A preparation method of autologous skull replantation matrix and its product

Abstract

The present invention relates to a preparation method of an autologous skull replantation matrix and its product, the preparation method comprising: sequentially subjecting the pretreated autologous skull to alkali treatment and enzyme treatment under ultrasonic conditions; subjecting the obtained autologous skull to perforation treatment, subjecting the obtained autologous skull to alkali treatment under ultrasonic conditions, and then subjecting the obtained autologous skull to degreasing and decellularization treatment to obtain the skull matrix; mixing with a solution containing BMPs, and incubating; mixing a solution containing collagen with a solution promoting BMPs adsorption under ultrasonic conditions, and then mixing and incubating with BMPs to obtain a first mixed solution, compounding the first mixed solution with the obtained skull, and freeze-drying. The replantation autologous skull matrix obtained by the preparation method of the present invention has good biocompatibility, no inflammatory reaction and no autologous absorption after surgery, and enhanced skull repair effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to a preparation method of an autologous skull replantation matrix and a product thereof. Background Art

[0002] The main function of the skull is to protect brain tissue and maintain normal physiological pressure inside and outside the skull. When decompressive craniectomy is required due to traffic accidents, tumor surgery, etc., the brain tissue loses its natural barrier, which may cause a series of problems such as local tissue bulging, changes in cerebrospinal fluid circulation and cerebral hemodynamics. At the same time, the appearance defects also affect the patient's mental health. Therefore, it is necessary to repair the skull to address the above problems. In cranioplasty, 3D shaping titanium mesh is widely used, but it has low bone integration ability, high postoperative skin ulceration and exposure risks, and high cost. The use of autologous skull reimplantation has the advantages of low biological homology rejection, bone healing with surrounding tissues, and low cost, and the appearance of the repair is highly satisfactory, and patients are psychologically easy to accept. However, low-temperature freezing of autologous bone is often used, which requires a long time to store, and has strict requirements for storage conditions, generally -80℃, requires proprietary equipment, and is prone to bone resorption after implantation, which limits its application in clinical cranioplasty.

[0003] In the existing treatment process, the normal structure of bones composed of organic matter and calcium salts is destroyed, which is equivalent to allogeneic bone transplantation to a certain extent, and the incidence of bone absorption is high. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing an autologous skull bone reimplantation matrix and a product thereof.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing an autologous skull bone reimplantation matrix, the preparation method comprising:

[0007] (1) The pre-treated autologous skull is subjected to alkali treatment and enzyme treatment in sequence under ultrasonic conditions;

[0008] (2) drilling the autologous skull obtained in step (1), treating it with alkali under ultrasonic conditions, and then performing defatting and decellularization to obtain a skull matrix;

[0009] (3) mixing the skull matrix obtained in step (2) with a solution containing BMPs and incubating;

[0010] (4) mixing the solution containing collagen with the solution promoting BMPs adsorption under ultrasonic conditions, and then mixing and incubating with BMPs to obtain a first mixed solution, and compounding the first mixed solution with the skull obtained in step (3), and freeze-drying to obtain;

[0011] The BMPs are BMP2 or BMP7.

[0012] BMPs are bone morphogenetic proteins. The method for preparing the autologous skull replantation matrix provided by the present invention has good biocompatibility, no inflammatory reaction and no autoabsorption after surgery, and enhanced skull repair effect. The above method has little effect on the normal structure of autologous bone, that is, it can provide a three-dimensional structure closer to natural tissue, provide a bionic microenvironment after replantation, promote cell ingrowth and survival, and is conducive to the formation of bone healing, and the two alkali treatments can reduce the content of endotoxin.

[0013] Preferably, the compounding method specifically includes: soaking the skull in the first mixed liquid for 1-10 h, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, 10 h, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0014] Preferably, the pretreatment includes washing the autologous skull with isotonic saline and removing soft tissue.

[0015] Preferably, the power of the ultrasound is independently 50-200 W, for example 50 W, 60 W, 70 W, 80 W, 90 W, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W, 190 W, 200 W, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0016] Preferably, the alkaline treatment specifically comprises mixing the autologous skull with a 1-6 mol / L sodium hydroxide solution for 2-6 h.

[0017] The molar concentration of the sodium hydroxide can be selected from 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, 5 mol / L, 5.5 mol / L, 6 mol / L, etc., and the time can be selected from 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0018] Preferably, the enzyme treatment specifically comprises mixing the autologous skull with a protease solution at 20-30° C. for 1-8 hours.

[0019] The temperature can be selected as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, etc. The time can be selected as 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc. Other specific point values ​​within the above numerical range can also be selected, so they will not be listed here one by one.

[0020] Preferably, the enzyme comprises trypsin and / or 1398 protease.

[0021] Preferably, the mass percentage of protease in the protease solution is 0.1-5%, for example 0.1%, 0.2%, 0.5%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, 5%, etc. Other specific points within the above numerical range can be selected, which will not be repeated here.

[0022] After the drilling process, the pores obtained have a diameter of 1-5 nm and a pore spacing of 5-10 nm.

[0023] The pore size can be selected as 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, etc., and the pore spacing can be selected as 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 9 nm, 9.5nm, 10 nm, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0024] Preferably, the alkali treatment is carried out at 0-20°C, for example, 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0025] Preferably, the incubation time in step (3) is 0.5-5 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, etc. Other specific points within the above numerical range can be selected and will not be described in detail here.

[0026] Preferably, the time for incubation with BMPs in step (4) is 0.5-5 h, for example, 0.5 h, 0.8 h, 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, 3.2 h, 3.4 h, 3.6 h, 3.8 h, 4 h, 4.2 h, 4.4 h, 4.6 h, 4.8 h, 5 h, etc. Other specific points within the above numerical range can be selected and will not be described one by one here.

[0027] Preferably, the concentration of collagen in the solution containing collagen is 10-60 mg / mL, for example, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0028] Preferably, the solution containing collagen also includes acetic acid.

[0029] Preferably, the mass percentage of acetic acid in the collagen-containing solution is 2%-20%, for example 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0030] Preferably, the solution for promoting BMPs adsorption comprises carbonate, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water.

[0031] Preferably, in the solution for promoting BMPs adsorption, the concentration of carbonate is 0.15-0.45 mol / L, the concentration of N-hydroxysuccinimide is 0.2-1 mg / mL, and the concentration of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide is 0.5-2 mg / mL.

[0032] The concentration of the carbonate can be selected from 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, etc., the concentration of the N-hydroxysuccinimide can be selected from 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, etc., and the concentration of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide can be selected from 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0033] The present invention finds that N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)-carbodiimide can promote the adsorption of BMPs, and adding carbonate under ultrasonic conditions can also improve the adsorption of BMPs.

[0034] Preferably, the volume ratio of the solution containing collagen to the solution promoting BMPs adsorption is (1-10):(1-10).

[0035] The specific point values ​​in (1-10) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be listed here one by one.

[0036] Preferably, the added amount of the BMPs is independently 0.005-0.03% of the skull mass, for example 0.005%, 0.008%, 0.01%, 0.012%, 0.015%, 0.018%, 0.02%, 0.022%, 0.025%, 0.028%, 0.03%, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0037] In a second aspect, the present invention provides an autologous skull bone reimplantation matrix prepared according to the method for preparing an autologous skull bone reimplantation matrix according to the first aspect.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The replantation matrix prepared by the method provided by the present invention has good biocompatibility after use, no inflammatory reaction and no autoabsorption after surgery, and enhanced skull repair effect. The above method has little effect on the normal structure of autologous bone, that is, it can provide a three-dimensional structure closer to natural tissue, provide a bionic microenvironment after replantation, promote cell ingrowth and survival, and is conducive to the formation of bone healing.

[0040] Studies have shown that bone morphogenetic proteins (BMPs) have the ability to induce undifferentiated mesenchymal stem cells to differentiate and proliferate into chondrocytes and osteoblasts, promote osteoblast differentiation and maturation, participate in the growth and development of bone and cartilage and their reconstruction, and thus accelerate the repair of bone defects. This application introduces a new method of BMPs compounding, which can promote the adsorption of factors on autologous bone and prolong the treatment time, thereby enhancing the repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a scanning electron microscopic image of the skull repaired according to the method of Example 1 6 months later.

[0042] Figure 2 This is a scanning electron microscope result of the skull repaired according to the method of Comparative Example 8 6 months later.

[0043] Figure 3 This is the skull thickness result graph.

[0044] Figure 4 This is the skull density result chart.

[0045] Figure 5 This is a general observation picture of the skull treated according to the method of Example 1 and the skull treated according to the method of Comparative Example 9 after 6 months of repair. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] The sources of the functional ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0048] BMP2 was purchased from Abcam;

[0049] BMP7 was purchased from Abcam;

[0050] Trypsin was purchased from Sigma-Aldrich;

[0051] 1398 protease was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0052] The degreasing treatment involved in the following content specifically includes: adding ethanol and ethyl acetate solution with a volume ratio of 1:1, soaking for 24 hours to degrease, ultrasonic, and deionized water cleaning 5 times, each time for 20 minutes.

[0053] The decellularization treatment specifically included: adding 0.1% SDS to soak for 10 h, and washing with deionized water 5 times, each time for 20 minutes. Example 1

[0054] This embodiment provides a method for preparing an autologous skull bone reimplantation matrix, the preparation method comprising:

[0055] (1) The autologous skull was cleaned with isotonic saline and the soft tissue was removed;

[0056] (2) mixing the autologous skull obtained in step (1) with a 3 mol / L sodium hydroxide solution under ultrasonic conditions for 4 h, and then mixing with a 2.5% protease solution for 5 h, wherein the protease solution includes trypsin and 1398 protease in a mass ratio of 1:1;

[0057] (3) The autologous skull obtained in step (2) was punched with a hole diameter of 3 mm and a hole spacing of 8 mm, and mixed with a 3 mol / L sodium hydroxide solution under ultrasonic conditions at 4°C for 4 h with an ultrasonic power of 150 W for degreasing and decellularization;

[0058] (4) Mixing the skull obtained in step (3) with a solution containing BMP2, the mass of which is 0.02% of the mass of the skull, and incubating for 3 h;

[0059] (5) An acetic acid solution containing 20 mg / mL collagen and a solution that promotes BMP2 adsorption (0.25 mol / L sodium carbonate, 0.6 mg / mL N-hydroxysuccinimide, 1.2 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water) in equal volumes were mixed under ultrasonic conditions, and then mixed with BMP2 and incubated for 2 h, with the mass of BMP2 being 0.02% of the mass of the skull, to obtain a first mixed solution. The first mixed solution and the skull obtained in step (3) were soaked at 25°C for 5 h, and freeze-dried to obtain the product. Example 2

[0060] This embodiment provides a method for preparing an autologous skull bone reimplantation matrix, the preparation method comprising:

[0061] (1) The autologous skull was cleaned with isotonic saline and the soft tissue was removed;

[0062] (2) mixing the autologous skull obtained in step (1) with a 6 mol / L sodium hydroxide solution under ultrasonic conditions for 2 h, and then mixing with a 1% trypsin solution for 8 h;

[0063] (3) The autologous skull obtained in step (2) was punched with a hole diameter of 5 mm and a hole spacing of 10 mm, and mixed with a 6 mol / L sodium hydroxide solution under ultrasonic conditions at 4°C for 1 h with an ultrasonic power of 150 W for degreasing and decellularization;

[0064] (4) Mix the skull obtained in step (3) with a solution containing 0.03% BMP7 and incubate for 5 h;

[0065] (5) An acetic acid solution containing 10 mg / mL collagen and a solution promoting BMP7 adsorption (0.15 mol / L sodium carbonate, 1 mg / mL N-hydroxysuccinimide and 2 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water) were mixed at a volume ratio of 5:1 under ultrasonic conditions, and then mixed with BMP7 and incubated for 3 h. The mass of BMP7 was 0.03% of the mass of the skull to obtain a first mixed solution. The first mixed solution and the skull obtained in step (3) were soaked at 25°C for 2 h and freeze-dried to obtain the obtained product. Example 3

[0066] This embodiment provides a method for preparing an autologous skull bone reimplantation matrix, the preparation method comprising:

[0067] (1) The autologous skull was cleaned with isotonic saline and the soft tissue was removed;

[0068] (2) mixing the autologous skull obtained in step (1) with a 1 mol / L sodium hydroxide solution under ultrasonic conditions for 6 h, and then mixing with a 5% 1398 protease solution for 1 h;

[0069] (3) The autologous skull obtained in step (2) was punched with a hole diameter of 1 mm and a hole spacing of 5 mm, and mixed with a 1 mol / L sodium hydroxide solution under ultrasonic conditions at 4°C for 6 h with an ultrasonic power of 150 W for degreasing and decellularization;

[0070] (4) Mix the skull obtained in step (3) with a solution containing 0.01% BMP2 and incubate for 0.5 h;

[0071] (5) An acetic acid solution containing 60 mg / mL collagen and a solution promoting BMP2 adsorption (0.4 mol / L sodium carbonate, 0.2 mg / mL N-hydroxysuccinimide and 0.5 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water) were mixed at a volume ratio of 1:5 under ultrasonic conditions, and then mixed with BMP2 and incubated for 0.5 h. The mass of BMP2 was 0.01% of the mass of the skull to obtain a first mixed solution. The first mixed solution and the skull obtained in step (3) were soaked at 25°C for 10 h and freeze-dried to obtain the product. Example 4

[0072] This embodiment provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Embodiment 1 only in that the solution for promoting BMP2 adsorption in step (5) does not contain N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, and other operations remain unchanged. Example 5

[0073] This embodiment provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Embodiment 1 only in that the solution for promoting BMP2 adsorption in step (5) does not contain sodium carbonate, and other operations remain unchanged. Comparative Example 1

[0074] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (2) is "mixing the autologous skull bone obtained in step (1) with a 2.5% protease solution under ultrasonic conditions for 5 h, wherein the protease solution comprises trypsin and 1398 protease in a mass ratio of 1:1", and other operations remain unchanged. Comparative Example 2

[0075] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (2) is "mixing the autologous skull bone obtained in step (1) with a 3 mol / L sodium hydroxide solution for 4 h, and then mixing with a 2.5% protease solution for 5 h, wherein the protease solution comprises trypsin and 1398 protease in a mass ratio of 1:1", and other operations remain unchanged. Comparative Example 3

[0076] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (3) is "perforating the autologous skull bone obtained in step (2) with a hole diameter of 3 mm and a hole spacing of 8 mm, and performing degreasing and decellularization treatment", and other operations remain unchanged. Comparative Example 4

[0077] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (3) is "the autologous skull bone obtained in step (2) is punched with a hole diameter of 3 mm and a hole spacing of 8 mm, mixed with a 3 mol / L sodium hydroxide solution at 4°C for 4 h, and ultrasonically powered at 150 W for degreasing and decellularization", and other operations remain unchanged. Comparative Example 5

[0078] (1) The autologous skull was cleaned with isotonic saline and the soft tissue was removed;

[0079] (2) mixing the autologous skull obtained in step (1) with a 2.5% protease solution for 5 h, wherein the protease solution comprises trypsin and 1398 protease in a mass ratio of 1:1;

[0080] (3) The autologous skull obtained in step (2) was punched with a hole diameter of 3 mm and a hole spacing of 8 mm, and ultrasonically treated for 4 h at 4°C and an ultrasonic power of 150 W for degreasing and decellularization;

[0081] (4) Mixing the skull obtained in step (3) with a solution containing BMP2, the mass of which is 0.01% of the mass of the skull, and incubating for 3 h;

[0082] (5) An acetic acid solution containing 20 mg / mL collagen and a solution that promotes BMP2 adsorption (0.25 mol / L sodium carbonate, 0.6 mg / mL N-hydroxysuccinimide, 1.2 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water) in equal volumes are mixed under ultrasonic conditions, and then mixed with BMP2 and incubated for 2 h. The mass of BMP2 is 0.01% of the mass of the skull to obtain a first mixed solution. The first mixed solution and the skull obtained in step (3) are soaked at 25°C for 5 h and freeze-dried to obtain the obtained product. Comparative Example 6

[0083] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (2) is "mixing the autologous skull bone obtained in step (1) with a 2.5% protease solution under ultrasonic conditions for 5 h, wherein the protease solution includes trypsin and 1398 protease in a mass ratio of 1:1, and then mixing with a 3 mol / L sodium hydroxide solution for 4 h", and other operations remain unchanged. Comparative Example 7

[0084] This comparative example provides a method for preparing an autologous skull bone reimplantation matrix, which differs from Example 1 only in that step (5) is "mixing equal volumes of an acetic acid solution containing 20 mg / mL collagen and a solution for promoting BMP2 adsorption (0.25 mol / L sodium carbonate, 0.6 mg / mL N-hydroxysuccinimide and 1.2 mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water), and then mixing and incubating with BMP2 for 2 h, wherein the mass of BMP2 is 0.01% of the mass of the skull bone, to obtain a first mixed solution, soaking the first mixed solution and the skull bone obtained in step (3) at 25°C for 5 h, and freeze-drying to obtain the matrix", and other operations remain unchanged. Comparative Example 8

[0085] This comparative example provides a method for preparing a skull reimplantation matrix, which differs from Example 1 only in that step (5) is "mixing an acetic acid solution containing 20 mg / mL collagen with BMP2 for 2 h, where the mass of BMP2 is 0.01% of the mass of the skull, to obtain a first mixed solution, soaking the first mixed solution and the skull obtained in step (3) at 25°C for 5 h, and freeze-drying to obtain the matrix", and other operations remain unchanged. Comparative Example 9

[0086] This comparative example provides a method for preserving an autologous skull, the method comprising:

[0087] Under sterile operation, the connective tissue adhering to the skull surface was removed with tissue forceps and repeatedly rinsed, and then soaked in physiological saline for 30 minutes. Then, it was wrapped with sterile medical gauze and placed in a medical sterilization bag and sealed. Finally, it was placed in an independent compartment in an ultra-low temperature refrigerator (-80°C) for refrigeration to obtain an ultra-low temperature preserved autologous skull.

[0088] Test Example 1

[0089] Endotoxin and fat content testing:

[0090] The skull bones obtained by the treatment were tested. The skull bones used in the embodiment and the comparative example were from the same skull bone and were in similar initial states. Endotoxin detection was performed according to the method specified in GB / T 14233.2-2005. Fat content was tested according to the second method of acid hydrolysis in GB 5009.6-2016. The results are shown in the following table:

[0091] Table 1

[0092]

[0093] It can be seen from the data in Table 1 that two alkaline treatments are very important for the removal of fat and endotoxins. The endotoxin and fat content of the skull obtained without any alkaline treatment are both high. The effect is better when combined with ultrasound during alkaline treatment, and the order of enzyme treatment and alkaline treatment cannot be reversed.

[0094] Test Example 2

[0095] Factor release test

[0096] Test method: Immerse the sample in an equal volume of PBS and place it on a shaker at 37°C. Collect 100μL of sustained-release solution at a specific time point and store it, and add 100μL of new PBS. Use enzyme-linked immunosorbent assay to detect the antibody reaction and then add a colorimetric substrate. Detect the OD value at a wavelength of 450nm and calculate the percentage of BMPs released by each sample after 7 days and 28 days. The results are shown in the following table:

[0097] Table 2

[0098]

[0099] The sustained release of BMPs can avoid the toxicity problem caused by the large-scale release of growth factors in a short period of time. At the same time, the longer the sustained release time, the more conducive to long-term repair. As can be seen from the data in Table 2, the BMPs will not be released in large quantities in a short period of time through the method described in this application. The carbonate, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)-carbodiimide in the solution that promotes the adsorption of BMPs are very important for the above effects, and mixing under ultrasonic conditions is very important for the effect.

[0100] Test Example 3

[0101] Replantation effect test

[0102] Test method: Beagle dog bilateral critical skull defect model Surgical procedure: Beagle dogs fasted for 12 hours before surgery, and surgical instruments were sterilized by high-temperature and high-pressure steam and dried for standby use. The experimental animals were anesthetized by intramuscular injection of 10 mg / kg of tiletamine hydrochloride / zolazepam compound anesthetic. After successful anesthesia induction, the animals were intubated and connected to a ventilator with continuous ECG monitoring. The dog's forearm cephalic vein was taken to establish an infusion channel, and cefazolin was diluted and intravenously dripped at a dose of 25 mg / kg to prevent perioperative infection. Propofol (100-200 μg / kg / min) was used to maintain anesthesia during surgery. The dog's skull was prepared on the frontal top, the surgical area and surrounding hair were removed, and the dog was fixed on the operating table in a prone position to protect the local joints and bone protrusions to avoid pressure sores. Routine iodine skin disinfection was performed, and a sterile surgical sheet was laid. A coronal incision was made to cut the skin, subcutaneous tissue and periosteum in full thickness to expose the skull bone surface. The periosteum was gradually separated from the skull using a periosteal stripper, forward to the superior orbital margin, and stripped along the superficial temporal fascia on both sides, as close to the superior edge of the zygomatic arch as possible, and exposed to the occipital tubercle backward. If exposure is difficult, the incision was expanded along the midsagittal line to form a T shape if necessary. The periosteal stripper was used to separate the temporalis muscle from the back to the front above the superior temporal line to expose the skull below the superior temporal line. To avoid damaging the sagittal sinus, a 5 mm opening was made beside the sagittal suture, and a quadrilateral skull block of approximately 20 mm × 20 mm was marked on both sides of the sagittal suture. Holes were drilled at the four corners of the osteotomy area using an electric grinder, and saline was used to rinse during grinding to avoid local overheating and damage to the surrounding brain tissue. The dura mater around the bone hole was separated using a nerve stripper, and then the wire saw guide was introduced into the wire saw from the bone hole under the inner plate of the skull. The skull was cut along the mark with a wire saw to obtain a bone flap of 20 mm × 20 mm. During the operation, the integrity of the dura mater was maintained and the sagittal sinus was not damaged. Hemorrhage from the bridging veins and arachnoid granulations in the surgical area was treated with bipolar electrocoagulation, and skull hemorrhage was sealed with a small amount of bone wax. After repeated flushing with normal saline, the surgical incision was sutured in layers after the temporalis muscle was properly fixed. The subcutaneous galea aponeurotica layer was intermittently sutured with 4-0 absorbable surgical sutures, and the scalp incision was intermittently sutured with 2-0 silk thread. After the operation, the beagle was returned to the breeding area after it was awake and recovered its spontaneous breathing. For two days after the operation, cefazolin (25 mg / kg) was intramuscularly injected twice a day to prevent infection. The experimental animals were fed with conventional feed, the wound was disinfected regularly, and the sutures were removed from the wound about 1 week later.

[0103] Reimplantation of skull bone flap: 2 months after the canine skull defect animal model was established, the reimplantation matrix treated by the methods described in Example 1 and Comparative Example 8 was implanted in situ into the canine skull defect. The preoperative preparation process, anesthesia, and skin disinfection during the operation are the same as the steps for establishing the animal model. During the repair process, at least the two sides of the bone flap are aligned with the cut surface formed when the surrounding bone window is cut. Holes are punched on the edges of the bone flap and the bone window, and non-absorbable silk thread is passed through the bone hole and then tied to fix. The surgical area is treated for hemostasis, the wound is sutured in layers, and the postoperative observation and treatment are the same as the modeling steps.

[0104] Three months after the skull repair, it was observed that the control group (Comparative Example 8) had obvious bone absorption at the edge of the bone flap, while the experimental group's bone flap maintained a relatively stable structure. Both groups of bone flaps formed a certain bone connection with the surrounding bone tissue. Six months after the repair, the skull was sampled and the samples were dehydrated and coated for scanning electron microscopy. The results showed that the experimental group (Example 1) had less fibrous tissue adhered to the outer plate of the skull, and some areas had exposed outer bone plates (see Figure 1 ), while the outer plate of the skull in the control group was covered with dense fibrous tissue (see Figure 2 ).

[0105] from Figure 3 and Figure 4 It can be seen that after 3 and 6 months of skull repair, the skull thickness and density of the experimental group were higher than those of the control group, and the data showed significant statistical differences. These results indicate that the matrix of the experimental group can better inhibit bone resorption.

[0106] The autologous skull preserved at ultra-low temperature (Comparative Example 9) was used as the control group and the replanted skull flap (experimental group) obtained by treatment in Example 1 was implanted in situ into the skull defect of dogs. The results after 6 months of repair are as follows: Figure 5 As shown. Obviously, compared with the experimental group (left), the control group (right) showed more obvious bone absorption. Therefore, the replanted skull flap obtained by the method of the present application is superior to the conventional ultra-low temperature preservation of the skull flap, significantly inhibiting autologous bone absorption and promoting bone formation.

[0107] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the preparation method of the autologous skull bone replantation matrix and its product, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0108] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0109] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing an autologous skull bone reimplantation matrix, characterized in that: The preparation method comprises: (1) The pre-treated autologous skull is subjected to alkali treatment and enzyme treatment in sequence under ultrasonic conditions; (2) drilling the autologous skull obtained in step (1), performing an alkali treatment under ultrasonic conditions, and then performing a degreasing and decellularizing treatment to obtain a skull matrix; (3) mixing the skull matrix obtained in step (2) with a solution containing BMPs and incubating; (4) mixing the solution containing collagen with the solution promoting BMPs adsorption under ultrasonic conditions, and then mixing and incubating with BMPs to obtain a first mixed solution, and compounding the first mixed solution with the skull obtained in step (3), and freeze-drying to obtain; The BMPs is BMP2 or BMP7; The collagen-containing solution also includes acetic acid; The alkaline treatment specifically comprises mixing the autologous skull with a 1-6 mol / L sodium hydroxide solution for 2-6 h; The solution for promoting BMPs adsorption comprises 0.15-0.45 mol / L of carbonate, 0.2-1 mg / mL of N-hydroxysuccinimide, 0.5-2 mg / mL of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and water.

2. The method for preparing the autologous skull bone reimplantation matrix according to claim 1, characterized in that: The enzyme treatment specifically includes mixing the autologous skull with a protease solution at 20-30° C. for 1-8 hours.

3. The method for preparing the autologous skull bone reimplantation matrix according to claim 1, characterized in that: The incubation time in step (3) is 0.5-5 h.

4. The method for preparing the autologous skull bone reimplantation matrix according to claim 1, characterized in that: The time of incubation with BMPs in step (4) is 0.5-5 h.

5. The method for preparing the autologous skull bone reimplantation matrix according to claim 1, characterized in that: The concentration of collagen in the solution containing collagen is 10-60 mg / mL.

6. The method for preparing the autologous skull bone reimplantation matrix according to claim 1, characterized in that: The amount of BMPs added in step (3) and step (4) is independently 0.005-0.03% of the skull mass.

7. The autologous skull reimplantation matrix prepared by the method for preparing an autologous skull reimplantation matrix according to any one of claims 1 to 6.

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