Low-endotoxin-level acellular dermal matrix and preparation method thereof

Abstract

The invention relates to the technical field of medical biological materials, in particular to a low-endotoxin-level acellular dermal matrix as well as a preparation method and application thereof. The acellular dermal matrix disclosed by the invention is prepared by decellularizing dermal tissues of aquatic animals, cleaning in a multi-stage manner and drying and forming. According to the scheme provided by the invention, the endotoxin of the aquatic-source acellular dermal matrix can be effectively controlled, so that the obtained acellular dermal matrix has a low endotoxin level while keeping a bionic structure of a raw material, and the problems of high endotoxin content of the acellular dermal matrix of aquatic animals, high biological safety risk in clinical application and the like are particularly solved; the material reaches an implantation level, and the application range of the acellular dermal matrix in the high-risk medical field is greatly expanded. In addition, the acellular dermal matrix also has the characteristic of low immunogenicity and good biological tolerance, and a treatment mode which is more consistent with the healing period of a patient can be customized in a repeated implantation mode according to requirements.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, and in particular to a decellularized dermal matrix with a low endotoxin level, and a preparation method and application thereof. Background Art

[0002] Acellular dermal matrix (ADM) is a type of acellular matrix. It refers to the extracellular matrix and fibrous structure of the dermis layer with relatively weak immunogenicity left after the skin of animals from different sources is treated by a cell-free process and the components with strong antigenicity in the skin tissue are removed. The ideal ADM material can not only provide a three-dimensional structural matrix, but also has good biocompatibility and plasticity, and has a degradation rate in the body that is adapted to tissue growth. Due to the removal of highly antigenic cell components, ADM can avoid acute rejection after transplantation, is suitable for cell migration, proliferation and growth, and has excellent tissue reconstruction function. In recent years, ADM materials have received widespread attention from the medical and material communities at home and abroad, and medical device products made from it have also been widely used in clinical practice. As a protein-based material, ADM's main components are fibrous proteins such as collagen and elastin. In addition, it also contains proteoglycans, growth factors and other components. Compared with protein materials with a single component, it is more biomimetic and has higher biological activity. But because of this, ADM materials are easily attached and contaminated by bacteria, resulting in excessive endotoxin levels in the materials after sterilization, causing biosafety risks.

[0003] ADM involves physical or chemical decellularization methods in its preparation process, and these treatment processes can more or less reduce the endotoxin level of the material. However, since ADM is a material with a microscopic scaffold structure, endotoxins can be adsorbed onto the amphiphilic protein scaffold wall through secondary bonds and other methods, resulting in the inability of simple decellularization methods to effectively remove endotoxins. High-intensity decellularization treatment methods will destroy the scaffold structure and beneficial components of ADM, which is a double-edged sword.

[0004] Patent CN118576777A discloses a porcine decellularized dermal matrix, which is treated with alkali, degreasing agent, and biological enzymes, followed by final cleaning with purified water, injection water, glycerol, and sodium lactate. The chemical reaction preparation in this patent has limited effect on endotoxin removal, and the cleaning process is difficult to effectively remove endotoxins adsorbed in the three-dimensional porous structure of the porcine skin.

[0005] Patent CN112618799A discloses a fish skin acellular dermal matrix, which is obtained by secondary peracetic acid virus inactivation, surfactant decellularization, and neutral salt and EDTA cleaning. The preparation described in the patent can only achieve cell removal effect, and the decellularization and removal process cannot effectively remove endotoxins inside the material.

[0006] Patent CN108187140A discloses a fish skin acellular dermal matrix, which uses plasma cleaning for disinfection, alkaline solution for degreasing, potassium permanganate and sodium bisulfite for decolorization, and is treated with high and low osmotic pressure, strong alkali and repeated freeze-thaw. Although the overall endotoxin level can be controlled to a certain extent, the strong oxidizing property of the preparation and the relatively complex process destroy the structure of the ADM material.

[0007] In summary, existing patents focus more on the physical and chemical characteristics of the material residual cell removal and the maintenance of the material structure stability, while ignoring the stringent requirements of medical biomaterials on the endotoxin level. In view of this, the present invention is proposed. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an implant-grade acellular dermal matrix with low endotoxin level and complete material bionic structure.

[0009] Specifically, the technical solution of the present invention is as follows: The present invention provides a low-endotoxin-level acellular dermal matrix, which is prepared by decellularization, multi-stage cleaning and drying of aquatic animal dermal tissue; the endotoxin level of the prepared acellular dermal matrix does not exceed 0.025 EU / mg; and has at least one of the following characteristics: (1) water absorption rate is 2-5 times of its own weight; (2) when fully infiltrated with physiological saline, the tensile strength is 3-30 N; the suture strength is 1.5-8N; (3) the thickness is 0.7-1.3mm.

[0010] Preferably, the preparation method of the decellularized dermal matrix with low endotoxin level comprises: preparing from aquatic animal dermal tissue by decellularization, multi-stage cleaning and drying and forming; the multi-stage cleaning comprises at least two of flushing, immersion, high-speed stirring, bubbling, rinsing, spray cleaning, ultrasonic cleaning, pulse electrolysis cleaning, and rotary cleaning; the cleaning solution of the first stage cleaning comprises one of phosphate buffer, borate buffer, acetate buffer, citric acid saline solution, saline, and purified water; the cleaning solution of the second stage cleaning comprises one of phosphate buffer, borate buffer, saline, purified water, and water for injection; the cleaning process avoids material agglomeration, and the cleaning process temperature is 2-10°C.

[0011] Preferably, after the multi-stage cleaning, dehydration is first performed by one or more of centrifugation, screening filtration, vacuum dehydration, and adsorption dehydration, and then drying and forming is performed.

[0012] Preferably, decellularization is performed by chemical methods; the chemical methods include one or more of biological enzymes, surfactants, acids and bases, alcohols, ketones, and hypertonic salts.

[0013] Preferably, when the chemical method comprises at least two methods, a washing step is further included between each two chemical reactions: non-acidic and alkaline reagents are washed with purified water; acidic and alkaline reagents are washed with purified water and neutral buffer salt; the washing time is 2 to 30 hours.

[0014] Preferably, the biological enzyme comprises one or more of pepsin, trypsin, elastase, type IV protease, papain, and tyrosinase, and the reaction time is preferably 15-40 h.

[0015] Preferably, the surfactant comprises nonionic and ionic surfactants, and in particular does not comprise sodium lauryl sulfate. The reaction time is preferably 12-35 h.

[0016] Preferably, the acid or base comprises one or more of hydrochloric acid, peracetic acid, sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and the reaction time is preferably 2-8 h.

[0017] Preferably, the alcohol comprises one or more of ethanol, methanol, pentanediol, n-butanol, isopentanol, and isopropanol, and the reaction time is 4-12 h.

[0018] Preferably, the ketone is particularly acetone and the reaction time is 1-6 h.

[0019] Preferably, the hypertonic salt comprises one or more of sodium chloride, phosphate, borate, acetate, carbonate, and formate, preferably at a concentration of 3-9%, and a reaction time of 8-24 h.

[0020] Preferably, decellularization can be performed by chemical methods in combination with physical methods; the physical methods include one or more of pressurization, freeze-thaw, ultrasound, and supercritical carbon dioxide.

[0021] Preferably, pressurizing comprises high net hydraulic pressurization.

[0022] Preferably, the freeze-thaw comprises high-frequency freeze-thaw, the number of freeze-thaw cycles is not less than 5 times, and the frequency is 3-10 MHz.

[0023] Preferably, the ultrasound comprises low-temperature low-frequency ultrasound with a temperature not exceeding 10° C. and a frequency of 0.05-0.075 MHz.

[0024] Preferably, the drying and molding is carried out by vacuum drying or freeze drying.

[0025] Preferably, sterilization is performed after drying and molding, and the sterilization is performed by one of irradiation sterilization and ethylene oxide sterilization.

[0026] Beneficial effects: The present invention provides a decellularized dermal matrix with low endotoxin levels and a preparation method thereof, wherein the decellularized dermal matrix is ​​prepared by decellularization, multi-stage cleaning and drying of aquatic animal dermal tissue. The scheme provided by the present invention can effectively control the endotoxins of aquatic decellularized dermal matrix, so that the obtained decellularized dermal matrix has a low endotoxin level while retaining the bionic structure of the raw material, and solves the problems of high endotoxin content in aquatic animal decellularized dermal matrix and high biosafety risk in clinical application. The material reaches the implantation level, which greatly expands the application scope of the decellularized dermal matrix in high-risk medical fields. The decellularized dermal matrix of the present invention also has the characteristics of low immunogenicity and good biological tolerance. It can be customized according to needs and through repeated implantation to a treatment method that is more consistent with the patient's healing period. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0028] Figure 1 This is the appearance of the acellular dermal matrix of the present invention.

[0029] Figure 2 The cross-sectional structure of the acellular dermal matrix of the present invention under an electron microscope. DETAILED DESCRIPTION

[0030] The invention provides an implantation-grade acellular dermal matrix with low endotoxin level and complete material bionic structure.

[0031] The acellular dermal matrix of the present invention is derived from the skin tissue of terrestrial animals or aquatic animals, preferably from the skin tissue of aquatic animals, and more preferably from the skin of marine or freshwater fish.

[0032] The acellular dermal matrix of the present invention includes but is not limited to sheet, mesh, porous, granular, and gel forms.

[0033] The acellular dermal matrix of the present invention has an endotoxin level of no more than 0.025 EU / mg.

[0034] Endotoxin levels in clean packaging materials that have passed packaging integrity verification can remain stable for at least 3 years.

[0035] The acellular dermal matrix of the present invention has a dry texture and an opaque white appearance. The acellular dermal matrix of the present invention has a water absorption rate of 2-5 times its own weight.

[0036] The acellular dermal matrix of the present invention has a tensile strength of 3-30 N and a suture strength of 1.5-8 N when fully infiltrated with physiological saline.

[0037] The acellular dermal matrix of the present invention has a thickness of 0.7-1.3 mm.

[0038] The acellular dermal matrix of the present invention has a natural animal dermal tissue structure and is prepared in a non-cross-linking manner.

[0039] According to a specific embodiment of the present invention, the acellular dermal matrix has no pyrogenic effect on rabbits.

[0040] According to a specific embodiment of the present invention, the acellular dermal matrix has an extremely low immune risk, and after being implanted into an animal, the proliferation level of lymphocytes of the animal is comparable to that of a negative control.

[0041] According to a specific embodiment of the present invention, the preparation method of the acellular dermal matrix is ​​as follows: (1) Pretreatment: Clean the animal dermis and remove impurities such as the epidermis and residual meat.

[0042] (2) Decellularization: Use chemical (or chemical combined with physical) methods to remove residual cells and some endotoxins inside and outside the animal dermis tissue in step (1).

[0043] (3) Precision cleaning: A multi-stage cleaning process is used to remove endotoxins inside and outside the animal dermis tissue in step (2).

[0044] (4) Drying and molding: The acellular dermis material obtained in step (3) is dried and molded into a certain shape.

[0045] (5) Packaging and sterilization: The acellular dermal matrix dry product obtained in step (4) is sealed and packaged, and the finished product is obtained after sterilization.

[0046] Wherein, the chemical method of step (2) comprises one or more of biological enzymes, surfactants, acids and bases, alcohols, ketones, and hypertonic salts. The biological enzymes include but are not limited to one or more of pepsin, trypsin, elastase, type IV protease, papain, and tyrosinase, and the reaction time is preferably 15-40 h; the surfactants include but are not limited to nonionic and ionic surfactants, especially excluding sodium dodecyl sulfate, and the reaction time is preferably 12-35 h; the acids and bases include but are not limited to one or more of hydrochloric acid, peracetic acid, sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and the reaction time is preferably 2-8 h; the alcohols include but are not limited to one or more of ethanol, methanol, pentanediol, n-butanol, isoamyl alcohol, and isopropanol, and the reaction time is 4-12 h; the ketones are especially acetone, and the reaction time is 1-6 h; the hypertonic salts include but are not limited to one or more of sodium chloride, phosphate, borate, acetate, carbonate, and formate, and the concentration is preferably 3-9%, and the reaction time is 8-24 h.

[0047] If multiple chemical reactions are used in step (2), a washing step is included between each two chemical reactions. If non-acidic or alkaline preparations are used during the chemical reaction, purified water is used for washing; if acidic or alkaline preparations are used during the chemical reaction, purified water and neutral buffer salt are used for washing; the washing time is 2 to 30 hours.

[0048] Step (2) may also be combined with a physical method. The physical method comprises one or more of high pressure, freeze-thaw, ultrasound, and supercritical carbon dioxide. In particular, high pressure comprises high net hydraulic pressurization; freeze-thaw comprises high frequency freeze-thaw, preferably the number of freeze-thaw cycles is not less than 5 times; ultrasound comprises low temperature and low frequency ultrasound, preferably the temperature does not exceed 10°C.

[0049] The multi-stage cleaning in step (3) includes but is not limited to at least two of flushing, immersion, high-speed stirring, bubbling, rinsing, spraying, ultrasonic cleaning, pulse electrolytic cleaning, and rotary cleaning. In particular, the cleaning process avoids material agglomeration, and the cleaning process temperature is 2-10°C.

[0050] In the multi-stage cleaning described in step (3), the cleaning solution for the first stage of cleaning includes but is not limited to one of phosphate buffer, borate buffer, acetate buffer, citric acid saline solution, saline solution, and purified water; the cleaning solution for the second stage of cleaning includes one of phosphate buffer, borate buffer, saline solution, purified water, and water for injection. If the multi-stage cleaning is more than two stages, the cleaning solution for the third stage and subsequent cleanings includes one of purified water and water for injection.

[0051] The multi-stage cleaning described in step (3) also includes dehydration after cleaning.

[0052] The dehydration in step (3) specifically comprises one or more of centrifugation, sieving and filtration, vacuum dehydration, and adsorption dehydration.

[0053] The drying in step (4) includes vacuum drying and freeze drying.

[0054] The sterilization in step (5) includes one of irradiation sterilization and ethylene oxide sterilization.

[0055] The acellular dermal matrix of the present invention can be used for, including but not limited to, wound damage repair, medical cosmetic coverage or filling, heart valves, bone defect filling or covering repair, dental postoperative coverage, cartilage defect repair, corneal repair, tissue thickening, residual cavity filling, hemostasis, nerve repair, etc.

[0056] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0058] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0059] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0060] Example 1 This example provides an acellular dermal matrix with a low endotoxin level, and investigates and compares the endotoxin level of the acellular dermal matrix.

[0061] The method for preparing the acellular dermal matrix with low endotoxin level provided in this embodiment is as follows: Take the fish skin of black carp, wash it with water and remove impurities such as epidermis and residual meat. Repeat the freezing and thawing of the fish skin 5 times at a frequency of 50 min / time, then add 0.5% concentration of peracetic acid to the fish skin and react at low temperature for 4 hours. After the reaction, wash it with purified water for 1 hour and rinse it with neutral phosphate buffer for 1 hour. Then add 0.3% concentration of Triton X-114 surfactant solution and continue to react at low temperature for 12 hours to remove residual cells. The decellularized fish skin was immersed in citric acid saline solution for high-speed stirring for 1 hour, immersed in phosphate buffer for 4 hours, and bubbled in injection water for 4 hours. After centrifugation to remove water, the fish skin was freeze-dried to obtain acellular dermal matrix dry product. The dry product was cut into a mesh, packaged in a paper-plastic bag, and sterilized with ethylene oxide to obtain the finished acellular dermal matrix.

[0062] The method for investigating the endotoxin level of the acellular dermal matrix in this example is as follows: take the finished acellular dermal matrix, add a certain volume of endotoxin test water at 37°C and extract for 1 h as the test solution according to the requirements of GB / T 16886.12. Test the endotoxin of the test sample according to the bacterial endotoxin test method in Part IV, 1143 of the Pharmacopoeia of the People's Republic of China (2020 edition).

[0063] Comparative Example 1.1: A fish skin acellular dermal matrix was prepared according to the method of Example 1 in patent CN112618799A, and the endotoxin of the sample was determined according to the endotoxin detection method of Example 1 of the present invention.

[0064] Comparative Example 1.2: A fish skin acellular dermal matrix was prepared according to the method of Example 1 in patent CN108478869A, and the endotoxin of the sample was determined according to the endotoxin detection method of Example 1 of the present invention.

[0065] The endotoxin test results of Example 1 and Comparative Examples 1.1 and 1.2 are shown in Table 1, which shows that the endotoxin of the acellular dermal matrix of the present invention is significantly better than that of the acellular dermal matrix of the disclosed technology.

[0066] Table 1 Endotoxin levels in acellular dermal matrix .

[0067] Example 2 This example provides an acellular dermal matrix with a low endotoxin level, and investigates the appearance, tensile strength, suture strength, thickness and endotoxin level of the acellular dermal matrix.

[0068] The method for preparing the acellular dermal matrix with low endotoxin level provided in this embodiment is as follows: Take the skin of Spanish mackerel, wash it with water and remove impurities such as epidermis and residual meat. The fish skin was treated with 100W ultrasound for 2 h, and then 0.003% trypsin was added to the fish skin for low temperature reaction for 24 h. After the reaction, it was washed with purified water for 30 h. Then 0.3% sodium hydroxide was added and continued to react at low temperature for 2 h to remove residual cells. The decellularized fish skin was rinsed with phosphate buffer for 10 min, bubbled in saline solution for 4 h, ultrasonically cleaned in purified water at 80W for 1 h, and sprayed in water for injection for 3 h. After sieving and filtering to remove moisture, the fish skin was freeze-dried to obtain acellular dermal matrix dry product. The dry product was cut into sheets, packaged in paper and plastic bags, and sterilized with ethylene oxide to obtain the finished acellular dermal matrix.

[0069] The method for investigating the appearance, tensile strength, suture strength and thickness of the acellular dermal matrix in this embodiment is as follows: Tensile strength test method: Cut the acellular dermal matrix into 54×10 mm strips, and clamp the upper and lower ends of the strips with a clamping depth of 9 mm. Set the clamp movement speed to 50 mm / min, start the tensile machine until it breaks, and record the maximum tensile force at the break.

[0070] Suture strength test method: Cut the acellular dermal matrix into 54×10 mm strips, pass through the middle of the upper end 4 mm from the edge with No. 2 suture, tie a knot 5 cm away from the perforation and fix it to the upper end of the tensile machine; the lower end of the strip is clamped with a clamp, and the clamping depth is 9 mm. Set the clamp movement speed to 100 mm / min, start the tensile machine until it breaks, and record the maximum tensile force at the break.

[0071] Thickness detection: Use a fully automatic thickness gauge to measure the thickness of the four corners and center of the sample.

[0072] Appearance inspection: visual inspection.

[0073] Endotoxin detection: refer to the method in Example 1.

[0074] See the appearance results for details. Figure 1 As can be seen from the figure, the acellular dermal matrix is ​​white and opaque. The tensile strength, suture strength, and thickness test results are shown in Table 2. From the table, it can be seen that the tensile strength of the acellular dermal matrix of the present invention is between 3-30 N, the suture strength is between 1.5-8 N, and the thickness of each point is between 0.7-1.3 mm. Endotoxin <0.025 EU / mg, reaching an extremely low level.

[0075] Table 2 Endotoxin, tensile strength, suture strength and thickness of acellular dermal matrix .

[0076] Example 3 This example provides an acellular dermal matrix with a low endotoxin level, and investigates the tensile strength, suture strength, thickness and endotoxin level of the acellular dermal matrix.

[0077] The method for preparing the acellular dermal matrix with low endotoxin level provided in this embodiment is as follows: Take the fish skin of grass carp, wash it with water and remove impurities such as epidermis and residual meat. Add 0.003% tyrosinase to the fish skin and react at low temperature for 15 hours. After the reaction, wash it with purified water for 20 hours. Add 0.1% ammonium hydroxide to the fish skin and react at low temperature for 2 hours, wash it with purified water for 2 hours, and wash it with neutral borate buffer for 6 hours. Then add 0.4% Triton X-100 and continue to react at low temperature for 16 hours to remove residual cells. The decellularized fish skin was rinsed with purified water for 12 minutes, washed with pulse electrolysis in injection water for 2 hours, and washed with injection water at high speed for 2 hours. After sieving and filtering to remove water, the fish skin was freeze-dried to obtain acellular dermal matrix dry product. The dry product was cut into sheets, packaged in paper and plastic bags, and sterilized with ethylene oxide to obtain the finished acellular dermal matrix.

[0078] The test methods for tensile strength, suture strength, thickness and endotoxin level are carried out in accordance with Example 2.

[0079] The tensile strength, suture strength and thickness test results are shown in Table 3. It can be seen from the table that the tensile strength of the acellular dermal matrix of the present invention is between 3-30 N, the suture strength is between 1.5-8 N, and the thickness at each point is between 0.7-1.3 mm. Endotoxin is less than 0.025 EU / mg, reaching an extremely low level.

[0080] Table 3 Endotoxin, tensile strength, suture strength and thickness of acellular dermal matrix .

[0081] Example 4 This example provides an acellular dermal matrix with a low endotoxin level, and investigates the water absorption rate and microstructure of the acellular dermal matrix.

[0082] The method for preparing the acellular dermal matrix with low endotoxin level provided in this embodiment is as follows: Take the skin of tilapia, wash it with water and remove impurities such as epidermis and residual meat. Add 0.001% elastase to the skin and react at low temperature for 36 hours. After the reaction, wash it with purified water for 16 hours. Then add 0.2% potassium hydroxide and continue to react at low temperature for 3 hours to remove residual cells. The decellularized fish skin was washed with borate buffer at high speed for 40 minutes, rotated in purification for 5 hours, and bubbled in injection water for 2 hours. After vacuum dehydration, the fish skin was freeze-dried to obtain acellular dermal matrix dry product. The dry product was cut into sheets, packaged in aluminum foil bags, and irradiated and sterilized to obtain the finished acellular dermal matrix.

[0083] The method for investigating the water absorption rate of the acellular dermal matrix in this embodiment is: Water absorption test: Weigh the sample and record the weight m1. Then put the sample into purified water at 37°C for 20 minutes, use tweezers to grab one corner of the sample and take it out of the water. Drain off excess water and weigh it again. Record the weight m2. The water absorption multiple is calculated as A= (m2-m1) / m2.

[0084] Comparative Example 4.1: The fish skin of tilapia was taken, washed with water, and then the skin, residual meat and other impurities were removed and then freeze-dried directly. The water absorption rate of the sample was measured according to the water absorption rate detection method of Example 3 of the present invention.

[0085] Comparative Example 4.2: A fish skin acellular dermal matrix was prepared according to the method of Example 1 of patent CN108187140A, and the water absorption rate of the sample was measured according to the water absorption rate detection method of Example 3 of the present invention.

[0086] The test results are shown in Table 4. From the results, it can be seen that after a series of process treatments, the final water absorption rate of the decellularized dermal matrix of the present invention is not significantly different from that of the fish skin (Comparative Example 4.1) that has not been processed, indicating that the overall microstructure of the decellularized dermal matrix of the present invention has not changed significantly. The water absorption rate of the decellularized dermal matrix of Comparative Example 4.2 is significantly higher than that of Example 4, indicating that excessive process treatment destroys the microstructure of the material, resulting in an increase in the pore size of the material and thus an increase in the water absorption rate. In addition, it can be seen from the results that the endotoxin level of the decellularized dermal matrix of the present invention is significantly better than that of the comparative example.

[0087] Table 4 Water absorption and endotoxin level of acellular dermal matrix .

[0088] The method for examining the microstructure of the acellular dermal matrix in this example is: the acellular dermal matrix of Example 4 is subjected to gold spraying and then observed using an electron microscope.

[0089] The electron microscopy results are shown in Figure 2 ,From the figure, we can see that the structure of the acellular dermal matrix is ​​intact, and there is no obvious collapse or broken structure.

[0090] Example 5 This example investigates the thermogenic response of the acellular dermal matrix of Example 4.

[0091] According to 3 cm 2 / mL extraction ratio, and the sample was extracted with physiological saline at 37℃ for 72 h to prepare the extract. Three healthy New Zealand rabbits aged 3 months were selected, and the 8 temperature tests were all in the range of 38.0~39.6℃, with a body temperature difference of ±0.4℃. The laboratory temperature was controlled at 20~25℃, and the test process was kept quiet and dim. The extract was injected into the rabbit's ear vein at a rate of (2~3)mL / min at 38℃. The rabbit's anal body temperature was measured once every 30 minutes, and the measurement was continued for 6 times. The elevated temperature calculation was expressed as the difference between the highest value of the 6 temperature tests and the normal body temperature value. According to Part IV of the Pharmacopoeia of the People's Republic of China, when the body temperature increase of the three rabbits is less than 0.6℃, and the total body temperature increase of the three rabbits is less than 1.3℃, it can be judged that the material has no pyrogenic effect.

[0092] The results of the pyrogenic test are shown in Table 5. From the results, it can be seen that the body temperature of the three rabbits increased by <0.6°C, and the total temperature increase was <1.3°C, indicating that the acellular dermal matrix of the present invention has no pyrogenic effect. It can also indirectly prove that the endotoxin and other pyrogen substances in the acellular dermal matrix of the present invention are controlled at an extremely low level.

[0093] Table 5 Pyrogenic test of acellular dermal matrix .

[0094] Example 6 This example investigates the immunogenicity of the acellular dermal matrix of Example 2.

[0095] Twelve Balb / c mice were selected and divided into two groups: a test group (S) and a negative group (N), with 6 mice in each group, half male and half female. Among them, one piece of the decellularized dermal matrix sample of the present invention soaked in physiological saline was implanted subcutaneously on the back of the mouse in the test group, and the negative group was a sham operation group. The animals were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and subcutaneous implantation was performed after shaving. The immunization cycle was 28 days. At the end of the immunization cycle, the animals were killed by cervical vertebrae removal, the spleen was removed aseptically, and the cell suspension was prepared by grinding. After centrifugation to remove the supernatant, 5 mL of red blood cell lysis buffer was added and incubated at 37°C for 15 min, 5 mL of RPMI1640 culture medium containing 10% FCS was added to terminate the reaction, the mixture was mixed and centrifuged to remove the supernatant, and then RPMI1640 culture medium containing 10% FCS was added to adjust the cell concentration to 2×10 6 cells / mL.

[0096] Splenic lymphocyte grouping: a) Non-stimulation group (-): spleen lymphocytes of test animals. b) Stimulation group (+): spleen lymphocytes of test animals + ConA, final concentration 5 μg / mL. Take the lymphocyte suspension of each animal mentioned above, wash twice with PBS, discard the supernatant, resuspend the cells with 2 mL of PBS solution containing 0.1% BSA, and add CFSE to a final concentration of 5 μmol / L. Incubate at 37℃ in the dark for 15 min. Add 3 mL of ice-cold RPMI1640 culture medium containing 2% FCS to terminate the staining reaction, mix gently, centrifuge at 400 g for 5 min, discard the supernatant, add 3 mL of RPMI1640 culture medium containing 10% FCS, and mix well.

[0097] According to the above grouping, the cell suspension was added to a 24-well plate at 1 mL / well. The corresponding ConA was added to the stimulation group at a final concentration of 5 μg / mL. The test plate was incubated at 37°C, 5% CO, and cultured for 3 days. The cells in each well were collected and stained with PE-labeled anti-mouse CD3 monoclonal antibody. After incubation at 4°C in the dark for 30 min, flow cytometry was used for analysis.

[0098] The test results are shown in Table 6. From the results, it can be seen that the acellular dermal matrix of the present invention, whether in the stimulation group or the non-stimulation group, has no significant difference with the group related to the negative control, which proves that the acellular dermal matrix of the present invention does not have an immune safety risk.

[0099] Table 6 Proliferation percentage and mean of spleen lymphocytes in each group Example 7 This example investigates the changes in endotoxin levels of the acellular dermal matrix of Example 1 over 3 years.

[0100] The decellularized dermal matrix of Example 1 was stored at 30±2°C and humidity (60±5)% for three years, and the endotoxin content of the samples was tested at 6 months, 12 months, 18 months, 24 months and 36 months. The endotoxin testing method was the same as that of Example 1.

[0101] The changes in the endotoxin levels of the samples within three years are shown in Table 7. From the results, it can be seen that the endotoxin level of the acellular dermal matrix of the present invention remains stable within three years.

[0102] Table 7 Endotoxin levels in acellular dermal matrix within three years .

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An acellular dermal matrix with low endotoxin level, characterized in that It is made from aquatic animal dermal tissue through decellularization, multi-stage cleaning and drying. The endotoxin level of the prepared decellularized dermal matrix does not exceed 0.025 EU / mg, and it also has at least one of the following characteristics: (1) the water absorption rate is 2-5 times of its own weight; (2) when fully infiltrated with physiological saline, the tensile strength is 3-30 N; the suture strength is 1.5-8N; (3) the thickness is 0.7-1.3mm.

2. The acellular dermal matrix with low endotoxin level according to claim 1, characterized in that: The preparation method comprises: preparing the dermal tissue of an aquatic animal through decellularization, multi-stage cleaning and drying and forming; the multi-stage cleaning comprises at least two of flushing, immersion, high-speed stirring, bubbling, rinsing, spraying, ultrasonic cleaning, pulse electrolysis cleaning and rotary cleaning; the cleaning solution of the first stage cleaning comprises one of phosphate buffer, borate buffer, acetate buffer, citric acid saline solution, saline solution and purified water; the cleaning solution of the second stage cleaning comprises one of phosphate buffer, borate buffer, saline solution, purified water and water for injection; the material is prevented from agglomerating during the cleaning process, and the temperature of the cleaning process is 2-10°C.

3. The acellular dermal matrix with low endotoxin level according to claim 2, characterized in that: After multi-stage cleaning, the product is first dehydrated by one or more of centrifugation, screening filtration, vacuum dehydration, and adsorption dehydration, and then dried and formed.

4. The acellular dermal matrix with low endotoxin level according to claim 2, characterized in that: Decellularization adopts chemical methods; the chemical methods include one or more of biological enzymes, surfactants, acids and bases, alcohols, ketones, and hypertonic salts.

5. The acellular dermal matrix with low endotoxin level according to claim 4, characterized in that: When the chemical method comprises at least two chemical reactions, a washing step is further included between each two chemical reactions: non-acidic and alkaline reagents are washed with purified water; acidic and alkaline reagents are washed with purified water and neutral buffer salt; and the washing time is 2 to 30 hours.

6. The acellular dermal matrix with low endotoxin level according to claim 4, characterized in that: The biological enzyme includes one or more of pepsin, trypsin, elastase, type IV protease, papain, and tyrosinase, and the reaction time is preferably 15-40 h; Alternatively, the surfactant comprises nonionic and ionic surfactants, and in particular does not comprise sodium lauryl sulfate, and the reaction time is preferably 12-35 h; Alternatively, the acid or base comprises one or more of hydrochloric acid, peracetic acid, sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and the reaction time is preferably 1-6 h; Alternatively, the alcohol includes one or more of ethanol, methanol, pentanediol, n-butanol, isopentanol, and isopropanol, and the reaction time is 4-12 h; Alternatively, the ketone is particularly acetone, and the reaction time is 1-6 h; Alternatively, the hypertonic salt comprises one or more of sodium chloride, phosphate, borate, acetate, carbonate, and formate, preferably at a concentration of 3-9%, and a reaction time of 8-24 h.

7. The acellular dermal matrix with low endotoxin level according to claim 4, characterized in that: Decellularization is carried out by using chemical methods in combination with physical methods; the physical methods include one or more of pressurization, freeze-thaw, ultrasound, and supercritical carbon dioxide.

8. The acellular dermal matrix with low endotoxin level according to claim 7, characterized in that: Pressurization includes high net hydraulic pressurization; Alternatively, the freeze-thaw comprises high-frequency freeze-thaw, the number of freeze-thaw cycles is not less than 5 times, and the frequency is 3-10 MHz; Alternatively, the ultrasound comprises low temperature low frequency ultrasound at a temperature not exceeding 10°C and a frequency of 0.05-0.075 MHz.

9. The acellular dermal matrix with low endotoxin level according to any one of claims 2 to 8, characterized in that: The drying and molding is performed by vacuum drying or freeze drying. After the drying and molding, sterilization is performed, and the sterilization is performed by irradiation sterilization or ethylene oxide sterilization.

10. The method for preparing the acellular dermal matrix with low endotoxin level according to any one of claims 1 to 9, characterized in that: include: The product is prepared from aquatic animal dermal tissue through decellularization, multi-stage cleaning and drying and forming; the multi-stage cleaning comprises at least two of flushing, immersion, high-speed stirring, bubbling, rinsing, spraying, ultrasonic cleaning, pulse electrolysis cleaning and rotary cleaning; the cleaning solution of the first stage cleaning comprises one of phosphate buffer, borate buffer, acetate buffer, citric acid saline solution, saline solution and purified water; the cleaning solution of the second stage cleaning comprises one of phosphate buffer, borate buffer, saline solution, purified water and water for injection; the cleaning process avoids material agglomeration, and the cleaning process temperature is 2-10°C.

Images