CN112618796A - Acellular dermal matrix material and preparation method and application thereof - Google Patents
Acellular dermal matrix material and preparation method and application thereof Download PDFInfo
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- CN112618796A CN112618796A CN202011280094.4A CN202011280094A CN112618796A CN 112618796 A CN112618796 A CN 112618796A CN 202011280094 A CN202011280094 A CN 202011280094A CN 112618796 A CN112618796 A CN 112618796A
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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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- A61L27/3683—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
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- 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
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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Abstract
The invention relates to an acellular dermal matrix material and a preparation method and application thereof. The method comprises the following steps: pretreating a dermis raw material; virus inactivation treatment: soaking a leather raw material in an alkaline solution in an ultrasonic cleaning machine, and then cleaning with purified water; carrying out cell removal treatment on the virus inactivation treated material by using an acid solution in an ultrasonic cleaning machine; repeating the step of cell removal treatment for 1-5 times after replacing the acid solution to obtain a cell removal treatment material; sequentially cleaning the acellular treatment material by using phosphate buffer solution and purified water in an ultrasonic cleaning machine to obtain acellular dermal tissue; freeze-drying the acellular dermal tissue; the material is cut and packaged in sequence, and ethylene oxide is adopted for sterilization to prepare the acellular dermal matrix material. The invention reduces the cytotoxicity risk of the product, can better reserve the three-dimensional structure and collagen of the material, avoids immunogenicity caused by enzyme residue, and is more favorable for maintaining good mechanical properties of the material.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an acellular dermal matrix material and a preparation method and application thereof.
Background
Acellular dermal matrix (acellular dermal matrix ADM) is prepared by completely removing epidermis and cell components in human or animal skin by physical and chemical methods and only retaining extracellular matrix containing collagen network frame in dermis. The acellular dermal matrix is generally free of rejection reactions due to the complete absence of the cellular components and the major immunological activities of the type i and type ii cytocompatible antigens. However, the acellular dermal matrix retains the three-dimensional structure of normal collagen and the extracellular matrix containing the collagen scaffold in the dermis can provide a good scaffold structure for the regeneration of tissue cells, and has the functions of wound surface covering, tissue defect filling, tissue regeneration guiding, scaffold guiding and the like.
According to different sources, the acellular dermal matrix is divided into an allogeneic acellular dermal matrix (mainly from cadaver skin) and a xenogeneic acellular dermal matrix (mainly from animal skin and mainly from pigskin and cow skin). The preparation process removes cells with antigenicity (epidermal cells, hair follicles, sebaceous glands, sweat glands, vascular endothelial cells in dermis, fibroblasts and the like) as far as possible, and retains the components and the basic tissue structure of collagen fibers. In the early days, people prepared ADM by using a repeated freeze-thaw method, a trypsinization method and the like, but because the methods can not completely remove cell components in the dermis, strong rejection reaction of a host is caused after the ADM is transplanted, and the preparation process is too complex, the ADM is not widely developed. The current ADM preparation methods mainly comprise a Dispase II-Triton method, a hypertonic salt-SDS method, a hypertonic salt-enzyme digestion method, a NaOH erosion method and the like.
Chinese patent application CN 104941008A discloses a preparation method and application of a guided tissue regeneration membrane; the method of the invention comprises the following steps: (1) soaking and treating the skin of the isolated animal by using a surfactant solution; (2) soaking the product of the step (1) by using an alkali solution; (3) soaking and treating the product of the step (2) by using a peroxide solution; (4) soaking and treating the product in the step (3) by using a radiation protection reagent solution; (5) soaking and treating the product of the step (4) by using a buffer solution; (6) and (5) sequentially carrying out freeze drying and irradiation sterilization on the product obtained in the step (5) to obtain the guided tissue regeneration membrane. Chinese patent application CN109364298A discloses a preparation method of an acellular dermal matrix material, which is characterized in that animal skins are fleshed and unhaired, then a piece of animal skin is taken, and the acellular dermal matrix is obtained by degreasing, unhairing, virus inactivation, acellular, crosslinking modification, cleaning and moisturizing, packaging and irradiation sterilization processes. However, the above method for preparing the acellular dermal matrix material has the following disadvantages: firstly, the production process is complicated and is not beneficial to the establishment of a large-scale production line; ionic or nonionic surfactants are used, the three-dimensional space structure of the material and collagen in the treatment engineering are correspondingly damaged, so that the mechanical property of the product is reduced, the surfactants have cytotoxicity, residues are always generated in the removal process, and the cytotoxicity risk of the product is increased; the processing time of various chemical reagents is long, and collagen denaturation and protein cracking of the material are easily caused; fourthly, when biological enzyme is used, the cells can not be completely removed in a short treatment time, and the cells are in a longer placeThe physical time can cause the content of collagen and elastin to be reduced, and further the mechanical strength of the matrix to be reduced; fifthly, during final sterilization, adopt60Co irradiation sterilization, high-dose irradiation is beneficial to sterilization and virus inactivation, but for collagen products, high dose can cause collagen denaturation and cracking of a collagen chain structure, so that the mechanical properties of the products are reduced, the degradation is too fast, the thermal change temperature is reduced, and the like, so that the product performance is poor; although the product irradiation protection link is added in part of the process, the complexity of the process is increased, and the product pollution risk is increased due to the addition and use of more reagents.
Disclosure of Invention
In order to solve one or more technical problems in the prior art, the invention provides an acellular dermal matrix material and a preparation method and application thereof. The method reduces the cytotoxicity risk of the product, has good acellular effect, can better retain the three-dimensional structure and collagen of the material, avoids immunogenicity caused by enzyme residue, and is more favorable for maintaining good mechanical properties of the material.
The present invention provides in a first aspect a method for preparing an acellular dermal matrix material, the method comprising the steps of:
(1) taking animal skin tissue materials, removing surface fat and epidermis, and cleaning with an ultrasonic cleaning machine to obtain a pretreated dermis raw material;
(2) soaking the pretreated dermal material obtained in the step (1) in an alkaline solution in an ultrasonic cleaning machine, and then cleaning the soaked dermal material with purified water to obtain a virus inactivation treated dermal material;
(3) carrying out cell removal treatment on the virus inactivation treated dermis material obtained in the step (2) by using an acid solution in an ultrasonic cleaning machine;
(4) repeating the step (3) for 1-5 times after replacing the acid solution to obtain a cell-free treatment material;
(5) cleaning the acellular treatment material obtained in the step (4) by using phosphate buffer solution and purified water in an ultrasonic cleaning machine in sequence to obtain acellular dermal tissue;
(6) freeze-drying the acellular dermal tissue obtained in the step (5);
(7) and (4) sequentially cutting and packaging the acellular dermal tissue subjected to freeze drying in the step (6), and then sterilizing by adopting ethylene oxide to prepare the acellular dermal matrix material.
Preferably, in step (1), the animal skin tissue material is selected from porcine, bovine or ovine skin tissue, and preferably, the animal skin tissue material is selected from porcine or bovine skin tissue.
Preferably, the frequency of the ultrasonic cleaning machine is 10-40 kHz, and preferably 25-40 kHz.
Preferably, in the step (2), the alkali solution is a KOH solution and/or a NaOH solution, the concentration of the alkali solution is 0.001-1 mol/L, and the volume usage of the alkali solution is 2-20 times of the volume of the pretreated dermal raw material; and/or in the step (2), the time of soaking treatment by using the alkali solution is 30-120 min.
Preferably, in the step (3), the acid solution is one or more of hydrochloric acid, sulfuric acid, nitric acid and formic acid, the concentration of the acid solution is 0.001-1 mol/L, and the volume of the acid solution is 2-20 times of the volume of the pretreated dermal raw material; and/or in the step (3), the time of the cell removing treatment by using the acid solution is 30-120 min.
Preferably, in the step (5), the pH value of the phosphate buffer solution is 7.0-7.4, and the reagent for preparing the phosphate buffer solution is NaCl, KCl or KH2PO4And Na2HPO4The volume usage amount of the phosphate buffer solution is 10-30 times of the volume of the pretreated dermis raw material; and/or in the step (5), the washing time by using a phosphate buffer solution is 30-120 min.
Preferably, in step (6), the freeze-drying comprises the following sub-steps:
(a) pre-freezing the acellular dermal tissue obtained in the step (5) at the temperature of minus 30 ℃ for 2 hours;
(b) treating the pre-frozen acellular dermal tissue for 10 hours under the vacuum condition at the temperature of minus 10 ℃, and then treating the acellular dermal tissue for 10 hours under the vacuum condition at the temperature of 0 ℃ to complete the first-stage sublimation process of the acellular dermal tissue;
(c) and (3) treating the acellular dermal tissue which finishes the first-stage sublimation process under the vacuum condition of 5 ℃ for 4 hours, then treating the acellular dermal tissue under the vacuum condition of 15 ℃ for 2 hours, then treating the acellular dermal tissue under the vacuum condition of 25 ℃ for 2 hours, and finally treating the acellular dermal tissue under the vacuum condition of 35 ℃ for 2 hours to finish the second-stage sublimation process of the acellular dermal tissue, thereby finishing the freeze drying of the acellular dermal tissue.
Preferably, in step (7), packaging is performed using a sterile tyvek packaging bag; and/or in the step (7), the concentration of the ethylene oxide is 600-700 mg/L, the sterilization temperature for sterilizing by adopting the ethylene oxide is 10-42 ℃, the sterilization humidity is 35-65% RH, and the sterilization time is 1-6 h.
In a second aspect, the present invention provides an acellular dermal matrix material produced by the production method according to the first aspect of the present invention.
In a third aspect, the invention provides the use of the acellular dermal matrix material prepared by the preparation method of the first aspect of the invention in the preparation of a product for dental implant or for assisting bone regeneration or guiding tissue growth.
Compared with the prior art, the invention at least has the following beneficial effects:
(1) the method provides a simple and effective preparation process of the acellular dermal matrix material, and is favorable for the enlarged production of the process; the process is simple and convenient, and is beneficial to the industrial production of products.
(2) In the preparation process, organic solvents with cytotoxicity such as surfactants and the like are not used, so that the cytotoxicity risk of the product is reduced.
(3) The invention adopts a mode of combining ultrasonic oscillation and reagent treatment in the preparation process, improves the treatment efficiency of the reagent, reduces the contact time of the material and the reagent, and better reserves the three-dimensional structure of the material and the collagen.
(4) In the treatment process, only three chemical reagents such as acid, alkali, salt and the like are adopted, so that the cleaning process is easy to remove, and the product pollution is avoided; reagents such as biological enzyme are not used, so that immunogenicity caused by enzyme residues is avoided.
(5) The terminal sterilization of the present invention employs ethylene oxide sterilization, with60Compared with Co irradiation sterilization, the Co irradiation sterilization has less damage to products and is more favorable for maintaining good mechanical properties.
Drawings
FIG. 1 is a photograph showing the microstructure of the acellular dermal matrix material according to example 1 of the present invention after HE staining under an optical microscope of 20X.
FIG. 2 is a photograph showing the microstructure of the acellular dermal matrix material prepared in example 2 of the present invention after HE staining under an optical microscope of 20 Xmagnification.
FIG. 3 is a photograph showing the microstructure of the acellular dermal matrix material according to example 3 of the present invention after HE staining under an optical microscope of 20X.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The present invention provides in a first aspect a method for preparing an acellular dermal matrix material, the method comprising the steps of:
(1) taking animal skin tissue materials, removing surface fat and epidermis, and cleaning with an ultrasonic cleaning machine to obtain a pretreated dermis raw material;
(2) soaking the pretreated dermal material obtained in the step (1) in an alkaline solution in an ultrasonic cleaning machine, and then cleaning the soaked dermal material with purified water to obtain a virus inactivation treated dermal material; in the invention, virus inactivation is carried out on the pretreated dermis raw material by adopting a mode of combining alkali solution treatment and ultrasonic oscillation of an ultrasonic cleaner;
(3) carrying out cell removal treatment on the virus inactivation treated dermis material obtained in the step (2) by using an acid solution in an ultrasonic cleaning machine; in the invention, the cell removing step adopts acid solution treatment to remove cells, on one hand, the acid can dissolve cell components to destroy the cells, and on the other hand, immunogenic substances such as DNA and the like are very unstable to the acid, so that the aim of completely removing the immunogenic substances can be fulfilled;
(4) repeating the step (3) for 1-5 times (for example, 1, 2, 3, 4 or 5 times) after replacing the acid solution to obtain a cell-free treatment material; in the present invention, replacing the acid solution means not replacing the kind of the acid solution, but repeating the step (3) after preparing the same acid solution in the step (3) again to perform the decellularization treatment; in the invention, the step (3) is repeated for 1-5 times, and through multiple acid treatments, on one hand, the cell membrane can be ensured to be fully cracked to expose cell nucleuses, and on the other hand, the exposed genetic materials such as DNA and the like can be fully eluted.
(5) Cleaning the acellular treatment material obtained in the step (4) by using phosphate buffer solution and purified water in an ultrasonic cleaning machine in sequence to obtain acellular dermal tissue;
(6) freeze-drying the acellular dermal tissue obtained in the step (5);
(7) and (4) sequentially cutting and packaging the acellular dermal tissue subjected to freeze drying in the step (6), and then sterilizing by adopting ethylene oxide to prepare the acellular dermal matrix material.
The method establishes a simple and effective preparation process of the acellular dermal matrix material, and is beneficial to the amplification production of the process; in the preparation process, organic solvents with cytotoxicity such as surfactants and the like are not adopted, so that the cytotoxicity risk of the product is reduced; in the preparation process, ultrasonic oscillation is utilized, the action efficiency of the reagent is improved, the treatment time of the reagent is reduced, the three-dimensional space structure and the collagen component of the matrix can be completely reserved while the cells are removed, and the material has good mechanical property; in the preparation process, only acid and alkali solutions are adopted for cell removal, biological methods such as biological enzyme and the like are not used, on one hand, the risk of enzyme residue is avoided, and on the other hand, the proper acid and alkali process can enable the material to have better biocompatibility; in the invention, ethylene oxide is adopted for sterilization during final sterilization, and the ethylene oxide can be used for sterilization of non-high temperature resistant and non-moisture resistant articles, has strong penetrability and can be used for sterilization of various parts which are difficult to permeate; the damage to the article is small, since the EO (ethylene oxide) sterilization microorganism utilizes the alkylation principle rather than the oxidation process, the damage to the article is very small. The method reduces the cytotoxicity risk of the product, has good acellular effect, can better retain the three-dimensional structure and collagen of the material, avoids immunogenicity caused by enzyme residue, and is more favorable for maintaining good mechanical properties of the material.
According to some embodiments, the preparation of the acellular dermal matrix material according to the present invention comprises the following steps:
pretreatment of a dermis raw material:
taking animal skin tissue materials, removing surface fat and epidermis, and cleaning with an ultrasonic cleaning machine to obtain a pretreated dermis raw material;
② virus inactivation treatment:
soaking the pretreated dermis raw material in an alkaline solution in an ultrasonic cleaning machine, and then cleaning the dermis raw material with purified water to obtain a virus inactivation treated dermis material;
③ acellular treatment:
carrying out cell removal treatment on the virus inactivation treated dermis material obtained in the step II by using an acid solution in an ultrasonic cleaning machine;
replacing the acid solution in the step III, and repeating the step III for 1-5 times to obtain a cell-free treatment material;
cleaning:
washing the acellular processing material obtained in the step (IV) by using phosphate buffer solution and purified water respectively in an ultrasonic washing machine to obtain acellular dermal tissue;
forming:
freeze-drying the acellular dermal tissue prepared in the fifth step;
and packaging and sterilizing:
cutting the freeze-dried acellular dermal tissue prepared in the step (sixty) into a required size, packaging the acellular dermal tissue by adopting an aseptic Tewei Qiang packaging bag, and sterilizing by adopting ethylene oxide to obtain the acellular dermal matrix material; in the present invention, the sterile barrier system employed is a tyvek bag.
According to some preferred embodiments, in step (1), the animal skin tissue material is selected from porcine, bovine or ovine skin tissue, preferably the animal skin tissue material is selected from porcine or bovine skin tissue.
According to some preferred embodiments, the frequency of the ultrasonic cleaning machine is 10 to 40kHz (e.g. 10, 15, 20, 25, 30, 35 or 40kHz), preferably 25 to 40kHz (e.g. 25, 30, 35 or 40 kHz).
According to some preferred embodiments, in the step (2), the alkali solution is a KOH solution and/or a NaOH solution, the concentration of the alkali solution is 0.001 to 1mol/L (e.g., 0.001, 0.005, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1mol/L), and the volume usage amount of the alkali solution is 2 to 20 times (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 times) of the volume of the pretreated dermal raw material; and/or in the step (2), the time of soaking treatment with the alkali solution is 30-120 min (for example, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 min). In the invention, the concentration of the alkali solution is preferably 0.001-1 mol/L, the volume usage of the alkali solution is preferably 2-20 times of the volume of the pretreated dermis raw material, and the soaking treatment time with the alkali solution is preferably 30-120 min; it has been found that if the concentration of the alkaline solution is too low, the amount of the alkaline solution is too small, or the treatment time is too short, the residual lipid impurities cannot be sufficiently removed, and if the concentration of the alkaline solution is too high, the amount of the alkaline solution is too large, or the treatment time is too long, collagen hydrolysis is caused, and the spatial structure of the dermal matrix is damaged.
According to some preferred embodiments, in the step (3), the acid solution is one or more of hydrochloric acid, sulfuric acid, nitric acid and formic acid, the concentration of the acid solution is 0.001 to 1mol/L (e.g., 0.001, 0.005, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1mol/L), and the volume of the acid solution is 2 to 20 times (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 times) of the volume of the pretreated dermal raw material; and/or in the step (3), the time for the decellularization treatment by the acid solution is 30-120 min (for example, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 min). In the invention, the concentration of the acid solution is preferably 0.001-1 mol/L, the volume of the acid solution is preferably 2-20 times of the volume of the pretreated dermis raw material, and the time of the acellular treatment by the acid solution is preferably 30-120 min; the invention discovers that if the concentration of the acid solution is too low, the using amount is too low or the processing time is too short, the cell membrane cannot be broken sufficiently, and the DNA cannot be eluted sufficiently; if the concentration of the acid solution is too high, the dosage is too high or the treatment time is too long, the protein is denatured, and the three-dimensional structure and the physical and chemical properties of the matrix material are damaged.
According to some preferred embodiments, in the step (5), the pH value of the phosphate buffer solution is 7.0 to 7.4 (e.g., 7.0, 7.1, 7.2, 7.3, or 7.4), and the reagent used for preparing the phosphate buffer solution is NaCl, KCl, KH2PO4And Na2HPO4The volume of the phosphate buffer solution is 10-30 times (for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 times) of the volume of the pretreated dermis raw material; and/or in the step (5), the washing time with the phosphate buffer solution is 30-120 min (for example, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 min). In the invention, the pH value of the phosphate buffer solution is preferably 7.0-7.4, and the volume consumption of the phosphate buffer solution is preferably 10-30 times of the volume of the pretreated dermis raw material, because the pH value of the extracellular fluid is 7.35-7.45, and the phosphate buffer solution with the pH value of 7.0-7.4 is selected to clean the material, so that the physiological environment of the tissue is simulated as much as possible, the residual reagent and impurities are easier to clean, the cleaning frequency is increased due to too small consumption of the phosphate buffer solution, and the waste of the phosphate buffer solution is caused due to too large consumption of the phosphate buffer solution.
According to some preferred embodiments, in step (5), the washing time with purified water is 30 to 120min (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 min).
According to some preferred embodiments, in step (6), the freeze-drying comprises the following sub-steps:
(a) pre-freezing the acellular dermal tissue obtained in the step (5) at the temperature of minus 30 ℃ for 2 hours;
(b) treating the pre-frozen acellular dermal tissue for 10 hours under the vacuum condition at the temperature of minus 10 ℃, and then treating the acellular dermal tissue for 10 hours under the vacuum condition at the temperature of 0 ℃ to complete the first-stage sublimation process of the acellular dermal tissue;
(c) and (3) treating the acellular dermal tissue which finishes the first-stage sublimation process under the vacuum condition of 5 ℃ for 4 hours, then treating the acellular dermal tissue under the vacuum condition of 15 ℃ for 2 hours, then treating the acellular dermal tissue under the vacuum condition of 25 ℃ for 2 hours, and finally treating the acellular dermal tissue under the vacuum condition of 35 ℃ for 2 hours to finish the second-stage sublimation process of the acellular dermal tissue, thereby finishing the freeze drying of the acellular dermal tissue.
In the present invention, it is preferable to perform the freeze-drying using a freeze-drying process including the above-described steps (a) to (c), and it has been found that the use of the above-described freeze-drying process is advantageous in maintaining the three-dimensional structure of the decellularized dermal tissue product.
According to some preferred embodiments, in step (7), packaging is performed using a sterile tyvek bag; and/or in step (7), the concentration of the ethylene oxide is 600-700 mg/L (for example, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690 or 700mg/L), the sterilization temperature for sterilization by using the ethylene oxide is 10-42 ℃ (for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 42 ℃), the sterilization humidity is 35-65% RH (relative humidity is 35%, 40%, 45%, 50%, 55%, 60% or 65% RH for example), and the sterilization time is 1-6 h (for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 h). A large number of creative tests prove that the optimized ethylene oxide sterilization parameters are as follows: the concentration of the ethylene oxide is 600-700 mg/L, the sterilization temperature for ethylene oxide sterilization is 10-42 ℃, the sterilization humidity is 35-65% RH, and the sterilization time is 1-6 h; the invention discovers that if the parameters such as ethylene oxide concentration, sterilization temperature, sterilization humidity, sterilization time and the like become small or less, the disqualification of sterilization can be caused; if the parameters are increased or the sterilization time is prolonged, the protein is denatured, the degradation rate of the product is increased, and the residual amount of ethylene oxide is increased.
In a second aspect, the present invention provides an acellular dermal matrix material produced by the production method according to the first aspect of the present invention.
In a third aspect, the invention provides the use of the acellular dermal matrix material prepared by the preparation method of the first aspect of the invention in the preparation of a product for dental implant or for assisting bone regeneration or guiding tissue growth. In some specific embodiments, the acellular dermal matrix material prepared by the invention is applied to dental implantation, and in dental implantation experiments, the acellular dermal matrix material prepared by the invention is found to have good bone tissue regeneration guiding performance through a section picture.
The invention will be further illustrated by way of example, but the scope of protection is not limited to these examples.
Example 1
Pretreatment of a dermis raw material:
taking animal (cattle) skin tissue materials, removing surface fat and epidermis, and cleaning with an ultrasonic cleaning machine to obtain a pretreated dermis raw material; wherein the frequency of the ultrasonic cleaning machine is 40 kHz.
② virus inactivation treatment:
soaking the pretreated dermis raw material in an alkaline solution in an ultrasonic cleaning machine, and then cleaning the dermis raw material with purified water to obtain a virus inactivation treated dermis material; wherein the frequency of the ultrasonic cleaning machine is 25kHz, the alkaline solution is NaOH solution with the concentration of 0.5mol/L, the volume consumption of the alkaline solution is 10 times of the volume of the dermal raw material pretreated in the step I, and the soaking treatment time of the alkaline solution is 60 min.
③ acellular treatment:
carrying out cell removal treatment on the virus inactivation treated dermis material obtained in the step II by using an acid solution in an ultrasonic cleaning machine; wherein the frequency of the ultrasonic cleaning machine is 25kHz, the acid solution is hydrochloric acid with the concentration of 0.5mol/L, the volume usage amount of the hydrochloric acid is 10 times of the volume of the dermal raw material pretreated in the step (i), and the time for carrying out the decellularization treatment by using the acid solution is 60 min.
Replacing the acid solution in the step III, and repeating the step III twice to obtain the cell-free processing material.
Cleaning:
washing the acellular processing material obtained in the step (IV) by using phosphate buffer solution and purified water respectively in an ultrasonic washing machine to obtain acellular dermal tissue; wherein the frequency of the ultrasonic cleaning machine is 40kHz, the pH value of the phosphate buffer solution is 7.2, and the reagents for preparing the phosphate buffer solution are NaCl, KCl and KH2PO4And Na2HPO4The volume usage amount of the phosphate buffer solution is 20 times of the volume of the pretreated dermis raw material; the washing time with phosphate buffer solution was 60min, and the washing time with purified water was 60 min.
Forming:
freeze-drying the acellular dermal tissue prepared in the fifth step; the freeze drying method comprises the following specific steps: prefreezing (temperature-30 ℃, time 2h) → first-stage sublimation (temperature-10 ℃, time 10h, evacuation) → (temperature 0 ℃, time 10h, evacuation) → second-stage sublimation (temperature 5 ℃, time 4h, evacuation) → (temperature 15 ℃, time 2h, evacuation) → (temperature 25 ℃, time 2h, evacuation) → (temperature 35 ℃, time 2h, evacuation) → freeze-drying completion.
And packaging and sterilizing:
cutting the freeze-dried acellular dermal tissue prepared in the step (sixty) into a required size, packaging the acellular dermal tissue by adopting an aseptic Tewei Qiang packaging bag, and sterilizing by adopting ethylene oxide to obtain the acellular dermal matrix material; wherein the concentration of the ethylene oxide is 670mg/L, the sterilization temperature is 37 ℃, the sterilization humidity is 55% RH, and the sterilization time is 2 h.
Example 2
Example 2 is essentially the same as example 1, except that:
in the second step, the volume usage amount of the alkaline solution is 5 times of the volume of the dermis raw material pretreated in the first step, and the soaking time of the alkaline solution is 120 min.
In the third step, the volume of the hydrochloric acid is 5 times of the volume of the dermis raw material pretreated in the first step, and the time for performing the decellularization treatment by using the acid solution is 120 min.
In the fourth step, the acid solution in the third step is replaced, and the third step is repeated for four times to obtain the acellular processing material.
In the fifth step, the volume usage amount of the phosphate buffer solution is 10 times of the volume of the dermis raw material pretreated in the first step; the washing time with phosphate buffer solution was 120min, and the washing time with purified water was 120 min.
In step (c), the sterilization temperature is 30 ℃, the sterilization humidity is 35% RH, and the sterilization time is 4 hours.
Example 3
Example 3 is essentially the same as example 1, except that:
in the second step, the volume usage amount of the alkaline solution is 20 times of the volume of the leather raw material pretreated in the first step, and the soaking time of the alkaline solution is 45 min.
In the third step, the volume of the hydrochloric acid is 20 times of the volume of the dermis raw material pretreated in the first step, and the time for performing the decellularization treatment by using the acid solution is 45 min.
In the fifth step, the volume usage amount of the phosphate buffer solution is 30 times of the volume of the dermis raw material pretreated in the first step; the washing time with phosphate buffer solution was 30min, and the washing time with purified water was 30 min.
In step (c), the sterilization temperature is 40 ℃, the sterilization humidity is 65% RH, and the sterilization time is 1 h.
The acellular dermal matrix materials prepared in examples 1 to 3 were subjected to HE staining under an optical microscope of 20 times and then observed for microstructure, and the results are respectively shown in FIG. 1, FIG. 2 and FIG. 3; the results show that the three-dimensional tissue structure of the animal skin is unchanged, the structure is complete, and impurities such as fat, hair roots and the like are not contained. Observing the decellularized dermis stroma HE stained sections prepared in example 1, example 2 and example 3 from fig. 1 to fig. 3, it can be seen that: the section has no intact cells, no nucleus material is found, and the structure of the connective tissue of the material is intact, which shows that the cell removing method can successfully remove the cells and retain the three-dimensional structure of the material.
Comparative example 1
Comparative example 1 is substantially the same as example 1 except that:
in the second step, the volume usage amount of the alkaline solution is 30 times of the volume of the leather raw material pretreated in the first step, and the soaking time of the alkaline solution is 150 min.
In the third step, the volume of the hydrochloric acid is 30 times of the volume of the dermis raw material pretreated in the first step, and the time for performing the decellularization treatment by using the acid solution is 150 min.
In the step IV, the acid solution in the step III is replaced, and the step III is repeated for six times to obtain the cell-free processing material.
In the fifth step, the volume usage amount of the phosphate buffer solution is 40 times of the volume of the dermis raw material pretreated in the first step; the washing time with phosphate buffer solution was 120min, and the washing time with purified water was 120 min.
In step (c), the sterilization temperature is 30 ℃, the sterilization humidity is 25% RH, and the sterilization time is 3 hours.
In the comparative example, the use amounts of the aqueous alkali and the hydrochloric acid are too large, and the soaking treatment time of the aqueous alkali and the cell removing treatment time of the acid solution are too long, so that the protein of the prepared material is denatured, even the acid and the alkali are hydrolyzed, and various performances of the material are damaged; also, the sterilization process in this comparative example may result in a sterile failure of the product.
Comparative example 2
Comparative example 2 is substantially the same as example 1 except that:
in the second step, the volume usage amount of the alkaline solution is 2 times of the volume of the leather raw material pretreated in the first step, and the soaking time of the alkaline solution is 20 min.
In the third step, the volume of the hydrochloric acid is 2 times of the volume of the dermis raw material pretreated in the first step, and the time for performing the decellularization treatment by using the acid solution is 20 min.
In the step (iv), the acid solution in the step (iii) is replaced, and the step (iii) is repeated 2 times to obtain the material for cell removal treatment.
In the fifth step, the volume usage amount of the phosphate buffer solution is 5 times of the volume of the dermis raw material pretreated in the first step; the washing time with phosphate buffer solution was 20min, and the washing time with purified water was 20 min.
In step (c), the sterilization temperature is 30 ℃, the sterilization humidity is 25% RH, and the sterilization time is 3 hours.
In the comparative example, the dosage of the alkali solution and the hydrochloric acid is too small, and the soaking treatment time of the alkali solution and the cell removing treatment time of the acid solution are too short, so that the cell removal of the material is incomplete, and impurities such as residual fat and the like are easy to generate; in addition, in the cleaning process, the dosage of phosphate buffer solution reagent is too small, the cleaning time is too short, the problems of unqualified pH value of the material, excessive residual DNA and the like can be caused, and various performances of the material are damaged; also, the sterilization process in this comparative example may result in a sterile failure of the product.
The acellular dermal matrix materials prepared in examples 1-3 and comparative examples 1-2 were subjected to performance testing.
1. Tear force detection
The tearing force detection method comprises the following steps: cutting the acellular dermal matrix material samples prepared in the examples 1-3 and the comparative examples 1-2 into long strips with the specification of 1cm multiplied by 3cm, penetrating the samples by a No. 4-0 suture at a position 3-5mm away from the edge of a short side, folding the suture in half, knotting the suture at a position 5cm away from a perforated position, and preventing the suture from falling off; and hydrating the sample with purified water for 3-5 min, fixing the end without threading to the lower part of a tensile testing machine, fixing the end with threading to the upper part of the tensile testing machine through a hook, starting detection until the sample is torn, and reading the maximum value of the tensile load force, namely the tearing force of the sample. 30 pieces of each sample were tested and the average of the measured tear forces is shown in Table 1.
2. Fat content detection the method for detecting the fat content is formulated according to the pancreatic enzyme fat detection in the second division of pharmacopoeia of the people's republic of China. The experiment was repeated three times, and 1g of each of the acellular dermal matrix materials in examples 1 to 3 and comparative examples 1 to 2 was taken, and the fat content was measured, and the results were averaged for the three times, as shown in table 2.
3. Detection of residual amount of DNA
The acellular dermal matrix materials prepared in examples 1 to 3 and comparative examples 1 to 2 were subjected to detection of the residual amount of DNA, by referring to part 25 of YY 0606.25-2014 tissue engineering medical products: the detection is carried out by a fluorescent staining method in animal-derived biomaterial DNA residue measurement. The experiment was repeated three times and the results were averaged over the three results as shown in table 3.
Table 1: tear force of the acellular dermal matrix materials prepared in examples 1-3 and comparative examples 1-2.
| Examples | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 |
| Tearing force | 9.89N | 10.04N | 9.95N | 5.82N | 7.36N |
Table 2: the fat content of the acellular dermal matrix materials prepared in examples 1 to 3 and comparative examples 1 to 2.
| Examples | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 |
| Fat content | 0.07％ | 0.07％ | 0.08％ | 0.19％ | 0.42％ |
Table 3: the DNA residue amounts of the acellular dermal matrix materials prepared in examples 1 to 3 and comparative examples 1 to 2.
| Examples | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 |
| Residual amount of DNA | 8.03ng/mg | 7.25ng/mg | 7.52ng/mg | 35.95ng/mg | 367.12ng/mg |
The data of DNA residue detection in the table 3 can intuitively express the acellular effect of the invention, the DNA residue of the acellular dermal matrix material prepared in the embodiments 1-3 is little, and the acellular effect is proved to be good, while the DNA residue of the material prepared in the comparative examples 1-2 is very large, and the acellular process is unsuccessful.
Finally, it should be noted that: 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 (10)
1. A method for preparing an acellular dermal matrix material, comprising the steps of:
(1) taking animal skin tissue materials, removing surface fat and epidermis, and cleaning with an ultrasonic cleaning machine to obtain a pretreated dermis raw material;
(2) soaking the pretreated dermal material obtained in the step (1) in an alkaline solution in an ultrasonic cleaning machine, and then cleaning the soaked dermal material with purified water to obtain a virus inactivation treated dermal material;
(3) carrying out cell removal treatment on the virus inactivation treated dermis material obtained in the step (2) by using an acid solution in an ultrasonic cleaning machine;
(4) repeating the step (3) for 1-5 times after replacing the acid solution to obtain a cell-free treatment material;
(5) cleaning the acellular treatment material obtained in the step (4) by using phosphate buffer solution and purified water in an ultrasonic cleaning machine in sequence to obtain acellular dermal tissue;
(6) freeze-drying the acellular dermal tissue obtained in the step (5);
(7) and (4) sequentially cutting and packaging the acellular dermal tissue subjected to freeze drying in the step (6), and then sterilizing by adopting ethylene oxide to prepare the acellular dermal matrix material.
2. The method of claim 1, wherein:
in the step (1), the animal skin tissue material is selected from porcine, bovine or ovine skin tissue, and preferably, the animal skin tissue material is selected from porcine or bovine skin tissue.
3. The method of claim 1, wherein:
the frequency of the ultrasonic cleaning machine is 10-40 kHz, and preferably 25-40 kHz.
4. The method of claim 1, wherein:
in the step (2), the alkali solution is a KOH solution and/or a NaOH solution, the concentration of the alkali solution is 0.001-1 mol/L, and the volume usage amount of the alkali solution is 2-20 times of the volume of the pretreated dermal raw material; and/or
In the step (2), the time of soaking treatment with the alkali solution is 30-120 min.
5. The method of claim 1, wherein:
in the step (3), the acid solution is one or more of hydrochloric acid, sulfuric acid, nitric acid and formic acid, the concentration of the acid solution is 0.001-1 mol/L, and the volume usage of the acid solution is 2-20 times of the volume of the pretreated dermis raw material; and/or
In the step (3), the time for carrying out the cell removing treatment by using the acid solution is 30-120 min.
6. The method of claim 1, wherein:
in the step (5), the pH value of the phosphate buffer solution is 7.0-7.4, and the reagents for preparing the phosphate buffer solution are NaCl, KCl and KH2PO4And Na2HPO4The volume usage amount of the phosphate buffer solution is 10-30 times of the volume of the pretreated dermis raw material; and/or
In the step (5), the washing time with phosphate buffer solution is 30-120 min.
7. The method of claim 1, wherein:
in step (6), the freeze-drying comprises the following sub-steps:
(a) pre-freezing the acellular dermal tissue obtained in the step (5) at the temperature of minus 30 ℃ for 2 hours;
(b) treating the pre-frozen acellular dermal tissue for 10 hours under the vacuum condition at the temperature of minus 10 ℃, and then treating the acellular dermal tissue for 10 hours under the vacuum condition at the temperature of 0 ℃ to complete the first-stage sublimation process of the acellular dermal tissue;
(c) and (3) treating the acellular dermal tissue which finishes the first-stage sublimation process under the vacuum condition of 5 ℃ for 4 hours, then treating the acellular dermal tissue under the vacuum condition of 15 ℃ for 2 hours, then treating the acellular dermal tissue under the vacuum condition of 25 ℃ for 2 hours, and finally treating the acellular dermal tissue under the vacuum condition of 35 ℃ for 2 hours to finish the second-stage sublimation process of the acellular dermal tissue, thereby finishing the freeze drying of the acellular dermal tissue.
8. The method of claim 1, wherein:
in the step (7), packaging by adopting an aseptic Terra-Wei-Qiang packaging bag; and/or
In the step (7), the concentration of the ethylene oxide is 600-700 mg/L, the sterilization temperature for ethylene oxide sterilization is 10-42 ℃, the sterilization humidity is 35-65% RH, and the sterilization time is 1-6 h.
9. An acellular dermal matrix material produced by the production method according to any one of claims 1 to 8.
10. Use of the acellular dermal matrix material prepared by the preparation method according to any one of claims 1 to 8 for the preparation of a product for dental implantation or for assisting bone regeneration or for guiding tissue growth.
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