Preparation method of allogeneic bone shaping material

Abstract

The invention discloses a preparation method of an allogeneic bone shaping material, which comprises the following steps: S1, bone pretreatment to obtain freeze-dried bone powder and demineralized bone powder; s2, bone gelatin extraction; s3, preparing an excipient, wherein the bone gelatin in the step S2 is added into water containing polyvinylpyrrolidone to be swelled; and S4, mixing the freeze-dried bone powder and the demineralized bone powder in the step S1 with the excipient in the step S3 in proportion to obtain the allogeneic bone shaping material. According to the preparation method, polyvinylpyrrolidone is added, the proportion of freeze-dried bone powder to demineralized bone powder in the mixed bone powder is adjusted, the degradation rate of the allogeneic bone powder can be adjusted, and the allogeneic bone powder can be effectively degraded on the basis that good bone induction and bone conduction performance of the allogeneic bone powder is reserved. And the material is endowed with good injectability and plasticity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and relates to a preparation method of an allogeneic bone shaped material, in particular to a preparation method of an allogeneic bone shaped material with adjustable degradation rate. BACKGROUND

[0002] In orthopedic clinical practice, the repair of bone defects caused by trauma, tumor resection or infection is a very challenging medical problem. Autologous bone transplantation is considered as the "gold standard", but its source is limited and there is a risk of complications in the donor area. Allogeneic bone is one of the most widely used bone repair materials in clinical application due to its excellent bone conduction and bone induction activity. According to different processing techniques, allogeneic bone powder is mainly divided into two categories: demineralized bone powder and freeze-dried bone powder. Demineralized bone powder removes calcium and phosphorus components (calcium content ≤8%) through acid treatment, retains collagen matrix and growth factors, and has significant bone induction activity, but its degradation rate is relatively fast (usually 4-8 weeks); freeze-dried bone powder (calcium content about 26%) has outstanding bone conduction due to the retention of mineralized components, but its degradation is slow (up to 6-12 months), and it lacks induction activity.

[0003] At present, in order to meet the needs of different defect sites for degradation rate, such as the need for rapid degradation in the cancellous bone area to promote the replacement of new bone, and the need for slow degradation in the weight-bearing area to maintain mechanical support, demineralized bone powder and freeze-dried bone powder are often used together. However, this method has two major technical bottlenecks: (1) Poor mixing uniformity: the demineralization process significantly reduces the density of demineralized bone powder, while freeze-dried bone powder has a higher density. Due to the density difference, freeze-dried bone powder quickly settles and demineralized bone powder floats during mixing, resulting in obvious stratification and affecting bone regeneration effect.

[0004] (2) Limited function of excipients: traditional excipients can improve the adhesion of bone powder, but their linear molecular structure cannot form a three-dimensional network to uniformly wrap the bone powder. Studies have shown that methyl cellulose-based materials are prone to swelling and degradation in body fluids (viscosity reduction >40%), further exacerbating uneven dispersion of bone powder; in addition, their degradation behavior is not coordinated with bone powder, and precise control of degradation rate cannot be achieved.

[0005] The existing technology has not effectively solved the core problems of uneven mixing of allogeneic bone powder and uncontrolled degradation. Therefore, it is urgent to develop a new preparation method. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a preparation method of an allogeneic bone shaped material. To solve the above technical problems, the specific technical scheme adopted by the present application is as follows: A method for preparing an allogeneic bone explant material includes the following steps: S1. Pre-treatment of bone to obtain freeze-dried bone powder and demineralized bone powder; S2. Bone gelatin extraction; S3. Preparation of excipients; In the preparation of excipients, the bone gelatin described in step S2 is added to water containing polyvinylpyrrolidone to swell; S4. The freeze-dried bone powder and demineralized bone powder obtained in step S1 are mixed with the excipient obtained in step S3 in a certain proportion to obtain the allogeneic bone excipient material.

[0007] In some specific implementations, in step S3, the mass ratio of the polyvinylpyrrolidone to the bone gelatin is 0.2~1:2~4.

[0008] In some specific implementations, in step S3, the particle size of the excipient particles is 0.1–2 mm.

[0009] In some specific implementations, in step S4, the mass ratio of the freeze-dried bone powder to the demineralized bone powder is 1:10 to 10:1, and the volume ratio of the mixed bone powder obtained from the freeze-dried bone powder and the demineralized bone powder to the excipient is 3:7 to 7:3.

[0010] In some specific implementations, step S1, the pre-bone treatment includes decellularization treatment, virus inactivation treatment, defatting treatment, and demineralization treatment.

[0011] In some specific implementations, the human bone selected in step S1 is chosen from one or both of cortical bone and cancellous bone.

[0012] In some specific implementations, the human bone selected in step S1 undergoes deep cryogenic pretreatment, wherein the deep cryogenic temperature is -80℃ to -40℃.

[0013] In some specific implementations, the decellularization treatment is carried out at a temperature of 30°C to 40°C and the shaking time is 12 to 24 hours.

[0014] In some specific implementations, the degreasing treatment is carried out at a temperature of 30°C to 40°C, and the liquid is changed 3 to 7 times.

[0015] In some specific implementations, the demineralization process is a gradient demineralization, with a volume ratio of human bone to hydrochloric acid solution of 1:5 to 1:10.

[0016] In some specific implementations, in the bone gelatin extraction described in step S2, the temperature is 30℃~40℃, the shaking time is 4~8h, and the supernatant is collected 4~8 times.

[0017] Preferably, a method for preparing an allogeneic bone prosthetic material includes the following specific steps: (1) Human bones frozen at deep low temperature were selected.

[0018] (2) Remove the soft tissue outside the bone tissue and clean the bone tissue. Cut the bone into small pieces and rinse the small pieces until there is no bone marrow or blood in them. Crush the small pieces to obtain bone powder with a particle size of 1-2 mm.

[0019] (3) Decellularization treatment: Soak bone meal in hydrogen peroxide solution and shake at a constant temperature. After soaking, wash bone meal with purified water until the washing solution is clear and transparent, and pour out hydrogen peroxide waste liquid.

[0020] (4) Virus inactivation treatment: Soak bone meal in ethanol solution for 2-4 hours and shake at constant temperature. After soaking, wash bone meal blocks with purified water until the washing solution is clear and transparent, and pour out ethanol waste liquid.

[0021] (5) Degreasing treatment: Soak bone meal in acetone solution and shake at a constant temperature. Change the solution every 12 hours until the acetone solution is clear and there is no oily substance floating on the surface. Pour out the acetone waste liquid.

[0022] (6) Remove impurities: Soak bone meal in trypsin solution for 8-12 hours to remove impurities. After soaking, pour out the waste solution of trypsin. Then soak in sodium dodecyl sulfate solution for 18 hours and change the solution 1-2 times until the soaking solution is clear and transparent. Pour out the waste solution of sodium dodecyl sulfate.

[0023] (7) Wash the bone powder with purified water until it is neutral; wash the bone powder with purified water until the wastewater after washing is neutral, divide the washed bone powder into two parts, store one part in a deep low temperature freezer, and then freeze-dry it to obtain freeze-dried bone powder.

[0024] (8) Another portion of the cleaned bone meal underwent gradient demineralization. The bone meal was mixed with hydrochloric acid solution and demineralized until no more bubbles were generated in the solution. The hydrochloric acid waste liquid was then poured out. Demineralization was continued using a low-concentration hydrochloric acid solution. The above steps were repeated until the bone meal became transparent. The hydrochloric acid waste liquid was then poured out, and the bone meal was washed with purified water until neutral. The bone meal after gradient demineralization was divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0025] (9) Another portion of the bone meal after gradient demineralization was mixed with sodium hydroxide solution and left to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 10-12 with sodium hydroxide solution.

[0026] (10) Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, shake at constant temperature, and collect the supernatant by centrifugation; add purified water of the same volume as the supernatant and repeat the above operation; combine the collected supernatants, put them into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0027] (11) The freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone and pre-swollen for 0.5-1h. Then it was transferred to glycerol at 50-70℃ and mixed evenly to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and placed at low temperature for 24h to condense and form a gel. The gel excipient was prepared into uniform granules using a homogenizer.

[0028] (12) Freeze-dried bone powder and demineralized bone powder are mixed evenly to form mixed bone powder. Then the mixed bone powder is mixed evenly with excipients to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. This application uses allogeneic bone as a substrate to prepare allogeneic bone-based materials with uniform mixing of bone powders of different densities and controllable degradation. These materials have excellent biocompatibility, good injectability and plasticity.

[0030] 2. The preparation method of this application prepares freeze-dried bone powder and demineralized bone powder through processes such as defatting, decellularization, virus inactivation, demineralization, and freeze-drying. Based on this, bone gelatin is extracted and excipients are prepared to give the allogeneic bone powder a good excipient effect and achieve uniform mixing of demineralized bone powder and freeze-dried bone powder.

[0031] 3. This application can achieve adjustable degradation rate of allogeneic bone meal by adjusting the ratio of freeze-dried bone meal and demineralized bone meal in the mixed bone meal.

[0032] 4. The allogeneic bone-derived material prepared in this application retains the good osteoinductive and osteoconductive properties of allogeneic bone powder, while giving the material good injectability and plasticity, effectively solving the problems of allogeneic bone powder being difficult to use and prone to drift in clinical practice.

[0033] 5. The allogeneic bone-associated material prepared in this application also has good biosafety, biocompatibility and biodegradability.

[0034] The following will further explain the concept and technical effects of the present invention in order to fully understand the purpose, features and effects of the present invention. Detailed Implementation

[0035] To make the technical means, inventive features, objectives and effects of the invention easier to understand, the invention is further described, but the invention is not limited to the following embodiments.

[0036] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, unless otherwise specified, the following embodiments and features in the embodiments can be combined with each other.

[0037] The method for preparing the allogeneic bone modeling material of this application specifically includes the following steps: S1. Pre-bone treatment, including decellularization, virus inactivation, defatting, and demineralization processes: (1) Select one or both of cortical bone and cancellous bone that have been cryogenically frozen at -80℃ to -40℃.

[0038] (2) Remove the soft tissue outside the bone tissue, clean the bone tissue with purified water, then cut the bone into small pieces, rinse the small pieces with a high-pressure water gun until there is no bone marrow or blood in the small pieces; mix the small pieces with dry ice at a mass ratio of 1:1, and then put them into a bone powder pulverizer to pulverize them to obtain bone powder with a particle size of 1~2mm.

[0039] (3) Decellularization treatment: The pulverized bone powder and 3% hydrogen peroxide solution are mixed in a volume ratio of 1:1. The bone powder is soaked in the 3% hydrogen peroxide solution and shaken at a constant temperature of 30℃~40℃ for 12~24h. After soaking, the bone powder is washed with purified water until the washing liquid is clear and transparent, and the hydrogen peroxide waste liquid is poured out.

[0040] (4) Virus inactivation treatment: Mix bone meal and 75% ethanol solution at a volume ratio of 1:2. Soak the bone meal in 75% ethanol solution for 2-4 hours and shake at a constant temperature. After soaking, wash the bone meal with purified water until the washing solution is clear and transparent, and pour out the ethanol waste liquid.

[0041] (5) Degreasing treatment: Mix bone meal and acetone solution at a volume ratio of 1:5, and soak the bone meal in the acetone solution under constant temperature and shaking. Change the acetone solution every 12 hours until the soaked acetone solution is clear and transparent and there is no oily substance floating on the surface. Pour out the acetone waste liquid. The soaking temperature is 30℃~40℃ and the number of liquid changes is 3~7 times.

[0042] (6) Impurity removal treatment: Soak bone meal in 0.25% trypsin solution at a volume ratio of 1:5 for 8-12 hours to remove impurities. After soaking, pour out the waste liquid of trypsin solution and soak in 0.25% sodium dodecyl sulfate solution for 18 hours, changing the solution 1-2 times until the soaking liquid is clear and transparent. Pour out the waste liquid of sodium dodecyl sulfate solution.

[0043] (7) Purified water washing treatment: Wash with purified water until the wastewater is neutral. The bone powder washed with purified water until neutral is divided into two parts. One part is stored in a deep low temperature freezer and then freeze-dried to obtain freeze-dried bone powder.

[0044] (8) Demineralization: Another portion of bone meal washed with purified water undergoes gradient demineralization. The bone meal is mixed with 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:5 to 1:10 until no more bubbles are generated in the solution, then the hydrochloric acid waste liquid is poured off. The above steps are then repeated with 0.6 mol / L hydrochloric acid solution to continue demineralization until the bone meal becomes transparent. The hydrochloric acid waste liquid is then poured off, and the bone meal is washed with purified water until neutral. The bone meal after gradient demineralization is divided into two parts. One part is stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0045] (9) Another portion of bone powder after gradient demineralization is treated with sodium hydroxide. The bone powder is mixed with a 10% mass fraction sodium hydroxide solution at a volume ratio of 3:10 to 3:15 and then left to stand for 24 hours. During this period, the pH of the reaction solution is adjusted to 10 to 12 with a 10% mass fraction sodium hydroxide solution.

[0046] It should be understood that the processing temperature, shaking time, soaking time, number of liquid changes, etc. in the processing steps can be selected according to the actual situation or the experience of those skilled in the art, as long as the purpose of cleaning can be achieved.

[0047] S2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, and keep it at a constant temperature of 30-40℃ with shaking for 4-8 hours. Centrifuge and collect the supernatant. Add purified water of the same volume as the supernatant, and repeat the above operation 4-8 times. Combine and collect the supernatants obtained from the repeated operations, put them into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0048] S3. Preparation of excipients: Freeze-dried bone gelatin was pre-swelled in purified water containing polyvinylpyrrolidone for 0.5–1 h, with a mass ratio of polyvinylpyrrolidone to bone gelatin of 0.2–1:2–4. The mixture was then transferred to glycerol and mixed thoroughly at 50–70 °C to form a sol. The sol was then placed in a vacuum drying oven to remove air bubbles and allowed to cool for 24 h to condense, resulting in a gel. An excipient with a particle size of 0.1–2 mm was prepared using a homogenizer and a vibrating sieve. This excipient not only prevents bone powder from settling and agglomerating due to gravity after mixing, but also reduces the friction between bone powder particles during subsequent use.

[0049] S4. Preparation of allogeneic bone-bone-associated materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 1:10 to 10:1 to form a mixed bone powder. The mixed bone powder and excipients are then mixed evenly at a volume ratio of 3:7 to 7:3 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0050] Example 1: Preparation of allogeneic bone adjuvant material with adjustable degradation rate 1. Pre-bone preparation: Cortical bone frozen at -80℃ was selected. The soft tissue surrounding the bone was removed, and the bone tissue was cleaned with purified water. The bone was then cut into small pieces and rinsed with a high-pressure water gun until no bone marrow or blood remained. The bone pieces were mixed with dry ice at a 1:1 mass ratio and then pulverized in a bone powder grinder to obtain bone powder with a particle size of 1-2 mm. The pulverized bone powder was mixed with a 3% hydrogen peroxide solution at a 1:1 volume ratio. The bone powder was then immersed in the 3% hydrogen peroxide solution and agitated at a constant temperature of 30℃ for 12 hours. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the hydrogen peroxide waste liquid was discarded. The bone powder was then mixed with a 75% ethanol solution at a 1:2 volume ratio. The bone powder was immersed in the 75% ethanol solution for 2 hours and agitated at a constant temperature. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the ethanol waste liquid was discarded. Bone meal and acetone solution were mixed at a volume ratio of 1:5. The acetone solution was then soaked in the bone meal under constant temperature and shaking at 30°C. The acetone solution was changed every 12 hours, for a total of 3 times, until the acetone solution was clear and transparent with no floating oily substances. The acetone waste liquid was then poured off. Bone meal was then soaked in 0.25% trypsin solution at a volume ratio of 1:5 for 8 hours to remove impurities. After soaking, the trypsin waste liquid was poured off. The bone meal was then soaked in 0.25% sodium dodecyl sulfate solution for 18 hours, with the solution changed 1-2 times until the soaking solution was clear and transparent. The sodium dodecyl sulfate waste liquid was then poured off. The bone meal was washed with purified water until neutral. The bone meal was then divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain freeze-dried bone meal.

[0051] Another portion of the bone meal washed with purified water underwent gradient demineralization. The bone meal was mixed with a 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:5 until no more bubbles were produced, at which point the hydrochloric acid waste liquid was poured off. This process was then repeated with a 0.6 mol / L hydrochloric acid solution until the bone meal became transparent. The hydrochloric acid waste liquid was then poured off, and the meal was washed with purified water until neutral. The demineralized bone meal was divided into two portions. One portion was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0052] Another portion of the bone meal after gradient demineralization was treated with sodium hydroxide. The bone meal was mixed with a 10% sodium hydroxide solution at a volume ratio of 3:10 and allowed to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 10 with a 10% sodium hydroxide solution.

[0053] 2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, keep the temperature at 40℃ and shake for 4 hours, centrifuge and collect the supernatant; add an equal volume of purified water to the supernatant, repeat the above operation 4 times, combine and collect the supernatant obtained from the repeated operation, put it into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0054] 3. Preparation of excipients: Freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone and pre-swollen for 0.5 h, wherein the mass ratio of polyvinylpyrrolidone to bone gelatin was 0.2:2; then it was transferred to glycerol and mixed evenly at 50 °C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and then placed at low temperature for 24 h to condense and form a gel. The excipient was homogenized into uniform granules with a particle size of 0.1–0.5 mm using a homogenizer.

[0055] 4. Preparation of allogeneic bone explant materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 1:10 to form mixed bone powder. Then, the mixed bone powder and excipients are mixed evenly at a volume ratio of 3:7 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0056] Example 2: Preparation of Allogeneic Bone Adhesive Material with Adjustable Degradation Rate (Part 2) 1. Pre-bone preparation: Cancellous bone frozen at -70℃ was selected. The external soft tissue was removed, and the bone tissue was cleaned with purified water. The bone was then cut into small pieces and rinsed with a high-pressure water gun until no bone marrow or blood components remained. The bone pieces were mixed with dry ice at a 1:1 mass ratio and then pulverized in a bone powder grinder to obtain bone powder with a particle size of 1-2 mm. The pulverized bone powder was mixed with a 3% hydrogen peroxide solution at a 1:1 volume ratio. The bone powder was then immersed in the 3% hydrogen peroxide solution and agitated at a constant temperature of 33℃ for 16 hours. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the hydrogen peroxide waste liquid was discarded. The bone powder was then mixed with a 75% ethanol solution at a 1:2 volume ratio. The bone powder was immersed in the 75% ethanol solution for 2.5 hours and agitated at a constant temperature. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the ethanol waste liquid was discarded. Bone meal and acetone solution were mixed at a volume ratio of 1:5. The acetone solution was then soaked in the bone meal under constant temperature and shaking at 35°C. The acetone solution was changed every 12 hours, for a total of 4 times, until the acetone solution was clear and transparent with no floating oily substances. The acetone waste liquid was then discarded. Bone meal was then soaked in a 0.25% trypsin solution at a volume ratio of 1:5 for 9 hours to remove impurities. After soaking, the trypsin waste liquid was discarded. The bone meal was then soaked in a 0.25% sodium dodecyl sulfate solution for 18 hours, with the solution changed 1-2 times until the soaking solution was clear and transparent. The sodium dodecyl sulfate waste liquid was then discarded. The bone meal was washed with purified water until neutral. The neutralized bone meal was then divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain freeze-dried bone meal.

[0057] Another portion of the bone meal washed with purified water underwent gradient demineralization. The bone meal was mixed with a 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:6 until no more bubbles were produced, at which point the hydrochloric acid waste liquid was poured off. This process was then repeated with a 0.6 mol / L hydrochloric acid solution until the bone meal became transparent. The hydrochloric acid waste liquid was then poured off, and the meal was washed with purified water until neutral. The demineralized bone meal was divided into two portions. One portion was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0058] Another portion of the bone meal after gradient demineralization was treated with sodium hydroxide. The bone meal was mixed with a 10% sodium hydroxide solution at a volume ratio of 3:11 and allowed to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 10.5 with a 10% sodium hydroxide solution.

[0059] 2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, keep the temperature at 38℃ and shake for 5 hours, centrifuge and collect the supernatant; add an equal volume of purified water to the supernatant, repeat the above operation 5 times, combine and collect the supernatant obtained from the repeated operation, put it into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0060] 3. Preparation of excipients: Freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone and pre-swollen for 0.5 h, wherein the mass ratio of polyvinylpyrrolidone to bone gelatin was 0.5:3; then it was transferred to glycerol and mixed evenly at 55 °C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and then placed at low temperature for 24 h to condense and form a gel. The excipient was homogenized into uniform granules with a particle size of 0.5–1 mm using a homogenizer.

[0061] 4. Preparation of allogeneic bone explant materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 1:5 to form mixed bone powder. Then, the mixed bone powder and excipients are mixed evenly at a volume ratio of 3:5 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0062] Example 3: Preparation of Allogeneic Bone Adaptive Materials with Adjustable Degradation Rate 1. Pre-bone preparation: Cancellous bone frozen at -60℃ was selected. The external soft tissue was removed, and the bone tissue was cleaned with purified water. The bone was then cut into small pieces and rinsed with a high-pressure water gun until no bone marrow or blood components remained. The bone pieces were mixed with dry ice at a 1:1 mass ratio and then pulverized in a bone powder grinder to obtain bone powder with a particle size of 1-2 mm. The pulverized bone powder was mixed with a 3% hydrogen peroxide solution at a 1:1 volume ratio. The bone powder was then immersed in the 3% hydrogen peroxide solution and agitated at a constant temperature of 35℃ for 18 hours. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the hydrogen peroxide waste liquid was discarded. The bone powder was then mixed with a 75% ethanol solution at a 1:2 volume ratio. The bone powder was immersed in the 75% ethanol solution for 3 hours and agitated at a constant temperature. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the ethanol waste liquid was discarded. Bone meal and acetone solution were mixed at a volume ratio of 1:5. The acetone solution was then soaked in the bone meal under constant temperature and shaking at 35°C. The acetone solution was changed every 12 hours, for a total of 5 times, until the acetone solution was clear and transparent with no floating oily substances. The acetone waste liquid was then discarded. Bone meal was then soaked in 0.25% trypsin solution at a volume ratio of 1:5 for 10 hours to remove impurities. After soaking, the trypsin waste liquid was discarded. The bone meal was then soaked in 0.25% sodium dodecyl sulfate solution for 18 hours, with the solution changed 1-2 times until the soaking solution was clear and transparent. The sodium dodecyl sulfate waste liquid was then discarded. The bone meal was washed with purified water until neutral. The bone meal was then divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain freeze-dried bone meal.

[0063] Another portion of the bone meal washed with purified water underwent gradient demineralization. The bone meal was mixed with a 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:7 until no more bubbles were generated in the solution, at which point the hydrochloric acid waste liquid was poured off. Then, the above steps were repeated with a 0.6 mol / L hydrochloric acid solution to continue demineralization until the bone meal became transparent. The hydrochloric acid waste liquid was then poured off, and the bone meal was washed with purified water until neutral. The bone meal after gradient demineralization was divided into two portions. One portion was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0064] Another portion of the bone meal after gradient demineralization was treated with sodium hydroxide. The bone meal was mixed with a 10% sodium hydroxide solution at a volume ratio of 3:13 and allowed to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 11 with a 10% sodium hydroxide solution.

[0065] 2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, and keep it at a constant temperature of 35°C with shaking for 6 hours. Centrifuge and collect the supernatant. Add an equal volume of purified water to the supernatant and repeat the above operation 6 times. Combine and collect the supernatants obtained from the repeated operations, put them into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0066] 3. Preparation of excipients: Freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone and pre-swollen for 0.8 h, wherein the mass ratio of polyvinylpyrrolidone to bone gelatin was 0.7:2.5; then it was transferred to glycerol and mixed evenly at 60 °C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and then placed at low temperature for 24 h to condense and form a gel. The excipient was homogenized into uniform granules with a particle size of 1-1.5 mm using a homogenizer.

[0067] 4. Preparation of allogeneic bone explant materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 1:1 to form mixed bone powder. Then, the mixed bone powder and excipients are mixed evenly at a volume ratio of 3:3 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0068] Example 4: Preparation of Allogeneic Bone Adhesive Materials with Adjustable Degradation Rate (Part 4) 1. Pre-bone preparation: Cancellous and cortical bone, cryogenically frozen at -50℃, was selected. The external soft tissue was removed, and the bone tissue was thoroughly cleaned with purified water. The bone was then cut into small pieces and rinsed with a high-pressure water gun until no bone marrow or blood components remained. The bone pieces were mixed with dry ice at a 1:1 mass ratio and then pulverized in a bone powder grinder to obtain bone powder with a particle size of 1-2 mm. The pulverized bone powder was mixed with a 3% hydrogen peroxide solution at a 1:1 volume ratio. The bone powder was then immersed in the 3% hydrogen peroxide solution and agitated at a constant temperature of 38℃ for 20 hours. After immersion, the bone powder was rinsed with purified water until the rinsing solution was clear and transparent, and the hydrogen peroxide waste liquid was discarded. The bone powder was then mixed with a 75% ethanol solution at a 1:2 volume ratio. The bone powder was immersed in the 75% ethanol solution for 3.5 hours and agitated at a constant temperature. After immersion, the bone powder was rinsed with purified water until the rinsing solution was clear and transparent, and the ethanol waste liquid was discarded. Bone meal and acetone solution were mixed at a volume ratio of 1:5. The acetone solution was then soaked in the bone meal under constant temperature and shaking at 40°C. The acetone solution was changed every 12 hours, for a total of 6 times, until the acetone solution was clear and transparent with no floating oily substances. The acetone waste liquid was then discarded. Bone meal was then soaked in a 0.25% trypsin solution at a volume ratio of 1:5 for 11 hours to remove impurities. After soaking, the trypsin waste liquid was discarded. The bone meal was then soaked in a 0.25% sodium dodecyl sulfate solution for 18 hours, with the solution changed 1-2 times until the soaking solution was clear and transparent. The sodium dodecyl sulfate waste liquid was then discarded. The bone meal was washed with purified water until neutral. The neutralized bone meal was then divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain freeze-dried bone meal.

[0069] Another portion of the bone meal washed with purified water underwent gradient demineralization. The bone meal was mixed with a 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:8 until no more bubbles were generated in the solution, at which point the hydrochloric acid waste liquid was poured off. Then, the above steps were repeated with a 0.6 mol / L hydrochloric acid solution to continue demineralization until the bone meal became transparent. The hydrochloric acid waste liquid was then poured off, and the bone meal was washed with purified water until neutral. The bone meal after gradient demineralization was divided into two portions. One portion was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0070] Another portion of the bone meal after gradient demineralization was treated with sodium hydroxide. The bone meal was mixed with a 10% sodium hydroxide solution at a volume ratio of 3:14 and allowed to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 11.5 with a 10% sodium hydroxide solution.

[0071] 2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, and keep it at a constant temperature of 33°C with shaking for 7 hours. Centrifuge and collect the supernatant. Add an equal volume of purified water to the supernatant and repeat the above operation 7 times. Combine and collect the supernatants obtained from the repeated operations, put them into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0072] 3. Preparation of excipients: Freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone for pre-swelling for 1 hour, wherein the mass ratio of polyvinylpyrrolidone to bone gelatin was 0.9:3; then it was transferred to glycerol and mixed evenly at 65°C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and then placed at low temperature for 24 hours to condense and form a gel. The excipient was homogenized into uniform granules with a particle size of 1.5–2 mm using a homogenizer.

[0073] 4. Preparation of allogeneic bone explant materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 2:1 to form mixed bone powder. Then, the mixed bone powder and excipients are mixed evenly at a volume ratio of 3:2 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0074] Example 5: Preparation of Allogeneic Bone Adaptive Materials with Adjustable Degradation Rate 1. Pre-bone preparation: Cortical bone frozen at -40℃ was selected. The surrounding soft tissue was removed, and the bone tissue was cleaned with purified water. The bone was then cut into small pieces and rinsed with a high-pressure water gun until no bone marrow or blood remained. The bone pieces were mixed with dry ice at a 1:1 mass ratio and then pulverized in a bone powder grinder to obtain bone powder with a particle size of 1-2 mm. The pulverized bone powder was mixed with a 3% hydrogen peroxide solution at a 1:1 volume ratio. The bone powder was then immersed in the 3% hydrogen peroxide solution and agitated at a constant temperature of 40℃ for 24 hours. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the hydrogen peroxide waste liquid was discarded. The bone powder was then mixed with a 75% ethanol solution at a 1:2 volume ratio. The bone powder was immersed in the 75% ethanol solution for 4 hours and agitated at a constant temperature. After immersion, the bone powder was washed with purified water until the washing solution was clear and transparent, and the ethanol waste liquid was discarded. Bone meal and acetone solution were mixed at a volume ratio of 1:5. The acetone solution was then soaked in the bone meal under constant temperature and shaking at 40°C. The acetone solution was changed every 12 hours, for a total of 7 times, until the acetone solution was clear and transparent with no floating oily substances. The acetone waste liquid was then discarded. Bone meal was then soaked in 0.25% trypsin solution at a volume ratio of 1:5 for 12 hours to remove impurities. After soaking, the trypsin waste liquid was discarded. The bone meal was then soaked in 0.25% sodium dodecyl sulfate solution for 18 hours, with the solution changed 1-2 times until the soaking solution was clear and transparent. The sodium dodecyl sulfate waste liquid was then discarded. The bone meal was washed with purified water until neutral. The bone meal was then divided into two parts. One part was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain freeze-dried bone meal.

[0075] Another portion of the bone meal washed with purified water underwent gradient demineralization. The bone meal was mixed with a 1.2 mol / L hydrochloric acid solution at a volume ratio of 1:10 until no more bubbles were generated in the solution, at which point the hydrochloric acid waste liquid was poured off. Then, the above steps were repeated with a 0.6 mol / L hydrochloric acid solution to continue demineralization until the bone meal became transparent. The hydrochloric acid waste liquid was then poured off, and the bone meal was washed with purified water until neutral. The bone meal after gradient demineralization was divided into two portions. One portion was stored in a deep cryogenic freezer and subsequently freeze-dried to obtain demineralized bone meal.

[0076] Another portion of the bone meal after gradient demineralization was treated with sodium hydroxide. The bone meal was mixed with a 10% sodium hydroxide solution at a volume ratio of 3:15 and allowed to stand for 24 hours. During this period, the pH of the reaction solution was adjusted to 12 with a 10% sodium hydroxide solution.

[0077] 2. Extraction of bone gelatin: Neutralize the pH of the above reaction solution with dilute hydrochloric acid solution to neutral, and keep it at a constant temperature of 30°C with shaking for 8 hours. Centrifuge and collect the supernatant. Add an equal volume of purified water to the supernatant and repeat the above operation 8 times. Combine and collect the supernatants obtained from the repeated operations, put them into a freeze-drying tray, and freeze-dry to obtain bone gelatin.

[0078] 3. Preparation of excipients: Freeze-dried bone gelatin was added to purified water containing polyvinylpyrrolidone for pre-swelling for 1 hour, wherein the mass ratio of polyvinylpyrrolidone to bone gelatin was 1:4; then it was transferred to glycerol and mixed evenly at 70°C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and then placed at low temperature for 24 hours to condense and form a gel. The excipient was homogenized into uniform granules with a particle size of 0.1–2 mm using a homogenizer.

[0079] 4. Preparation of allogeneic bone explant materials: Freeze-dried bone powder and demineralized bone powder are mixed evenly at a mass ratio of 10:1 to form mixed bone powder. Then, the mixed bone powder and excipients are mixed evenly at a volume ratio of 7:3 to obtain an allogeneic bone excipient material with adjustable degradation rate.

[0080] Comparative Example 1 Similar to the bone pretreatment procedure in Example 1, cortical bone frozen at -80°C was used. First, demineralized bone powder was prepared, followed by bone gelatin. The freeze-dried bone gelatin was added to purified water for 0.5 hours to pre-swell, with sodium carboxymethyl cellulose added to the purified water at a mass ratio of 0.2:2. Then, it was transferred to glycerol and mixed thoroughly at 50°C to form a sol. The sol was then placed in a vacuum drying oven to remove air bubbles and allowed to condense at low temperature for 24 hours, resulting in a gel. A homogenizer was used to obtain uniform granules with a particle size of 0.1–2 mm. The demineralized bone powder and the excipient were mixed thoroughly at a volume ratio of 3:7 to obtain the allogeneic bone excipient material of Comparative Example 1.

[0081] Comparative Example 2 Similar to the bone pretreatment procedure in Example 5, cortical bone frozen at -40°C was used to first obtain freeze-dried bone powder. Then, demineralized bone powder and bone gelatin were prepared according to the method in Example 5. The freeze-dried bone gelatin was added to purified water for pre-swelling for 0.5 hours, with hydroxypropyl methylcellulose added to the purified water at a mass ratio of 1:4 to the bone gelatin. It was then transferred to glycerol and mixed evenly at 50°C to form a sol. The sol was placed in a vacuum drying oven to remove air bubbles and allowed to condense at low temperature for 24 hours, resulting in a gel. A homogenizer was used to obtain uniform granular excipients with a particle size of 0.1–2 mm. The mixed bone powder obtained from the freeze-dried bone powder and demineralized bone powder was mixed with the excipient at a volume ratio of 3:7 to obtain the allogeneic bone excipient material of Comparative Example 2.

[0082] Test Example 1: Injectability Test The injectability of the materials was tested by measuring the extrusion force of the allogeneic bone-derived materials from Examples 1-5 and Comparative Examples 1-2 from a syringe. The testing equipment was a universal mechanical testing machine; the syringe tip opening had an inner diameter of 3 mm, and the syringe volume was 3 mL; the testing temperature was room temperature, the extrusion speed was 30 mm / min, and the testing distance was the complete extrusion of 3 mL. Based on the convenience of clinical use, injection was easier when the extrusion force was below 20 N, while the extrusion operation became more difficult when the force was above 20 N. The test results are shown in Table 1.

[0083] Test Example 2: Plasticity Test The plasticity of the allogeneic bone-derived materials from Examples 1-5 and Comparative Examples 1-2 was tested by measuring the disintegration time in physiological saline. A constant-temperature water bath was used, with the water temperature set at 37°C and the oscillation frequency at 30 rpm. The samples were prone to disintegration and may shift at the implantation site; generally, a longer disintegration time was preferable. The test results are shown in Table 1.

[0084] Test Example 3: Tests for osteoconductivity and osteoinductive properties Osteoconductivity and osteoinductive properties were demonstrated through animal experiments. The osteoinduction test was designed according to "YY / T1680-2020 Evaluation of In vivo osteogenic induction performance of allogeneic repair materials and demineralized bone materials", while the osteoconductivity test was designed according to "YY / T 0511-2009 Test methods for in vivo degradation and osteogenic performance evaluation of porous bioceramics". In the osteoconductivity and osteoinductive tests, the degradation performance was assessed by observing the residual amount and degradation time of the implanted materials in Examples 1-5 and Comparative Examples 1-2. The test results are shown in Table 1.

[0085] Table 1 As shown in Table 1, the extrusion force of the allogeneic bone-derived materials in Examples 1-5 prepared by the method of this application is all below 20N, which can stably produce allogeneic bone-derived materials that are convenient for clinical use. The allogeneic bone-derived materials in Examples 1-5 have a longer disintegration time and better plasticity. However, osteoinductive properties and degradation rate need to be considered comprehensively. Materials with osteoinductive properties are conducive to osteogenesis, and generally speaking, the osteogenesis rate is faster, and the degradation rate is also faster. If the osteogenesis rate is slower than the degradation rate, pores will form at the implantation site, which is not conducive to osteogenesis in the later stages. If the sample does not have osteoinductive properties and the product only has osteoconductive properties, the degradation and osteogenesis rates will be slower, but it will prolong the bone tissue repair time at implantation sites where rapid osteogenesis is required. Therefore, the bone powder in Comparative Example 1 is demineralized bone powder. Although it has good injectability and osteoinductive properties, it degrades rapidly in the early stages, which may affect later osteogenesis. Comparative Example 2 has a large proportion of freeze-dried bone powder, and the excipient's shaping effect is poor, resulting in a high extrusion force, which is not conducive to filling operations. At the same time, the proportion of demineralized bone powder is low, resulting in poor osteoinductive properties, slow degradation and osteogenic rates, and a long bone tissue repair time. The preparation method of this application yields a relatively stable allogeneic bone excipient material with low extrusion force, moderate degradation rate, certain osteoinductive properties, and good osteoconductivity. The allogeneic bone excipient material combining these characteristics can not only effectively mix bone powders of different densities, but also regulate the degradation rate of allogeneic bone powder, solving problems in clinical use.

[0086] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A method of preparing an allogeneic bone shaped material, characterized in that, The method comprises the following steps: S1. bone pretreatment to obtain freeze-dried bone powder and demineralized bone powder; S2. bone gelatin extraction; S3. excipient preparation, wherein the bone gelatin in step S2 is added to water containing polyvinylpyrrolidone to swell; S4. mixing the freeze-dried bone powder and demineralized bone powder in step S1 with the excipient in step S3 in a certain proportion to obtain the allogeneic bone excipient material.

2. The method for preparing allogeneic bone excipients according to claim 1, characterized in that, In step S3, the mass ratio of the added polyvinylpyrrolidone to the bone gelatin is 0.2-1:2-4.

3. The method for preparing allogeneic bone explant material according to claim 1, characterized in that, In step S3, the particle size of the excipient particles is 0.1-2 mm.

4. The method for preparing allogeneic bone explant material according to claim 1, characterized in that, In step S4, the mass ratio of the freeze-dried bone powder to the demineralized bone powder is 1:10-10:1, and the volume ratio of the mixed bone powder obtained from the freeze-dried bone powder and the demineralized bone powder to the excipient is 3:7-7:

3.

5. The method for preparing allogeneic bone excipients according to claim 1, characterized in that, In step S1, the bone pretreatment comprises decellularization treatment, virus inactivation treatment, defatting treatment and demineralization treatment.

6. The method of claim 5, wherein the allogeneic bone-shaped material is prepared by the steps of: In step S1, the human bone is selected from one or both of cortical bone and cancellous bone.

7. The method of claim 6, wherein the allogeneic bone-shaped material is prepared by the steps of: In step S1, the human bone is pretreated by deep low temperature freezing, and the deep low temperature is-80℃ to-40℃.

8. The method for preparing allogeneic bone explant material according to claim 5, characterized in that, The temperature of the decellularization treatment is 30-40℃, and the oscillation time is 12-24 h. The temperature of the defatting treatment is 30-40℃, and the number of liquid changes is 3-7 times.

9. The method for preparing allogeneic bone excipient material according to claim 5, characterized in that, The demineralization treatment is gradient demineralization, and the volume ratio of the human bone to hydrochloric acid solution is 1:5-1:

10.

10. The method for preparing allogeneic bone-bone modeling material according to claim 1, characterized in that, In step S2, the temperature of the bone gelatin extraction is 30-40℃, the oscillation time is 4-8 h, and the number of repeated collection of supernatant is 4-8 times.

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