CN107412867B - Preparation method of heterogenous acellular dermal matrix - Google Patents
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Abstract
The invention relates to a preparation method of a heterogenous acellular dermal matrix, which comprises the following steps: the method comprises the steps of taking Bama miniature pig skin as a skin source, reversely taking a leather drum into a faulted skin sheet, carrying out steps of peeling, decellularization and the like to obtain acellular dermis, further reacting the acellular dermis with carboxymethyl chitosan to prepare an acellular dermis matrix, and finally carrying out treatment by a softening agent and a humectant to enable the prepared acellular dermis matrix to be closer to the natural state of human skin, so that the use compliance of the acellular dermis matrix is improved. The heterogenous acellular dermal matrix prepared by the method has low immunogenicity, has a three-dimensional structure for keeping the dermal collagen fiber, improves the stability of the three-dimensional structure of the dermal collagen fiber, and has better biocompatibility.
Description
Technical Field
The invention belongs to the technical field of tissue engineering skin, and particularly relates 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. Therefore, the search for a heterogeneous dermis which can be implanted into a human body, has good tissue compatibility and low immunity and is used for burns, plastic cosmetology and used as a tissue filler is a hot spot of the current domestic and foreign researches.
An Acellular Dermal Matrix (ADM) is a tissue engineering material which is used for removing epidermis and cells, has no bacterial growth, no toxicity, no irritation, no immunological rejection, elasticity, soft texture and no breaking, and is mainly applied to the repair of tissue wound surfaces. The acellular dermal matrix is made of natural skin tissues, and components which can cause immunologic rejection reaction after human implantation in the tissues are removed through special physicochemical treatment, and meanwhile, the three-dimensional scaffold structure of the original tissues is kept, so that fibroblast and vascular endothelial cells can be induced to grow in, the acellular dermal matrix has the effect of inducing tissue regeneration, and the acellular dermal matrix is an effective method for treating full-layer skin defect needing skin grafting. However, the existing acellular dermal matrix production process has the following disadvantages: can not effectively remove xenodermal cells, reduces xenodermal antigenicity and simultaneously retains the three-dimensional structure of intact dermal collagen fibers; in the preparation process of the acellular dermal matrix, extracellular amorphous matrix components such as hyaluronic acid, dermatan sulfate, heparin sulfate and the like are washed out along with cell components, so that the original external environment protection of the collagen scaffold is lost, and the collagen scaffold is degraded quickly after being transplanted on a wound surface and has local dissolution tendency; in addition, acellular dermal matrices are prone to wound infection when used. Therefore, a skin substitute with low immunogenicity and available for transplantation is urgently needed clinically.
Chinese patent application No. CN 102580153B, "a method for preparing acellular dermal matrix medical tissue patch", is to prepare a medical tissue patch of acellular dermal matrix from animal skin by processes of preparing a section skin sheet, inactivating viruses, degreasing, decellularizing, freeze-drying, cutting, molding, packaging and sterilizing by irradiation, and has the advantages of complete decellularization and light damage to natural structure of dermal extracellular matrix.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method of a heterogenous acellular dermal matrix.
The technical scheme provided by the invention is as follows:
the invention provides a preparation method of a heterogenous acellular dermal matrix, which comprises the following steps:
(1) selecting Bama miniature pig skin as a skin source, unhairing and disinfecting a skin taking area, taking off a large piece of skin by using a skin taking machine, cleaning the skin by using sterile normal saline, taking a leather drum back into a faulted skin piece with the thickness of 0.25-0.5 mm, and drilling holes on the faulted skin piece by using a laser drilling technology for later use;
(2) soaking the faulted skin sheet prepared in the step (1) in 0.5-1.0% (m/v) of sodium o-phenylphenol aqueous solution, treating for 6-8 min, washing for 3-4 times by using sterile normal saline, then adding 1-2% (m/v) of trihydroxymethyl methylamino ethanesulfonic acid aqueous solution, carrying out ultrasonic oscillation treatment for 6-12 h, and replacing the solution every 2h, wherein the weight ratio of the added trihydroxymethyl methylamino ethanesulfonic acid aqueous solution to the faulted skin sheet is 10:1, so as to obtain the derm without epidermis;
(3) placing the de-epidermis dermis obtained in the step (2) in 0.2-0.4% TritonX-100(v/v) solution, oscillating and washing for 6-12 h at room temperature, washing for 3 times by using 4000U/L gentamicin-containing normal saline solution, adding an acellular treatment solution, acting for 12-24 h at 37 ℃, wherein the weight ratio of the acellular treatment solution to the de-epidermis dermis is 10:1, washing for 3 times by using 4000U/L gentamicin-containing normal saline solution, and washing for 3-4 times by using sterile normal saline to obtain the de-epidermis;
(4) putting the acellular dermis obtained in the step (3) into a rotary drum, adding a carboxymethyl chitosan solution with the temperature of 35 ℃ and the pH of 5.7 in several times, reacting for 30min, adjusting the pH value to 5.0, reacting for 75min, adjusting the pH value to 6.8, discharging waste liquid after the treatment is finished, washing the treated material with sterile physiological salt for 2 times, each time for 15min, and then draining the water to obtain the acellular dermis matrix;
(5) soaking the acellular dermal matrix obtained by the treatment in the step (4) in a glutaraldehyde solution with the volume concentration of 0.1-0.3% (v/v) for crosslinking for 15-30 min, washing with sterile normal saline for 10-15 min, then placing in a softening agent solution for soaking for 30-60 min, cleaning, then placing in a humectant solution, preserving heat at 37 ℃ for 30-60 min, and finally fully washing with sterile normal saline to remove residues;
(6) and (4) placing the acellular dermal matrix obtained by the treatment in the step (5) into a freezing chamber of a freeze dryer for freeze drying, forming and packaging, and then performing gamma ray irradiation for disinfection and sterilization to obtain a finished product.
Further, the o-phenylphenol sodium aqueous solution in the step (2) contains 0.01-0.1% (v/v) of dipropylene glycol dimethyl ether; the aqueous solution of trihydroxymethyl methylamino ethanesulfonic acid contains 1-3% (m/v) sodium tripolyphosphate, 0.01-0.04% (v/v) dipropylene glycol dimethyl ether and 0.1-0.4% (m/v) polyethylene glycol 15-hydroxystearate.
The cell-removing treatment solution in the step (3) contains 0.025% (m/v) of pancreatin, 0.02% (m/v) of EDTA, 0.4-1.0% (m/v) of sodium lauroyl glycinate and 0.1-1.0% (m/v) of polyethylene glycol 15-hydroxystearate, and the concentration of the aqueous solution of trimethylol methylamino ethanesulfonic acid is 1-2% (m/v).
Further, the weight ratio of the decellularized dermis in the step (4) to the carboxymethyl chitosan solution is 1: 2-4.
Further, the concentration of the softening agent solution in the step (5) is 1-5% (m/v), and the softening agent is at least one selected from the group consisting of cetyltrimethyl ammonium chloride, cetyltrimethyl ammonium bromide, heptadecyl-N-aminoethyl imidazoline propylene oxide and octadecyl dimethyl benzyl ammonium chloride.
Further, in the step (5), the concentration of the humectant solution is 0.2-1% (m/v), and the humectant is at least one selected from glycerol, hyaluronic acid, sodium pyrrolidone carboxylate, dimethyl proline and octadecyl dimethyl amine oxide.
The Bama miniature pig skin fault skin sheet is treated by the o-phenylphenol sodium aqueous solution containing the dipropylene glycol dimethyl ether, so that bacteria and viruses on the surface of the skin sheet can be effectively killed, and the o-phenylphenol sodium is deep under sebaceous glands of the skin sheet under the action of the dipropylene glycol dimethyl ether, so that a better anti-corrosion and fresh-keeping effect is exerted.
The invention takes 1-3% (m/v) sodium tripolyphosphate, 0.01-0.04% (v/v) dipropylene glycol dimethyl ether and 0.1-0.4% (m/v) polyethylene glycol 15-hydroxystearate, and takes 1-2% (m/v) trihydroxymethyl methylamino ethanesulfonic acid aqueous solution as the treatment solution for removing the epidermis of the fault leather sheet, so that the transparent keratin layer of the leather sheet can be fully peeled off, the adhesion between the peeled transparent keratin layer and the dermis can be prevented, and the epidermis of the leather sheet can be fully removed. Meanwhile, the polyethylene glycol 15-hydroxystearate can effectively prevent collagen fibers, elastic fibers, reticular fibers and other components on the exposed dermis layer after the epidermis is removed from being hydrolyzed under the action of sodium tripolyphosphate to cause the three-dimensional structure collapse phenomenon of the dermis collagen fibers, and a better three-dimensional structure is kept.
In addition, the invention uses 0.025% (m/v) pancreatin, 0.02% (m/v) EDTA, 0.4-1.0% (m/v) sodium lauroyl glycinate and 0.1-1.0% (m/v) polyethylene glycol 15-hydroxystearate, and 1-2% (m/v) trihydroxymethyl aminomethane sulfonic acid aqueous solution as the acellular treatment solution of dermis, and the dermis is digested by the pancreatin with lower concentration under the action of the sodium lauroyl glycinate, so as to promote the full cell shedding, prevent the digestive dissolution of amorphous matrix of the dermis, such as hyaluronic acid, dermatan sulfate and heparin sulfate, by the pancreatin with high concentration, and improve the stability of the three-dimensional structure of the collagen fiber of the dermis. The polyethylene glycol 15-hydroxystearate can effectively prevent the exfoliated dermal cells from agglomerating, keeps better suspension property and is beneficial to cleaning and removing the exfoliated cells.
Furthermore, the prepared acellular dermis reacts with the carboxymethyl chitosan, the prepared acellular dermis matrix has better biocompatibility, can effectively promote wound healing and simultaneously exert better antibacterial property, improves the application effect of the acellular dermis matrix, and simultaneously treats the prepared acellular dermis matrix with the softening agent and the humectant, so that the acellular dermis matrix is closer to natural skin texture, and the use compliance is improved.
The present inventors also claim a xenogenic acellular dermal matrix produced according to the above-mentioned method for producing a xenogenic acellular dermal matrix.
The preparation method of the xenogenic acellular dermal matrix provided by the invention is also suitable for preparing the xenogenic acellular dermal matrix by taking sheep skin, cow skin, rabbit skin and the like as raw materials.
Compared with the prior art, the invention has the advantages that:
the preparation method of the heterogenous acellular dermal matrix provided by the invention can ensure that the transparent keratin layer of the faulted skin piece is fully peeled off, prevent the peeled transparent keratin layer from being adhered with the dermis, fully remove the epidermis of the skin piece, fully peel off the dermal cells of the epidermis and reduce the immunogenicity. Meanwhile, collagen fibers, elastic fibers and reticular fibers on the exposed dermis layer after the epidermis is removed can be effectively prevented from being hydrolyzed to cause the phenomenon of three-dimensional structure collapse of the dermis collagen fibers, a better three-dimensional structure is kept, and the stability of the three-dimensional structure of the dermis collagen fibers is improved. The prepared acellular dermal matrix has better biocompatibility, can effectively promote wound healing and simultaneously exert better antibacterial property, improves the application effect of the acellular dermal matrix, is closer to natural skin texture and improves the use compliance.
Detailed Description
The present invention will be further described below by way of specific embodiments, but the present invention is not limited to only the following examples.
Example 1
A preparation method of a heterogenic acellular dermal matrix comprises the following steps:
(1) selecting Bama miniature pig skin as a skin source, depilating and disinfecting a skin taking area, taking off a large piece of skin by using a skin taking machine, cleaning the skin by using sterile normal saline, taking a 0.30mm fault skin piece by a drum, and making holes on the fault skin piece by using a laser drilling technology for later use;
(2) soaking the faulted skin sheet prepared in the step (1) in 0.02% (v/v) dipropylene glycol dimethyl ether and 0.8% (m/v) sodium o-phenylphenol aqueous solution, treating for 6min, washing for 3 times by using sterile normal saline, then adding a solution containing 2% (m/v) sodium tripolyphosphate, 0.02% (v/v) dipropylene glycol dimethyl ether and 0.2% (m/v) polyethylene glycol 15-hydroxystearate, and 2% (m/v) trimethylolpropane amine ethanesulfonic acid aqueous solution, carrying out ultrasonic oscillation treatment for 8h, and replacing the solution every 2h, wherein the weight ratio of the added trimethylolpropane amine ethanesulfonic acid aqueous solution to the faulted skin sheet is 10:1, so as to obtain the dermized dermis with epidermis removed;
(3) placing the de-epidermal dermis obtained in the step (2) in 0.25% TritonX-100(v/v) solution, shaking and washing for 8h at room temperature, washing for 3 times by using 4000U/L gentamicin-containing physiological saline solution, adding 2% (m/v) trihydroxymethyl aminomethane sulfonic acid aqueous solution containing 0.025% (m/v) pancreatin, 0.02% (m/v) EDTA, 0.4% (m/v) sodium lauroyl glycinate and 0.6% (m/v) polyethylene glycol 15-hydroxystearate, acting for 24h at 37 ℃, wherein the weight ratio of the de-cytotic treatment solution to the de-epidermal dermis is 10:1, washing for 3 times by using 4000U/L gentamicin-containing physiological saline solution, and washing for 4 times by using sterile physiological saline to obtain the de-epidermal dermis;
(4) placing the acellular dermis obtained in the step (3) into a rotary drum, adding a carboxymethyl chitosan solution with the temperature of 35 ℃ and the pH value of 5.7 in a fractional manner, wherein the weight ratio of the acellular dermis to the carboxymethyl chitosan solution is 1:3, reacting for 30min, adjusting the pH value to 5.0, reacting for 75min, adjusting the pH value to 6.8, discharging waste liquid after the treatment, washing the treated material with sterile physiological salt for 2 times, each time for 15min, and draining the washed material to obtain an acellular dermis matrix;
(5) soaking the acellular dermal matrix obtained by the treatment in the step (4) in a glutaraldehyde solution with the volume concentration of 0.2% (v/v) for crosslinking for 20min, washing with sterile physiological saline for 15min, then placing the acellular dermal matrix in a hexadecyltrimethylammonium chloride solution with the concentration of 2% (m/v) for soaking for 40min, washing, then placing the acellular dermal matrix in a dimethyl amino acid proline solution with the concentration of 0.5% (m/v), preserving heat at 37 ℃ for 40min, and finally fully washing with sterile physiological saline to remove residues;
(6) and (4) placing the acellular dermal matrix obtained by the treatment in the step (5) into a freezing chamber of a freeze dryer for freeze drying, forming and packaging, and then performing gamma ray irradiation for disinfection and sterilization to obtain a finished product.
Example 2
A preparation method of a heterogenic acellular dermal matrix comprises the following steps:
(1) selecting Bama miniature pig skin as a skin source, depilating and disinfecting a skin taking area, taking off a large piece of skin by using a skin taking machine, cleaning the skin by using sterile normal saline, taking a 0.35mm fault skin piece by a drum, and making holes on the fault skin piece by using a laser drilling technology for later use;
(2) soaking the faulted skin sheet prepared in the step (1) in 0.04% (v/v) dipropylene glycol dimethyl ether-containing o-phenylphenol sodium aqueous solution with the concentration of 0.6% (m/v), treating for 8min, washing for 4 times by using sterile normal saline, then adding a solution containing 3% (m/v) sodium tripolyphosphate, 0.02% (v/v) dipropylene glycol dimethyl ether and 0.1% (m/v) polyethylene glycol 15-hydroxystearate with the concentration of 2% (m/v) trimethylol methylamino ethanesulfonic acid aqueous solution, carrying out ultrasonic oscillation treatment for 12h, and replacing the solution every 2h, wherein the weight ratio of the added trimethylol methylamino ethanesulfonic acid aqueous solution to the faulted skin sheet is 10:1, so as to obtain the derm with epidermis removed;
(3) placing the de-epidermal dermis obtained in the step (2) in 0.2% TritonX-100(v/v) solution, shaking and washing for 12h at room temperature, washing for 3 times by using 4000U/L gentamicin-containing physiological saline solution, adding 2% (m/v) trihydroxymethyl aminomethane sulfonic acid aqueous solution containing 0.025% (m/v) pancreatin, 0.02% (m/v) EDTA, 0.8% (m/v) sodium lauroyl glycinate and 0.6% (m/v) polyethylene glycol 15-hydroxystearate, acting for 12h at 37 ℃, wherein the weight ratio of the de-cytotic treatment solution to the de-epidermal dermis is 10:1, washing for 3 times by using 4000U/L gentamicin-containing physiological saline solution, and washing for 4 times by using sterile physiological saline to obtain the de-epidermal dermis;
(4) placing the acellular dermis obtained in the step (3) into a rotary drum, adding a carboxymethyl chitosan solution with the temperature of 35 ℃ and the pH value of 5.7 in a fractional manner, wherein the weight ratio of the acellular dermis to the carboxymethyl chitosan solution is 1:2, reacting for 30min, adjusting the pH value to 5.0, reacting for 75min, adjusting the pH value to 6.8, discharging waste liquid after the treatment, washing the treated material with sterile physiological salt for 2 times, each time for 15min, and draining after washing to obtain the acellular dermis matrix;
(5) soaking the acellular dermal matrix obtained by the treatment in the step (4) in a glutaraldehyde solution with the volume concentration of 0.2% (v/v) for crosslinking for 20min, washing with sterile normal saline for 15min, then placing in an octadecyl dimethyl benzyl ammonium chloride solution with the concentration of 4% (m/v) for soaking for 30min, washing, then placing in a pyrrolidone sodium carboxylate solution with the concentration of 0.8% (m/v), carrying out heat preservation treatment at 37 ℃ for 30min, and finally fully washing with sterile normal saline to remove residues;
(6) and (4) placing the acellular dermal matrix obtained by the treatment in the step (5) into a freezing chamber of a freeze dryer for freeze drying, forming and packaging, and then performing gamma ray irradiation for disinfection and sterilization to obtain a finished product.
Comparative example 1
The method of preparing the xenogenic acellular dermal matrix according to comparative example 1 is substantially the same as in example 1, except that the liquid for exfoliating the skin sheets in the step (2) does not contain polyethylene glycol 15-hydroxystearate, and the liquid for exfoliating the skin sheets contains 1 to 3% (m/v) sodium tripolyphosphate and 0.01 to 0.04% (v/v) dipropylene glycol dimethyl ether, and an aqueous solution of 1 to 2% (m/v) trimethylol-methylaminoethanesulfonic acid.
Comparative example 2
The method for preparing the xenogenic acellular dermal matrix according to comparative example 2 is substantially the same as in example 1, except that the dermal cell treatment solution in step (3) does not contain sodium lauroyl glycinate, and an aqueous solution of 1 to 2% (m/v) trimethylol methylaminoethanesulfonic acid, which contains 0.025% (m/v) pancreatin, 0.02% (m/v) EDTA, and 0.1 to 1.0% (m/v) polyethylene glycol 15-hydroxystearate, is used as the dermal acellular treatment solution.
Comparative example 3
The method for preparing the xenogenic acellular dermal matrix according to comparative example 3 is substantially the same as in example 1, except that the dermal cell treatment solution in step (3) does not contain polyethylene glycol 15-hydroxystearate, i.e., an aqueous solution of trimethylol methylaminoethanesulfonic acid having a concentration of 2% (m/v) containing pancreatin of 0.025% (m/v), EDTA of 0.02% (m/v) and sodium lauroyl glycinate of 0.4% (m/v) is used as the dermal acellular treatment solution.
Test example one, biomechanical examination of the different acellular dermal matrix
An INSTRON 4302 stretcher is adopted, a special chuck for clamping the specimen is adopted, a sensor of 100N is applicable, the load range is set to be 20N, the acellular dermal matrix prepared in the embodiment 1-2 and the comparative example 1-3 is respectively subjected to strength and stress tests, wherein the strength test breaks the acellular dermal matrix at the speed of 10mm/min, the force at the moment is recorded, and the strength of each specimen is represented by dividing the force at the moment when the specimen is broken by the initial sectional area of the specimen; and (3) stress testing: the acellular dermal matrix is unloaded when the acellular dermal matrix is pulled to 2.0MPa koch stress, preset for 3 times, the 4 th time is recorded, the corresponding elastic modulus (strain) is measured when the stress (300kPa) in a physiological range is measured, a corresponding stress-strain curve is drawn, and the elongation coefficient parameter is obtained through the stress-strain curve. The elongation of the test piece is the intersection of the elastic modulus slope and the strain axis at a stress of 300kPa, and the results are shown in Table 1 below.
TABLE 1 biomechanical test results
| Group of | Strength (MPa) | Elongation (%) |
| Example 1 | 5.842±0.254*#▽ | 1.078±0.038*#▽ |
| Example 2 | 5.906±0.312*#▽ | 1.092±0.045*#▽ |
| Comparative example 1 | 5.275±0.220 | 0.896±0.032 |
| Comparative example 2 | 5.321±0.192 | 0.905±0.027 |
| Comparative example 3 | 5.280±0.226 | 0.874±0.030 |
Note: in comparison with comparative example 1,*P<0.05, compared with comparative example 2,#P<0.05, compared with comparative example 3,▽P<0.05。
as can be seen from the above table, the acellular dermal matrixes prepared in comparative examples 1-3 have different reductions in strength and elongation compared with examples 1-2, which indicates that the acellular dermal matrixes prepared in comparative examples 1-3 have different degrees of degradation, resulting in the reduction in strength of the matrixes, the destruction of the collagen fiber structures, and the substantial reduction in elongation of the matrixes.
Test example two, detection of cell compatibility of different acellular dermal matrices
Co-culturing cartilage cells and bone marrow stromal cells from New Zealand white rabbit in a ratio of 3:7 to 3 rd generation as seed cells, inoculating to the acellular dermal matrix prepared in example 1-2 and comparative example 1-3, respectively, at 37 deg.C and 5% CO2Culturing for 48h under the condition of saturated humidity, and observing the adhesion condition of the cells on the matrix by a scanning electron microscope.
Meanwhile, seed cells inoculated on an acellular dermal matrix for culture are used as an experimental group, pure seed cells are used as a control group, after 2 weeks of culture, the mRNA expression of type II collagen is detected by RT-PCR, and primers are synthesized by Boo biotechnology, Inc. The primer sequence is as follows: type II collagen upstream primer: 5'-ACCAAAGGGACAGAAAG-3', downstream primer: 5'-ACAGCATAACATGGGGCTTC-3', product length: 470 bp; beta-actin upstream primer: 5'-AGTGCGACGTGGACATCCTTG-3', downstream primer: 5'-TGGCTCTAACAGTCCGCCTAGAGCTA-3', the length of the product is 302 bp. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles; extension at 72 ℃ for 5 min. 5mL of the PCR product was subjected to 1% agarose gel electrophoresis, and then scanned and photographed by a gel imaging system. The ScnImage image analysis software measured the grayscale values, and the ratio of the grayscale values of type II collagen to β -actin was used as the relative expression level of type II collagen mRNA. Each set of 6 samples. The results are shown in Table 2 below.
TABLE 2 results of cytocompatibility assays
Note: comparison with comparative example 1，*P<0.05, compared with comparative example 2,#P<0.05, compared with comparative example 3,▽P<0.05。
as can be seen from the above table, the acellular dermal matrices prepared in examples 1-2 of the present invention can effectively maintain the growth state of the co-cultured seed cells, and the difference from the growth state of the seed cells without co-culture (control group) is not significant. The acellular dermis mediums prepared in the comparative examples 1-3 have certain influence on the growth state of co-culture seed cells, a small amount of cells float, and individual cells have irregular shapes, unclear outlines and bad cell growth states.
Test example III detection of antigen component of foreign acellular dermal matrix
Immunohistochemical semi-quantitative analysis of highly immunogenic components of the xenogenic acellular dermal matrices prepared in examples 1-2 of the present invention and comparative examples 1-3: IV collagen, fibronectin, laminin, the results are shown in tables 3-5 below.
TABLE 3 IV collagen immunohistochemical staining semi-quantitative test results (n)
| Grade | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| - | 10 | 10 | 6 | 5 | 5 |
| + | 0 | 0 | 3 | 4 | 3 |
| ++ | 0 | 0 | 1 | 1 | 2 |
| +++ | 0 | 0 | 0 | 0 | 0 |
TABLE 4 results of semi-quantitative detection of fibronectin (N) layer by immunohistochemical staining
| Grade | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| - | 10 | 10 | 7 | 6 | 5 |
| + | 0 | 0 | 2 | 3 | 3 |
| ++ | 0 | 0 | 1 | 1 | 2 |
| +++ | 0 | 0 | 0 | 0 | 0 |
TABLE 5 laminin immunohistochemical staining semi-quantitative detection results (n)
As can be seen from the above table, the acellular dermal matrix prepared in examples 1-2 of the present invention substantially completely removes the highly immunogenic components such as iv collagen, fibronectin, laminin, etc., and simultaneously maintains the normal tissue structure of the dermal collagen scaffold, thereby reducing the immunogenicity of the dermal matrix to a certain extent and improving the safety of the dermal matrix in use.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (7)
1. A preparation method of a heterogenous acellular dermal matrix is characterized by comprising the following steps:
(1) selecting Bama miniature pig skin as a skin source, unhairing and disinfecting a skin taking area, taking off a large piece of skin by using a skin taking machine, cleaning the skin by using sterile normal saline, taking a leather drum back into a faulted skin piece with the thickness of 0.25-0.5 mm, and drilling holes on the faulted skin piece by using a laser drilling technology for later use;
(2) soaking the faulted skin sheet prepared in the step (1) in 0.5-1.0% (m/v) of sodium o-phenylphenol aqueous solution, treating for 6-8 min, washing for 3-4 times by using sterile normal saline, then adding 1-2% (m/v) of trihydroxymethyl methylamino ethanesulfonic acid aqueous solution, carrying out ultrasonic oscillation treatment for 6-12 h, and replacing the solution every 2h, wherein the weight ratio of the added trihydroxymethyl methylamino ethanesulfonic acid aqueous solution to the faulted skin sheet is 10:1, so as to obtain the derm without epidermis;
(3) placing the de-epidermis dermis obtained in the step (2) in 0.2-0.4% TritonX-100(v/v) solution, oscillating and washing for 6-12 h at room temperature, washing for 3 times by using 4000U/L gentamicin-containing normal saline solution, adding an acellular treatment solution, acting for 12-24 h at 37 ℃, wherein the weight ratio of the acellular treatment solution to the de-epidermis dermis is 10:1, washing for 3 times by using 4000U/L gentamicin-containing normal saline solution, and washing for 3-4 times by using sterile normal saline to obtain the de-epidermis;
(4) putting the acellular dermis obtained in the step (3) into a rotary drum, adding a carboxymethyl chitosan solution with the temperature of 35 ℃ and the pH of 5.7 in several times, reacting for 30min, adjusting the pH value to 5.0, reacting for 75min, adjusting the pH value to 6.8, discharging waste liquid after the treatment is finished, washing the treated material with sterile physiological salt for 2 times, each time for 15min, and then draining the water to obtain the acellular dermis matrix;
(5) soaking the acellular dermal matrix obtained by the treatment in the step (4) in a glutaraldehyde solution with the volume concentration of 0.1-0.3% (v/v) for crosslinking for 15-30 min, washing with sterile normal saline for 10-15 min, then placing in a softening agent solution for soaking for 30-60 min, cleaning, then placing in a humectant solution, preserving heat at 37 ℃ for 30-60 min, and finally fully washing with sterile normal saline to remove residues;
(6) placing the acellular dermal matrix obtained by the treatment in the step (5) into a freezing chamber of a freeze dryer for freeze-drying, forming and packaging, and then performing gamma-ray irradiation sterilization to obtain a finished product;
the aqueous solution of the trihydroxymethyl methylamino ethanesulfonic acid in the step (2) contains 1-3% (m/v) of sodium tripolyphosphate, 0.01-0.04% (v/v) of dipropylene glycol dimethyl ether and 0.1-0.4% (m/v) of polyethylene glycol 15-hydroxystearate;
the cell removing treatment solution in the step (3) contains 0.025% (m/v) of pancreatin, 0.02% (m/v) of EDTA, 0.4-1.0% (m/v) of sodium lauroyl glycinate and 0.1-1.0% (m/v) of polyethylene glycol 15-hydroxystearate, and the concentration of the trihydroxymethyl methylamino ethanesulfonic acid solution is 1-2% (m/v).
2. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the aqueous solution of sodium o-phenylphenol in the step (2) contains 0.01 to 0.1% (v/v) of dipropylene glycol dimethyl ether.
3. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the weight ratio of the acellular dermis and the carboxymethyl chitosan solution in the step (4) is 1: 2-4.
4. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the softening agent in the step (5) is at least one selected from the group consisting of cetyltrimethyl ammonium chloride, cetyltrimethyl ammonium bromide, heptadecyl-N-aminoethyl imidazoline propylene oxide, and octadecyldimethylbenzylammonium chloride.
5. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the concentration of the softening agent solution in the step (5) is 1 to 5% (m/v).
6. The method for preparing a dermal matrix according to claim 1, wherein the moisturizer in the step (5) is at least one selected from the group consisting of glycerol, hyaluronic acid, sodium pyrrolidone carboxylate, dimethyl proline, and octadecyl dimethyl amine oxide.
7. The method for preparing a xenogenic acellular dermal matrix according to claim 1, wherein the concentration of the moisturizer solution in the step (5) is 0.2 to 1% (m/v).
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