pore to be convenient to drain,, realizes from the body cutization for promoting that " passing " dermis scaffold from the body surface skin provides passage; 5) safe and reliable, avirulence is avoided pathophoresis; 6) the character softness is moistening, adhesiveness good, is convenient to combine closely with wound surface; 7) the permanent reservation growth of skin corium support prevents to cause cicatrization overweight because of the skin corium disappearance; 8) cheap, be suitable for wide clinical application.
Summary of the invention
At the existing in prior technology shortcoming, purpose of the present invention is exactly the preparation method that proposes a kind of tissue engineering xenoskin, makes the xenogenesis skin of preparation can satisfy above-mentioned requirements, is suitable for the skin injury that clinical treatment burn and wound cause.
The preparation method of the tissue engineering xenoskin that the present invention proposes, adopting the abundant piglet in source is the skin source, through bark fetching, epidermis fix, cut, remove the skin corium cell, modify three-dimensional collagen structure, the step that changes physical behavior, sterilization packaging obtains finished product; Concrete operations may further comprise the steps:
Step 1 is got the qualified Corii Sus domestica of quarantine and is cleaned, and soaks 15～30min with 0.1% bromo geramine, and water fully cleans; Again Corii Sus domestica epidermis side single face being adopted volumetric concentration is that 1～2% glutaraldehyde solution is fixed 30～60min, fully washes with normal saline, and it is residual to remove glutaraldehyde; Remove corium face superabundant fats and connective tissue again, from the cutting of corium face, the excision hair follicle stays pore during cutting, keeps the Corii Sus domestica of 0.2～0.4mm gauge strips epidermal area and skin corium and pore with splitter; Soak 15～30min with 0.1% bromo geramine at last, aquesterilisa fully cleans;
Step 2, the corium face single face of the Corii Sus domestica that will handle through step 1 adopts 0.25～0.5% (W/V) pancreatin solution soaking of normal saline preparation, 80～100 rev/mins of 37 ℃ of constant temperature shaking tables, and digestion 45～60min, behind ultrasonic wave concussion 30～60min, clean repeatedly with aquesterilisa; Insert in 0.5～1%TritonX-100 (TritonX) solution that contains (V/V) 0.5% gentamycin sulfate again, clean 24h for 80～100 rev/mins in 37 ℃ of constant temperature shaking tables, ultrasonic wave concussion 30～60min removes residual component, and water fully cleans; Insert in 0.5～1%SDS (dodecyl sodium sulfate) solution that contains (V/V) 0.5% gentamycin sulfate again, clean 24h for 80～100 rev/mins in 37 ℃ of constant temperature shaking tables, ultrasonic wave concussion 30～60min removes residual component, cleans with aquesterilisa; Again it is immersed in 0.5～1mol/L NaOH solution,, remove residual composition, clean with aquesterilisa in 80～100 rev/mins of basification 15min of constant temperature shaking table;
Step 3 will be crosslinked 15～30min in 0.2% the glutaraldehyde solution in fresh volumetric concentration through Corii Sus domestica two-sided immersion that step 2 is handled, and normal saline fully washes; Reduce immunogenicity by this processing, and made the crosslinked once more fusion of collagen fibril recover original three-dimensional collagen scaffold structure, modified the space structure of collagen; Again Corii Sus domestica is put into softening agents solution and soaked 30～60min and carry out softening, put into wetting agent solution after the cleaning again, preserve moisture for 37 ℃ and handle 30～60min, the abundant wash and remove residual of normal saline;
Step 4 will be handled the product that obtains through step 3, be cut to different size according to clinical needs, vacuum packaging, and cobalt 60 irradiations can be preserved more than 2 years under 2～10 ℃ of environment.
Processing method to Corii Sus domestica single face (epidermis side/corium face) of the present invention can adopt Corii Sus domestica is simultaneously sticked on the flat board, puts into treatment fluid, can carry out the processing of exposed surface, is called the pasting board method; Also can adopt the Corii Sus domestica doubling,, put into treatment fluid, can carry out the processing of exposed surface, be called the contained side method with the uncovered limit of clip/clamp clamping doubling.
Described softening agents (consumption is 10～15%, and the colleague knows) can be selected any of hexadecyltrimethylammonium chloride or cetyl trimethyl ammonium bromide or heptadecyl-N aminoethyl imidazoline expoxy propane (softening agent ES) or stearyl dimethyl benzyl ammonium chloride; Described wetting agent (consumption is 5～10%, and the colleague knows) can be selected any of glycerol or hyaluronic acid (HA) pyrrolidone sodium carboxylate (being called for short PCA-NA) mixed solution or natural dimethylamino proline acid or octadecyl dimethyl amine oxide.
Adopt the tissue engineering xenoskin of the inventive method preparation, compare with technology with existing product with clinical practice, have the following advantages: 1) use the complete fixed table cortex of glutaraldehyde to make it become protecting film, can prevent that body fluids from losing by zoopery; Thoroughly removing the skin corium cell makes skin corium become the support of growing from the body skin.2) in splitter cutting Corii Sus domestica, can reach and remove Pilus Sus domestica and hair follicle effect in the lump, utilize the natural pore of Corii Sus domestica can promote " to pass " dermis scaffold, realize accelerating the speed of growth of skin new vessels from the body cutization from the body surface skin as micropore.Give allograft skin, artificial Composite Skin, the many employings of pigskin dressing punching laser boring in the prior art artificially, and laser produces calcination to collagen structure, forms small Cauterize crust, makes the original structure of collagen imperfect, is unfavorable for the growth of cell; The invention solves the drawback that laser boring exists.3) single face of Corii Sus domestica is handled, and can avoid fixedly skin corium of glutaraldehyde when the fixed table cortex, can avoid damaging epidermal area when removing the skin corium cell, removal skin corium cell when being implemented in complete reservation table cortex.4) skin corium cell, Triton and SDS clean and twice glutaraldehyde cross-linking reduced immunogenicity jointly by removing.5) use softening agents and wetting agent to increase the flexibility and the wettability of xenogenesis skin of the present invention, compare with existing artificial skin with existing dried, hard pigskin dressing, more just adhere to closely with wound surface, accelerate the speed of growth of skin new vessels, shorten wound healing time.6) be applicable to burn, skin injury, have to from the similar double-layer structure of body skin structure, safe and reliable, nontoxic, avoid pathophoresis; The permanent reservation of dermis scaffold can be avoided accelerating the skin new vessels speed of growth simultaneously because of the skin corium disappearance causes cicatrization overweight, shortens wound healing time.7) life cycle of the product of the present invention's preparation is short, the holding time is long and cheap, is suitable for wide clinical application.
The specific embodiment
Below by specific embodiment technical solution of the present invention is described further.
Step 1 selects the biological rank of pure strain to raise the piglet of 3 months sizes, body weight 30～50Kg as the skin source.The Corii Sus domestica of getting quarantine qualified (not containing PRRS virus, pig circular ring virus, Pseudorabies virus, epidemic encephalitis B virus, swine fever virus, pig parvoviral, foot and mouth disease virus) is shaved hair and is cleaned, soak 25min with 0.1% bromo geramine (production of Fourth Ring, Beijing health medical instruments factory), water fully cleans; Corii Sus domestica epidermis side (adopting the pasting board method) is carried out single face to be handled, the epidermis side of Corii Sus domestica is soaked into crosslinked fixedly 40min in freshly prepared 2% glutaraldehyde solution (be preferably under the stirring condition and carry out), with normal saline flushing (taking off from pasting board), the flush away glutaraldehyde is residual.Remove corium face superabundant fats and connective tissue afterwards, equably from the cutting of corium face, excise hair follicle during cutting in the lump and stay pore, keep the Corii Sus domestica of 0.3mm gauge strips epidermal area and skin corium and pore with splitter.After soaking 25min with 0.1% bromo geramine at last, clean with sterilized water for injection.
Step 2 through the Corii Sus domestica (adopting the contained side method) that step 1 is handled, adopts corium face single face 0.3% (V/W) pancreatin solution soaking of normal saline preparation, in 37 ℃ of constant temperature water bath shaking tables 100 rev/mins, digestion 50min behind the ultrasonic wave concussion 40min, cleans repeatedly with sterilized water for injection; Insert in the 1%TritonX-100 solution that contains (V/V) 0.5% gentamycin sulfate, 80 rev/mins are cleaned 24h in 37 ℃ of constant temperature water bath shaking tables again, and ultrasonic wave concussion 50min removes residual component, and water fully cleans; Insert in the 0.8%SDS solution that contains (V/V) 0.5% gentamycin sulfate again, prevent germ contamination in the processing procedure, clean 24h for 80 rev/mins in 37 ℃ of constant temperature water bath shaking tables, ultrasonic wave concussion 40min removes residual component, cleans with aquesterilisa; Again it is immersed in the 1mol/L NaOH solution,, remove residual composition, clean (opening contained side) with sterilized water for injection in 80 rev/mins of basification 15min of constant temperature shaking table.
Step 3 will be crosslinked 20min in 0.2% the glutaraldehyde solution in freshly prepared volumetric concentration through Corii Sus domestica two-sided immersion that step 2 is handled, fully wash flush away glutaraldehyde debris with normal saline; Reduce immunogenicity by this processing, and made the crosslinked once more fusion of collagen fibril recover original three-dimensional collagen scaffold structure, modified the space structure of collagen; The softening agents solution of again Corii Sus domestica being put into 10% (hexadecyltrimethylammonium chloride) soaks 40min and carries out softening, put into (hyaluronic acid pyrrolidone sodium carboxylate mixed solution) wetting agent solution of 8% after the cleaning again, preserve moisture for 37 ℃ and handle 30min, at last with the abundant wash and remove residual of normal saline.
Step 4 will be handled the product that obtains through step 3, be cut to different size according to clinical needs, the evacuation packing, and (60 irradiations of 15～25KGy) cobalts can be preserved more than 2 years under 2～10 ℃ of environment.
Prove through zoopery and clinical practice, prepared xenogenesis skin solved limited, Production Time of costing an arm and a leg, originate of existing artificial skin product long, the holding time is short, the secondary skin-grafting brings the shortcoming of damage for body; Also solved pigskin dressing table of absence skin structure, antigenicity strong, still need when using the secondary self-skin transplant form again new damage, contamination rate higher, be used for repelling behind the wound surface early form wound surface exposed, easily dry, easily bring out infection, elasticity is relatively poor, water penetration is high, can not with the wound surface tight adhesion, do not have to promote growth of body epidermis cell and hypodermal cell and the poky shortcoming of skin new vessels.
Claims (4)
1, a kind of preparation method of tissue engineering xenoskin is characterized in that, may further comprise the steps:
Step 1 is got the qualified Corii Sus domestica of quarantine and is cleaned, and soaks 15～30min with 0.1% bromo geramine, and water fully cleans; Again Corii Sus domestica epidermis side single face being adopted volumetric concentration is that 1～2% glutaraldehyde solution is fixed 30～60min, fully washes with normal saline; Remove corium face superabundant fats and connective tissue again, from the cutting of corium face, the excision hair follicle stays pore, keeps the Corii Sus domestica of 0.2～0.4mm gauge strips epidermal area and skin corium and pore with splitter; Soak 15～30min with 0.1% bromo geramine at last, aquesterilisa cleans;
Step 2, the corium face single face of the Corii Sus domestica that will handle through step 1 adopts normal saline 0.25～0.5% pancreatin solution soaking of volume ratio preparation by weight, 80～100 rev/mins of 37 ℃ of constant temperature shaking tables, digestion 45～60min behind ultrasonic wave concussion 30～60min, cleans repeatedly with aquesterilisa; Insert again in 0.5～1% TritonX-100 solution that contains volume ratio 0.5% gentamycin sulfate, clean 24h for 80～100 rev/mins in 37 ℃ of constant temperature shaking tables, ultrasonic wave concussion 30～60min, water fully cleans; Insert in the 0.5～1%SDS solution that contains volume ratio 0.5% gentamycin sulfate again, clean 24h for 80～100 rev/mins in 37 ℃ of constant temperature shaking tables, ultrasonic wave concussion 30～60min cleans with aquesterilisa; Again it is immersed in 0.5～1mol/L NaOH solution,, clean with aquesterilisa in 80～100 rev/mins of basification 15min of constant temperature shaking table;
Step 3 will be crosslinked 15～30min in 0.2% the glutaraldehyde solution in fresh volumetric concentration through Corii Sus domestica two-sided immersion that step 2 is handled, and normal saline fully washes; Corii Sus domestica is put into softening agents solution again and soaked 30～60min and carry out softening, put into wetting agent solution after the cleaning again, preserve moisture for 37 ℃ and handle 30～60min, normal saline fully washes;
Step 4 will be handled the product that obtains through step 3, according to clinical needs cutting, and vacuum packaging, cobalt 60 irradiations are preserved under 2～10 ℃ of environment.
2, preparation method according to claim 1 is characterized in that, pasting board method or contained side method are adopted in described processing to the Corii Sus domestica single face.
3, preparation method according to claim 1, it is characterized in that the softening agents in the described step 3 is any of hexadecyltrimethylammonium chloride or cetyl trimethyl ammonium bromide or heptadecyl-N aminoethyl imidazoline expoxy propane or stearyl dimethyl benzyl ammonium chloride.
4, preparation method according to claim 1, it is characterized in that the wetting agent in the described step 3 is any of glycerol or hyaluronic acid pyrrolidone sodium carboxylate mixed solution or natural dimethylamino proline acid or octadecyl dimethyl amine oxide.
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| CN101947147A (en) * | 2010-06-23 | 2011-01-19 | 乐普（北京）医疗器械股份有限公司 | Method for chemically treating biological tissue materials |
| CN101954116A (en) * | 2010-08-31 | 2011-01-26 | 长沙达瑞奇实业有限公司 | Manufacturing method of xenoskin used for burns and scalds |
| CN102631706A (en) * | 2012-04-13 | 2012-08-15 | 武岩 | Method for preparing low-immunogenicity pig dermal support |
| CN103349794A (en) * | 2013-05-08 | 2013-10-16 | 中国人民解放军军事医学科学院野战输血研究所 | Method for clearing non-human animal skin heterologous antigen, and low immunogenicity non-human animal skin preparation method |
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| CN107412867A (en) * | 2017-05-10 | 2017-12-01 | 广州润虹医药科技股份有限公司 | A kind of preparation method of Xenogenic acellular dermal matrix |
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| CN101947147A (en) * | 2010-06-23 | 2011-01-19 | 乐普（北京）医疗器械股份有限公司 | Method for chemically treating biological tissue materials |
| CN101954116A (en) * | 2010-08-31 | 2011-01-26 | 长沙达瑞奇实业有限公司 | Manufacturing method of xenoskin used for burns and scalds |
| CN102631706A (en) * | 2012-04-13 | 2012-08-15 | 武岩 | Method for preparing low-immunogenicity pig dermal support |
| CN102631706B (en) * | 2012-04-13 | 2014-01-29 | 武岩 | Method for preparing low-immunogenicity pig dermal support |
| CN104095692A (en) * | 2013-04-08 | 2014-10-15 | 天津市塑料研究所有限公司 | Method for manufacturing bovine jugular vein valved conduit |
| CN103349794A (en) * | 2013-05-08 | 2013-10-16 | 中国人民解放军军事医学科学院野战输血研究所 | Method for clearing non-human animal skin heterologous antigen, and low immunogenicity non-human animal skin preparation method |
| CN103349794B (en) * | 2013-05-08 | 2015-08-19 | 中国人民解放军军事医学科学院野战输血研究所 | Restructuring alpha-galactosidase removes method and the product of xenogeneic skin antigen |
| CN106256382A (en) * | 2015-06-18 | 2016-12-28 | 北京朗嘉仪生物技术有限公司 | Pericystium biological support and its production and use |
| CN107412867A (en) * | 2017-05-10 | 2017-12-01 | 广州润虹医药科技股份有限公司 | A kind of preparation method of Xenogenic acellular dermal matrix |
| CN107412868A (en) * | 2017-08-10 | 2017-12-01 | 广州润虹医药科技股份有限公司 | A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix |
| CN111330079A (en) * | 2020-03-31 | 2020-06-26 | 江苏白衣缘生物工程有限公司 | Artificial dura mater composite patch |
| CN111330079B (en) * | 2020-03-31 | 2021-12-03 | 江苏白衣缘生物工程有限公司 | Artificial dura mater composite patch |
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