Method for preparing mineralized collagen material from swine-derived tissue

Abstract

The invention provides a method for preparing a mineralized collagen material from pig-derived tissues. The prepared bone repair material can be used for bone defect filling repair and spinal fusion surgery. The method comprises the steps of pretreatment, enzymolysis, collagen fibrosis, mineralization, washing, slurry preparation, freeze drying, crosslinking, packaging and sterilization and the like. The obtained material has rapid liquid absorbability and adjustable porosity. The material can be mixed with bone fragments of a patient to form stable paste, components and structures similar to those of surrounding bone tissues are formed after bone defects are filled, and osseointegration is promoted. The pore structure can be adjusted to meet different operation requirements, and the integration effect with bone tissue is improved. The material provided by the invention can stably integrate a bioactive agent while keeping the structural integrity of collagen, does not influence the functions of the bioactive agent, improves the clinical effects of osseointegration and spinal fusion, and has wide application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, in particular to the preparation of mineralized collagen materials from porcine tissues for use in bone repair, especially in bone defect filling and repair and spinal fusion surgery. The material is intended to address challenges in the bone regeneration process, provide rapid liquid absorption and adjustable porosity, and form a stable paste or paste when mixed with blood and bone fragments to enhance surgical effects. Background Art

[0002] In orthopedic surgery, especially spinal fusion surgery, the use of bone graft replacement materials is crucial. Collagen-based materials, especially those that are mineralized to mimic natural bone structure, have been widely explored. However, existing materials face several limitations: 1. Absorption and integration issues: Many bone graft materials with collagen as the main component cannot effectively and quickly absorb blood or other fluids, which may affect their ability to form a cohesive and stable structure during surgery. They are easily dispersed by blood and their structure is unstable.

[0003] 2. Porosity and mechanical strength: Although some materials offer adjustable porosity, they often fail to maintain mechanical integrity when the design is modified, resulting in difficult handling and reduced osseointegration.

[0004] 3. Additive compatibility: In modern regenerative medicine, the integration of antibiotics, growth factors or other bioactive agents is essential to prevent infection and promote healing. However, many collagen-based bone repair materials lose stability or degrade when combined with these additives, making them unreliable in complex surgeries.

[0005] 4. Biosafety: Extracting collagen from bovine Achilles tendon poses a risk of transmissible spongiform encephalopathy (TSE), and control and monitoring measures must be strictly followed when using these materials. Bone transplant substitute materials prepared from bovine collagen are difficult to inactivate viruses and cannot effectively eliminate the risk of TSE infection.

[0006] The present invention aims to solve the following main problems: 1. Adjustable porosity to adapt to bone defect repair in different application scenarios: Controlling the porosity of the material is crucial to meet specific surgical needs. High porosity can improve fluid absorption and integration with bone, but often compromises mechanical strength. The present invention provides a preparation method for adjusting porosity while maintaining the structural stability and function of the material.

[0007] 2. Stable paste formation: In spinal fusion and other bone repair surgeries, it is critical that the material forms a stable paste or paste when mixed with broken bone fragments. This paste must maintain consistency under surgical conditions to ensure proper placement and integration. In spinal fusion surgery, the ability to quickly absorb blood is critical to forming a paste that is easy to operate. The mineralized collagen material of the present invention has rapid liquid absorption and can absorb liquid equivalent to 2 to 10 times its own volume within 1 minute, which is beneficial to surgical operations and bone healing processes.

[0008] 3. Absorption with bioactive agents: Modern surgical procedures often require the use of antibiotic drugs to prevent infection and growth factors to stimulate bone regeneration. Many existing materials lose structural integrity when mixed with these agents, resulting in poor performance. The present invention solves this problem by ensuring that the mineralized collagen material can stably integrate these bioactive substances without compromising their function.

[0009] 4. Bionics of bone structure: To ensure biocompatibility and promote natural bone healing, the mineral composition of the material is closely aligned with that of human bone. Existing bone graft substitute materials are either over-mineralized, resulting in rigidity, or under-mineralized, resulting in insufficient structural support. The present invention mimics the mineral content of human bones to achieve optimal performance in bone regeneration. The collagen fibers produced by the pretreatment, enzymatic hydrolysis, and collagen fibrosis treatment of porcine materials have a typical fiber structure compared to collagen materials obtained by other processes, and the mineralized complex is closer to bone composition and structure.

[0010] 5. Better biosafety: Through traceable porcine tissues, such as pig skin, pig pericardium and pig peritoneum, which are rich in collagen fibers, de-telopeptide collagen is obtained through pretreatment and enzymatic hydrolysis, and collagen fibers are assembled through alcohol treatment. This process route provides collagen fibers with high purity and better stability. Compared with collagen extracted from traditional bovine tissues, it has higher biosafety and no TSE risk. Summary of the invention

[0011] In view of the deficiencies in the prior art, the present invention provides a method for preparing a mineralized collagen material from porcine tissue, the method comprising the steps of pretreatment, enzymatic hydrolysis, collagen fibrosis, mineralized collagen, secondary washing, slurry preparation, freeze drying, cross-linking treatment, packaging and sterilization. The material prepared by this method has rapid liquid absorption and adjustable porosity, and is suitable for bone defect repair and spinal fusion surgery. The material is also compatible with bioactive agents, and the preparation process does not lose the integrity of the collagen fiber structure.

[0012] The method for preparing the mineralized collagen material from porcine tissue comprises the following steps: (1) Pretreatment: Take porcine tissue and wash it with alcohol, alkali and acid to obtain pretreated tissue; the porcine tissue includes any one or more of porcine skin, porcine small intestinal submucosa (SIS), porcine pericardium and porcine peritoneum; the alcohol washing uses ethanol or isopropanol; the alkali washing uses 2% sodium hydroxide or 5% sodium carbonate solution; the acid washing uses acetic acid or hydrochloric acid or phosphoric acid solution with a pH of 2 to 3; the alcohol washing, alkali washing and acid washing steps have a washing time of 1 to 6 hours each time, and the washing process can be repeated 1 to 5 times; (2) Enzymatic hydrolysis: The pretreated tissue blocks are crushed at 2-8°C and then enzymatically hydrolyzed using pepsin, with the pH adjusted to 2-3 and the temperature set to 2-37°C, or using trypsin, with the pH adjusted to 6-8 and the temperature set to 25-37°C. The enzymatic hydrolysis time is 2-72 hours, and the enzymatic hydrolyzate is filtered to obtain the collagen solution; (3) Collagen fibrosis: salting out the collagen solution with a high-concentration salt solution, filtering to obtain a filter cake, and repeating the salting out step once or multiple times; the salt solution is a high-concentration salt solution made of one or more of sodium chloride, sodium sulfate, or disodium hydrogen phosphate; adding ethanol or isopropanol solution to the filter cake and stirring and dehydrating for more than 30 minutes to obtain a fibrous precipitate, filtering, and repeating the dehydration for more than 2 times to obtain a collagen fiber filter cake; the collagen fiber filter cake can be dried at low temperature and crushed to obtain collagen fibers; (4) Mineralized collagen: Take collagen fibers or collagen fiber filter cake, add water and stir to dissolve, then add calcium chloride solution, phosphoric acid solution and sodium hydroxide solution in sequence, adjust the pH to 11-12, and precipitate and mature at 20-37°C for more than 12 hours; wash and filter to obtain a mineralized collagen bionic apatite filter cake; dry and crush the mineralized collagen bionic apatite filter cake to obtain bionic apatite powder; (5) Secondary washing: Take the collagen fiber filter cake of step (3), add 4 to 10 times the mass of ethanol or isopropanol solution, then add the mineralized collagen bionic apatite filter cake or bionic apatite powder of step (4), and stir for more than 20 minutes; after filtering and draining the solvent, the mineralized collagen bionic apatite filter cake or bionic apatite powder can be added repeatedly in batches; repeat the ethanol or isopropanol washing for more than 2 times; and vacuum dry to remove the solvent; (6) preparing a slurry: mixing the mineralized collagen fibers prepared in step (5) with the biomimetic apatite powder, and adjusting the mass ratio of water added to obtain a slurry having a water content of 50% to 90%; (7) Freeze drying: loading into a mold, pre-freezing and then freeze drying, the pre-freezing temperature range being -10 to -20°C; (8) Cross-linking treatment: vacuum heat cross-linking treatment is performed, wherein the vacuum heat cross-linking temperature ranges from 105 to 160° C. and the cross-linking time ranges from 3 to 72 hours; (9) Packaging and sterilization: According to the designed size, trimming, shaping, packaging, and radiation sterilization are carried out to obtain the mineralized collagen material.

[0013] Furthermore, the method for preparing bionic apatite powder from mineralized collagen comprises the following steps: (1) Solution preparation: 175 g of anhydrous calcium chloride dissolved in 1.5 kg of purified water, 160 g of sodium hydroxide dissolved in 1.0 kg of purified water, 105 g of 85% phosphoric acid dissolved in 0.9 kg of purified water, and 6.00 g of collagen dissolved in 6.0 kg of purified water; (2) Precipitation reaction: add the calcium chloride solution prepared above to the collagen solution in sequence, stir for more than 30 minutes, add the phosphoric acid solution, stir for more than 10 minutes, add the sodium hydroxide solution, adjust the pH to 11-11.5, and keep the pH value within this range and stir for more than 2 hours; (3) Maturation: raising the temperature of the reaction system to 35-37°C and leaving it to stand for more than 16 hours to promote the aging of the bionic apatite precipitation; (4) Washing: Stir and wash with a large amount of purified water. Repeat the washing operation for more than 3 times. Detect the TDS of the supernatant. When the TDS is less than 200 ppm, the washing is considered complete. (5) Drying and crushing, filtering to obtain a filter cake, low-temperature drying, crushing and sieving the dried filter cake to obtain bionic apatite powder.

[0014] Furthermore, the preparation method makes full use of the characteristics of collagen fibers made from porcine tissues, and through 40-80% ethanol or isopropanol cleaning and bionic apatite composite treatment, achieves efficient utilization of materials and ensures the integrity of the collagen fiber structure.

[0015] Furthermore, the main components of the mineralized collagen material prepared by the preparation method are collagen and apatite. The collagen exists in the form of fibers, maintains structural integrity, and enhances the ability of the material to form a cohesive structure during surgery. It has rapid liquid absorbency (absorbs 2 to 10 times the liquid within 1 minute) and adjustable porosity (50% to 95%).

[0016] The mineralized collagen material of the present invention not only has the ability to quickly absorb blood and form a paste that is easy to operate in surgery, but also has a pore structure that can be adjusted according to specific surgical needs to promote better integration with bone tissue. In addition, while maintaining structural integrity, the material is compatible with bioactive agents such as antibiotics and growth factors, and does not lose its structural integrity when integrating these bioactive agents.

[0017] The present invention also provides methods of using the mineralized collagen material in spinal fusion surgery by mixing with the patient's bone fragments and blood to form a stable paste that aids in bone integration.

[0018] The present invention also provides a bone repair material made of mineralized gelatin prepared from porcine tissue as raw material, wherein the bone repair material prepared from the mineralized gelatin material has a bulk density of 0.1-0.5 g / cm 3 characteristics.

[0019] The mineralized collagen material of the present invention has a wide range of application potentials and can improve the clinical effects of spinal fusion surgery and other bone defect repair surgeries.

[0020] Compared with the prior art, the present invention has the following advantages: 1. Biocompatibility: The porcine tissue is pre-treated by alcohol washing, alkali washing and acid washing, combined with enzymatic hydrolysis, and the extracted collagen has an atelopeptide structure, which not only ensures the integrity of the fiber structure, but also has good biocompatibility and low immunogenicity, which means that it will not cause a strong immune response when used in the human body and is suitable as an implant repair material.

[0021] 2. The bulk density of bone repair materials is 0.1~0.5g / cm 3 Adjustable, fast liquid absorbency (absorbs 2~10 times of liquid in 1 minute), suitable for bone defect repair and spinal fusion surgery.

[0022] 3. Mineralized collagen material is obtained by treating collagen fibers with biomimetic apatite during the cleaning process with 40-80% ethanol or isopropanol, so as to achieve efficient utilization and ensure the integrity of the collagen fiber structure to promote better integration with bone tissue.

[0023] 4. The material is compatible with bioactive agents and does not lose the structural integrity of collagen fibers during integration, providing a new material to improve the clinical effects of spinal fusion surgery and other bone repair surgeries. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a photo of the sample prepared in Example 3 of the present invention.

[0025] Figure 2 This is a photograph of the sample prepared in Example 3 of the present invention after being mixed with simulated blood.

[0026] Figure 3 This is a photo of a sample prepared in Example 4 of the present invention.

[0027] Figure 4 This is a photo of the sample prepared in Example 5 of the present invention.

[0028] Figure 5 This is a photo of the sample prepared in Example 6 of the present invention.

[0029] Figure 6 This is a photograph of the sample prepared in Example 6 of the present invention after absorbing water. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Example 1: Preparation of collagen fibers

[0031] step: 1. Preprocessing: Take 10 kg of pig skin and wash it with 100 L of ethanol, 40 kg of 2% sodium hydroxide, and 40 kg of 0.1 M acetic acid solution in sequence. Wash it for 1 hour each time and repeat the washing process 3 times. 2. Enzymatic hydrolysis: The pretreated pig skin was added to purified water, cooled to 2-8°C, crushed at low temperature and then enzymatically hydrolyzed, 100 g of pepsin (1:10000 NF) was added, the pH was adjusted to 2-3, the temperature was set to 25°C, the enzymatic hydrolysis time was 16 h, and the enzymatic hydrolyzate was filtered to obtain a collagen solution; 3. Collagen fibrosis: The collagen solution was added to a 7% NaCl solution for salting out, and the filter cake was filtered out and the salting out step was repeated once; the filter cake was added to an ethanol solution and stirred for dehydration for more than 30 min to obtain a fibrous precipitate, which was filtered and dehydrated for more than 2 times to obtain a collagen fiber filter cake; the collagen fiber filter cake was dried at low temperature for 12 h and crushed to obtain 485 g of collagen fibers. Example 2: Preparation of biomimetic apatite powder

[0032] step: 1. Solution preparation: Add 175 g of anhydrous calcium chloride to 1.5 kg of purified water, stir to dissolve and cool to room temperature.

[0033] Add 160 g of sodium hydroxide to 1.0 kg of purified water, stir to dissolve and cool to room temperature.

[0034] Add 105 g of 85% phosphoric acid to 0.9 kg of purified water, stir to dissolve and cool to room temperature.

[0035] Add 6.00 g collagen and 6.0 kg purified water into a 25 L bucket and stir at room temperature until the collagen is completely dissolved.

[0036] 2. Precipitation reaction: Add the calcium chloride solution prepared above into a 25L bucket and stir for 30 minutes.

[0037] The phosphoric acid solution prepared above was added and stirred for 10 minutes.

[0038] The sodium hydroxide solution prepared above was used to adjust the pH to 11-11.5, and the pH value was kept within this range, and the reaction was stirred for 2 h.

[0039] The pH value was continuously monitored during the reaction to ensure that it remained between 11 and 11.5.

[0040] 3. Ripening: After the reaction is completed, the reaction system is heated to 35-37°C and allowed to stand for 16 h to promote the precipitation and maturation of the bionic apatite.

[0041] 4. Washing: In a 25L barrel, remove the supernatant, add 15L of purified water, stir for 5 minutes and let it settle.

[0042] Repeat the washing operation 3 times, then detect the TDS of the supernatant. When the TDS is lower than 200ppm, the washing is considered complete; if it does not meet the conditions, continue washing.

[0043] 5. Drying and crushing: The mixture was filtered using a Buchner funnel and the filter cake was dried at 33-36°C.

[0044] The dried filter cake was crushed and sieved (60 meshes) to obtain bionic apatite powder, and 150 g of bionic apatite powder was obtained.

[0045] Example 3: Preparation of mineralized collagen material (bulk density = 0.1 g / cm 3 ) step: 1. Pretreatment: Cut fresh pigskin into 2 mm slices for pretreatment. Take 20 g of the pigskin and wash it with isopropanol for 2 h. Then wash it with 5% sodium carbonate solution for 2 h. Repeat this step twice. Wash the pigskin with pH=2 phosphoric acid solution for 2 h. Repeat this step once.

[0046] 2. Enzymatic hydrolysis: Add the washed pig skin into purified water, cool to 4°C and then grind at low temperature, add 1g pepsin (1:10000NF), adjust the pH to 2-3, stir and enzymolyze at 2-8°C for 2h, and filter to obtain the atelocollagen solution.

[0047] 3. Collagen fibrosis: Add the collagen solution to disodium hydrogen phosphate solution for salting out, stir and filter to obtain the filter cake, repeat the salting out step once, add the filter cake to ethanol solution, stir for more than 30 minutes, filter, and repeat the dehydration for more than 2 times to obtain the collagen fiber filter cake.

[0048] 4. Mixing and secondary washing: Disperse the collagen fiber filter cake in a 40% isopropanol aqueous solution, add 1.00 g of biomimetic apatite powder (Example 1), stir for more than 20 minutes, and filter to obtain a filter cake. Wash the filter cake in a 40% isopropanol aqueous solution and filter to obtain a filter cake. Wash it twice in 800 ml of isopropanol and filter to obtain a filter cake. Dry it under vacuum at 37°C to remove the solvent and obtain 2.98 g of mineralized collagen fibers.

[0049] 5. Prepare slurry: weigh mineralized collagen fibers and biomimetic apatite powder, add 9 times the weight of purified water and mix to obtain a slurry with a water content of 90%.

[0050] 6. Freeze drying: After stirring evenly, put it into a mold, pre-freeze at -12℃, and freeze dry for 48 hours.

[0051] 7. Cross-linking treatment: vacuum heat cross-linking treatment, cross-linking at 105℃ for 3h.

[0052] 8. Packaging and sterilization: Packaging, radiation sterilization, and obtain a bulk density of 0.1 g / cm 3 of mineralized collagen. test:

[0053] (1) Liquid absorbency: 8.9 times the liquid absorption in 1 minute (2) Porosity (Archimedes drainage method) = 91% The sample can quickly absorb simulated blood and maintain the fiber structure of collagen after rubbing. Figure 2 shown.

[0054] Example 4: Preparation of mineralized collagen material (bulk density = 0.2 g / cm 3 ) step: 1. Pretreatment: Cut fresh pig skin into thin slices, take 200g, and stir and wash with 2000ml isopropanol for 3h. Filter to remove isopropanol, wash the pig skin with 2000ml 2% sodium hydroxide solution for 3h, repeat this step twice. Wash the pig skin with 2000ml acetic acid solution with pH=2.5 for 2h, repeat this step twice.

[0055] 2. Enzymatic hydrolysis: Add pig skin to 4L purified water, cool to 4℃ and grind with a colloid mill to form a viscous liquid. Add 2.0g pepsin (1:10000NF) and stir at 20℃ for 2h to form a de-telopeptide collagen solution.

[0056] 3. Collagen fibrosis: Adjust the pH to neutral, add the collagen solution to 4L 20% sodium chloride solution, stir and perform salting out, filter and take the filter cake, repeat the salting out step once. Add the filter cake to the ethanol solution and stir for more than 30 minutes, filter, and repeat the dehydration for more than 2 times.

[0057] 4. Mixing and secondary washing: Disperse the filter cake in 2L of 60% ethanol aqueous solution, add 12.0g of biomimetic apatite powder (Example 1), stir and wash, and filter to obtain the filter cake. Wash the filter cake thoroughly in 60% ethanol aqueous solution, and remove the filter cake after dehydration. Dehydrate twice in 10L of anhydrous ethanol, and remove the filter cake after filtration. Vacuum dry to remove the solvent, and obtain 36.03g of mineralized collagen fiber.

[0058] 5. Prepare slurry: weigh mineralized collagen fibers and biomimetic apatite powder, mix with 4 times purified water to obtain a slurry with a water content of 80%.

[0059] 6. Freeze drying: After stirring evenly, put it into a mold, pre-freeze at -20℃, and freeze dry for 72 hours.

[0060] 7. Cross-linking treatment: vacuum heat cross-linking treatment, cross-linking at 105℃ for 24h.

[0061] 8. Packaging and sterilization: Packaging, radiation sterilization, and obtain a bulk density of 0.2 g / cm 3 of mineralized collagen. test:

[0062] (1) Liquid absorbency: 4.6 times the liquid absorption in 1 minute (2) Porosity (Archimedes drainage method) = 78% Example 5: Preparation of mineralized collagen material (bulk density = 0.5 g / cm 3 ) step: 1. Pretreatment: Slice fresh pigskin, take 2kg, and stir and wash with 20L isopropanol for 4h. Filter to remove isopropanol, wash the pigskin with 20L 2% sodium hydroxide solution for 3h, repeat this step once. Wash the pigskin with 20L 2.5% sodium carbonate solution for 2h. Wash the pigskin with 20L pH=2 phosphoric acid solution for 2h, filter and repeat this step once. Wash the pigskin with pH=2.5 acetic acid solution for 2h.

[0063] 2. Enzymatic hydrolysis: Add pig skin to 40L purified water, cool to 5℃ and crush to form a viscous liquid. Add 10.0g pepsin (1:10000NF) and stir at 15℃ for 4h to form a de-telopeptide collagen solution.

[0064] 3. Collagen fibrosis: Add the collagen solution to 40L of 15% sodium chloride solution, stir, filter and take the filter cake, repeat the salting out step once. Add the filter cake to the ethanol solution and stir for more than 30 minutes, filter, and repeat the dehydration for more than 2 times.

[0065] 4. Mixing and secondary washing: Disperse the filter cake in 20L of 60% ethanol aqueous solution, add 160g of hydroxyapatite (Example 1), stir and wash, and filter to obtain the filter cake. Wash the filter cake thoroughly in 60% ethanol aqueous solution, dehydrate and obtain the filter cake. Dehydrate in 20L of anhydrous ethanol, filter and obtain the filter cake, and repeat 4 times. Vacuum dry at 37°C to obtain 390g of mineralized collagen fiber.

[0066] 5. Prepare slurry: Mix mineralized collagen fiber, biomimetic apatite powder and purified water in a ratio of 1:1 to obtain a slurry with a water content of 50%.

[0067] 6. Freeze drying: After stirring evenly, inject into the mold, compact, pre-freeze at -10℃, and freeze dry for 72 hours.

[0068] 7. Cross-linking treatment: vacuum heat cross-linking treatment, vacuum cross-linking at 105℃ for 4h.

[0069] 8. Packaging and sterilization: Packaging, radiation sterilization, and obtain a bulk density of 0.5 g / cm 3 of mineralized collagen. test:

[0070] (1) Liquid absorbency: 2.1 times the liquid absorption in 1 minute (2) Porosity (Archimedes drainage method) = 53% Example 6: Continuous process for preparing mineralized collagen material from porcine tissue (bulk density = 0.1 g / cm 3 ) 1. Preprocessing Take 10 kg of pig skin, slice it, stir and wash it with 100L isopropanol for 4 hours, remove and drain. Add 40 kg of purified water, add 800 g of NaOH and stir for 1 hour, remove and drain, repeat 3 times. Add purified water and acetic acid to adjust the pH to 2-3, stir for 30 minutes, remove and drain after the end, repeat 3 times. Add 40 kg of purified water each time, and add acetic acid to adjust the pH to 2-3.

[0071] 2. Enzymatic hydrolysis Add 90 L of purified water to a 100 L reactor, cool to 2-8°C, and grind mechanically at low temperature. Add 100 g of pepsin (1:10000 NF) and stir to dissolve, add acetic acid to adjust the pH to 2-3, and perform enzymolysis at 20-25°C for 24 hours. Filter the enzymolysis solution to obtain the collagen solution.

[0072] 3. Collagen fibrosis Add 6 kg NaCl and 40 kg water to the collagen solution, salt out, stir for 0.5 h to obtain a precipitate, and filter to obtain a filter cake. Repeat the salting out step once. Add the filter cake to the ethanol solution and stir for 30 min, filter, and repeat the dehydration twice to obtain a collagen fiber filter cake. The collagen fiber filter cake is vacuum dried at room temperature for 72 h and crushed to obtain collagen fibers.

[0073] 4. Mineralized collagen Take 80 g of collagen fiber, add it to 50 L of purified water, and stir overnight to dissolve. Add 1750 g of anhydrous CaCl to 15 kg of purified water. 2 , add 1.6 kg NaOH to 10 kg purified water, add 1050 g phosphoric acid (85%) to 9 kg purified water, stir and dissolve. 2 The solution was added to the collagen solution and stirred for 30 minutes; the phosphoric acid solution was added to the above solution and stirred for 10 minutes; sodium hydroxide solution was added to the above solution in batches, the pH was adjusted to 11-11.5, and the pH value was kept in this range and stirred for more than 2 hours; the temperature was raised to 33.5°C (35-37°C), stirring was stopped and allowed to stand overnight for aging. After standing and aging for 18 hours, the supernatant was removed, purified water was added, stirred, washed and filtered. The TDS of the supernatant was detected. When the TDS was less than 200ppm, the washing was considered complete. After filtration, the filter cake was vacuum dried at 30°C and crushed to obtain biomimetic apatite powder (<60 mesh), about 1.5kg.

[0074] 5. Secondary washing Take 1.40 kg of collagen fiber filter cake after salting out and add it to 5.6 kg of 60% anhydrous ethanol (4 times the mass), then add 150 g of dried and crushed biomimetic apatite powder (<60 mesh), stir for 20 min; filter and drain the solvent, then add 50 g of hydroxyapatite (<60 mesh) and 4.2 kg of 60% anhydrous ethanol (3 times), stir for 20 min; repeat the anhydrous ethanol washing twice. After vacuum drying for 18 h, collect 581.75 g of the mixture.

[0075] 6. Prepare slurry According to the design of mass ratio of collagen: apatite ≈ 1:1, 172 g of the mixture was taken, and 50 g of biomimetic apatite (<60 mesh) was added; 9 times the mass of purified water was added, and the mixture was stirred and mixed evenly to obtain a slurry with a water content of 90%.

[0076] 7. Freeze Drying Put it into a mold, pre-freeze it at -10℃ for 6 hours, and freeze-dry it. Take it out after freeze-drying for 48 hours.

[0077] 8. Cross-linking The vacuum heat cross-linking treatment was carried out at 105°C for 3 h.

[0078] 9. Packaging After cutting, double-layer packaging is performed, and according to the designed size, trimming and shaping are performed, packaging is performed, and radiation sterilization is performed to obtain the mineralized collagen material. test:

[0079] (1) Liquid absorption 8.3 times in 1 minute (2) Porosity (Archimedes drainage method) = 86% The sample absorbs water and is kneaded into shape, which is suitable for bone defect filling and spinal fusion surgery, such as Figure 6 shown.

[0080] This embodiment provides a complete scheme for extracting collagen fibers from pig skin and preparing mineralized collagen materials. Through the above preparation method, high-purity, high-biocompatible bone repair materials are prepared. Compared with the commonly used method of preparing bone repair materials with collagen composite hydroxyapatite, which takes 30 to 45 days, the entire process of this scheme only takes 9 to 12 days, greatly saving manufacturing time.

[0081] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a mineralized collagen material from porcine tissue, characterized in that The method comprises the following steps: (1) Pretreatment: taking porcine tissue, and washing it with alcohol, alkali and acid to obtain a pretreated decellularized tissue block; the porcine tissue includes any one or more of porcine skin, porcine small intestinal submucosa (SIS), porcine pericardium and porcine peritoneum; the alcohol washing uses ethanol or isopropanol; the alkali washing uses 2% sodium hydroxide or 5% sodium carbonate solution; the acid washing uses acetic acid or hydrochloric acid or phosphoric acid solution with a pH of 2 to 3; the alcohol washing, alkali washing and acid washing steps each take 1 to 6 hours, and the washing process can be repeated 1 to 5 times; (2) Enzymatic hydrolysis: The pretreated tissue blocks are crushed at 2-8°C and then enzymatically hydrolyzed using pepsin, with the pH adjusted to 2-3 and the temperature set to 2-37°C; or using trypsin, with the pH adjusted to 6-8 and the temperature set to 25-37°C. The enzymatic hydrolysis time is 2-72 hours, and the enzymatic hydrolyzate is filtered to obtain the collagen solution; (3) Collagen fibrosis: salting out the collagen solution with a high-concentration salt solution, filtering to obtain a filter cake, and repeating the salting out step once or multiple times; the salt solution is a high-concentration salt solution made of one or more of sodium chloride, sodium sulfate, or disodium hydrogen phosphate; adding ethanol or isopropanol solution to the filter cake and stirring and dehydrating for more than 30 minutes to obtain a fibrous precipitate, filtering, and repeating the dehydration for more than 2 times to obtain a collagen fiber filter cake; the collagen fiber filter cake can be dried at low temperature and crushed to obtain collagen fibers; (4) Mineralized collagen: Take collagen fibers or collagen fiber filter cake, add water and stir to dissolve, then add calcium chloride solution, phosphoric acid solution and sodium hydroxide solution in sequence, adjust the pH to 11-12, and precipitate and mature at 20-37°C for more than 12 hours; wash and filter to obtain a mineralized collagen bionic apatite filter cake; dry and crush the mineralized collagen bionic apatite filter cake to obtain bionic apatite powder; (5) Secondary washing: Take the collagen fiber filter cake of step (3), add 4 to 10 times the mass of ethanol or isopropanol solution, then add the mineralized collagen bionic apatite filter cake or bionic apatite powder of step (4), and stir for more than 20 minutes; after filtering and draining the solvent, the mineralized collagen bionic apatite filter cake or bionic apatite powder can be added repeatedly in batches; repeat the ethanol or isopropanol washing for more than 2 times; and vacuum dry to remove the solvent; (6) preparing a slurry: mixing the mineralized collagen fibers prepared in step (5) with the biomimetic apatite powder, and adjusting the mass ratio of water added to obtain a slurry having a water content of 50% to 90%; (7) Freeze drying: loading into a mold, pre-freezing and then freeze drying, the pre-freezing temperature range being -10 to -20°C; (8) Cross-linking treatment: vacuum heat cross-linking treatment is performed, wherein the vacuum heat cross-linking temperature ranges from 105 to 160° C. and the cross-linking time ranges from 3 to 72 hours; (9) Packaging and sterilization: According to the designed size, trimming, shaping, packaging, and radiation sterilization are carried out to obtain the mineralized collagen material.

2. The method according to claim 1, characterized in that The method for preparing mineralized collagen in step (4) comprises the following steps: (1) Solution preparation: Prepare the solution in the following proportions: 175 g of anhydrous calcium chloride dissolved in 1.5 kg of purified water, 160 g of sodium hydroxide dissolved in 1.0 kg of purified water, 105 g of 85% phosphoric acid dissolved in 0.9 kg of purified water, and 6.00 g of collagen dissolved in 6.0 kg of purified water; (2) Precipitation reaction: add the calcium chloride solution prepared above to the collagen solution in sequence, stir for more than 30 minutes, add the phosphoric acid solution, stir for more than 10 minutes, add the sodium hydroxide solution, adjust the pH to 11-11.5, and keep the pH value within this range and stir for more than 2 hours; (3) Ripening: The reaction system was heated to 35-37 °C and allowed to stand for more than 16 h to promote the aging of the bionic apatite precipitation; (4) Washing: Stir and wash with a large amount of purified water, repeat the washing operation for more than 3 times, and detect the TDS of the supernatant. When the TDS is less than 200ppm, the washing is considered complete; (5) Drying and crushing: Filter to obtain a filter cake, dry it at low temperature, and crush and sieve the dried filter cake to obtain bionic apatite powder.

3. The method according to claim 1, characterized in that The step (5) makes full use of the characteristics of the collagen fibers made from porcine tissues, and achieves efficient use of the materials by washing with 40-80% ethanol or isopropanol and compounding with bionic apatite, thereby ensuring the integrity of the collagen fiber structure.

4. A mineralized collagen material prepared from porcine tissue, characterized in that The material is prepared by the method according to claim 1, and its main components are collagen and apatite. The collagen exists in the form of fibers, maintains structural integrity, plays a role in enhancing the ability of the material to form a cohesive structure during surgery, has rapid liquid absorption (absorbs 2 to 10 times the liquid within 1 minute) and adjustable porosity (50% to 95%).

5. The mineralized collagen material according to claim 4, characterized in that It can quickly form a stable paste when mixed with blood, which is suitable for bone defect filling and repair and spinal fusion surgery.

6. The mineralized collagen material according to claim 4, characterized in that It is compatible with bioactive agents such as antibiotics and growth factors and can incorporate these bioactive agents without losing its structural integrity.

7. A method for using the mineralized collagen material according to any one of claims 4 to 6 in spinal fusion surgery, characterized in that The material is mixed with the patient’s own bone fragments to form a stable paste that aids in osseointegration.

8. A bone repair material made from mineralized gel prepared from porcine tissue, characterized in that Prepared from the mineralized collagen material according to any one of claims 1 to 7, having a bulk density of 0.1-0.5 g / cm 3 characteristics.

Images