CN102218162A - Preparation method of homologous acellular dermal matrix - Google Patents
Preparation method of homologous acellular dermal matrix Download PDFInfo
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- CN102218162A CN102218162A CN2011101345119A CN201110134511A CN102218162A CN 102218162 A CN102218162 A CN 102218162A CN 2011101345119 A CN2011101345119 A CN 2011101345119A CN 201110134511 A CN201110134511 A CN 201110134511A CN 102218162 A CN102218162 A CN 102218162A
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Abstract
The invention belongs to the technical field of medical biomaterials, and particularly relates to a preparation method of a homologous acellular dermal matrix. The preparation method mainly solves the defects that the existing preparation method of the acellular dermal matrix has inconvenient preparation processes, long time consumption, non-thorough removal of cell chippings, an amount of remaining quantity of chemical substances, and the like. In the invention, the preparation method for the homologous acellular dermal matrix comprises the following steps of obtaining skins; removing cuticular layers: placing and oscillating a split thickness skin graft in hyperosmotic salt solution; and removing the cuticular layers to obtain dermal matrixs; removing cell components: placing the dermal matrixes in sodium dodecyl sulfate aseptic saline solution to carry out ultrasonic treatment so as to break the cells, thus obtaining the acellular dermal matrixes; cleaning; decorating spatial structure: placing the cleaned acellular dermal matrixes in trypsinase aseptic saline solution to carry out ultrasonic cleaning; cleaning for a second time; and sterilizing. The preparation method has the advantages of having a simple method, short time consumption, thoroughly removing the cell components, improving vascularization speed after implantation, and the like.
Description
Technical field
The invention belongs to medical biotechnology material technology field, be specifically related to a kind of acellular dermal matrix preparation method of the same race.
Background technology
Acellular dermal matrix is with animal skins or people's corpse skin, through a series of physics, chemical method, remove the cell component in epidermis and the corium, keep the dermal matrix composition of utmost point reduced immunogenicity and get, it is the earliest as a kind of dermal substitute is used to burn, wound causes the damaged alternative reparation of corium, but discover this product thereafter except that can be used for burning, wound and wound surface, also can be used for the repairing and the soft tissue filling of mucosa, as reconstruction of oral defect repairing, gingival atrophy reparation, nasal septum repairing, perforation of ear drum repairing and aesthetic medicine field etc.
Notification number is CN201350162Y, name is called a kind of patent of taking off cell allosome dermal matrix tissue patch, disclosing a kind of is basement membrane to take off cell allosome dermal matrix, it is immersed drug solns in the astragaloside, make at least one side of basement membrane that the astragaloside Chinese traditional medicine layer be arranged, but this patent is not done detailed description to the preparation method of taking off cell allosome corium basement membrane; Notification number is CN100372511C, name is called a kind of acellular dermal matrix, this patent adopts mammal skin, use the sodium hydroxide solution repeated treatments, remove cytogenetics matter DNA in the tissue, the reuse detergent is removed the lipid material in the cell membrane, though this method can make acellular dermal matrix, but its preparation process is loaded down with trivial details, can not effectively remove the cell debris composition; Notification number is CN1268401C, name is called a kind of acellular dermal matrix preparation methods, it is raw material that this method adopts the healthy animal skin, number of chemical mass treatment such as applying composite enzyme collagen inorganic agent and alkali make acellular dermal matrix, but this method easily causes the number of chemical material residual, influence is implanted back fiber vascular tissue and is grown into, and preparation manipulation process complexity, and required time is long; Publication number is CN1176725C, name is called micropore allosome (kind) acellular dermis substitute, disclose the method that adopts traditional enzymic digestion, detergent to soak people's cadaver skin and prepared cell-less corium ground substance, it is not thorough that but this method intracellular matter is removed, and causes that easily immunogenic substance is residual.Current scholar thinks a little less than the acellular dermal matrix immunogenicity of the same race, can retain for a long time in host and tissue reconstruction takes place, and xenogenesis acellular dermal matrix antigenicity obviously is better than acellular dermal matrix of the same race.
Existing loaded down with trivial details, the consuming time length of acellular dermal matrix patent preparation process, cell debris is removed not thorough.The xenogenesis acellular dermal matrix has stronger immunogenicity.
Summary of the invention
The present invention is primarily aimed at that present acellular dermal matrix preparation method exists that loaded down with trivial details, the consuming time length of preparation process, cell debris are removed thoroughly, the residual deficiencies such as many of chemical substance, simple, the consuming time weak point of a kind of method, cell component are removed thoroughly, chemical substance is less residual and increase the collagen fiber gap and provide, and improve the acellular dermal matrix preparation method of the same race of implanting back vascularization speed.
A kind of acellular dermal matrix preparation method of the same race of the present invention may further comprise the steps:
1) bark fetching: selecting people's cadaver skin for use is the skin source, gets the split-thickness skin graft that has epidermal area and skin corium;
2) take off epidermal area: split-thickness skin graft is put into hypertonic saline solution, in 35～40 ℃ of constant temperature shaking tables, shook 16～24 hours, take out skin graft, throw off epidermal area, get dermal matrix;
3) remove cell component: dermal matrix is put into sodium lauryl sulphate physiological saline solution solution, and at room temperature supersound process is 0.5～1 hour, and smudge cells, and the cell component in the removal corium get acellular dermal matrix;
4) clean:, clean 2～5 times with physiological saline solution with acellular dermal matrix;
5) modify space structure: the acellular dermal matrix after will cleaning, put into trypsin physiological saline solution solution, ultrasonic cleaning 20～30 minutes;
6) secondary cleaning:, clean with sterile phosphate buffer solution with the acellular dermal matrix of step (5) gained;
7) sterilization: the acellular dermal matrix after the secondary cleaning is drawn in the net, encapsulates, and the sterilization of cobalt-60 gamma-ray irradiation makes acellular dermal matrix of the same race.
Wherein said hypertonic saline solution is that concentration is the sodium chloride solution of 0.5～2mol/L.
The concentration of described sodium dodecyl sulfate solution is 2.5～7.5g/L.
The concentration of described trypsin solution is 1.0～2.5g/L.
Described cobalt-60 gamma-ray irradiation sterilizing dose is 15～20kGy.
For further showing the effect of the acellular dermal matrix of the same race that the inventive method prepares, the present invention is to (people) of the same race acellular dermal matrix and draw in the net that acellular dermal matrix behind the irradiation has carried out the picture contrast and to light microscopic (100 times) observation of application on human skin histological structure light microscopic (100 times) and (people) of the same race acellular dermal matrix histological structure, the result shows, the prepared acellular dermal matrix of this method has been removed cell component in epidermis and the corium fully, keeps complete collagen scaffold structure; The present invention has also carried out (the PAS dyeing of basement membrane om observation to application on human skin and (people) of the same race acellular dermal matrix, 200 times), the result shows in the acellular dermal matrix that the present invention prepares and contains abundant neutral mucopolysaccharide that PAS dyeing is positive, and does not destroy its basement membrane structure; The present invention simultaneously also carries out elastic fiber light microscopic (Verhoeff ' s dyeing, 200 times) to application on human skin and (people) of the same race acellular dermal matrix and observes, and the result shows that acellular dermal matrix inner elastomeric fibre structure is intact.
The present invention compared with prior art has following beneficial effect:
1) the present invention adopts the method for certain ultrasound condition smudge cells and ultrasonic cleaning in the acellular dermal matrix preparation process, the acellular dermal matrix cell component of preparation is removed thoroughly, and kept complete collagen scaffold structure, reduces its immunogenicity;
2) the present invention modifies its space structure with trypsin solution and certain ultrasound condition combination, increases acellular dermal matrix collagen fiber gap, improves it and implants back vascularization speed;
3) contain abundant neutral mucopolysaccharide in the acellular dermal matrix of the inventive method preparation, and its inner elastomeric fibre structure is intact, PAS dyeing is positive, and does not destroy its basement membrane structure,
4) simple, the consuming time weak point of preparation method of the present invention.
Description of drawings
Fig. 1 is (people) of the same race acellular dermal matrix figure;
Fig. 2 be draw in the net, (people) of the same race acellular dermal matrix figure of irradiation;
Fig. 3 normal skin tissue that behaves learns structure optics fibrescope and observes (HE * 100) figure;
Fig. 4 is (people) of the same race acellular dermal matrix histological structure observation by light microscope (HE * 100) figure;
Fig. 5 people's normal skin basement membrane optical fiber sem observation (PAS * 200) figure;
Fig. 6 is (people) of the same race acellular dermal matrix basement membrane optical fiber sem observation (PAS * 200) figure;
Fig. 7 people's normal skin elastic fiber optical fiber sem observation (Verhoeff ' s * 200) figure;
Fig. 8 is (people) of the same race acellular dermal matrix elastic fiber optical fiber sem observation (Verhoeff ' s * 200) figure.
The specific embodiment
Embodiment 1
A kind of (people) of the same race acellular dermal matrix preparation method may further comprise the steps:
1) bark fetching: get healthy people's fresh cadaver skin of donation, get the split-thickness skin graft that thickness is 0.6mm with drum dermatome;
2) take off epidermal area: split-thickness skin graft is cut to 5cm * 5cm～10cm * 10cm size, puts into the sodium chloride solution that concentration is 0.5mol/L, concussion is 16 hours in 35 ℃ of constant temperature shaking tables, takes out skin graft, throws off epidermal area, gets dermal matrix;
3) remove cell component: dermal matrix is put into the sodium lauryl sulphate physiological saline solution solution that concentration is 2.5g/L, at room temperature using output is 250～500W, frequency is the Ultrasound Instrument supersound process 0.5 hour of 20～26.5KHz, smudge cells, and the cell component in the removal corium, get acellular dermal matrix;
4) clean:, clean 2 times with physiological saline solution with acellular dermal matrix;
5) modify space structure: the acellular dermal matrix after will cleaning, putting into concentration is the trypsin physiological saline solution solution of 1.0g/L, the use output is 250～500W, frequency is the Ultrasound Instrument ultrasonic cleaning 20 minutes of 20～26.5KHz, further remove cell debris, increase acellular dermal matrix collagen fiber gap, modify its space structure;
6) secondary cleaning:, clean with sterile phosphate buffer solution with the acellular dermal matrix of step (5) gained;
7) sterilization: the acellular dermal matrix after the secondary cleaning is drawn in the net, encapsulates, at the 15kGy irradiation sterilization, make acellular dermal matrix product of the same race with cobalt-60 gamma-rays.
Fig. 1 is (people) of the same race acellular dermal matrix figure of present embodiment preparation; Fig. 2 draws in the net acellular dermal matrix figure behind the irradiation; Fig. 3 skin histology's structure light microscopic (100 times) of behaving is observed figure, Fig. 4 is that the light microscopic (100 times) of (people) of the same race acellular dermal matrix histological structure is observed figure, the result shows, the prepared acellular dermal matrix of this method has been removed cell component in epidermis and the corium fully, keeps complete collagen scaffold structure; (the PAS dyeing of Fig. 5 application on human skin basement membrane om observation, 200 times) figure, Fig. 6 is (the PAS dyeing of (people) of the same race acellular dermal matrix basement membrane light microscopic, 200 times) observe and scheme, the result shows that the present embodiment skin basement membrane is because of containing abundant neutral mucopolysaccharide, PAS dyeing is positive, and the cell-less corium ground substance preparation process is not destroyed its basement membrane structure; Fig. 7 behave the elastic fibers of skin light microscopic (Verhoeff ' s dyeing, 200 times) observe and scheme, Fig. 8 is that (people) of the same race acellular dermal matrix elastic fiber light microscopic (Verhoeff ' s dyeing, 200 times) is observed figure, and the result shows that acellular dermal matrix inner elastomeric fibre structure is intact.
Embodiment 2
A kind of (people) of the same race acellular dermal matrix preparation method may further comprise the steps:
1) bark fetching: get healthy people's fresh cadaver skin of donation, get the split-thickness skin graft that thickness is 0.4mm with motor dermatome;
2) take off epidermal area: split-thickness skin graft is cut to 5cm * 5cm～10cm * 10cm size, puts into the sodium chloride solution that concentration is 1mol/L, concussion is 20 hours in 38 ℃ of constant temperature shaking tables, takes out skin graft, throws off epidermal area, gets dermal matrix;
3) remove cell component: dermal matrix is put into the sodium lauryl sulphate physiological saline solution solution that concentration is 5.0g/L, at room temperature using output is 250～500W, frequency is the Ultrasound Instrument supersound process 0.8 hour of 20～26.5KHz, smudge cells, and the cell component in the removal corium, get acellular dermal matrix;
4) clean:, clean 3 times with physiological saline solution with acellular dermal matrix;
5) modify space structure: the acellular dermal matrix after will cleaning, putting into concentration is the trypsin physiological saline solution solution of 1.5g/L, the use output is 250～500W, frequency is the Ultrasound Instrument ultrasonic cleaning 25 minutes of 20～26.5KHz, further remove cell debris, increase acellular dermal matrix collagen fiber gap, modify its space structure;
6) secondary cleaning:, clean with sterile phosphate buffer solution with the acellular dermal matrix of step (5) gained;
7) sterilization: the acellular dermal matrix after the secondary cleaning is drawn in the net, encapsulates, at the 15kGy irradiation sterilization, make acellular dermal matrix product of the same race with cobalt-60 gamma-rays.
Embodiment 3
A kind of (people) of the same race acellular dermal matrix preparation method may further comprise the steps:
1) bark fetching: get healthy people's fresh cadaver skin of donation, get the split-thickness skin graft that thickness is 0.8mm with drum dermatome;
2) take off epidermal area: split-thickness skin graft is cut to 5cm * 5cm～10cm * 10cm size, puts into the sodium chloride solution that concentration is 0.5mol/L, concussion is 24 hours in 40 ℃ of constant temperature shaking tables, takes out skin graft, throws off epidermal area, gets dermal matrix;
3) remove cell component: dermal matrix is put into the sodium lauryl sulphate physiological saline solution solution that concentration is 7.5g/L, at room temperature using output is 250～500W, frequency is the Ultrasound Instrument supersound process 0.5 hour of 20～26.5KHz, smudge cells, and the cell component in the removal corium, get acellular dermal matrix;
4) clean:, clean 5 times with physiological saline solution with acellular dermal matrix;
5) modify space structure: the acellular dermal matrix after will cleaning, putting into concentration is the trypsin physiological saline solution solution of 2.5g/L, the use output is 250～500W, frequency is the Ultrasound Instrument ultrasonic cleaning 30 minutes of 20～26.5KHz, further remove cell debris, increase acellular dermal matrix collagen fiber gap, modify its space structure;
6) secondary cleaning:, clean with sterile phosphate buffer solution with the acellular dermal matrix of step (5) gained;
7) sterilization: the acellular dermal matrix after the secondary cleaning is drawn in the net, encapsulates, at the 20kGy irradiation sterilization, make acellular dermal matrix product of the same race with cobalt-60 gamma-rays.
Claims (5)
1. acellular dermal matrix preparation method of the same race is characterized in that may further comprise the steps:
1) bark fetching: selecting people's cadaver skin for use is the skin source, gets the split-thickness skin graft that has epidermal area and skin corium;
2) take off epidermal area: split-thickness skin graft is put into hypertonic saline solution, in 35～40 ℃ of constant temperature shaking tables, shook 16～24 hours, take out skin graft, throw off epidermal area, get dermal matrix;
3) remove cell component: dermal matrix is put into sodium lauryl sulphate physiological saline solution solution, and at room temperature supersound process is 0.5～1 hour, and smudge cells, and the cell component in the removal corium get acellular dermal matrix;
4) clean:, clean 2～5 times with physiological saline solution with acellular dermal matrix;
5) modify space structure: the acellular dermal matrix after will cleaning, put into trypsin physiological saline solution solution, ultrasonic cleaning 20～30 minutes;
6) secondary cleaning:, clean with sterile phosphate buffer solution with the acellular dermal matrix of step (5) gained;
7) sterilization: the acellular dermal matrix after the secondary cleaning is drawn in the net, encapsulates, and the sterilization of cobalt-60 gamma-ray irradiation makes acellular dermal matrix of the same race.
2. a kind of acellular dermal matrix preparation method of the same race according to claim 1 is characterized in that described hypertonic saline solution is that concentration is the sodium chloride solution of 0.5～2mol/L.
3. a kind of acellular dermal matrix preparation method of the same race according to claim 1, the concentration that it is characterized in that described sodium dodecyl sulfate solution is 2.5～7.5g/L.
4. a kind of acellular dermal matrix preparation method of the same race according to claim 1, the concentration that it is characterized in that described trypsin solution is 1.0～2.5g/L.
5. a kind of acellular dermal matrix preparation method of the same race according to claim 1 is characterized in that described cobalt-60 gamma-ray irradiation sterilizing dose is 15～20kGy.
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| CN103436489A (en) * | 2013-08-30 | 2013-12-11 | 重庆大清医诚生物技术有限公司 | Reagent for acellular processing of animal skin tissue, and processing method thereof |
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| CN111671974A (en) * | 2020-04-26 | 2020-09-18 | 上海亚朋生物技术有限公司 | Acellular dermal matrix tissue filler with water absorption induced shape memory and preparation method thereof |
| CN112190764A (en) * | 2020-09-17 | 2021-01-08 | 上海亚朋生物技术有限公司 | Allogeneic skin acellular method |
| CN112957532A (en) * | 2021-02-26 | 2021-06-15 | 山西奥瑞生物材料有限公司 | Preparation method of biological membrane for dental implantation |
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Application publication date: 20111019 |