CN104587530A - Medical purified pig dermis and preparation method thereof - Google Patents
Medical purified pig dermis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses medical purified pig dermis and a preparation method thereof. The preparation method comprises the following steps: selecting traceable and healthy fresh pigskin as a raw material; removing non-collagen ingredients and micromolecular substances in the pigskin by means of repeated cleaning, step-by-step alkyl polyglycoside degreasing, hydrogen peroxide depilating and alternate treatment purification with various enzyme preparations and various basic materials; and fully separating and loosening collagen fiber to obtain the medical purified pig dermis which has a complete three-dimensional reticulate structure, favorable biocompatibility, favorable mechanical property and appropriate biodegradability and bioactivity. The medical purified pig dermis can be used as a raw material for preparing medical collagen, body surface wound dressings, tissue engineering scaffold material, biological filling material, restoration and reconstruction material and the like.
Description
Technical field
The present invention relates to medical purification pig dermis and preparation method thereof, belong to the preparation field of biomedical material.
Background technology
Collagen is present in mammiferous bone, tendon, cartilage and skin and other connective tissue widely, accounts for 1/3 of mammalian proteins total amount, is the extracellular matrix components that formation body weight for humans is wanted.Collagen has excellent biocompatibility, more weak antigenicity, can promote adhesion and the propagation of cell, good anthemorrhagic performance, good degradability, and good hydrophilic etc.Therefore, for a long time, people utilize the performance characteristics of collagen, research and develop serial collagen base biological medical material, as biological dressing, artificial skin, biological medicinal membrane, sthptic sponge, acellular dermal matrix material, Injectable Collagen, collagen-based composite medical fiber and tissue engineering bracket material etc., part collagen base biological medical material has been applied to clinical, and be proved to be and there is certain curative effect (but defend China, Wang Kunyu, Zeng Rui, Deng. the medical application of collagen and development prospect thereof. biomedical engineering and clinical .2004, 8(1): 45-48).
Collagen is the renewable animal organism matter resource that Resources Reserve is maximum in the world, wide material sources, cheap and easy to get, has very high medical science value.The Land use systems of collagen mainly contains two kinds: (1) extracts collagen: from the tissue (as beef tendon, Mus tail tendon) containing collagen, extract collagen (albumen), prepare various medical material with the collagen obtained; (2) body utilizes: directly process is containing the tissue (Corii Sus domestica, Corii Caprae seu Ovis, Corii Bovis seu Bubali, pericardium etc.) of collagen, removing non-collagen tissue, then apply (but time, but defend China, Zeng Rui, Deng. the Land use systems of animal skins and development prospect thereof. Chinese leather, 2007,36(1): 38-40).
Regrettably, collagen, in medical application, because the biocompatibility of collagen base biological medical material is restive in the fabrication process, causes material in use usually to occur the problem of reliability and safety.Occur that the basic reason of these problems is exactly because the purity of used collagen does not reach requirement.According to our research, the key solving the biocompatibility of collagen base biological medical material is: the purity improving collagen tissue, removes non-collagen tissue, small-molecule substance in raw material to greatest extent, keeps the triple helix structure of collagen simultaneously; In the process of the purification of collagen, the biological activity of collagen should be guaranteed, remove antigenic substance again, eliminate its immunogenicity.In fact, no matter be the first Land use systems, or the second Land use systems, all relate to this key issue of purification (but time, but defend China, vast stretch of wooded country, Deng. genipin is cross-linked the research of acellular allodermis matrix material. functional material .2012,21(43): 2966-2969; Wu Xia, but defend China, Xiao Shiwei. extract the research of pigskin collagen under different condition. Chinese leather .2011,41(3): 33-36; Liu Xinhua, Lu Cuijuan, but time, etc. from the leftover pieces of acellular allodermis matrix material, extract type i collagen. Chinese leather, 2013,42 (13): 22-27; But time, Liu Xinhua, but defend China, etc. the preparation of pig source type i collagen/chitosan composite sponge. leather science and engineering .2014,24(3): 17-22).If collagen purification is not in place, the safety of body application is difficult to ensure, the collagen of extraction also affects its performance containing impurity unavoidably.
China is pig-breeding big country, and have abundant Corii Sus domestica resource, Corii Sus domestica is cheap and easy to get.Corii Sus domestica histologically has certain homology with human body skin, is desirable natural collagen raw material.Except containing except collagen in Corii Sus domestica, also containing more non-collagen tissue, as epidermis, fleece, oils and fats, cell etc.
In existing collagen extraction techniques, the non-collagen tissue in some use sodium chloride removing raw materials, then carry out purifying (Fu Yanfeng, Shen Yuexin, Yang Chenggang, etc. the extraction of fresh water fish skin collagen. Shanghai Aquatic Products Univ. 9CN) journal .2004,13(6): 146-150; Zhang Yan, Li Guoying, Miao Yuqing, etc. the extraction of grass carp skin collagen and the preliminary study of performance. aquatic science, 2006,25(3): 136-139); Some technology be then carry out some simple pretreatment just carry out extracting (Li Kaixiong, Zhao Zhiyuan, Liu Xia. the extraction and application of Collagen Protein From Pig Skin. meat research .1996,4:43-48; Yang Bo, Li Guoying. the extraction of non-degeneration I-type collagen and performance characterization in bullfrog skin. leather science and engineering, 2012,22(5): 5-8,19).These simple methods cannot remove the multiple non-collagen tissue in Corii Sus domestica, are difficult to the needs meeting Corii Sus domestica purification.
In the preparation of acellular dermal, the normal method adopted has (Song Bing. the present Research of acellular dermal matrix. Binzhou Medical College journal .2007,30 (1): 53-54):
(1) DispaseII-Triton method: utilize from neutral protease DispaseII, epidermal area is separated with skin corium, then nonionic surfactant Triton X-100 are utilized, process residue dermal tissue, destroy hypodermal cell cell membrane, and then hypodermal cell and organelle thereof are dissolved, obtain after abundant rinsing.
(2) height oozes salt-SDS method: first skin sample is soaked in 1mol/L NaCl solution by this method, acts on 24 h at 37 DEG C, is separated epidermis and corium, then under rupture of membranes agent sodium lauryl sulphate (SDS) room temperature using 0.5%
Hatch 1 h remove the cell component in corium and obtain.
(3) height oozes salt-enzyme digestion: in the solution of 1mol/L, and 37 DEG C are soaked 24-48h, hatches 90min, remove cell component in corium and obtain after removing epidermis with 0.25% trypsin under adding 0.1%EDTA chelating agen 37 DEG C of constant temperature.
(4) NaOH ablation method: in the solution of 1mol/L, 37 DEG C are soaked 24h and remove epidermis, then use 4% NaOH solution ablation, and Type B ultrasonic cleaning instrument is interrupted oscillation cleaning, changes ablation liquid once, ablation 16-18 h every 4 h.Skin graft after process embathes 24 more than h in distilled water.Finally skin graft is dipped in normal saline (dripping penicillin and gentamycin solution), 4 DEG C of storages.
Processing method relatively more conventional at present, as the preparation method of de-cell xenogenesis (pig) dermal matrix prepared by Qidong, Jiangsu biomaterial institute, detailed process is as follows: with the healthy little Bai pig of about 50kg without dermatopathy, live after killing and shave hair, take off spready hide skin, the split-thickness skin graft that 0.3-0.5mm made by skin machine is split with electronic, after liquor hibitane sterilization, 24 hours are soaked with aseptic hypertonic saline (30g/L), slough epidermis, after glutaraldehyde (2g/L soaks 10 minutes) is crosslinked, process with the de-Cell sap being primary raw material containing trypsin, normal saline flushing, make netted packaging again and preserve (Li Kaitong, Liu Daen. de-cell xenogenesis (pig) dermal matrix clinical practice and progress. Chinese community doctor medical speciality, 2012, 14(6): 13-14.).
These methods have focused on removing epidermis and cell, and method is difficult to the cell that effectively removes completely in tissue, do not relate to or the removing of other composition in shorter mention skin.In some preparation method, adopt the method for shaving hair, removing fleece, but Rhizoma Imperatae still remains in skin.Some method adopts Na
2s dissolves hair, can make to remain the larger S of toxicity in product
2-, very adverse influence is produced to biocompatibility, and containing S in waste water
2-, cause serious environmental pollution.
In sum, prior art has the following disadvantages:
1. extract collagen with the relatively limited beef tendon of resource, Mus tail tendon for raw material, have ignored the selection of raw material and purification thereof for the critical importance preparing medical collagen, preparation cost is high, is difficult to the needs meeting ordinary consumer;
2. be that raw material extracts collagen with cattle tissue, there is the risk of carrying crazy heifer disease virus.And Corii Sus domestica organizes homology good, wide material sources, cheap and easy to get.
Method in the past focuses on removing epidermis and cell, and method is difficult to the cell that effectively removes completely in tissue.Also fail effectively to remove to oils and fats, fleece etc., be difficult to the needs meeting multiple non-collagen tissue in removing Corii Sus domestica, the product purity obtained is difficult to meet medical requirement, more cannot meet the requirement of medical embedded material.
Summary of the invention
Medical purification pig dermis has following performance (content is in over dry base):
(1) outward appearance: white or close white;
(2) protein content: >=95%(mass fraction);
(3) tensile strength: >=4.0Mpa;
(4) elongation at break: >=15%;
(5) acid-base value: pH value 5.5-8.0;
(6) content of beary metal :≤10 μ g/ml;
(7) dichloromethane extract :≤2%(mass fraction);
(8) ash :≤2%(mass fraction);
(9) hydroxyproline content: the 5%(mass fraction that should be not less than total amino acid content);
(10) histological examination: HE staining dyeing after tissue slice, light microscopy, noresidue cell;
(11) vitro cytotoxicity: cell-cytotoxic reaction is not more than 1 grade;
(12) sensitization test (STT): should without delayed hypersensitivity;
(13) Intradermal stimulates: constitutional stimulates P II to be less than 0.4;
(14) pyrogen: without thermal source.
2. medical purification pig dermis according to claim 1 and preparation method thereof, comprises the following steps:
(1) raw material skin prepares: the fresh porcine skin stripped from tractability, healthy pig; Adopt fleshing machine to carry out fleshing immediately, repair the positions such as tail of decaptitating, abdomen the part between the ribs and the hips, extremity with cutter, retain Corii Sus domestica identity position, often open skin and adopt 500-2000 grams of Sal to pickle rear freezen protective; Complete to freezen protective from stripping, isolated time is no more than 4h;
(2) weigh: the freezing Corii Sus domestica taking 100 weight portions;
(3) first time cleaning: the regulator solution that adds water, than 2.0-4.0, soaks 30-60min, after thawing completely, then rotates 30-120min, controls dry waste liquid, the regulator solution that adds water is than 2.0-4.0, and soda 0.5-1.2% rotates 30-120min, controls dry waste liquid;
(4) second time cleaning: the regulator solution that adds water, than 2.0-4.0, rotates 30-120min, controls dry waste liquid, the regulator solution that adds water, than 2.0-4.0, rotates 30-120min, controls dry waste liquid;
(5) third time cleaning: add the hot water that temperature is 35-45 DEG C, regulator solution is than 2.0-4.0, and bath temperature 30-35 DEG C, adds alkyl polyglycoside 0.2-3.0 weight portion, rotates 30-180min, controls dry waste liquid;
(6) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(7) soak: the regulator solution that adds water, than 2.0-6.0, adds alkyl polyglycoside 0.2-3.0 weight portion, Sal 1.0-6.0 weight portion, rotate 30-180min, stop drum and spend the night; Secondary morning rotates 30-60min, controls dry waste liquid;
(8) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(9) first time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(10) second time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(11) third time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(12) wash: add the hot water that temperature is 35-40 DEG C, regulator solution is than 2.0-6.0, and vexed washing 15-30min, discards waste liquid; Then add the hot water that temperature is 35-40 DEG C, regulator solution is than 2.0-6.0, and vexed washing 10-20min, discards waste liquid; Add the hot water that temperature is 35-40 DEG C again, regulator solution than 2.0-6.0, vexed washing 10-20min; Discard waste liquid; Finally, the regulator solution that adds water is than 2.0-6.0, and flowing water washes 30min;
(13) hydrogen peroxide depilation: the water adding 30-50 weight portions, then the H adding that purity is 30%
2o
22.0-6.0 weight portions, then add after cooling in the water NaOH of 3.0-8.0 weight portions being dissolved in 10-30 weight portions, rotate 20-40min, then skin is put in rotary drum, rotate 30-120min.Check depilation situation, after hair purifies, expand liquor ratio to 2.0-5.0, rotate 120min, stop drum and spend the night; Next day rotates 30-60min, controls dry waste liquid;
(14) wash: the regulator solution that adds water is than 2.0-6.0, and vexed washing 10-30min, controls dry waste liquid; Then, the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min, controls dry waste liquid;
(15) first time dealkalize: add temperature be the hot water regulator solution of 38-42 DEG C than 1.0-4.0, bath temperature 30-35 DEG C, add lactic acid 1.0-5.0 weight portion, alkyl polyglycoside 0.3-1.5 weight portion, rotation 30-90min;
(16) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 30-90min, controls dry waste liquid.Regulator solution add water again than 2.0-6.0, and flowing water washes 30-90min, controls dry waste liquid;
(17) second time dealkalize: add temperature be the hot water regulator solution of 38-42 DEG C than 1.0-4.0, bath temperature 30-35 DEG C, add lactic acid 1.0-5.0 weight portion, alkyl polyglycoside 0.3-1.5 weight portion, rotation 30-90min;
(18) wash: add normal-temperature water regulator solution than 2.0-6.0, flowing water washes 30-90min, controls dry waste liquid; Add normal-temperature water regulator solution again than 2.0-6.0, flowing water washes 30-90min, controls dry waste liquid;
(19) enzymic digestion: add the hot water that temperature is 40-44 DEG C, preheating, regulator solution ratio is to 1.0-3.0, bath temperature is 30-36 DEG C, then, adds compound enzyme C 0.10-2.00 weight portion, alkyl polyglycoside 0.3-3.0 weight portion, after rotating 60-180min, stops drum and spends the night; Next day rotates 15-45min, controls dry waste liquid;
(20) clean: add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid, namely obtain medical purification pig dermis;
(21) roll salt: add sodium chloride 5.0-20.0 weight portion, rotate 20-60min, go out drum;
(22) split: be cut into rectangle according to the large young pathbreaker's purified pigskin of hide, and then be divided into the fritter rectangle of 10.0-80.0cm × 10.0-80.0cm;
(23) pack, freezen protective: the purified pigskin of pack is put into and freezes storehouse, in temperature-5---freezen protective under the condition of 15 DEG C.
3. the manufacture method of medical purification pig dermis according to claim 2, is characterized in that rotary drum is rustless steel rotary drum.
4. compound enzyme C according to claim 2, its formula and preparation method are:
Take AS1.398 neutral protease (50,000 unit) 100 weight portions, add pancreatin (1:25) 10-100 weight portion, mix homogeneously.
5. alkyl polyglycoside according to claim 2, sodium hydroxide, lactic acid, sodium chloride etc., and AS1.398 neutral protease in claim 4 and pancreatin are commercially available prod.
the present invention has the following advantages:
1. the Corii Sus domestica of, wide material sources good, cheap and easy to get with homology prepares purified pigskin for raw material, both reduces material cost, turn avoid and uses beef tendon, all deficiencies existing for Mus tail tendon;
2. adopt surfactant alkyl polyglycoside of new generation to be fat-free material, efficiency is high, and safety is good;
3. adopt hydrogen peroxide-alkaline process depilation, eliminate fleece for a full due, overcome and use Na
2the toxic and side effects that S depilation produces biomaterial, decreases environmental pollution;
4. adopt compound enzyme C digestion, together with method above, thoroughly can remove the non-collagen tissue in Corii Sus domestica, effectively reduce antigenicity.
5. achieve the efficiently purifying of Corii Sus domestica, purified pigskin not only as extracting the raw material preparing medical collagen, but also can be applicable to other plurality of medical purposes;
6. this method also can be used for the body efficiently purifying of other animal skins;
7. production process is pollution-free, has significant environmental benefit and economic benefit.
four, detailed description of the invention
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is; the present embodiment is only used to further illustrate the present invention; and the restriction of the protection domain to invention can not be interpreted as, the person skilled in the art in this field content according to the present invention can make some nonessential improvement and adjustment.
embodiment 1
(1) raw material skin prepares: the fresh porcine skin stripped from tractability, healthy pig.Adopt fleshing machine fleshing, remove the hypodermis layer of animal skins.
(2) weigh: the freezing Corii Sus domestica taking 100 weight portions;
(3) first time cleaning: the regulator solution that adds water, than 2.0, soaks 30min, after thawing completely, then rotates 30min, controls dry waste liquid, the regulator solution that adds water is than 2.0, and soda 0.5%, rotates 30min, control dry waste liquid;
(4) second time cleaning: the regulator solution that adds water, than 2.0, rotates 30min, controls dry waste liquid, the regulator solution that adds water, than 2.0, rotates 30min, controls dry waste liquid;
(5) third time cleaning: add the hot water that temperature is 35 DEG C, regulator solution is than 2.0, and bath temperature 30 DEG C, adds alkyl polyglycoside 0.2 weight portion, rotates 30min, controls dry waste liquid;
(6) wash: the regulator solution that adds water is than 2.0, and flowing water washes 10min;
(7) soak: the regulator solution that adds water, than 2.0, adds lauryl glucoside 0.2 weight portion, Sal 1.0 weight portion, rotate 30min, stop drum and spend the night; Secondary morning rotates 30min, controls dry waste liquid;
(8) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(9) first time defat: add the hot water regulator solution of 40 DEG C than 1.0, bath temperature is 30 DEG C, adds lauryl glucoside 0.8 weight portion, rotates 60min, controls dry waste liquid;
(10) second time defat: add the hot water regulator solution of 40 DEG C than 1.0, bath temperature is 30 DEG C, adds lauryl glucoside 1.0 weight portion, rotates 60min, controls dry waste liquid;
(11) third time defat: add the hot water regulator solution of 40 DEG C than 1.0, bath temperature is 30 DEG C, adds lauryl glucoside 1.0 weight portion, rotates 60min, controls dry waste liquid;
(12) wash: add the hot water that temperature is 35 DEG C, regulator solution is than 2.0, and vexed washing 15min, discards waste liquid; Then add the hot water that temperature is 35 DEG C, regulator solution is than 2.0, and vexed washing 10min, discards waste liquid; Add the hot water that temperature is 35 DEG C again, regulator solution than 2.0, vexed washing 10min; Discard waste liquid; Finally, the regulator solution that adds water is than 2.0, and flowing water washes 30min;
(13) hydrogen peroxide depilation: the water adding 30 weight portions, then the H adding that purity is 30%
2o
24.0 weight portions, then add after cooling in the water NaOH of 6.0 weight portions being dissolved in 30 weight portions, rotate 30min, then skin is put in rotary drum, rotate 90min.Check depilation situation, after hair purifies, expand liquor ratio to 2.0, rotate 120min, stop drum and spend the night; In morning next day, rotate 30min, control dry waste liquid;
(14) wash: the regulator solution that adds water is than 2.0, and vexed washing 10min, controls dry waste liquid; Then, the regulator solution that adds water is than 2.0, and flowing water washes 10min, controls dry waste liquid;
(15) first time dealkalize: add temperature be the hot water regulator solution of 38 DEG C than 1.0, bath temperature 30 DEG C, add lactic acid 1.0 weight portion, lauryl glucoside 0.3 weight portion, rotation 30min;
(16) wash: the regulator solution that adds water is than 2.0, and flowing water washes 30min, controls dry waste liquid.Regulator solution add water again than 2.0, and flowing water washes 30min, controls dry waste liquid;
(17) second time dealkalize: add temperature be the hot water regulator solution of 38 DEG C than 1.0, bath temperature 30 DEG C, add lactic acid 1.0 weight portion, lauryl glucoside 0.3 weight portion, rotation 30min;
(18) wash: add normal-temperature water regulator solution than 2.0, flowing water washes 30min, controls dry waste liquid; Add normal-temperature water regulator solution again than 2.0, flowing water washes 30min, controls dry waste liquid;
(19) enzymic digestion: add the hot water that temperature is 40 DEG C, preheating, regulator solution ratio is to 1.0, and bath temperature is 30 DEG C, then, adds compound enzyme C 0.10 weight portion, lauryl glucoside 0.3 weight portion, after rotating 60min, stops drum and spends the night; Next day rotates 15min, controls dry waste liquid;
(20) clean: add room temperature pure water regulator solution than 2.0, add sodium chloride 2.0 weight portion, rotate 90min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0, add sodium chloride 2.0 weight portion, rotate 90min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0, add sodium chloride 2.0 weight portion, rotate 90min, control dry waste liquid, namely obtain medical purification pig dermis;
(21) roll salt: add sodium chloride 5.0 weight portion, rotate 20min, go out drum;
(22) split: be cut into rectangle according to the large young pathbreaker's purified pigskin of hide, and then be divided into the fritter rectangle of 10.0-80.0cm × 10.0-80.0cm;
(23) pack, freezen protective: the purified pigskin of pack is put into and freezes storehouse, in temperature-5---freezen protective under the condition of 15 DEG C.
embodiment 2
(1) raw material skin prepares: the fresh porcine skin stripped from tractability, healthy pig; Adopt fleshing machine fleshing, remove the hypodermis layer of animal skins;
(2) weigh: the freezing Corii Sus domestica taking 100 weight portions;
(3) first time cleaning: the regulator solution that adds water, than 3.0, soaks 60min, after thawing completely, then rotates 120min, controls dry waste liquid, the regulator solution that adds water is than 4.0, and soda 0.8%, rotates 80min, control dry waste liquid;
(4) second time cleaning: the regulator solution that adds water, than 3.0, rotates 75min, controls dry waste liquid, the regulator solution that adds water, than 3.0, rotates 90min, controls dry waste liquid;
(5) third time cleaning: add the hot water that temperature is 42 DEG C, regulator solution is than 3.0, and bath temperature 35 DEG C, adds alkyl polyglycoside 1.5 weight portion, rotates 90min, controls dry waste liquid;
(6) wash: the regulator solution that adds water is than 4.0, and flowing water washes 20min;
(7) soak: the regulator solution that adds water, than 3.0, adds alkyl polyglycoside (APG1216) 1.5 weight portion, Sal 3 weight portion, rotate 90min, stop drum and spend the night; Secondary morning rotates 45min, controls dry waste liquid;
(8) wash: the regulator solution that adds water is than 4.0, and flowing water washes 20min;
(9) first time defat: add the hot water regulator solution of 42 DEG C than 2.5, bath temperature is 35 DEG C, adds alkyl polyglycoside (APG1216) 1.5 weight portion, rotates 120min, controls dry waste liquid;
(10) second time defat: add the hot water regulator solution of 402 DEG C than 2.5, bath temperature is 35 DEG C, adds alkyl polyglycoside (APG1216) 1.5 weight portion, rotates 120min, controls dry waste liquid;
(11) third time defat: add the hot water regulator solution of 402 DEG C than 2.5, bath temperature is 35 DEG C, adds alkyl polyglycoside (APG1216) 1.5 weight portion, rotates 120min, controls dry waste liquid;
(12) wash: add the hot water that temperature is 38 DEG C, regulator solution is than 4.0, and vexed washing 25min, discards waste liquid; Then add the hot water that temperature is 38 DEG C, regulator solution is than 4.0, and vexed washing 20min, discards waste liquid; Add the hot water that temperature is 38 DEG C again, regulator solution than 4.0, vexed washing 20min; Discard waste liquid; Finally, the regulator solution that adds water is than 4.0, and flowing water washes 30min;
(13) hydrogen peroxide depilation: the water adding 40 weight portions, then the H adding that purity is 30%
2o
25.0 weight portions, then add after cooling in the water NaOH of 6.0 weight portions being dissolved in 30 weight portions, rotate 40min, then skin is put in rotary drum, rotate 90min.Check depilation situation, after hair purifies, expand liquor ratio to 4.0, rotate 120min, stop drum and spend the night; In morning next day, rotate 40min, control dry waste liquid;
(14) wash: the regulator solution that adds water is than 4.0, and vexed washing 20min, controls dry waste liquid; Then, the regulator solution that adds water is than 4.0, and flowing water washes 20min, controls dry waste liquid;
(15) first time dealkalize: add temperature be the hot water regulator solution of 40 DEG C than 2.5, bath temperature 38 DEG C, add lactic acid 3.0 weight portion, alkyl polyglycoside (APG1216) 0.9 weight portion, rotation 60min;
(16) wash: the regulator solution that adds water is than 4.0, and flowing water washes 60min, controls dry waste liquid.Regulator solution add water again than 4.0, and flowing water washes 60min, controls dry waste liquid;
(17) second time dealkalize: add temperature be the hot water regulator solution of 40 DEG C than 2.5, bath temperature 32 DEG C, add lactic acid 3.0 weight portion, alkyl polyglycoside (APG1216) 0.9 weight portion, rotation 60min;
(18) wash: add normal-temperature water regulator solution than 4.0, flowing water washes 30min, controls dry waste liquid; Add normal-temperature water regulator solution again than 4.0, flowing water washes 60min, controls dry waste liquid;
(19) enzymic digestion: add the hot water that temperature is 42 DEG C, preheating, regulator solution ratio is to 2.0, and bath temperature is 34 DEG C, then, adds compound enzyme C 1.00 weight portion, alkyl polyglycoside (APG1216) 1.5 weight portion, after rotating 90min, stops drum and spends the night; Next day rotates 30min, controls dry waste liquid;
(20) clean: add room temperature pure water regulator solution than 3.0, add sodium chloride 3.0 weight portion, rotate 120min, control dry waste liquid.Add room temperature pure water regulator solution than 3.0, add sodium chloride 3.5 weight portion, rotate 120min, control dry waste liquid.Add room temperature pure water regulator solution than 3.0, add sodium chloride 3.5 weight portion, rotate 120min, control dry waste liquid, namely obtain medical purification pig dermis;
(21) roll salt: add sodium chloride 10.0 weight portion, rotate 40min, go out drum;
(22) split: be cut into rectangle according to the large young pathbreaker's purified pigskin of hide, and then be divided into the fritter rectangle of 10.0-80.0cm × 10.0-80.0cm;
(23) pack, freezen protective: the purified pigskin of pack is put into and freezes storehouse, in temperature-5---freezen protective under the condition of 15 DEG C.
embodiment 3
(1) raw material skin prepares: the fresh porcine skin stripped from tractability, healthy pig.Adopt fleshing machine fleshing, remove the hypodermis layer of animal skins;
(2) weigh: the Corii Sus domestica taking 100 weight portions;
(3) first time cleaning: the regulator solution that adds water, than 4.0, soaks 60min, and after thawing completely, then rotate 120min, control dry waste liquid, the regulator solution that adds water is than 4.0, and soda 1.2% rotates 120min, controls dry waste liquid;
(4) second time cleaning: the regulator solution that adds water, than 4.0, rotates 120min, controls dry waste liquid, the regulator solution that adds water, than 4.0, rotates 120min, controls dry waste liquid;
(5) third time cleaning: add the hot water that temperature is 45 DEG C, regulator solution is than 4.0, and bath temperature 35 DEG C, adds alkyl polyglycoside (APG0810) 0.2-3.0 weight portion, rotates 30-180min, controls dry waste liquid;
(6) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(7) soak: the regulator solution that adds water, than 2.0-6.0, adds alkyl polyglycoside 0.2-3.0 weight portion, Sal 6.0 weight portion, rotate 180min, stop drum and spend the night; Secondary morning rotates 60min, controls dry waste liquid;
(8) wash: the regulator solution that adds water is than 6.0, and flowing water washes 130min;
(9) first time defat: add the hot water regulator solution of 45 DEG C than 4.0, bath temperature is 38 DEG C, adds alkyl polyglycoside (APG0810) 3.0 weight portion, rotates 180min, controls dry waste liquid;
(10) second time defat: add the hot water regulator solution of 45 DEG C than 4.0, bath temperature is 38 DEG C, adds alkyl polyglycoside (APG0810) 3.0 weight portion, rotates 180min, controls dry waste liquid;
(11) third time defat: add the hot water regulator solution of 45 DEG C than 4.0, bath temperature is 38 DEG C, adds alkyl polyglycoside (APG0810) 3.0 weight portion, rotates 180min, controls dry waste liquid;
(12) wash: add the hot water that temperature is 40 DEG C, regulator solution is than 6.0, and vexed washing 30min, discards waste liquid; Then add the hot water that temperature is 40 DEG C, regulator solution is than 6.0, and vexed washing 20min, discards waste liquid; Add the hot water that temperature is 40 DEG C again, regulator solution than 6.0, vexed washing 20min; Discard waste liquid; Finally, the regulator solution that adds water is than 6.0, and flowing water washes 30min;
(13) hydrogen peroxide depilation: the water adding 50 weight portions, then the H adding that purity is 30%
2o
26.0 weight portions, then add after cooling in the water NaOH of 8.0 weight portions being dissolved in 30 weight portions, rotate 20-40min, then skin is put in rotary drum, rotate 120min.Check depilation situation, after hair purifies, expand liquor ratio to 5.0, rotate 120min, stop drum and spend the night; In morning next day, rotate 60min, control dry waste liquid;
(14) wash: the regulator solution that adds water is than 6.0, and vexed washing 30min, controls dry waste liquid; Then, the regulator solution that adds water is than 6.0, and flowing water washes 30min, controls dry waste liquid;
(15) first time dealkalize: add the hot water regulator solution ratio-4.0 that temperature is 42 DEG C, bath temperature 35 DEG C, adds lactic acid 5.0 weight portion, alkyl polyglycoside (APG0810) 1.5 weight portion, rotates 90min;
(16) wash: the regulator solution that adds water is than 6.0, and flowing water washes 90min, controls dry waste liquid.Regulator solution add water again than 6.0, and flowing water washes 90min, controls dry waste liquid;
(17) second time dealkalize: add temperature be the hot water regulator solution of 42 DEG C than 4.0, bath temperature 35 DEG C, add lactic acid 5.0 weight portion, alkyl polyglycoside (APG0810) 1.5 weight portion, rotation 90min;
(18) wash: add normal-temperature water regulator solution than 6.0, flowing water washes 90min, controls dry waste liquid; Add normal-temperature water regulator solution again than 6.0, flowing water washes 90min, controls dry waste liquid;
(19) enzymic digestion: add the hot water that temperature is 44 DEG C, preheating, regulator solution ratio is to 3.0, and bath temperature is 36 DEG C, then, adds compound enzyme C 2.00 weight portion, alkyl polyglycoside (APG0810) 3.0 weight portion, after rotating min, stops drum and spends the night; Next day rotates 45min, controls dry waste liquid;
(20) clean: add room temperature pure water regulator solution than 5.0, add sodium chloride 5.0 weight portion, rotate 180min, control dry waste liquid.Add room temperature pure water regulator solution than 5.0, add sodium chloride 5.0 weight portion, rotate 180min, control dry waste liquid.Add room temperature pure water regulator solution than 5.0, add sodium chloride 5.0 weight portion, rotate 180min, control dry waste liquid, namely obtain medical purification pig dermis;
(21) roll salt: add sodium chloride 20.0 weight portion, rotate 60min, go out drum;
(22) split: be cut into rectangle according to the large young pathbreaker's purified pigskin of hide, and then be divided into the fritter rectangle of 10.0-80.0cm × 10.0-80.0cm;
(23) pack, freezen protective: the purified pigskin of pack is put into and freezes storehouse, in temperature-5---freezen protective under the condition of 15 DEG C.
Claims (5)
1. medical purification pig dermis has following performance (content is in over dry base):
(1) outward appearance: white or close white;
(2) protein content: >=95%(mass fraction);
(3) tensile strength: >=4.0Mpa;
(4) elongation at break: >=15%;
(5) acid-base value: pH value 5.5-8.0;
(6) content of beary metal :≤10 μ g/ml;
(7) dichloromethane extract :≤2%(mass fraction);
(8) ash :≤2%(mass fraction);
(9) hydroxyproline content: the 5%(mass fraction that should be not less than total amino acid content);
(10) histological examination: HE staining dyeing after tissue slice, light microscopy, noresidue cell;
(11) vitro cytotoxicity: cell-cytotoxic reaction is not more than 1 grade;
(12) sensitization test (STT): should without delayed hypersensitivity;
(13) Intradermal stimulates: constitutional stimulates P II to be less than 0.4;
(14) pyrogen: without thermal source.
2. medical purification pig dermis according to claim 1 and preparation method thereof, comprises the following steps:
(1) raw material skin prepares: the fresh porcine skin stripped from tractability, healthy pig; Adopt fleshing machine to carry out fleshing immediately, repair the positions such as tail of decaptitating, abdomen the part between the ribs and the hips, extremity with cutter, retain Corii Sus domestica identity position, often open skin and adopt 500-2000 grams of Sal to pickle rear freezen protective; Complete to freezen protective from stripping, isolated time is no more than 4h;
(2) weigh: the freezing Corii Sus domestica taking 100 weight portions;
(3) first time cleaning: the regulator solution that adds water, than 2.0-4.0, soaks 30-60min, after thawing completely, then rotates 30-120min, controls dry waste liquid, the regulator solution that adds water is than 2.0-4.0, and soda 0.5-1.2% rotates 30-120min, controls dry waste liquid;
(4) second time cleaning: the regulator solution that adds water, than 2.0-4.0, rotates 30-120min, controls dry waste liquid, the regulator solution that adds water, than 2.0-4.0, rotates 30-120min, controls dry waste liquid;
(5) third time cleaning: add the hot water that temperature is 35-45 DEG C, regulator solution is than 2.0-4.0, and bath temperature 30-35 DEG C, adds alkyl polyglycoside 0.2-3.0 weight portion, rotates 30-180min, controls dry waste liquid;
(6) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(7) soak: the regulator solution that adds water, than 2.0-6.0, adds alkyl polyglycoside 0.2-3.0 weight portion, Sal 1.0-6.0 weight portion, rotate 30-180min, stop drum and spend the night; Secondary morning rotates 30-60min, controls dry waste liquid;
(8) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min;
(9) first time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(10) second time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(11) third time defat: add the hot water regulator solution of 40-45 DEG C than 1.0-4.0, bath temperature is 30-38 DEG C, adds alkyl polyglycoside 0.3-3.0 weight portion, rotates 60-180min, controls dry waste liquid;
(12) wash: add the hot water that temperature is 35-40 DEG C, regulator solution is than 2.0-6.0, and vexed washing 15-30min, discards waste liquid; Then add the hot water that temperature is 35-40 DEG C, regulator solution is than 2.0-6.0, and vexed washing 10-20min, discards waste liquid; Add the hot water that temperature is 35-40 DEG C again, regulator solution than 2.0-6.0, vexed washing 10-20min; Discard waste liquid; Finally, the regulator solution that adds water is than 2.0-6.0, and flowing water washes 30min;
(13) hydrogen peroxide depilation: the water adding 30-50 weight portions, then the H adding that purity is 30%
2o
22.0-6.0 weight portions, then add after cooling in the water NaOH of 3.0-8.0 weight portions being dissolved in 10-30 weight portions, rotate 20-40min, then skin is put in rotary drum, rotate 30-120min.Check depilation situation, after hair purifies, expand liquor ratio to 2.0-5.0, rotate 120min, stop drum and spend the night; Next day rotates 30-60min, controls dry waste liquid;
(14) wash: the regulator solution that adds water is than 2.0-6.0, and vexed washing 10-30min, controls dry waste liquid; Then, the regulator solution that adds water is than 2.0-6.0, and flowing water washes 10-30min, controls dry waste liquid;
(15) first time dealkalize: add temperature be the hot water regulator solution of 38-42 DEG C than 1.0-4.0, bath temperature 30-35 DEG C, add lactic acid 1.0-5.0 weight portion, alkyl polyglycoside 0.3-1.5 weight portion, rotation 30-90min;
(16) wash: the regulator solution that adds water is than 2.0-6.0, and flowing water washes 30-90min, controls dry waste liquid.Regulator solution add water again than 2.0-6.0, and flowing water washes 30-90min, controls dry waste liquid;
(17) second time dealkalize: add temperature be the hot water regulator solution of 38-42 DEG C than 1.0-4.0, bath temperature 30-35 DEG C, add lactic acid 1.0-5.0 weight portion, alkyl polyglycoside 0.3-1.5 weight portion, rotation 30-90min;
(18) wash: add normal-temperature water regulator solution than 2.0-6.0, flowing water washes 30-90min, controls dry waste liquid; Add normal-temperature water regulator solution again than 2.0-6.0, flowing water washes 30-90min, controls dry waste liquid;
(19) enzymic digestion: add the hot water that temperature is 40-44 DEG C, preheating, regulator solution ratio is to 1.0-3.0, bath temperature is 30-36 DEG C, then, adds compound enzyme C 0.10-2.00 weight portion, alkyl polyglycoside 0.3-3.0 weight portion, after rotating 60-180min, stops drum and spends the night; Next day rotates 15-45min, controls dry waste liquid;
(20) clean: add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid.Add room temperature pure water regulator solution than 2.0-5.0, add sodium chloride 2.0-5.0 weight portion, rotate 90-180min, control dry waste liquid, namely obtain medical purification pig dermis;
(21) roll salt: add sodium chloride 5.0-20.0 weight portion, rotate 20-60min, go out drum;
(22) split: be cut into rectangle according to the large young pathbreaker's purified pigskin of hide, and then be divided into the fritter rectangle of 10.0-80.0cm × 10.0-80.0cm;
(23) pack, freezen protective: the purified pigskin of pack is put into and freezes storehouse, in temperature-5---freezen protective under the condition of 15 DEG C.
3. the manufacture method of medical purification pig dermis according to claim 2, is characterized in that rotary drum is rustless steel rotary drum.
4. compound enzyme C according to claim 2, its formula and preparation method are:
Take AS1.398 neutral protease (50,000 unit) 100 weight portions, add pancreatin (1:25) 10-100 weight portion, mix homogeneously.
5. alkyl polyglycoside according to claim 2, sodium hydroxide, lactic acid, sodium chloride etc., and AS1.398 neutral protease in claim 4 and pancreatin are commercially available prod.
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