CN105727367B - Preparation method of heterogenous acellular dermal matrix - Google Patents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention discloses a preparation method of a heterogenous acellular dermal matrix, which specifically comprises the following steps: skin taking, epidermis layer removing, cell removing, laser drilling and sterilization. The preparation method of the xenogenic acellular dermal matrix provided by the invention has the advantages of clean removal of cell components, low antigenicity, maintenance of a dermal collagen structure, good histocompatibility, short treatment time, low treatment cost and good social and economic benefits, and the whole preparation process only takes about 60 hours.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a preparation method of a heterogenous acellular dermal matrix.
Background
Scar hyperplasia and contracture deformity are often left in skin defects caused by factors such as burns, wounds, epithelial cancer excision and skin diseases, and the appearance and the function are seriously influenced. At present, the repair materials for repairing the wound surface mainly comprise allogenic skin, xenogenic skin and various artificially synthesized wound surface coverings besides autologous skin and skin flaps. In the treatment of patients with extensive burns, the patient's own skin source is limited, and these skin substitutes need to be applied; the source of the xenogenic skin is limited, the xenogenic skin has the risk of disease infection and is restricted by ethics; the artificial synthetic material technology is not mature, the anti-infection capability is poor, and the price is high. Compared with the traditional Chinese medicine preparation, the xenogeneic skin is subjected to acellular denaturation treatment, the antigenicity is reduced, the source is wide, the price is low, most effective tissue components and framework structures in the dermal tissue are preserved, the tissue structure of human skin is simulated to the maximum extent, and the growth of host cells and the reconstruction of skin collagen can be better induced.
At present, the preparation method of the heterogenic acellular dermal matrix mainly comprises the following steps: (1) repeated freeze thawing method: repeatedly freezing and thawing fresh skin for more than 3 times; (2) a trypsin digestion method: digesting cells in the dermis by an exogenous protease; (3) a phosphate buffer solution soaking method: effective removal of cellular components from the skin by activation of endogenous enzymes in the dermis; (4) the DispaseII-Triton method: firstly, treating dermis by using DispaseII, and then treating dermis by using TritonX-100; (5) hypertonic salt-SDS method: firstly, treating dermis by adopting hypertonic salt, and then thoroughly removing cell components in the dermis by adopting a film breaking agent of Sodium Dodecyl Sulfate (SDS); (6) high-permeability salt-NaOH corrosion method: firstly, the epidermis is removed by hypertonic salt, and then the cells are dehydrated and killed by the water absorption effect of NaOH so as to completely remove the cell components in the dermis. Although the preparation of the existing xenogenic acellular dermal matrix is mature, there are some problems in its preparation and application, such as: the cell components are not completely removed, the antigenicity is high, the histocompatibility is poor, and the skin grafting failure is easy to cause; the structure of the basement membrane is damaged greatly, which is not beneficial to the adhesion and growth of epithelial cells; the preparation process is complex, and the treatment time is too long; the preparation cost is high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a heterogenous acellular dermal matrix with short treatment period and small damage to a dermal extracellular matrix structure.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a preparation method of a heterogenous acellular dermal matrix specifically comprises the following steps:
1) skin taking: selecting Bama miniature pig skin as a skin source, depilating and sterilizing a skin taking area, cutting a fault skin sheet with a skin layer and a dermis layer by using a skin taking knife to obtain a required size, soaking the fault skin sheet with the skin layer and the dermis layer in a benzalkonium chloride solution of 1 per thousand for 20-40 min, and degreasing and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃, peeling off the epidermis layer, and washing the residual part with a sterile PBS (phosphate buffer solution) for 3-5 times;
3) and (3) cell removal treatment: putting the dermal matrix obtained in the step 2) into 0.5% Triton X-100 solution, performing ultrasonic treatment at room temperature for 3-7 min, then shaking table vibration for 5-6 h, performing ultrasonic treatment again for 3-7 min, then shaking table vibration for 3-4 h, taking out the dermal matrix, putting the dermal matrix into sterile PBS solution, shaking table vibration for 1-2 h, repeating the steps of 1 time of the ultrasonic vibration-sterile PBS solution vibration of the Triton X-100 solution, taking out the dermal matrix, performing ultrasonic cleaning for 3-7 min by using 4% sodium deoxycholate solution, shaking table vibration for 2-3 h, then shaking table vibration for 2-3 h in the sterile PBS solution, crushing cell components in dermis, and eluting to obtain a decellularized dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, and washing for 3-5 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and performing Co-60 gamma ray irradiation sterilization to obtain a finished product.
Preferably, the sterile PBS solution is prepared from 8.00g of NaCl + 0.20g of KCl + KH2PO4 0.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
Preferably, the thickness of the fault skin slice cut in the step 1) is 0.3-0.5 mm.
Preferably, the soaking time in the step 2) is 22-24 hours.
Preferably, the aperture in the step 4) is 0.1-1.3 mm, and the hole spacing is 0.5-5 mm.
Preferably, the irradiation time in the step 5) is 10-12 h.
The invention has the beneficial effects that: the preparation method of the xenogenic acellular dermal matrix provided by the invention has the advantages of clean removal of cell components, low antigenicity, maintenance of a dermal collagen structure, no obvious irritation to tissues, good histocompatibility, and a microporous structure, is favorable for draining wound exudate, promoting angiogenesis, improving the survival rate of skin sheets, reducing scar formation, has good technical effect, has the whole preparation process only for about 60 hours, short treatment time, low treatment cost, and good social benefit and economic benefit.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a preparation method of a heterogenous acellular dermal matrix specifically comprises the following steps:
1) skin taking: selecting Bama miniature pig skin as skin source, depilating and sterilizing the skin area, cutting 0.3mm of fault skin with epidermis layer and dermis layer with skin-taking knife, cutting into required size, soaking in 1 ‰ benzalkonium chloride solution for 30min, defatting and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃ for 23 hours, peeling off the epidermal layer, and washing the residual part with sterile PBS solution for 4 times;
3) and (3) cell removal treatment: ultrasonically cleaning the dermal matrix obtained in the step 2) by using 0.5% Triton X-100 solution at room temperature for 5min, shaking table vibration for 6h, ultrasonically cleaning again for 5min, shaking table vibration for 4h, finally transferring to sterile PBS solution, shaking table vibration for 2h, repeating the steps of ultrasonically cleaning the Triton X-100 solution, shaking, cleaning, shaking and vibrating the sterile PBS solution for 1 time, ultrasonically cleaning the dermal matrix by using 4% sodium deoxycholate solution for 5min, shaking table vibration for 3h, then shaking table vibration in the sterile PBS solution for 3h, crushing cell components in the dermis, and eluting to obtain a decellularized dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, wherein the aperture is 0.1mm, the hole interval is 1mm, and washing the acellular dermal matrix for 4 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and performing Co-60 gamma ray irradiation for 11h for disinfection and sterilization to obtain a finished product.
Wherein the sterile PBS solution used in the preparation method is prepared from 8.00g of NaCl, 0.20g of KCl and KH2PO40.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
Example 2:
a preparation method of a heterogenous acellular dermal matrix specifically comprises the following steps:
1) skin taking: selecting Bama miniature pig skin as skin source, depilating and sterilizing the skin area, cutting 0.3mm slice with epidermis layer and dermis layer with skin-taking knife, cutting into required size, soaking in 1 ‰ benzalkonium chloride solution for 20min, defatting and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃ for 22 hours, peeling off the epidermis layer, and washing the residual part with sterile PBS solution for 5 times;
3) and (3) cell removal treatment: ultrasonically cleaning the dermal matrix obtained in the step 2) by using 0.5% Triton X-100 solution at room temperature for 7min, shaking table vibration for 6h, ultrasonically cleaning again for 7min, shaking table vibration for 4h, finally transferring to sterile PBS solution, shaking table vibration for 2h, repeating the steps of ultrasonically cleaning the Triton X-100 solution, shaking, cleaning, shaking and vibrating the sterile PBS solution for 1 time, ultrasonically cleaning the dermal matrix by using 4% sodium deoxycholate solution for 7min, shaking table vibration for 3h, then shaking table vibration in the sterile PBS solution for 3h, crushing cell components in the dermis, and eluting to obtain a decellularized dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, wherein the aperture is 0.3mm, the hole interval is 2mm, and washing the acellular dermal matrix for 3 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and performing Co-60 gamma ray irradiation for 10 hours for sterilization to obtain a finished product.
Wherein the sterile PBS solution used in the preparation method is prepared from 8.00g of NaCl, 0.20g of KCl and KH2PO40.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
Example 3:
a preparation method of a heterogenous acellular dermal matrix specifically comprises the following steps:
1) skin taking: selecting Bama miniature pig skin as skin source, depilating and sterilizing the skin area, cutting 0.5mm slice with epidermis layer and dermis layer with skin-taking knife, cutting into required size, soaking in 1 ‰ benzalkonium chloride solution for 40min, defatting and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃ for 24 hours, peeling off the epidermal layer, and washing the residual part with sterile PBS solution for 3 times;
3) and (3) cell removal treatment: ultrasonically cleaning the dermal matrix obtained in the step 2) by using 0.5% Triton X-100 solution at room temperature for 3min, shaking table vibration for 5h, ultrasonically cleaning again for 3min, shaking table vibration for 3h, finally transferring to sterile PBS solution, shaking table vibration for 1h, repeating the steps of ultrasonically cleaning the Triton X-100 solution, shaking, cleaning, shaking and vibrating the sterile PBS solution for 1 time, ultrasonically cleaning the dermal matrix by using 4% sodium deoxycholate solution for 3min, shaking table vibration for 2h, then shaking table vibration for 2h in the sterile PBS solution, crushing cell components in the dermis, and eluting to obtain a decellularized dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, wherein the aperture is 1.3mm, the hole interval is 5mm, and washing the acellular dermal matrix for 5 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and sterilizing by Co-60 gamma ray irradiation for 12h to obtain a finished product.
Wherein the sterile PBS solution used in the preparation method is prepared from 8.00g of NaCl, 0.20g of KCl and KH2PO40.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
Example 4:
a preparation method of a heterogenous acellular dermal matrix specifically comprises the following steps:
1) skin taking: selecting Bama miniature pig skin as skin source, depilating and sterilizing the skin area, cutting 0.4mm of fault skin with epidermis layer and dermis layer with skin-taking knife, cutting into required size, soaking in 2 ‰ benzalkonium chloride solution for 30min, defatting and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃ for 23 hours, peeling off the epidermal layer, and washing the residual part with sterile PBS solution for 3 times;
3) and (3) cell removal treatment: ultrasonically cleaning the dermal matrix obtained in the step 2) by using 0.5% Triton X-100 solution at room temperature for 4min, shaking the shaking table for 5.5h, ultrasonically cleaning again for 4min, shaking the shaking table for 3.5h, finally transferring the dermal matrix into sterile PBS solution, shaking the shaking table for 1.5h, repeating the steps of ultrasonically cleaning the Triton X-100 solution, shaking the cleaning, shaking the sterile PBS solution for 1 time, ultrasonically cleaning the dermal matrix by using 4% sodium deoxycholate solution for 4min, shaking the shaking table for 2.5h, then oscillating the dermal matrix in the sterile PBS solution for 2.5h, crushing cell components in the dermis, and eluting to obtain an acellular dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, wherein the aperture is 0.7mm, the hole spacing is 2.5mm, and washing the acellular dermal matrix for 4 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and performing Co-60 gamma ray irradiation for 11h for disinfection and sterilization to obtain a finished product.
Wherein the sterile PBS solution used in the preparation method is prepared from 8.00g of NaCl, 0.20g of KCl and KH2PO40.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (3)
1. A preparation method of a heterogenous acellular dermal matrix is characterized by comprising the following steps:
1) skin taking: selecting Bama miniature pig skin as a skin source, depilating and sterilizing a skin taking area, cutting a fault skin sheet with a skin layer and a dermis layer by using a skin taking knife to obtain a required size, soaking the fault skin sheet with the skin layer and the dermis layer in a benzalkonium chloride solution of 1 per thousand for 20-40 min, and degreasing and sterilizing;
2) removing the epidermis layer: soaking the faulted skin slice obtained in the step 1) with 0.25% trypsin solution at the constant temperature of 4 ℃ for 22-24 hours, peeling off the epidermis layer, and washing the residual part with a sterile PBS (phosphate buffer solution) for 3-5 times;
3) and (3) cell removal treatment: putting the dermal matrix obtained in the step 2) into 0.5% TritonX-100 solution, performing ultrasonic treatment at room temperature for 3-7 min, then performing shaking table vibration for 5-6 h, performing ultrasonic treatment again for 3-7 min, then performing shaking table vibration for 3-4 h, taking out the dermal matrix, putting the dermal matrix into sterile PBS solution, performing shaking table vibration for 1-2 h, repeating the steps of the ultrasonic vibration-sterile PBS solution vibration of the TritonX-100 solution for 1 time, taking out the dermal matrix, performing ultrasonic cleaning for 3-7 min by using 4% deoxycholate solution, performing shaking table vibration for 2-3 h in the sterile PBS solution, crushing cell components in dermis, and eluting to obtain an acellular dermal matrix;
4) laser drilling: intermittently perforating the acellular dermal matrix obtained in the step 3) by using laser, and washing for 3-5 times by using a sterile PBS (phosphate buffer solution);
5) and (3) sterilization: packaging the washed acellular dermal matrix obtained in the step 4), and performing Co-60 gamma ray irradiation sterilization to obtain a finished product, wherein the irradiation time is 10-12 hours;
the sterile PBS solution is prepared from 8.00g of NaCl, 0.20g of KCl and KH2PO4 0.20g+Na2HPO41.56g was dissolved in a 1000mL volumetric flask and the volume was fixed.
2. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the thickness of the section skin sheet cut in the step 1) is 0.3 to 0.5 mm.
3. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the pore size in the step 4) is 0.1 to 1.3mm, and the pore pitch is 0.5 to 5 mm.
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| 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 |
| CN106310352A (en) * | 2016-11-01 | 2017-01-11 | 广东泰宝医疗科技股份有限公司 | Preparation method of antibacterial acellular dermal matrix dressing material |
| CN107412867B (en) * | 2017-05-10 | 2020-09-01 | 广州润虹医药科技股份有限公司 | Preparation method of heterogenous acellular dermal matrix |
| CN107412868A (en) * | 2017-08-10 | 2017-12-01 | 广州润虹医药科技股份有限公司 | A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix |
| CN109675112B (en) * | 2019-02-22 | 2021-08-17 | 上海仁康科技有限公司 | Preparation method of human-derived acellular dermal matrix |
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| CN112957534A (en) * | 2021-04-06 | 2021-06-15 | 海南大学 | Preparation method of acellular dermal matrix material |
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| 梁黎明等.激光打孔制备微孔猪脱细胞真皮基质.《中国激光医学杂志》.2007,第16卷(第2期),第88-91页. * |
| 激光打孔制备微孔猪脱细胞真皮基质;梁黎明等;《中国激光医学杂志》;20070430;第16卷(第2期);第89页 * |
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