A two-component osteoinductive collagen membrane and its preparation method

Abstract

The present invention discloses a two-component bone-inducing collagen membrane and a preparation method thereof. The collagen membrane is prepared by blending and cross-linking collagen extracted from the skin or tendon tissue of pigs or cattle with bone morphogenetic protein-2, and then injecting the mixture into a prefabricated mold and freeze-drying. The specific preparation method comprises cutting the animal tissue into thin slices, performing enzymatic hydrolysis, filtering, and precipitation to obtain collagen, which is then redissolved, and EDC and NHS cross-linking agents are added in batches. The collagen is then blended with bone morphogenetic protein-2, and finally injection-molded, freeze-dried, and irradiated for sterilization to obtain a bone morphogenetic protein + collagen two-component structure membrane. The collagen membrane not only has excellent spatial maintenance ability, but also can control the amount of BMP added and the degradation time of the product, and has good medical and practical value.

Description

Technical Field

[0001] The present invention relates to the field of biological repair materials, and in particular to a two-component bone-inducing collagen membrane and a preparation method thereof. Background Art

[0002] Oral collagen membrane is a thin film-like material primarily composed of collagen, used as a filling material in dentistry. It is a natural material that is fully absorbed by the human body and does not produce an immune rejection reaction. The primary function of oral collagen membrane is to promote the healing and regeneration of oral soft tissue. During oral surgery, doctors can use it to fill defects in alveolar bone or periodontal tissue to promote the regeneration and repair of bone tissue. It can also be used in dental implant surgery as a support material for the soft tissue surrounding the implant, promoting the regeneration and fixation of soft tissue. The advantages of oral collagen membrane are safety, reliability, and ease of use. It can effectively promote the regeneration and healing of oral soft tissue and reduce the occurrence of surgical complications. At the same time, oral collagen membrane is relatively inexpensive and can meet the needs of different patients.

[0003] Currently, the predominant oral collagen membrane used on the market is acellular matrix membrane. Decellularized membranes are biomedical materials whose cellular components have been removed during the preparation process. This is typically achieved through chemical, physical, or biological methods to remove cells and extracellular matrix components from tissues, preserving the three-dimensional structure, bioactivity, and mechanical properties of the extracellular matrix. This approach reduces immune rejection and infection risks, while also enhancing the biocompatibility and bioactivity of the material. However, decellularized membranes lack spatial retention and typically require cross-linking to enhance their mechanical strength and stability. For example, when used in combination with autologous bone or other bone augmentation materials, the degradation time of such products is difficult to control and predict, complicating subsequent applications. Furthermore, the amount of cross-linker used can affect their bioactivity, mechanical strength, and stability. For example, excessive cross-linking can produce large amounts of byproducts and cross-linking products that are toxic and immunogenic to biomolecules. This can compromise the regenerative efficacy of the collagen membrane, resulting in poor osteogenic performance and a detrimental effect on bone regeneration.

[0004] Bone morphogenetic protein (BMP) is a secretory multifunctional protein produced by a variety of cells. It promotes osteoblast growth and proliferation, inducing undifferentiated mesenchymal cells to differentiate into chondrocytes in vitro and in vivo, and promoting rapid new bone formation through osteoinduction. Research has shown that the combination of BMP and collagen membranes can be used in bone tissue engineering and bone defect repair. For example, U.S. Patent No. US20050074459A1 discloses an Escherichia coli O157:H7 epithelial adhesin, which combines bone morphogenetic protein-2 (BMP-2) with collagen membranes to create a composite material. The specific method involves extracting collagen from animal skin and preparing a collagen membrane through acid treatment, salting out, and freeze-drying. The biodegradable polymer polylactic acid (PLA) is used as a BMP-2 carrier. BMP-2 is uniformly dispersed in a PLA solution and prepared by solution blending. The BMP-2 carrier is then physically adsorbed onto the collagen membrane to create the BMP-2 / collagen membrane composite. This composite material was used to repair skull defects in rats, promoting the adhesion and proliferation of bone cells and accelerating the formation and repair of bone tissue. However, this approach presents at least the following challenges: 1) PLA as a carrier has a slow biodegradation rate, making it difficult to control and predict degradation time; 2) the physical adsorption of BMP to the collagen membrane results in weak binding, and the amount of BMP adsorbed on the collagen membrane surface is limited, preventing long-term bone regeneration and limiting the repair effect; 3) BMP adsorption on the collagen membrane surface provides limited improvement in its mechanical support, and the product structure depends on the collagen membrane structure, limiting its application scenarios.

[0005] Therefore, how to balance the biocompatibility and mechanical properties of the material while controlling its degradation rate and further improving the osteogenic properties of the collagen membrane remains a technical problem that needs to be solved urgently. Summary of the Invention

[0006] To address the aforementioned issues with existing acellular membranes and their integration with bone morphogenetic protein (BMP), the present invention provides a method for preparing an osteoinductive collagen membrane. Collagen and BMP are prepared as separate solutions, cross-linked using a suitable crosslinking agent, and then injection molded and freeze-dried to produce the desired BMP + collagen dual-component membrane. This membrane not only exhibits excellent spatial retention but also allows for control of BMP dosage and product degradation time. The specific technical solution is as follows:

[0007] First, the present invention provides a two-component bone-inducing collagen membrane, which is prepared by mixing and cross-linking collagen extracted from pig or cow skin or tendon tissue with bone morphogenetic protein-2, and then injecting the mixture into a prefabricated mold and freeze-drying.

[0008] The preparation method of the two-component osteoinductive collagen membrane comprises the following steps:

[0009] 1) Slicing: Clean the pig or cow skin or tendon tissue and cut it into slices of a certain thickness to obtain animal tissue slices;

[0010] 2) Enzymatic hydrolysis: Place the obtained animal tissue slices in acetic acid or hydrochloric acid solution and add protease for enzymatic hydrolysis to obtain crude protein solution;

[0011] 3) Filtration: Filter out the particulate matter in the crude protein solution to obtain a protein solution;

[0012] 4) Precipitation: Add sodium hydroxide solution to the protein solution to precipitate the protein and obtain collagen;

[0013] 5) Redissolution: Dissolve the obtained collagen in acetic acid to obtain a collagen solution;

[0014] 6) Cross-linking: EDC and NHS cross-linkers were added to the collagen solution in batches under stirring to obtain a collagen-EDC intermediate.

[0015] 7) Blending: Slowly add bone morphogenetic protein-2 to the collagen-EDC intermediate, stir and blend, to obtain a bone morphogenetic protein / collagen blend solution;

[0016] 8) Injection molding: injecting the obtained bone morphogenetic protein / collagen blend solution into a pre-made mold;

[0017] 9) Freeze-drying: The BMP / collagen blend solution after injection molding is sent together with the mold into a freeze dryer for freeze-drying to form a collagen film;

[0018] 10) Sterilization: The freeze-dried collagen membrane is sterilized by irradiation to obtain a finished collagen membrane.

[0019] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, the thickness of the animal tissue slice in step 1) is 1 to 3 mm.

[0020] In the aforementioned method for preparing a two-component osteoinductive collagen membrane, the concentration of the acetic acid or hydrochloric acid solution in step 2) is 0.1% to 5%; the added protease is trypsin, pepsin, and / or ficin, and the added amount is 8% to 15% of the weight of the animal tissue slice. The enzymatic hydrolysis conditions are: stirring at 2 to 8°C for 6 to 36 hours.

[0021] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, the filtration in step 3) is performed by using a 20-40 mesh stainless steel mesh to filter out particulate matter.

[0022] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, the concentration of the sodium hydroxide solution added for precipitation in step 4) is 0.5-4 mol / L.

[0023] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, in the redissolution in step 5), the ratio of the precipitated protein to the acetic acid solution is 1-50:1-100.

[0024] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, the cross-linking process in step 6) is as follows:

[0025] 6-1) Crosslinker Preparation: Prepare EDC and NHS crosslinkers at a 1:1 mass ratio. Divide the EDC into 6 equal portions and the NHS into 3 equal portions, and set aside.

[0026] 6-2) First reactivation: Add the collagen solution to a blender, adjust its pH to 6.0, and stir at 400 rpm for 10 minutes. Then, add three portions of EDC crosslinker sequentially while stirring. Each portion of EDC crosslinker is stirred at 400 rpm for 10 minutes before adding the next portion. This allows the carboxylic acid groups on the collagen to form amide bonds with the EDC, yielding collagen-EDC intermediate I.

[0027] 6-3) First level protection: Adjust the pH of collagen-EDC intermediate I to 7.2, control the stirring speed to 300 rpm, and sequentially add two portions of NHS crosslinker while stirring. Stir for 10 minutes after adding the first portion of NHS crosslinker before adding the second portion to stabilize collagen-EDC intermediate I, thereby obtaining collagen-EDC / NHS intermediate II.

[0028] 6-4) Second activation: Adjust the pH of collagen-EDC intermediate II to 6.5, maintain the stirring speed at 300 rpm, and add the remaining 3 portions of EDC crosslinker. Stir each portion at 300 rpm for 20 minutes before adding the next portion to activate the unreacted carboxylic acid groups on the collagen to obtain collagen-EDC intermediate III.

[0029] 6-5) Second protection: Adjust the pH of collagen-EDC intermediate I to 7.0, control the stirring speed to 200 rpm, add the remaining 1 part of NHS cross-linker, and continue stirring for 20 minutes to obtain collagen-EDC intermediate IV for blending with bone morphogenetic protein-2.

[0030] In the aforementioned method for preparing a two-component osteoinductive collagen membrane, in step 7), the material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate is 1-2:2.8-6.8; the blending speed is 200 rpm-600 rpm, and the stirring time is 3-5 hours.

[0031] In the aforementioned method for preparing the two-component osteoinductive collagen membrane, the sterilization dosage in step 10) is 15.2-25 KGY.

[0032] The beneficial effects of the present invention are:

[0033] 1) The present invention prepares collagen and bone morphogenetic protein solutions separately, cross-links them with a suitable cross-linking agent, and then performs injection molding and freeze-drying to obtain the desired bone morphogenetic protein + collagen two-component structure membrane. This membrane not only has excellent spatial maintenance ability but also can control the amount of BMP added and the degradation time of the product.

[0034] 2) In the present invention, collagen and bone morphogenetic protein are prepared into solutions respectively for cross-linking, and the collagen and bone morphogenetic protein are evenly mixed, the proteins are fully compounded, and the mechanical strength and stability thereof are effectively improved.

[0035] 3) In the present invention, collagen and bone morphogenetic protein are prepared into solutions and mixed and cross-linked, so that the composite amounts of collagen and bone morphogenetic protein can be controlled respectively, and their supporting effect and degradation time can be better controlled.

[0036] 4) The present invention uses EDC in combination with NHS. EDC can directly combine the active carboxylic acid and amine functional groups through amide bonds, while NHS can make the active ester generated by the reaction more stable in water, thereby avoiding hydrolysis. The efficiency and specificity of the reaction are improved through dual activation protection. At the same time, because the intermediate generated by the reaction of EDC and NHS is reversible, the cross-linking reaction step is controllable, which is conducive to controlling the dosage of the drug, the dosage of the cross-linking agent, and the degree of cross-linking. In addition, the combination of EDC and NHS can increase the reaction speed and the reaction conditions are mild, which facilitates experimental operation.

[0037] 5) The present invention refines the cross-linking step, adds EDC and NHS in batches, and controls the reaction conditions of EDC, collagen, and NHS to ensure uniform bonding between collagen and EDC, thereby ensuring the concentration of the generated active ester and the effect of subsequent cross-linking with bone morphogenetic protein-2, while avoiding the generation of toxic and immunogenic byproducts due to the reaction of excessive EDC and NHS.

[0038] 6) The collagen membrane prepared by the present invention can be used to prefabricate a collagen membrane mold according to its function to prepare a collagen membrane of a suitable form. At the same time, cross-linking agents and bone morphogenetic proteins can be added in appropriate amounts according to the expected support strength and degradation time of the collagen membrane to obtain a special collagen membrane that takes into account both mechanical properties and degradation speed. It has good promotion and application prospects and market economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The degradation of the two-component bone-inducing collagen membrane of product 2 of the present invention was observed after 0 days, 5 days, 10 days, and 15 days.

[0040] Figure 2 This is the degradation of the two-component bone-inducing collagen membrane of product 2 of the present invention for 20 days, 25 days, 30 days, and 35 days. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings.

[0042] Example 1

[0043] This embodiment is a two-component osteoinductive collagen membrane. The collagen membrane is prepared by mixing and cross-linking collagen extracted from pig or bovine skin or tendon tissue with bone morphogenetic protein-2, injecting the mixture into a prefabricated mold, and freeze-drying. The preparation method of the two-component osteoinductive collagen membrane includes the following steps:

[0044] 1) Slicing: Wash the pig or cow skin or tendon tissue and cut it into slices 1 to 3 mm thick to obtain animal tissue slices;

[0045] 2) Enzymatic hydrolysis: Place the obtained animal tissue slices in a 0.1% to 5% acetic acid or hydrochloric acid solution, add trypsin, pepsin, and / or ficin for enzymatic hydrolysis (the amount added is 8% to 15% of the weight of the animal tissue slices), and stir at 2 to 8°C for 6 to 36 hours to obtain a crude protein solution;

[0046] 3) Filtration: Use a 20-40 mesh stainless steel mesh to filter out particulate matter in the crude protein solution to obtain a protein solution;

[0047] 4) Precipitation: Add a sodium hydroxide solution with a concentration of 0.5-4 mol / L to the protein solution to precipitate the protein and obtain collagen;

[0048] 5) Redissolving: Dissolving the obtained collagen in acetic acid at a protein to acetic acid ratio of 1-50:1-100 to obtain a collagen solution;

[0049] 6) Cross-linking: Under stirring conditions, EDC and NHS cross-linking agents are added to the collagen solution in batches to obtain a collagen-EDC intermediate. The concentration of the EDC and NHS cross-linking agents is 5 mol / L, and the amount of each cross-linking agent is 0.5% to 2% of the mass of the collagen solution. The cross-linking process is as follows:

[0050] 6-1) Crosslinker Preparation: Prepare EDC and NHS crosslinkers at a 1:1 mass ratio. Divide the EDC into 6 equal portions and the NHS into 3 equal portions, and set aside.

[0051] 6-2) First reactivation: Add the collagen solution to a blender, adjust its pH to 6.0, and stir at 400 rpm for 10 minutes. Then, add three portions of EDC crosslinker sequentially while stirring. Each portion of EDC crosslinker is stirred at 400 rpm for 10 minutes before adding the next portion. This allows the carboxylic acid groups on the collagen to form amide bonds with the EDC, yielding collagen-EDC intermediate I.

[0052] 6-3) First level protection: Adjust the pH of collagen-EDC intermediate I to 7.2, control the stirring speed to 300 rpm, and sequentially add two portions of NHS crosslinker while stirring. Stir for 10 minutes after adding the first portion of NHS crosslinker before adding the second portion to stabilize collagen-EDC intermediate I, thereby obtaining collagen-EDC / NHS intermediate II.

[0053] 6-4) Second activation: Adjust the pH of collagen-EDC intermediate II to 6.5, maintain the stirring speed at 300 rpm, and add the remaining 3 portions of EDC crosslinker. Stir each portion at 300 rpm for 20 minutes before adding the next portion to activate the unreacted carboxylic acid groups on the collagen to obtain collagen-EDC intermediate III.

[0054] 6-5) Second protection: Adjust the pH of collagen-EDC intermediate I to 7.0, control the stirring speed to 200 rpm, add the remaining 1 part of NHS cross-linker, and continue stirring for 20 minutes to obtain collagen-EDC intermediate IV for blending with bone morphogenetic protein-2.

[0055] 7) Blending: Slowly adding bone morphogenetic protein-2 to the collagen-EDC intermediate, with the material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate being 1-2:2.8-6.8; stirring and blending at a blending speed of 200 rpm to 600 rpm for 3 to 5 hours to obtain a bone morphogenetic protein / collagen blend solution;

[0056] 8) Injection molding: injecting the obtained bone morphogenetic protein / collagen blend solution into a pre-made mold;

[0057] 9) Freeze-drying: The BMP / collagen blend solution after injection molding is sent together with the mold into a freeze dryer for freeze-drying to form a collagen film;

[0058] 10) Sterilization: The freeze-dried collagen membrane is irradiated and sterilized at a sterilization dose of 15.2 to 25 kg / m³ to obtain a finished collagen membrane.

[0059] Example 2

[0060] This example uses the method of Example 1 to prepare a two-component osteoinductive collagen membrane. The membrane is prepared by cross-linking collagen extracted from pig or bovine skin or tendon tissue with bone morphogenetic protein-2, injecting it into a prefabricated mold, and freeze-drying. The preparation method of the two-component osteoinductive collagen membrane includes the following steps:

[0061] 1) Slicing: Wash the pig skin tissue and cut it into slices with a thickness of 1 to 3 mm;

[0062] 2) Enzymatic hydrolysis: Place the pigskin slices in a 1.5% acetic acid or hydrochloric acid solution, add 10% trypsin powder by weight of the pigskin slices, and stir at 4°C for 32 hours to obtain a crude protein solution;

[0063] 3) Filtration: Use a 36-mesh stainless steel mesh to filter out particulate matter in the crude protein solution to obtain a protein solution;

[0064] 4) Precipitation: Add 2.5 mol / L sodium hydroxide solution to the protein solution to precipitate the protein and obtain collagen;

[0065] 5) Redissolution: Dissolve the obtained collagen in acetic acid at a protein to acetic acid ratio of 20:68 to obtain a collagen solution;

[0066] 6) Cross-linking: Under stirring conditions, EDC cross-linker and NHS cross-linker were added to the collagen solution in batches. The concentration of EDC cross-linker and NHS cross-linker were both 5 mol / L, and the amount was 0.75% of the mass of the collagen solution to obtain a collagen-EDC intermediate. The cross-linking process was as follows:

[0067] 6-1) Crosslinker Preparation: Prepare EDC and NHS crosslinkers at a 1:1 mass ratio. Divide the EDC into 6 equal portions and the NHS into 3 equal portions, and set aside.

[0068] 6-2) First reactivation: Add the collagen solution to a blender, adjust its pH to 6.0, and stir at 400 rpm for 10 minutes. Then, add three portions of EDC crosslinker sequentially while stirring. Each portion of EDC crosslinker is stirred at 400 rpm for 10 minutes before adding the next portion. This allows the carboxylic acid groups on the collagen to form amide bonds with the EDC, yielding collagen-EDC intermediate I.

[0069] 6-3) First level protection: Adjust the pH of collagen-EDC intermediate I to 7.2, control the stirring speed to 300 rpm, and sequentially add two portions of NHS crosslinker while stirring. Stir for 10 minutes after adding the first portion of NHS crosslinker before adding the second portion to stabilize collagen-EDC intermediate I, thereby obtaining collagen-EDC / NHS intermediate II.

[0070] 6-4) Second activation: Adjust the pH of collagen-EDC intermediate II to 6.5, maintain the stirring speed at 300 rpm, and add the remaining 3 portions of EDC crosslinker. Stir each portion at 300 rpm for 20 minutes before adding the next portion to activate the unreacted carboxylic acid groups on the collagen to obtain collagen-EDC intermediate III.

[0071] 6-5) Second protection: Adjust the pH of collagen-EDC intermediate I to 7.0, control the stirring speed to 200 rpm, add the remaining 1 part of NHS cross-linker, and continue stirring for 20 minutes to obtain collagen-EDC intermediate IV for blending with bone morphogenetic protein-2.

[0072] 7) Blending: Slowly add bone morphogenetic protein-2 to the collagen-EDC intermediate, with the material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate being 1:5; stir and blend at a blending speed of 300 rpm for 4.5 hours to obtain a bone morphogenetic protein / collagen blend solution;

[0073] 8) Injection molding: injecting the obtained bone morphogenetic protein / collagen blend solution into a pre-made mold;

[0074] 9) Freeze-drying: The BMP / collagen blend solution after injection molding is placed in a freeze-dryer together with the mold for freeze-drying to form a collagen membrane; the pre-freezing stage temperature is set to -45°C and the pre-freezing time is maintained for at least 7 hours; the sublimation stage temperature is set to -20°C and the sublimation time is maintained for at least 50 hours; the final drying stage temperature is 20°C and the holding time is 8 hours. It has been verified that the eutectic point of the oral collagen membrane prepared in this embodiment is -15°C. The key factors affecting the freeze-drying effect during the freeze-drying process are (1) the final temperature and time of the pre-freezing stage. The pre-freezing time is maintained for at least 7 hours. If it is less than 7 hours, liquid water will still exist in the middle of the collagen membrane; (2) the processing temperature and time of the eutectic point. If the sublimation time is less than 50 hours, the sample will still have a wet area after the sublimation is completed; (3) the final temperature of the drying stage. In this embodiment, the specific freeze-drying parameters are shown in Table 1.

[0075] Table 1: Freeze-drying procedure of BMP / collagen blend solution after injection molding

[0076]

[0077] 10) Sterilization: The freeze-dried collagen membrane is irradiated and sterilized at a sterilization dose of 20 kg / m³ to obtain a finished collagen membrane.

[0078] Example 3

[0079] This example investigates the effect of the amount of bone morphogenetic protein-2 added on the spatial maintenance capacity of a prepared two-component osteoinductive collagen membrane. This example examines both pure collagen membranes and two-component osteoinductive collagen membranes supplemented with different amounts of bone morphogenetic protein-2. Each collagen membrane was prepared using the same method as in Example 2, except for the amount of bone morphogenetic protein-2 added. The specific proportions are shown in Table 2. Because excessive BMP can negatively impact multiple fibrodysplasia ossificans and connective tissue ossification, the maximum amount of bone morphogenetic protein-2 added in this example was 75%.

[0080] Table 2: Amount of bone morphogenetic protein-2 added

[0081]

[0082] This embodiment uses three dimensions as indicators to evaluate the spatial maintenance capacity of the collagen membrane: ① alveolar bone height and width; ② vertical and labial-palatal bone absorption; and ③ peri-implant bone absorption.

[0083] The study process was as follows: 40 patients requiring anterior tooth extraction were recruited and divided equally into five groups. Gender, age, and tooth position were controlled for, with no statistically significant differences between the groups (P>0.05). All patients completed basic periodontal treatment 2 weeks before surgery, as indicated, and all met the criteria for implantation of the membrane. Products 1-5 were then implanted, with sutures removed 2 weeks after surgery. Six months later, after the sockets had healed, implant surgery was performed. Postoperative healing of the extraction sites was compared among the five groups, as were alveolar bone height and width before and 6 months after surgery. CBCT scans were performed before and after surgery, and alveolar bone height and width were measured using software. Alveolar bone density, vertical bone loss, and labial-palatal bone loss were recorded in both groups before and 6 months after surgery. Three measurements were repeated, and the average value was calculated. The experimental results for each group were recorded, and the average values ​​are shown in Table 3.

[0084] Table 3: Effect of BMP-2 addition on product spatial maintenance ability

[0085]

[0086] The results showed that the implantation of the two-component bone-inducing collagen membrane of the present invention can slow down the decline in alveolar bone height, width and bone density after tooth extraction, has certain clinical value in retaining bone mass, and can effectively improve the repair effect of the patient's alveolar bone.

[0087] Example 4

[0088] This example examines the effects of the added amounts of EDC and NHS crosslinkers on the spatial maintenance capacity of prepared two-component osteoinductive collagen membranes. The collagen membranes in this example were prepared using the same method as in Example 2, except for the different amounts of EDC and NHS crosslinkers. Specific ratios and contents are shown in Table 4.

[0089] Table 4: Addition amount of EDC crosslinker and NHS crosslinker

[0090]

[0091] This embodiment also uses three dimensions as indicators to evaluate the spatial maintenance ability of the collagen membrane: ① alveolar bone height and width; ② vertical and labial-palatal bone absorption; and ③ peri-implant bone absorption.

[0092] In this experiment, 25 patients who needed to have their anterior teeth extracted were recruited and divided into five groups. The specific experimental process was the same as in Example 3. Each group of patients was implanted with product 6-10. The specific test results of each group of patients are shown in Table 5.

[0093] Table 5: Effect of EDC and NHS crosslinker addition on product dimensional maintenance

[0094]

[0095] Example 5

[0096] This example investigates the degradation performance of collagen membranes with different bone morphogenetic protein-2 contents. The objects of investigation in this example are products 1-5 in Example 3. The degradation test method is as follows:

[0097] 1. Prepare phosphate buffer by mixing 18.2% of solution a) and 81.8% of solution b) (volume fractions); a) 1 / 15 mol / L potassium dihydrogen phosphate, b) 1 / 15 mol / L sodium dihydrogen phosphate.

[0098] 2. Prepare phosphate buffered saline with collagenase type I at a concentration of 0.02 mg / mL.

[0099] 3. Calculate the required amount of enzyme based on the amount of phosphate buffer and add it to the phosphate buffer, stirring slowly to dissolve the enzyme;

[0100] 4. Take multiple samples of products 1-5 and place them in clean 30×50 weighing bottles. Label them and add 20ml of phosphate buffered saline with type I collagenase at a concentration of 0.02mg / mL to each weighing bottle. Press gently to completely immerse the sample. Cover the weighing bottle and place it in a 37℃ incubator for degradation. On the 5th, 10th, 15th, 20th, 25th, 30th and 35th day after degradation, randomly select one sample from the experimental group at each time point to observe and record its degradation status, such as Figure 1 and Figure 2 The degradation process of product 2 is shown, and a sample was randomly taken to measure the degradation rate. The results are shown in Table 6.

[0101] Table 6: Degradation rate of collagen membranes with different bone morphogenetic protein-2 contents

[0102]

[0103] The results showed that with the increase of bone morphogenetic protein-2 content, the degradation time of the prepared collagen membrane was prolonged. When the material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate was less than 15%, its degradation time could not reach 35 days, which was difficult to meet the alveolar bone repair time and support requirements. The degradation time was prolonged with the increase of bone morphogenetic protein-2 content, but the amount of bone morphogenetic protein-2 added needed to be limited to avoid side effects. Overall, it is more appropriate to control the material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate at 1~2:2.8~6.8.

[0104] In general, the present invention prepares separate solutions of collagen and bone morphogenetic protein, crosslinks them with a suitable crosslinking agent, and then performs injection molding and freeze-drying to obtain the desired bone morphogenetic protein + collagen two-component structure membrane. This membrane not only has excellent spatial maintenance capabilities, but also allows for control of the amount of BMP added and the degradation time of the product. By preparing separate solutions of collagen and bone morphogenetic protein and then crosslinking them, the collagen and bone morphogenetic protein are evenly mixed, and the proteins are fully complexed, which can effectively improve the mechanical strength and stability of the membrane. Furthermore, by controlling the combined amount of collagen and bone morphogenetic protein, the support effect and degradation time can be better controlled.

[0105] In addition, the present invention utilizes EDC in combination with NHS. EDC can directly bond the active carboxylic acid and amine functional groups through amide bonds, while NHS can make the resulting active ester more stable in water, thereby preventing hydrolysis. This dual activation protection improves the efficiency and specificity of the reaction. Furthermore, because the intermediate generated by the combined reaction of EDC and NHS is reversible, the cross-linking reaction step is controllable, facilitating control of drug dosage, cross-linker dosage, and degree of cross-linking. Furthermore, the combination of EDC and NHS can increase the reaction rate and provide mild reaction conditions, facilitating experimental procedures. During the preparation process, the cross-linking step is refined, and EDC and NHS are added in batches. The reaction conditions of EDC, collagen, and NHS are controlled to ensure uniform bonding between collagen and EDC, ensuring the concentration of the generated active ester and the effectiveness of subsequent cross-linking with bone morphogenetic protein-2. This also prevents excessive EDC and NHS from reacting to generate toxic and immunogenic byproducts.

[0106] In addition, the collagen membrane prepared by the present invention can be used to prefabricate a collagen membrane mold according to its function to prepare a collagen membrane of a suitable form. At the same time, cross-linking agents and bone morphogenetic proteins can be added in appropriate amounts according to the expected support strength and degradation time of the collagen membrane to obtain a special collagen membrane that takes into account both mechanical properties and degradation speed. It has good promotion and application prospects and market economic value.

[0107] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from all perspectives. In addition, it should be understood that although this specification is described in terms of implementation methods, it does not contain only one technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should read the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing a two-component osteoinductive collagen membrane, characterized by: The collagen membrane is prepared by mixing and cross-linking collagen extracted from pig or cow skin or tendon tissue with bone morphogenetic protein-2, injecting the mixture into a prefabricated mold, and freeze-drying. The preparation method specifically comprises the following steps: 1) Slicing: Wash the pig or cow skin or tendon tissue and cut it into slices 1 to 3 mm thick to obtain animal tissue slices; 2) Enzymatic hydrolysis: Place the obtained animal tissue slices in acetic acid or hydrochloric acid solution and add protease for enzymatic hydrolysis to obtain crude protein solution; 3) Filtration: Filter out the particulate matter in the crude protein solution to obtain a protein solution; 4) Precipitation: Add sodium hydroxide solution to the protein solution to precipitate the protein and obtain collagen; 5) Redissolution: Dissolve the obtained collagen in acetic acid to obtain a collagen solution; 6) Cross-linking: EDC and NHS cross-linkers were added to the collagen solution in batches under stirring to obtain a collagen-EDC intermediate. The cross-linking process was as follows: 6-1) Crosslinker Preparation: Prepare EDC and NHS crosslinkers at a 1:1 mass ratio. Divide the EDC into 6 equal portions and the NHS into 3 equal portions, and set aside. 6-2) First reactivation: Add the collagen solution to a blender, adjust its pH to 6.0, and stir at 400 rpm for 10 minutes. Then, add three portions of EDC crosslinker sequentially while stirring. Each portion of EDC crosslinker is stirred at 400 rpm for 10 minutes before adding the next portion. This allows the carboxylic acid groups on the collagen to form amide bonds with the EDC, yielding collagen-EDC intermediate I. 6-3) First level protection: Adjust the pH of collagen-EDC intermediate I to 7.2, control the stirring speed to 300 rpm, and sequentially add two portions of NHS crosslinker while stirring. Stir for 10 minutes after adding the first portion of NHS crosslinker before adding the second portion to stabilize collagen-EDC intermediate I, thereby obtaining collagen-EDC / NHS intermediate II. 6-4) Second activation: Adjust the pH of collagen-EDC intermediate II to 6.5, maintain the stirring speed at 300 rpm, and add the remaining 3 portions of EDC crosslinker. Stir each portion at 300 rpm for 20 minutes before adding the next portion to activate the unreacted carboxylic acid groups on the collagen to obtain collagen-EDC intermediate III. 6-5) Second protection: Adjust the pH of collagen-EDC intermediate I to 7.0, control the stirring speed to 200 rpm, add the remaining 1 part of NHS crosslinker, and continue stirring for 20 minutes to obtain collagen-EDC intermediate IV, ready for blending with bone morphogenetic protein-2; 7) Blending: Slowly add bone morphogenetic protein-2 to collagen-EDC intermediate IV, stir and blend, to obtain a bone morphogenetic protein / collagen blend solution; The material-liquid ratio of bone morphogenetic protein-2 to collagen-EDC intermediate IV is 1-2:2.8-6.8; the blending speed is 200 rpm-600 rpm, and the stirring time is 3-5 hours; 8) Injection molding: injecting the obtained bone morphogenetic protein / collagen blend solution into a pre-made mold; 9) Freeze-drying: The BMP / collagen blend solution after injection molding is sent together with the mold into a freeze dryer for freeze-drying to form a collagen film; 10) Sterilization: The freeze-dried collagen membrane is sterilized by irradiation to obtain a finished collagen membrane.

2. The method for preparing a two-component osteoinductive collagen membrane according to claim 1, wherein: The concentration of the acetic acid or hydrochloric acid solution in step 2) is 0.1% to 5%. The added protease is trypsin, pepsin and / or ficin, and the added amount is 8% to 15% of the weight of the animal tissue slice. The enzymatic hydrolysis conditions are: stirring at 2 to 8°C for 6 to 36 hours.

3. The method for preparing a two-component osteoinductive collagen membrane according to claim 1, wherein: The filtration in step 3) is to use a 20-40 mesh stainless steel mesh to filter out particulate matter.

4. The method for preparing a two-component osteoinductive collagen membrane according to claim 1, wherein: The concentration of the sodium hydroxide solution added for precipitation in step 4) is 0.5 to 4 mol / L.

5. The method for preparing a two-component osteoinductive collagen membrane according to claim 1, wherein: In the re-dissolution in step 5), the ratio of the precipitated protein to the acetic acid solution is 1-50:1-100.

6. The method for preparing a two-component osteoinductive collagen membrane according to claim 1, wherein: The sterilization dose in step 10) is 15.2 to 25 kGy.

Images