CN109675112B - Preparation method of human-derived acellular dermal matrix - Google Patents
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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
- 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
- A61L27/362—Skin, e.g. dermal papillae
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- 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
- 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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- 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
- 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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Abstract
The invention discloses a preparation method of a human acellular dermal matrix, which comprises the following specific steps: taking stored human skin; removing subcutaneous fat components; cleaning and disinfecting with iodophor and alcohol, and repeatedly soaking in hypotonic solution to clean skin; treating with Dispase II solution overnight; removing epidermis, and cleaning with normal saline to obtain dermis; treating with hypertonic salt solution; washing with normal saline; treating the obtained product in a dialysis device by using an acellular liquid to obtain an acellular dermal matrix; washing with physiological saline to obtain human acellular dermal matrix. The preparation method is simple and convenient, the production process is simple and reliable, the production efficiency is improved, the production cost is saved, the environmental pollution can be reduced to the greatest extent, the obtained acellular dermal matrix keeps a skin basement membrane and is more beneficial to cell adhesion proliferation, and meanwhile, the acellular dermis contains rich growth factors, has good biocompatibility and is more beneficial to supporting the growth of cells.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a preparation method of a human-derived acellular dermal matrix.
Background
The dermal substitute has an important function in the wound healing process, can provide a dermal scaffold at the bottom of the wound, improves the survival rate of cultured epidermal membranes, and reduces wound contraction and scar formation. Skin grafting is the best treatment for patients with extensive burns or wounds. The Acellular Dermal Matrix (ADM) has an internal structure similar to that of the dermis of the skin, and is applied to both experiments and clinics at home and abroad as a dermal substitute.
At present, acellular dermal matrixes are mainly divided into two types: allogenic acellular dermal matrices and xenogenic acellular dermal matrices. Compared with a heterogenous acellular dermal matrix, practice and clinical use prove that the heterogenous acellular dermal prepared from the human cadaver skin is most ideal as a dermal substitute, has mechanical properties close to those of normal skin, has better histocompatibility and smaller immunological rejection, and is more suitable for repairing skin defects.
The aim of acellular dermal matrix preparation is to remove as much as possible the cellular components in the skin tissue (including epidermal cells, hair follicles, sebaceous glands, sweat glands, vascular endothelial cells in the dermis, fibroblasts, etc.), and to retain as much as possible the extracellular matrix components and the intact three-dimensional structure of the dermal tissue. Thus, the biological scaffold for guiding tissue regeneration can be provided, and the purpose of repairing tissue defect to the maximum extent is achieved. Although a plurality of methods for preparing the acellular dermal matrix exist at present, the acellular dermal matrix has certain problems: the existing preparation methods of the acellular dermal matrix are very complicated, require enzyme digestion, alkali liquor treatment and the like, have complicated steps and complex process, and greatly increase the production cost; most of the existing methods for preparing the acellular dermal matrix use chemical fixing agents such as glutaraldehyde and the like, which greatly changes the natural structure of the acellular dermal matrix and hinders the degradation of the acellular dermal matrix in organisms; meanwhile, the residual glutaraldehyde causes larger cell and genetic toxicity of a decellularized product, and the clinical use is greatly influenced; at present, a plurality of methods for preparing the acellular dermal matrix introduce the treatment of alkali solution and organic solvent, which not only influences the structure of the acellular dermal matrix, but also increases the possibility of environmental pollution, and increases the cost by treating waste liquid; for example, the method of repeated freeze thawing belongs to a physical method for cell removal treatment, has long time and long period, and is not used basically at present.
Therefore, it is an urgent problem to provide a method for preparing an acellular dermal matrix of human origin.
Disclosure of Invention
In view of the above, the invention provides a preparation method of a human-derived acellular dermal matrix, which ensures that the obtained acellular dermal matrix has low cytotoxicity, low genetic toxicity and high biocompatibility, and is mainly used for repairing skin and mucosa defects.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a human acellular dermal matrix comprises the following specific steps:
(1) taking stored human skin;
(2) removing subcutaneous fat components;
(3) removing the subcutaneous skin with fat components in the step (2), cleaning and disinfecting the skin with iodophor and alcohol, and repeatedly soaking and cleaning the skin with hypotonic solution;
(4) treating the skin cleaned in the step (3) with 2.5mg/ml Dispase II solution overnight;
(5) discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
(6) applying Ca-containing composition to the dermis obtained in step (5)2+Treating with hypertonic salt solution for 12-24 h;
(7) after the hypertonic saline solution is discarded, cleaning the solution by using normal saline;
(8) placing the dermis cleaned in the step (7) in prepared cell removal liquid, and treating for 2-6 h in a dialysis device to obtain a cell removal dermis matrix;
(9) and (3) washing the acellular dermal matrix obtained in the step (8) with physiological saline for 15min each time to obtain the human-derived acellular dermal matrix.
Further, step (3), the skin with subcutaneous fat components removed in step (2) is cleaned and disinfected by 2% iodophor, deiodinated by 75% alcohol, and repeatedly soaked in sterile distilled water to clean the skin.
Further, step (6), the dermis obtained in step (5) is treated with a Ca content of 0.5-3mol/L2+Is treated by hypertonic salt solution for 12 to 24 hours, Ca2+The final concentration of (b) is 1-10 mM/L.
Further, the hypertonic salt solution contains 0.5-3mol/L of Ca2+Sodium chloride solution of (2), Ca2+The final concentration of (b) is 1-10 mM/L.
Further, the cell removal solution in the step (8) comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.5mol/L，Ca2+1-10mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20mmol/L。
Further, the cell removal solution in the step (8) comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.6mol/L，Ca2+1-10mmol/L；Ca2+From CaCl2。
According to the technical scheme, compared with the prior art, the invention discloses and provides the preparation method of the human-derived acellular dermal matrix, the acellular dermal matrix is derived from human skin, has an internal structure similar to skin dermis, and is applied to both domestic and foreign experiments and clinics as a dermal substitute; the immune rejection reaction of human skin after the decellularization treatment is extremely small, the mechanical property is close to that of normal skin, the satisfactory repairing effect can be obtained, and the method has higher practical application value in the medical field;
the invention utilizes novel cell-removing liquid containing Ca2+The components such as detergent, NaCl and the like, and the dermis structure is kept unchanged to the maximum extent while cells are removed; ca2+As an essential ion for cell culture, the compound can not only stabilize the structure of the acellular dermal matrix under a proper concentration, but also enhance the mechanical, physical and chemical properties, biocompatibility, hemostatic performance and the like of the material in the acellular treatment process; meanwhile, a dialysis device is innovatively introduced, harmful cell removal reagents are removed while cell removal is carried out, and low cytotoxicity, low genetic toxicity and high biocompatibility of the product are guaranteed;
the reagent adopted by the invention is cheap and easy to obtain, is non-toxic and harmless, can kill pathogens such as bacteria and viruses and simultaneously remove cells to the maximum extent, retains the components of a cell matrix, and the immune rejection reaction of the obtained product is extremely small;
the preparation method is simple and convenient, the production process is simple and reliable, the production efficiency is improved, the production cost is saved, the method provided by the invention can reduce the environmental pollution to the maximum extent, the obtained acellular dermal matrix reserves a skin basement membrane and is more beneficial to cell adhesion proliferation, and the acellular dermis contains rich growth factors, has good biocompatibility and is more beneficial to supporting the growth of cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic view of a dialysis device according to the present invention;
FIG. 2 is a photograph of an acellular dermal matrix obtained by the present invention;
FIG. 3 is an electron micrograph of the internal structure of the acellular dermal matrix of the present invention;
FIG. 4 is a graph showing the results of HE staining of normal skin tissue according to the present invention;
FIG. 5 is a graph showing the results of HE staining of the acellular dermal matrix according to the present invention;
FIG. 6 is a graph showing the staining of the acellular dermal matrix HE according to the present invention without the use of an acellular liquid and without the use of a dialysis device;
FIG. 7 is a graph showing the results of the cell proliferation assay of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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 human acellular dermal matrix comprises the following specific steps:
(1) taking stored human skin;
(2) removing subcutaneous fat components;
(3) removing the subcutaneous skin with fat components in the step (2), cleaning and disinfecting with 2% iodophor, deiodinating with 75% alcohol, and repeatedly soaking and cleaning the skin with sterile distilled water;
(4) treating the skin cleaned in the step (3) with 2.5mg/ml Dispase II solution overnight;
(5) discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
(6) mixing the corium obtained in step (5) with 2mol/L Ca2+Is treated with sodium chloride solution for 18h, Ca2+The final concentration of (3) is 5 mM/L;
(7) discarding 2mol/L sodium chloride solution, and then washing with normal saline;
(8) placing the dermis cleaned in the step (7) into a prepared cell removal solution, wherein the cell removal solution comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.5mol/L，Ca2+1-10mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20 mmol/L; treating in a dialysis device for 4h to obtain acellular dermal matrix; the dialysis device is schematically shown in FIG. 1;
(9) washing the acellular dermal matrix obtained in step 8 with physiological saline for 3 times, each time for 15min, to obtain human-derived acellular dermal matrix, as shown in fig. 2.
A dialysis device: the allogeneic skin is placed in a dialysis bag, the acellular fluid enters from one end and flows out from the other end of the device, and harmful ions in the acellular fluid are dialyzed and removed while the acellular fluid is acellular, so that the cytotoxicity and the genetic toxicity of the acellular dermal matrix are reduced to the maximum extent while the acellular effect is ensured, and the biocompatibility is effectively improved.
Example 2 internal Structure of acellular dermal matrix
(1) Cutting the obtained acellular dermal matrix into a size of 1cm multiplied by 1cm, and sticking the acellular dermal matrix on a metal tray; hardening and embrittling the sectional acellular dermal matrix by adopting a liquid nitrogen quenching method, and then breaking; the section of the adhesive is upward and is adhered on a metal tray.
(2) After spraying gold in the sample preparation room, the sample is loaded on the machine, observed by a scanning electron microscope and photographed, and the result is shown in fig. 3.
As a result, no cell residue was observed in the tissue, and the collagen structure was intact, similar to that of normal skin.
Example 3 acellular dermal matrix HE staining
(1) Respectively placing normal skin tissues and acellular dermal matrixes in 4% paraformaldehyde fixing solution for fixing for 24 hours;
(2) dehydrating with alcohol, and making xylene transparent;
(3) dipping wax for 30min, embedding, and making a 5-micron thick sagittal paraffin section;
(4) dewaxing by gradient alcohol, dehydrating and HE dyeing;
(5) the results of HE staining of normal skin tissue by transparency, mounting and observation with a light microscope are shown in fig. 4, and the results of HE staining of acellular dermal matrix are shown in fig. 5.
As a result, no cell residue remained in the skin tissue.
Example 4
A preparation method of a human acellular dermal matrix comprises the following specific steps:
(1) taking stored human skin;
(2) removing subcutaneous fat components;
(3) removing the subcutaneous skin with fat components in the step (2), cleaning and disinfecting with 2% iodophor, deiodinating with 75% alcohol, and repeatedly soaking and cleaning the skin with sterile distilled water;
(4) treating the skin cleaned in the step (3) with 2.5mg/ml Dispase II solution overnight;
(5) discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
(6) mixing the corium obtained in step (5) with 2mol/L Ca2+Is treated with sodium chloride solution for 18h, Ca2+The final concentration of (3) is 5 mM/L;
(7) after discarding the sodium chloride solution, the mixture was washed with physiological saline for 15min 3 times to obtain an acellular dermal matrix of human origin, as shown in fig. 6.
Example 5 cell proliferation assay (MTT)
(1) Collecting logarithmic phase Hacat cells, adding 100ul of Hacat cells into each hole, and paving to adjust the density of the cells to be detected to 1000-10000 cells/hole; ADM group: laying sterile ADM with the same size as the hole to the bottom of the hole in advance; WG-ADM group: laying sterile WG-ADM with the same size as the hole to the bottom of the hole in advance;
(2)5％CO2incubation at 37 ℃, and performing MTT detection at 12h, 1d, 2d and 3d respectively;
(3) adding 20ul MTT solution (5mg/ml, namely 0.5% MTT) into each well, and continuing culturing for 2 h;
(4) terminating the culture, and carefully sucking out the culture solution in the holes;
(5) 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker for 10min at a low speed to dissolve the crystals sufficiently, and the absorbance of each well was measured at OD490nm in an ELISA detector.
The results are shown in FIG. 7, for ADM group: an acellular dermal matrix group; group B: blank group; group C: control group (no ADM, same ADM group for other experimental conditions); WG-ADM group: without addition of Ca2+And an acellular dermal matrix group prepared without using a dialysis device.
As can be seen from MTT experiments, ADM prepared by the method has no cytotoxicity, can effectively promote cell proliferation compared with other groups, and is beneficial to cell proliferation.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. A preparation method of a human acellular dermal matrix is characterized by comprising the following specific steps:
(1) taking stored human skin;
(2) removing subcutaneous fat components;
(3) removing the subcutaneous skin with fat components in the step (2), cleaning and disinfecting the skin with iodophor and alcohol, and repeatedly soaking and cleaning the skin with hypotonic solution;
(4) treating the skin cleaned in the step (3) with 2.5mg/ml Dispase II solution overnight;
(5) discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
(6) applying Ca-containing composition to the dermis obtained in step (5)2+Treating with hypertonic salt solution for 12-24 h;
(7) after the hypertonic saline solution is discarded, cleaning the solution by using normal saline;
(8) placing the dermis cleaned in the step (7) in prepared cell removal liquid, and treating for 2-6 h in a dialysis device to obtain a cell removal dermis matrix;
(9) washing the acellular dermal matrix obtained in the step (8) with physiological saline for 3 times, and each time for 15min to obtain a human-derived acellular dermal matrix;
the cell removal solution in the step (8) comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+ 0.1-0.5 mol/L，Cl- 0.1-0.5 mol/L，Ca2+ 1-10 mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20 mmol/L; or comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+ 0.1-0.5 mol/L，Cl- 0.1-0.6 mol/L，Ca2+ 1-10 mmol/L；Ca2+From CaCl2；
The dialysis apparatus according to step (8): the allogeneic skin is placed in a dialysis bag, the acellular fluid enters from one end and flows out from the other end of the device, and harmful ions in the acellular fluid are dialyzed and removed while the acellular fluid is acellular, so that the cytotoxicity and the genetic toxicity of the acellular dermal matrix are reduced to the maximum extent while the acellular effect is ensured, and the biocompatibility is effectively improved.
2. The method for preparing the human-derived acellular dermal matrix according to claim 1, wherein in the step (3), the skin with subcutaneous fat components removed in the step (2) is washed and sterilized with 2% iodophor, deiodinated with 75% alcohol, and repeatedly soaked in sterile distilled water to wash the skin.
3. The method for preparing the human acellular dermal matrix according to claim 1, wherein in the step (6), the dermis obtained in the step (5) is used at 0.5-3mol/L and contains Ca2+Is treated by hypertonic salt solution for 12 to 24 hours, Ca2+The final concentration of (b) is 1-10 mM/L.
4. The method for preparing the human acellular dermal matrix according to claim 3, wherein the hypertonic saline solution contains 0.5 to 3mol/L Ca2+Sodium chloride solution of (2), Ca2+The final concentration of (b) is 1-10 mM/L.
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