CN1268401C - Method for preparing acellular dermal matrix material - Google Patents
Method for preparing acellular dermal matrix material Download PDFInfo
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- CN1268401C CN1268401C CN 200410022506 CN200410022506A CN1268401C CN 1268401 C CN1268401 C CN 1268401C CN 200410022506 CN200410022506 CN 200410022506 CN 200410022506 A CN200410022506 A CN 200410022506A CN 1268401 C CN1268401 C CN 1268401C
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Abstract
The present invention discloses a method for preparing acellular dermal matrix materials, which is characterized in that a healthy animal is selected, a raw hide is physically and chemically processed, such as wash, degreasing, etc. within 3 hours after the healthy animal is butchered and stripped; the raw hide is then depilated and purified by utilizing chemical substances, such as a hide collagen processing agent of compound enzymes, alkali, etc. so that the acellular dermal matrix materials with favorable biocompatibility and a proper biodegradation speed are obtained. The materials can be used for the medical purposes of dermal substitutes, repairing ulcer skin, plastic beautification, etc.
Description
One, technical field
The present invention relates to a kind of manufacture method of acellular dermal matrix material, belong to the manufacture field of bio-medical material.
Two, background technology
No matter be war or peacetime, always have the wound surface of leaving over because of burn, wound or war wound, so wound repair just become the eternal theme of medical treatment.Along with the continuous development and progress of science and technology, the material that can be used for wound repair day by day increases, as from body skin, allograft skin, artificial skin and biological dressing etc.Though existing wound repair material has certain rehabilitation efficacy, but still can not satisfy clinical needs.
(acellular dermal matrix is a kind of dermal replacement that obtained clinical practice in recent years in burn and field of orthopedic surgery ADM) to the acellular dermal matrix material, and its raw material is allograft skin and xenogenesis skin.
The AlloDerm that U.S. Lifecell company produces be seen in report the earliest take off cell allosome corium.Krejci etc. with cadaver skin behind three freeze-thaw cycle, radiation gamma 12min with 500rad, in 37 ℃ PBS (phosphoric acid formula salt buffer solution), soaked 7-14 days then, adopt mechanical means to remove epidermis again, then, placing temperature is that 37 ℃ PBS soaked for 3 weeks, till tissues observed section under the light microscopic is acellular.Usefulness trypsin such as Matoskiva are 37 ℃ in temperature and handle Corii Sus domestica 2h, after removing epidermis, corium is thoroughly cleaned in PBS, the reuse trypsin is handled 1h down at 37 ℃, rinsing in culture fluid is then immersed and is soaked at least 3 days in the water to remove cell and antibiotic remaining composition.Wainwright etc. induce anchor shape fiber to separate from the basal cell hemi desmosome cadaver skin overnight incubation in the RPMI1640 culture fluid under the tension force condition, not changing collagen or destroying under the situation of basement membrane, remove the fibroblast in the dermal matrix.Elder generations such as Livesey spend the night Corii Sus domestica in the RPMI1640 culture fluid, be to hatch under 37 ℃ in temperature in 1mol/LNaCl then, again 0.5%SDS (Sodium dodecyl suphate is hatched 1h in SDS), up to distinguish under the light microscopic misknow cell till.Medalie etc. with the cadaver skin fast freeze-thaw after, in PBS, hatch again with the preparation acellular dermal matrix material (ADM).Usefulness 20% formalin such as domestic Sun Yong China carry out crosslinked to the extracellular matrix of rat skin, use trypsin and EDTA chelating agen to remove epidermis, keep basement membrane, reuse DNA enzyme, RNA enzyme and other chemical reagent and hypotonic chemical compound buffer and EDTA chelating agen etc. can cause the cell component of host cell recognition reaction and handle to intradermal.1998, Sun Yonghua also reported to utilize and has taken off cell allosome corium with successful from body thin skin sheet cograft.In addition, also have the method for usefulness Dispase such as Custafson and each 12h of TritonX-100 processing cadaver skin to prepare the report of ADM.
Because allosome acellular dermal matrix source is limited, costs an arm and a leg, and has the danger of communicate illness, scientists begins to turn one's attention to the development of xenogenesis acellular dermal matrix material.Domesticly take the lead in releasing xenogenesis (pig) acellular dermal matrix material by Qidong, Jiangsu medical material institute, it is superior to do manual work, and thickness is even, is crowned with complete success after clinical use, and its transplantation effect is second to hardly and takes off cell allosome corium.The base preparation method is: after the healthy white piglets work of no dermatopathy about 50Kg is killed, shave the clean skin of hair, peel spready hide, on electric skin splitting device, make the split-thickness skin graft that thickness is 0.3-0.5mm, be cut into the skin graft of 4cm * 5cm size, after the liquor hibitane sterilization, immerse hypertonic saline 24h, tear epidermis gently off, behind the glutaraldehyde cross-linking, with containing trypsin is that the Cell sap that takes off of main component removes cell, and normal saline flushing is made behind the mesh standby with special net making machine.
Put it briefly, existing acellular dermal matrix material preparation method mainly comprises epidermis, takes off three basic steps such as hypodermal cell and lyophilization processing, and two kinds of situations are arranged.(1) cadaver skin is handled through hypertonic saline and sodium lauryl sulphate (SDS), forms NaCl-SDS ADM; (2) after DispaseII handles, handle through TritonX-100 again, form Dispase-Triton ADM.
Existing acellular dermal matrix material generally is in the experimentation stage, does not see the report of large-scale industrial production as yet, and has the following disadvantages:
1. complicated operation, the cycle is long, and is time-consuming;
2. mostly be manual operations, labor intensity is big;
3. there are a large amount of Rhizoma Imperataes and other impurity in the host material;
4. do not have standard preparation technology and special equipment, material, can't guarantee the quality and the performance of target material.
Three, summary of the invention
A kind of acellular dermal matrix preparation methods of providing at the deficiencies in the prior art is provided, be characterized in that adopting healthy animal skins is raw material, chemical substances such as applying composite enzyme collagen inorganic agent and alkali, preparation has the acellular dermal matrix material of good biocompatibility and suitable biodegradation rate.
Purpose of the present invention is realized that by following technical measures the umber of wherein said raw and auxiliary material is parts by weight except that specified otherwise.
The acellular dermal matrix preparation methods:
1. clean: choose healthy animal,, peel its skin, get 100 parts of fresh animal skins, put into rotary drum, add normal-temperature water 200-500 part, cover the rotary drum door, rotate 30-150min, control dried waste liquid through butchering.Add normal-temperature water 200-500 part, cover the rotary drum door, rotate 30-150min, control dried waste liquid, so repeatable operation is 2-6 time, then fully washing.
2. pre-defat:, make that the temperature of liquid reaches 36-42 ℃ in the rotary drum with the hot water preheating homoiothermic of 40-50 ℃ of temperature.Control is done.The hot water 200-500 part that adds 34-42 ℃ of temperature adds peregal 0.5-10.0 part, soda 0.5-12.0 part and penetrating agent JFC 0.2-1.0 part, rotates 30-120min.Control dried waste liquid.Add 30-36 ℃ of hot water 200-600 of temperature part, rotate 20-120min, control dried waste liquid.Add 30-36 ℃ again, hot water 200-600 part is rotated 20-120min.Control dried waste liquid, so repeatable operation is 2-6 time.
3. secondary degreasing: with temperature is 40-46 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 36-42 ℃ in the rotary drum.Control is done.Add hot water 200-500 part of 36-42 ℃, add degreasing agent PT-101 1.0-10.0 part, peregal 0.2-8.0 part, detergent 0.2-10.0 part is rotated 30-240min, controls dried waste liquid.Add 30-36 ℃ of hot water 200-600 part again, rotate 20-120min.Control dried waste liquid, so repeatable operation is 2-6 time.Then, control dried waste liquid.
4. preheating: with temperature is 40-46 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 24-30 ℃ in the rotary drum.Control dried waste liquid.Take out animal skins, put into special-purpose constant temperature cabinet immediately, leave standstill dewatering 2-8h.Require room temperature identical with temperature in the special-purpose constant temperature cabinet, temperature controllable is 24-32 ℃.
5. being coated with enzyme banks up: the water content that dewatering reaches animal skins is about 60-65%, and the enzyme for preparing is in advance stuck with paste the flesh noodles that evenly is applied to animal skins.The prescription that enzyme is stuck with paste is: compound enzyme collagen inorganic agent E-894 20-100 part, water 10-50 part.After coating, flesh noodles is neat to the flesh noodles tiling, covers completely with plastic sheeting.All operation is all carried out under constant temperature, and time 18-50h banks up.
6. manual unhairing: after being coated with enzyme and reaching requirement, take out pin hair and fine hair on the dermatotome by hand.
7. clean: manual plucked animal skins is put into rotary drum, add normal-temperature water 200-600 part, cover the rotary drum door, rotate 30-180min.Control dried waste liquid.Add normal-temperature water 200-600 part, cover the rotary drum door, rotate 30-180min, control dried waste liquid, so repeatable operation is 2-6 time.Add NaCl5-30 part, add normal-temperature water 100-400 part, rotate 30-240min.Control dried waste liquid.Add normal-temperature water 200-600 part, cover the rotary drum door, rotate 30-180min.Control dried waste liquid.Repetitive operation 2-6 time as stated above again.Then, fully washing.Control dried waste liquid.
8. epilation: after cleaning, washing, adding temperature is 20-30 ℃ of water 100-400 part, adds sodium hydroxide 0.5-10.0 part, rotates 30-240min.Remove pin hair and the fine hair that remains on every block animal skin with tweezers.
9. cut open layer: residual pin hair on the animal skins and fine hair cut open layer after removing totally fully on accurate splitting machine, cut open layer thickness: it is 0.4-1.0mm that the first layer lether of band grain cuts open layer thickness; Not cuing open layer thickness with the double-layer fur of grain is 0.4-1.2mm.Remainder reclaims as the raw material that extracts collagen.
10. dealkalize: get the first layer lether of band grain or not with 100 parts of the double-layer furs of grain, add lactic acid 0.2-3.0 part, ammonium chloride 1.0-6.0 part, or ammonium sulfate 0.6-6.0 part are rotated 30-180min.Waste liquid is removed in control.
11. clean: add normal-temperature water 200-600 part, NaCl 5-30 part is rotated 30-240min.Control dried waste liquid.Add normal-temperature water 200-600 part, rotate 30-150min, control dried waste liquid.Repetitive operation is 2-6 time as stated above.Then, fully dried waste liquid is controlled in washing.Skin is taken out from rotary drum, use centrifuge dewatering.Again skin is put into rotary drum, add deionized water 200-600 part, rotate 30-180min.Control dried waste liquid.Repetitive operation according to said method 2-6 time.Then, skin is taken out from rotary drum, use centrifuge dewatering.
12. will through the dehydration animal skins, put into the refrigerating chamber lyophilizing of freezer dryer after, be 6.0-30.0KGy/h's through dosage again
60The gamma-rays width of cloth that Co produced is finished product according to sterilization, formed package.
In the acellular dermal matrix preparation methods of the present invention used animal skins be fresh virus-free infection, do not have dermatosis and do not have the animal skins of medical history, if pig, cattle, sheep domestic animal skin then should be chosen pig, cattle, the sheep domestic animal of not using forage feed.
Used rotary drum is suspension type wood rotary drum and rustless steel rotary drum in the acellular dermal matrix preparation methods of the present invention.
Adopt the prepared acellular dermal matrix material of this method, can satisfy the requirement of following medical application:
1. as the succedaneum of corium, be used for the reparation of burn wound.For shallow II degree burn and dark III degree burn significant curative effect is arranged, obvious curative effects is arranged for the III burn.
2. be used for the reparation of ulcer skin, be particularly useful for the treatment of the skin ulcer that causes because of diabetes.
3. enlarge the bosom, grand lip and augmentation rhinoplasty.After target material being carried out the processing of suitable body modification, can be used for enlarging the bosom, medical operatings such as grand lip and augmentation rhinoplasty.
4. can be used for the reparation of ophthalmology shaping, facial soft tissue defects etc.
The present invention has the following advantages:
1. keeping under the constant prerequisite of animal skins three-dimensional tissue structure, thoroughly remove non-collagen tissue such as cell, cell debris and other extracellular matrix components in the animal skins, obtained having the corium of complete three-dimensional tissue's structure.
2. very easy, the easy row of method is economical, reasonable, safe, reliable.
3. whole technical process belongs to and cleans production, environmentally safe.
Four, the specific embodiment
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the foregoing invention content.
Embodiment 1
(1) cleans: choose healthy pig,, peel skin, get fresh porcine skin 100kg, put into rotary drum, add normal-temperature water 200kg, cover the rotary drum door, rotate 60min through butchering.Control dried waste liquid.Add normal-temperature water 200kg, cover the rotary drum door, rotate 60min, control dried waste liquid, so repeatable operation is 3 times.Fully washing then.
(2) pre-defat: with temperature is 40 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 36 ℃ in the rotary drum.Control is done.The adding temperature is 36 ℃ hot water 200kg, adds peregal 3.0kg, soda 2.5kg and penetrating agent JFC 0.4kg, rotates 40min.Control dried waste liquid.The adding temperature is 30 ℃ hot water 200kg, rotates 40min, controls dried waste liquid.The hot water 300kg that adds 30 ℃ again rotates 40min.Control dried waste liquid, so repeatable operation is 3 times.
(3) secondary degreasing: with temperature is 40 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 36 ℃ in the rotary drum, controls dried waste liquid.The hot water 200kg that adds 36 ℃ adds degreasing agent PT-1013.0kg, peregal 2.0kg, and detergent 2.5kg rotates 90min, controls dried waste liquid.The adding temperature is 30 ℃ hot water 300kg, rotates 70min, controls dried waste liquid.Adding temperature again is 30 ℃ of hot water 300kg, rotates 70min, controls dried waste liquid.So repeatable operation is 2 times.Then, control dried waste liquid.
(4) preheating: with temperature is 40 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 24 ℃ in the rotary drum, controls dried waste liquid.Take out Corii Sus domestica, put into special-purpose constant temperature cabinet immediately, leave standstill dewatering 2h.Require room temperature identical, can be controlled in 24 ℃ with temperature in the special-purpose constant temperature cabinet.
(5) being coated with enzyme banks up: after the water content that dewatering reaches Corii Sus domestica is about 60-65%, the enzyme for preparing is in advance stuck with paste the flesh noodles that is applied to animal skins equably.The prescription that enzyme is stuck with paste is compound enzyme collagen inorganic agent E-89420kg, water 10kg.After coating, flesh noodles is neat to the flesh noodles tiling, covers completely with plastic sheeting.All operation is all carried out under constant temperature, and time 18h banks up.
(6) manual unhairing: after being coated with enzyme and reaching requirement, take out pin hair and fine hair on the dermatotome by hand.
(7) clean: manual plucked skin is put into rotary drum, add normal-temperature water 300kg, cover the rotary drum door, rotate 60min, control dried waste liquid.Add normal-temperature water 300kg, cover the rotary drum door, rotate 60min.Control dried waste liquid.So repeatable operation is 3 times.Add NaCl 15kg, add normal-temperature water 200kg, rotate 60min.Control dried waste liquid.Repetitive operation 3 times as stated above again.Then, fully dried waste liquid is controlled in washing.
(8) epilation: after cleaning, washing finish, add 20 ℃ of water 200kg, add sodium hydroxide 3.0kg, rotate 90min.Take out pin hair and the fine hair that remains on every block animal skin with tweezers.
(9) cut open layer: remain in pin hair on the animal skins or fine hair remove fully clean after, on accurate splitting machine, cut open layer, cut open layer thickness: it is 0.50mm that the first layer lether of band grain cuts open layer thickness; Not cuing open layer thickness with the double-layer fur of grain is 0.60mm, and remainder reclaims as the raw material that extracts collagen.
(10) dealkalize: get the first layer lether of band grain or not with the double-layer fur 100kg of grain, add lactic acid 1.0kg, ammonium chloride or ammonium sulfate 2.0kg rotate 120min.Waste liquid is removed in control.
(11) clean: add normal-temperature water 300kg, NaCl 10kg rotates 120min, controls dried waste liquid.Add normal-temperature water 300kg, rotate 90min, control dried waste liquid.Repetitive operation is 3 times as stated above.Then, fully dried waste liquid is controlled in washing.Corii Sus domestica is taken out from rotary drum, use centrifuge dewatering.Again Corii Sus domestica is put into rotary drum, add deionized water 300kg, rotate 120min, control dried waste liquid.Repetitive operation according to said method 3 times.Then, Corii Sus domestica is taken out from rotary drum, use centrifuge dewatering.
(12) will through the dehydration Corii Sus domestica, put into the refrigerating chamber lyophilizing of freezer dryer after, be 10.0KGy/h's through dosage again
60The gamma-rays width of cloth that Co produced is finished product according to sterilization, formed package.
Embodiment 2
(1) cleans: choose healthy goat,, peel skin, get fresh goat skin 100kg, put into rotary drum, add normal-temperature water 350kg, cover the rotary drum door, rotate 90min through butchering.Control dried waste liquid.Add normal-temperature water 350kg, cover the rotary drum door, rotate 90min, control dried waste liquid, so repeatable operation is 4 times.Fully washing then.
(2) pre-defat: with temperature is 45 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 38 ℃ in the rotary drum.Control is done.The adding temperature is 38 ℃ hot water 350kg, adds peregal 7.0kg, soda 6.5kg and penetrating agent JFC 0.7kg, rotates 75min.Control dried waste liquid.The adding temperature is 34 ℃ hot water 400kg, rotates 75min, controls dried waste liquid.The hot water 400kg that adds 34 ℃ again rotates 75min.Control dried waste liquid, so repeatable operation is 4 times.
(3) secondary degreasing: with temperature is 44 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 38 ℃ in the rotary drum, controls dried waste liquid.The hot water 350kg that adds 38 ℃ adds degreasing agent PT-1017.0kg, peregal 5.0kg, and detergent 6.5kg rotates 120min, controls dried waste liquid.The adding temperature is 33 ℃ hot water 400kg, rotates 90min, controls dried waste liquid.Adding temperature again is 33 ℃ of hot water 400kg, rotates 90min, controls dried waste liquid.So repeatable operation is 4 times.Then, control dried waste liquid.
(4) preheating: with temperature is 43 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 27 ℃ in the rotary drum, controls dried waste liquid.Take out goat skin, put into special-purpose constant temperature cabinet immediately, leave standstill dewatering 5h.Require room temperature identical, can be controlled in 27 ℃ with temperature in the special-purpose constant temperature cabinet.
(5) being coated with enzyme banks up: after the water content that dewatering reaches goat skin is about 60-65%, the enzyme for preparing is in advance stuck with paste the flesh noodles that is applied to animal skins equably.The prescription that enzyme is stuck with paste is compound enzyme collagen inorganic agent E-894 60kg, water 30kg.After coating, flesh noodles is neat to the flesh noodles tiling, covers completely with plastic sheeting.All operation is all carried out under constant temperature, and time 36h banks up.
(6) manual unhairing: after being coated with enzyme and reaching requirement, take out pin hair and fine hair on the dermatotome by hand.
(7) clean: manual plucked goat skin is put into rotary drum, add normal-temperature water 450kg, cover the rotary drum door, rotate 120min, control dried waste liquid.Add normal-temperature water 450kg, cover the rotary drum door, rotate 120min.Control dried waste liquid.So repeatable operation is 4 times.Add NaCl 20kg, add normal-temperature water 300kg, rotate 180min.Control dried waste liquid.Repetitive operation 4 times as stated above again.Then, fully dried waste liquid is controlled in washing.
(8) epilation: after cleaning, washing finish, add 25 ℃ of water 300kg, add sodium hydroxide 6.5kg, rotate 180min.Take out pin hair and the fine hair that remains on every block animal skin with tweezers.
(9) cut open layer: remain in pin hair on the animal skins or fine hair remove fully clean after, on accurate splitting machine, cut open layer, cut open layer thickness: it is 0.50mm that the first layer lether of band grain cuts open layer thickness; Not cuing open layer thickness with the double-layer fur of grain is 0.60mm, and remainder reclaims as the raw material that extracts collagen.
(10) dealkalize: get the first layer lether of band grain or not with the double-layer fur 100kg of grain, add lactic acid 2.5kg, ammonium chloride or ammonium sulfate 4.0kg rotate 150min.Waste liquid is removed in control.
(11) clean: add normal-temperature water 450kg, NaCl 15kg rotates 180min, controls dried waste liquid.Add normal-temperature water 450kg, rotate 120min, control dried waste liquid.Repetitive operation is 4 times as stated above.Then, fully dried waste liquid is controlled in washing.Goat skin is taken out from rotary drum, use centrifuge dewatering.Again goat skin is put into rotary drum, add deionized water 450kg, rotate 150min, control dried waste liquid.Repetitive operation according to said method 4 times.Then, goat skin is taken out from rotary drum, use centrifuge dewatering.
(12) will through the dehydration goat skin, put into the refrigerating chamber lyophilizing of freezer dryer after, be 14.8KGy/h's through dosage again
60The gamma-rays width of cloth that Co produced is finished product according to sterilization, formed package.
Embodiment 3
(1) cleans: choose healthy cattle,, peel skin, get fresh cattle hide 100kg, put into rotary drum, add normal-temperature water 500kg, cover the rotary drum door, rotate 150min through butchering.Control dried waste liquid.Add normal-temperature water 500kg, cover the rotary drum door, rotate 150min, control dried waste liquid, so repeatable operation is 5 times.Fully washing then.
(2) pre-defat: with temperature is 48 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 42 ℃ in the rotary drum.Control is done.The adding temperature is 42 ℃ hot water 500kg, adds peregal 10.0kg, soda 12.0kg and penetrating agent JFC 1.0kg, rotates 120min.Control dried waste liquid.The adding temperature is 36 ℃ hot water 600kg, rotates 120min, controls dried waste liquid.The hot water 600kg that adds 36 ℃ again rotates 120min.Control dried waste liquid, so repeatable operation is 5 times.
(3) secondary degreasing: with temperature is 46 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 42 ℃ in the rotary drum, controls dried waste liquid.The hot water 500kg that adds 42 ℃ adds degreasing agent PT-10110.0kg, peregal 8.0kg, and detergent 10.0kg rotates 180min, controls dried waste liquid.The adding temperature is 36 ℃ hot water 600kg, rotates 120min, controls dried waste liquid.Adding temperature again is 36 ℃ of hot water 600kg, rotates 120min, controls dried waste liquid.So repeatable operation is 5 times.Then, control dried waste liquid.
(4) preheating: with temperature is 46 ℃ hot water preheating homoiothermic, makes that the temperature of liquid reaches 30 ℃ in the rotary drum, controls dried waste liquid.Take out cattle hide, put into special-purpose constant temperature cabinet immediately, leave standstill dewatering 8h.Require room temperature identical, can be controlled in 30 ℃ with temperature in the special-purpose constant temperature cabinet.
(5) being coated with enzyme banks up: after the water content that dewatering reaches goat skin is about 60-65%, the enzyme for preparing is in advance stuck with paste the flesh noodles that is applied to cattle hide equably.The prescription that enzyme is stuck with paste is compound enzyme collagen inorganic agent E-894 80kg, water 40kg.After coating, flesh noodles is neat to the flesh noodles tiling, covers completely with plastic sheeting.All operation is all carried out under constant temperature, and time 50h banks up.
(6) manual unhairing: after being coated with enzyme and reaching requirement, take out pin hair and fine hair on the dermatotome by hand.
(7) clean: manual plucked cattle hide is put into rotary drum, add normal-temperature water 600kg, cover the rotary drum door, rotate 180min, control dried waste liquid.Add normal-temperature water 600kg, cover the rotary drum door, rotate 180min.Control dried waste liquid.So repeatable operation is 6 times.Add NaCl 30kg, add normal-temperature water 400kg, rotate 240min.Control dried waste liquid.Repetitive operation 5 times as stated above again.Then, fully dried waste liquid is controlled in washing.
(8) epilation: after cleaning, washing finish, add 30 ℃ of water 400kg, add sodium hydroxide 10.0kg, rotate 240min.Take out pin hair and the fine hair that remains on the every cattle hide with tweezers.
(9) cut open layer: remain in pin hair on the animal skins or fine hair remove fully clean after, on accurate splitting machine, cut open layer, cut open layer thickness: it is 0.50mm that the first layer lether of band grain cuts open layer thickness; Not cuing open layer thickness with the double-layer fur of grain is 0.60mm, and remainder reclaims as the raw material that extracts collagen.
(10) dealkalize: get the first layer lether of band grain or not with the double-layer fur 100kg of grain, add lactic acid 3.0kg, ammonium chloride or ammonium sulfate 5.0kg rotate 150min.Waste liquid is removed in control.
(11) clean: add normal-temperature water 600kg, NaCl 25kg rotates 180min, controls dried waste liquid.Add normal-temperature water 600kg, rotate 150min, control dried waste liquid.Repetitive operation is 5 times as stated above.Then, fully dried waste liquid is controlled in washing.Cattle hide is taken out from rotary drum, use centrifuge dewatering.Again cattle hide is put into rotary drum, add deionized water 600kg, rotate 180min, control dried waste liquid.Repetitive operation according to said method 5 times.Then, cattle hide is taken out from rotary drum, use centrifuge dewatering.
(12) will through the dehydration cattle hide, put into the refrigerating chamber lyophilizing of freezer dryer after, be 20.0KGy/h's through dosage again
60The gamma-rays width of cloth that Co produced is finished product according to sterilization, formed package.
Used compound enzyme collagen inorganic agent is that Chengdu enlightening Australia collagen Hitek Ltd produces, enzyme-added auxiliary liming agent EA produces for the auxiliary reagent factory, Shanghai for the chemical plant production of Guangdong Pericarpium Citri tangerinae leather, peregal, detergent and penetrating agent JFC among the present invention, and degreasing agent PT-101 produces for the detergent factory, Guilin.All the other chemical reagent are commercially available analysis pure preparation.
Claims (3)
1. acellular dermal matrix preparation methods is characterized in that:
(1) cleans: get fresh and healthy animal skins 100 weight portions, put into rotary drum, add normal-temperature water 200-500 weight portion, cover the rotary drum door, rotate 30-150min, control dried waste liquid, add normal-temperature water 200-500 weight portion, cover the rotary drum door, rotate 30-150min, control dried waste liquid, so repeatable operation is 2-6 time, fully washing then
(2) pre-defat: the hot water preheating homoiothermic that with temperature is 40-50 ℃, make that the temperature of liquid reaches 36-42 ℃ in the rotary drum, control is done, the hot water 200-500 weight portion that adds 34-42 ℃, add peregal 0.5-10.0 weight portion, soda 0.5-12.0 weight portion and penetrating agent JFC 0.2-1.0 weight portion, rotate 30-120min, control dried waste liquid, the adding temperature is 30-36 ℃ a hot water 200-600 weight portion, rotates 20-120min, control dried waste liquid, add temperature again and be 30-36 ℃ hot water 200-600 weight portion, rotate 20-120min, control dried waste liquid, so repeatable operation is 2-6 time
(3) secondary degreasing: with 40-46 ℃ hot water preheating homoiothermic, make that the temperature of liquid reaches 36-42 ℃ in the rotary drum, control is done, the adding temperature is 36-42 ℃ a hot water 200-500 weight portion, adds degreasing agent PT-1011.0-10.0 weight portion, peregal 0.2-8.0 weight portion, detergent 0.2-10.0 weight portion rotates 30-240min, controls dried waste liquid, adding temperature again is 30-36 ℃ of hot water 200-600 weight portion, rotates 20-120min, controls dried waste liquid, so repeatable operation is 2-6 time, then, control dried waste liquid
(4) preheating: with 40-46 ℃ hot water preheating homoiothermic, make that the temperature of liquid reaches 24-30 ℃ in the rotary drum, control dried waste liquid, take out animal skins, put into special-purpose constant temperature cabinet immediately, leave standstill dewatering 2-8h, require room temperature identical with temperature in the special-purpose constant temperature cabinet, temperature controllable is 24-32 ℃
(5) being coated with enzyme banks up: the water content that dewatering reaches animal skins is 60-65%, the enzyme for preparing is in advance stuck with paste the flesh noodles that evenly is applied to animal skins, the prescription that enzyme is stuck with paste is: compound enzyme collagen inorganic agent E-894 20-100 weight portion, water 10-50 weight portion, after coating, flesh noodles is neat to the flesh noodles tiling, cover completely with plastic sheeting, all operation is all carried out under constant temperature, and time 18-50h banks up
(6) manual unhairing: after being coated with enzyme and reaching requirement, take out pin hair and fine hair on the dermatotome by hand,
(7) clean: manual plucked animal skins is put into rotary drum, add normal-temperature water 200-600 weight portion, cover the rotary drum door, rotate 30-180min, control dried waste liquid, add normal-temperature water 200-600 weight portion, cover the rotary drum door, rotate 30-180min, control dried waste liquid, so repeatable operation is 2-6 time, add the NaCl5-30 weight portion, add normal-temperature water 100-400 weight portion, rotate 30-240min, control dried waste liquid, add normal-temperature water 200-600 weight portion, cover the rotary drum door, rotate 30-180min, control dried waste liquid, repetitive operation 2-6 time as stated above again, then, fully washing, control dried waste liquid
(8) epilation: after cleaning, washing, adding temperature is 20-30 ℃ of water 100-400 weight portion, adds sodium hydroxide 0.5-10.0 weight portion, rotates 30-240min, removes pin hair and the fine hair that remains on every block animal skin with tweezers,
(9) cut open layer: residual pin hair on the animal skins and fine hair cut open layer after removing totally fully on accurate splitting machine, cut open layer thickness: it is 0.4-1.0mm that the first layer lether of band grain cuts open layer thickness; Not cuing open layer thickness with the double-layer fur of grain is 0.4-1.2mm, and remainder reclaims as the raw material that extracts collagen,
(10) dealkalize: get the first layer lether of band grain or not with double-layer fur 100 weight portions of grain, add lactic acid 0.2-3.0 weight portion, ammonium chloride 1.0-6.0 weight portion, or ammonium sulfate 0.6-6.0 weight portion rotate 30-180min, and waste liquid is removed in control,
(11) clean: add normal-temperature water 200-600 weight portion, NaCl 5-30 weight portion rotates 30-240min, control dried waste liquid, add normal-temperature water 200-600 weight portion, rotate 30-150min, control dried waste liquid, repetitive operation is 2-6 time as stated above, then, and fully washing, control dried waste liquid, skin is taken out from rotary drum, use centrifuge dewatering, again skin is put into rotary drum, add deionized water 200-60 weight portion, rotate 30-180min, control dried waste liquid, repetitive operation according to said method 2-6 time, then, skin is taken out from rotary drum, use centrifuge dewatering
(12) will through the dehydration animal skins, put into the refrigerating chamber lyophilizing of freezer dryer after, be 6.0-30.0KGy/h through dosage again
60Gamma-ray irradiation sterilization, formed package that Co produced are finished product.
2. according to the described acellular dermal matrix preparation methods of claim 1, it is characterized in that animal skins be fresh virus-free infection, do not have dermatosis and do not have the animal skins of medical history, when adopting pig, cattle, sheep domestic animal skin, then should choose pig, cattle, the sheep domestic animal of not using forage feed.
3. according to the described acellular dermal matrix preparation methods of claim 1, it is characterized in that used rotary drum is suspension type wood rotary drum or rustless steel rotary drum.
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| 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 |
| 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 |
| CN102580153B (en) * | 2012-03-09 | 2013-11-06 | 潘银根 | Method for preparing allograft acellular dermal matrixes |
| CN105268022B (en) * | 2014-08-01 | 2018-04-20 | 山东大学 | A kind of preparation method of xenogenesis cell-less corium ground substance |
| CN104587530B (en) * | 2015-01-15 | 2017-07-07 | 四川大学 | Medical purifying pig dermis and preparation method thereof |
| CN106474547B (en) * | 2015-08-28 | 2019-07-26 | 北京华信佳音医疗科技发展有限责任公司 | A kind of biologic bracket material and preparation method thereof of suitable cell growth |
| CN105664254A (en) * | 2015-12-09 | 2016-06-15 | 北京大学第三医院 | Application and use method of acellular dermal matrix |
| CN105497982A (en) * | 2015-12-09 | 2016-04-20 | 北京大学第三医院 | Purpose and use method of acellular dermal matrix |
| CN107198794B (en) * | 2016-03-18 | 2020-02-14 | 中国科学院上海硅酸盐研究所 | Natural polymer bioactive wound repair material with active ion release function and preparation method thereof |
| CN105963782A (en) * | 2016-04-28 | 2016-09-28 | 陕西瑞盛生物科技有限公司 | Biological membrane and preparation method thereof |
| CN106267312A (en) * | 2016-09-21 | 2017-01-04 | 广东泰宝医疗科技股份有限公司 | A kind of preparation method of xenogenesis high-performance acellular dermal matrix dressing |
| CN107890586B (en) * | 2017-10-27 | 2021-06-01 | 百澳瑞派（天津）生物科技有限公司 | Preparation method of allogeneic biological breast patch |
| CN112474552A (en) * | 2020-10-27 | 2021-03-12 | 广州创尔生物技术股份有限公司 | Cleaning method of beef tendon slices |
| CN115990290A (en) * | 2023-03-23 | 2023-04-21 | 北赛泓升(北京)生物科技有限公司 | Cockscomb oil tissue acellular matrix material and preparation method thereof |
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