A biodegradable bone scaffold composite material and its preparation method

Abstract

This invention provides a biodegradable bone scaffold composite material and its preparation method, comprising the following steps: (1) swelling wet collagen fibers in an acidic solution and then adding inorganic materials and stirring to obtain a mixed liquid; wherein the wet collagen fibers are obtained by mixing pretreated animal tissue derivatives and water, followed by crushing and centrifugation; (2) freezing-drying the mixed liquid and then mixing it with a crosslinking agent solution to obtain a collagen skeleton material; (3) immersing the collagen skeleton material in a biodegradable polymer solution and then freezing-drying it to obtain the biodegradable bone scaffold composite material. The bone scaffold composite material of this invention has both excellent mechanical and biological properties, which can effectively meet the multiple requirements for cell ingrowth, material transport and mechanical matching during bone repair; at the same time, the composite material can regulate the local pH during the scaffold degradation process, effectively avoiding inflammatory reactions caused by acidic environment, thereby ensuring the normal healing of bone tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a biodegradable bone scaffold composite material and its preparation method. Background Technology

[0002] Bone is a natural composite material composed of collagen and minerals, providing the human body with essential rigidity and strength. Clinically, bone defects frequently occur due to trauma, tumor resection, or infection, leading to an increasing demand for bone repair materials. Currently, commonly used bone graft materials, besides autologous bone as the gold standard, can be categorized into four main types from a material perspective: natural bio-derived materials (such as decellularized and decalcified bone matrix, derived from allogeneic or xenogeneic bone), synthetic inorganic materials (such as hydroxyapatite (HA), tricalcium β-phosphate (TCP), and bioactive glass), synthetic organic materials (such as polylactic acid and its copolymers), and synthetic composite materials (such as polylactic acid-TCP and other organic-inorganic complexes).

[0003] Polylactic acid (PLA) is widely used in bone repair due to its good biocompatibility and biodegradability. However, its degradation products are acidic, which can easily cause local inflammatory reactions, leading to foreign body reactions and chronic inflammation, resulting in tissue redness, swelling, and pain. Meanwhile, traditional polymer-inorganic composites (such as PLA / HA) possess certain mechanical properties, but they generally suffer from high hardness and brittleness, making it difficult to shape them according to the bone defect during surgery, thus limiting their application in complex defects. Summary of the Invention

[0004] This invention provides a biodegradable bone scaffold composite material and its preparation method, which can solve the problems of poor biocompatibility and poor toughness of existing bone repair materials.

[0005] In a first aspect, a method for preparing a biodegradable bone scaffold composite material, the method comprising the following steps: (1) After the wet collagen fibers are swollen in an acid solution, inorganic materials are added and stirred to obtain a mixed liquid; wherein the wet collagen fibers are obtained by mixing pretreated animal tissue derivatives and water, followed by crushing and centrifugation; (2) The mixed liquid is freeze-dried and then mixed with a crosslinking agent solution to obtain a collagen skeleton material; (3) The collagen skeleton material is immersed in a biodegradable polymer solution and then freeze-dried to obtain the biodegradable bone scaffold composite material.

[0006] Preferably, in step (1), the animal tissue derivative is dermal material, peritoneal tissue, or bladder basement membrane material.

[0007] Preferably, the pretreatment includes virus inactivation treatment, decellularization treatment, and defatting treatment in sequence.

[0008] More preferably, in step (1), the animal tissue derivative is inactivated by peracetic acid solution; wherein the virus treatment time is 1-3 h and the mass concentration of peracetic acid solution is 0.1-0.5%.

[0009] More preferably, a strongly alkaline solution is used to decellularize the animal tissue derivative; wherein the strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution with a concentration of 0.1~2 mol / L, and the treatment time is 2~5 h.

[0010] More preferably, the animal tissue derivative is degreased under ultrasonic conditions using an organic solvent; wherein the organic solvent is at least one of methanol, chloroform, acetone or isopropanol; the treatment temperature is 30~40℃ and the treatment time is 1~3h.

[0011] Preferably, in step (1), the pulverization temperature is 1~10℃ and the pulverization is performed 2~3 times.

[0012] Preferably, the wet collagen fiber has a length of 0.05~2mm and a diameter of 10~200μm.

[0013] Preferably, the solid content of the wet collagen fiber is 10-25%.

[0014] Preferably, in step (1), the inorganic material is hydroxyapatite, bioactive glass, or β-tricalcium phosphate.

[0015] Preferably, the mass ratio of the collagen fiber to the inorganic material is 1:(1~2).

[0016] Preferably, in step (1), an acid solution and water are added to the wet collagen fibers, and a collagen swelling solution is obtained after stirring; wherein the acid solution is a hydrochloric acid solution or an acetic acid solution.

[0017] Preferably, in the collagen swelling solution, the dry weight of collagen fibers is 5-15 wt%, and the concentration of the acid solution is 0.01-0.1 mol / L.

[0018] Preferably, in step (2), the solute of the crosslinking agent solution is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or glutaraldehyde, and the solvent is anhydrous ethanol.

[0019] Preferably, in step (2), the mass concentration of the crosslinking agent solution is 0.02~4%.

[0020] Preferably, in step (2), the reaction temperature is 25~30℃ and the time is 24~48h.

[0021] Preferably, in step (2), the freeze-drying temperature is -4℃ to -2℃, the vacuum degree is less than 5Pa, and the time is 24 to 30h.

[0022] Preferably, in step (3), the solute in the biodegradable polymer solution is polylactic acid or polycaprolactone, and the solvent is 1,4-dioxane, chloroform or dichloromethane.

[0023] Preferably, in step (3), the mass concentration of the biodegradable polymer solution is 5-15%.

[0024] Preferably, in step (3), the collagen skeleton material is immersed in a biodegradable polymer solution under vacuum; wherein the vacuum degree is 0.04~0.06MPa and the immersion time is 2~5min.

[0025] Secondly, embodiments of the present invention also provide a biodegradable bone scaffold composite material, which is prepared by any of the preparation methods described in the first aspect above.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: In this invention, wet collagen fibers are first prepared using animal tissue derivatives as raw materials. Pretreatment effectively removes immunogenic components from the tissue while retaining some active factors, which helps enhance cell function and promote angiogenesis. Then, the obtained decellularized collagen fibers are swollen in an acidic solution and uniformly mixed with inorganic materials. After freeze-drying and cross-linking, a collagen framework material is obtained. This framework possesses good elasticity and toughness, and the cross-linking treatment further enhances its structural stability, allowing it to deform within a certain range without breaking under stress. Finally, the collagen framework material is immersed in a biodegradable polymer solution and freeze-dried again to obtain a biodegradable bone scaffold composite material. The introduction of inorganic and polymeric materials effectively improves the compressive strength of the composite scaffold. Furthermore, by controlling the ratio of inorganic to polymeric materials, the local pH during scaffold degradation can be regulated, preventing inflammatory reactions caused by an acidic environment and ensuring normal bone tissue healing. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1This is a scanning electron microscope image of a biodegradable bone scaffold composite material provided in Embodiment 1 of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Therefore, embodiments of the present invention provide a method for preparing a biodegradable bone scaffold composite material, the method comprising the following steps: (1) After the wet collagen fibers are swollen in an acid solution, inorganic materials are added and stirred to obtain a mixed liquid; wherein the wet collagen fibers are obtained by mixing pretreated animal tissue derivatives and water, followed by crushing and centrifugation; (2) The mixed liquid is freeze-dried and then mixed with a crosslinking agent solution to obtain a collagen skeleton material; (3) The collagen skeleton material is immersed in a biodegradable polymer solution and then freeze-dried to obtain the biodegradable bone scaffold composite material.

[0031] In this embodiment of the invention, wet collagen fibers are first prepared using animal tissue derivatives as raw materials. Pretreatment effectively removes immunogenic components from the tissue while retaining some active factors, which helps enhance cell function and promote angiogenesis. Then, the obtained decellularized collagen fibers are swollen in an acidic solution and uniformly mixed with inorganic materials. After freeze-drying and cross-linking, a collagen framework material is obtained. This framework possesses good elasticity and toughness, and the cross-linking treatment further enhances its structural stability, allowing it to deform within a certain range without breaking under stress. Finally, the collagen framework material is immersed in a biodegradable polymer solution and freeze-dried again to obtain a biodegradable bone scaffold composite material. The introduction of inorganic and polymeric materials effectively improves the compressive strength of the composite scaffold. Furthermore, by controlling the ratio of inorganic to polymeric materials, the local pH during scaffold degradation can be regulated, preventing inflammatory reactions caused by an acidic environment and ensuring normal bone tissue healing.

[0032] According to some preferred embodiments, in step (1), the animal tissue derivative is dermal material, peritoneal tissue or bladder basement membrane material.

[0033] According to some preferred embodiments, in step (1), the pretreatment sequentially includes virus inactivation treatment, decellularization treatment, and defatting treatment; the animal tissue derivative is subjected to virus inactivation treatment using peracetic acid solution; wherein the virus treatment time is 1-3 hours (e.g., 1 hour, 2 hours, or 3 hours), and the mass concentration of the peracetic acid solution is 0.1-0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%); the animal tissue derivative is subjected to decellularization treatment using a strongly alkaline solution; wherein the strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution with a concentration of 0. The concentration of the solvent is 1-2 mol / L (e.g., 0.1 mol / L, 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, or 2 mol / L), and the treatment time is 2-5 h (e.g., 2 h, 3 h, 4 h, or 5 h). The animal tissue derivative is defatted using an organic solvent under ultrasonic conditions; wherein the organic solvent is at least one of methanol, chloroform, acetone, or isopropanol; the treatment temperature is 30-40°C (e.g., 30°C, 32°C, 35°C, 38°C, or 40°C), and the treatment time is 1-3 h (e.g., 1 h, 2 h, or 3 h).

[0034] In this embodiment of the invention, animal tissue derivatives are sequentially placed in a peracetic acid solution of a certain concentration, a strongly alkaline solution, and an organic solvent for virus inactivation, decellularization, and defatting treatment, respectively. This effectively removes immunogenic components, inactivates potential viruses, and clears adipose tissue, while retaining some active factors, such as vascular endothelial growth factor and transforming growth factor-β. These factors have anti-inflammatory effects, help enhance cell function, and promote angiogenesis. Ultimately, decellularized collagen fibers with intact structure and high biosafety are obtained.

[0035] It should be noted that in the embodiments of the present invention, after virus inactivation treatment, decellularization treatment and defatting treatment, purified water is used to wash the animal tissue to remove residual treatment reagents and avoid adverse effects on the biocompatibility of the subsequent composite material and the retention of active factors.

[0036] According to some preferred embodiments, in step (1), the pulverization temperature is 1~10℃ (e.g., 1℃, 3℃, 5℃, 8℃ or 10℃), and the pulverization times are 2~3 times (e.g., 2 times or 3 times); the length of the wet collagen fiber is 0.05~2mm (e.g., 0.05mm, 0.1mm, 0.5mm, 0.8mm, 1mm, 1.5mm or 2mm), and the diameter is 10~200μm (e.g., 10μm, 20μm, 50μm, 80μm, 100μm, 150μm or 200μm); the solid content of the wet collagen fiber is 10~25% (e.g., 10%, 15%, 20% or 25%).

[0037] In this embodiment of the invention, the pretreated animal tissue derivative is added to cold water at 1-10°C for pulverization. This not only helps to protect the natural structural integrity of collagen fibers but also allows the fibers to be fully dispersed, forming a uniformly dispersed collagen fiber liquid. Furthermore, by controlling the pulverization parameters, the length and diameter of the collagen fibers are kept within a suitable range, which helps to ensure that the final composite scaffold has good mechanical and biological properties. After pulverization, the material is centrifuged at a speed of 2000-5000 r / min for 2-5 min, and the supernatant is removed to obtain a wet collagen fiber material with a solid content of 10-25%.

[0038] According to some preferred embodiments, in step (1), an acid solution and water are added to the wet collagen fibers, and a collagen swelling solution is obtained after stirring; wherein, the acid solution is a hydrochloric acid solution or an acetic acid solution; in the collagen swelling solution, the dry basis of the collagen fibers is 5~15wt% (for example, it can be 5wt%, 8wt%, 10wt%, 12wt% or 15wt%), and the concentration of the acid solution is 0.01~0.1mol / L (for example, it can be 0.01mol / L, 0.05mol / L, 0.08mol / L or 0.1mol / L).

[0039] According to some preferred embodiments, in step (1), the inorganic material is hydroxyapatite, bioactive glass or β-tricalcium phosphate; the mass ratio of the collagen fiber dry base to the inorganic material is 1:(1~2) (for example, it can be 1:1, 1:1.5 or 1:2).

[0040] In this embodiment of the invention, wet collagen fibers are first added to a water and acid solution for swelling, and then mixed with the aforementioned inorganic materials. This helps ensure that the inorganic particles are evenly distributed in the collagen swelling solution, avoiding aggregation and thus ensuring the uniformity and mechanical stability of the collagen scaffold structure. Simultaneously, by controlling the mass ratio of collagen fibers to inorganic materials, the overall performance of the composite scaffold can be optimized. For example, if the collagen fiber content is too high, it enhances the flexibility and elasticity of the scaffold, but it is difficult to provide sufficient mechanical support, and it leads to a faster degradation rate, affecting the structural stability in the early stages of bone defect repair. Conversely, if the collagen fiber content is too low, the proportion of inorganic materials is too large, increasing the scaffold's brittleness and making it prone to brittle fracture under stress. Furthermore, the rapid release of alkaline ions during degradation may lead to excessively high local pH, which is detrimental to cell growth and tissue repair.

[0041] According to some preferred embodiments, in step (2), the solute of the crosslinking agent solution is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or glutaraldehyde, and the solvent is anhydrous ethanol; the mass concentration of the crosslinking agent solution is 0.02~4% (for example, it can be 0.02%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4%), and the mass ratio of collagen fiber dry base to solute (polymer material) in the crosslinking agent solution is (5~500):1 (for example, it can be 5:1, 10:1, 50:1, 10...). The ratios are 0:1, 200:1, 300:1, 400:1, or 500:1; the reaction temperature is 25~30℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃), and the time is 24~48h (e.g., 24h, 30h, 36h, 40h, or 48h); the freeze-drying temperature is -4℃~-2℃ (e.g., -4℃, -3℃, or -2℃), the vacuum degree is less than 5Pa, and the time is 24~30h (e.g., 24h, 25h, 26h, 27h, 28h, 29h, or 30h).

[0042] In this embodiment of the invention, a white viscous mixture obtained by mixing collagen fibers with inorganic materials is placed in a mold and then placed in a freeze dryer. First, it is pre-frozen at a low temperature (below -20°C) for a certain period of time, then evacuated to below 5 Pa and freeze-dried for 3-5 hours, followed by a gradient temperature increase to -4°C to -2°C for 10-12 hours of freeze-drying. By adjusting the freeze-drying process parameters and controlling the component ratio, a collagen scaffold material with suitable porosity, a median pore size of 50-250 μm, and a porosity of 50%-90% can be obtained. The freeze-dried sample is then placed in a container containing a cross-linking agent solution, and the container is placed in a vacuum chamber and evacuated to a low vacuum, allowing the sample to fully absorb the cross-linking agent solution and sink below the liquid surface. Cross-linking is then performed at room temperature for a period of time. Simultaneously, by controlling the ratio of collagen fibers to the cross-linking agent, a covalent cross-linked network is formed between the collagen molecular chains, enhancing the structural stability and mechanical properties of the collagen skeleton while preserving the integrity of the porous structure. After cross-linking is completed, in order to completely remove the unreacted residual cross-linking agent and byproducts and avoid their adverse effects on cell activity and tissue compatibility, the cross-linked sample is soaked in water for a period of time, then centrifuged, and the above steps are repeated multiple times. After cleaning, the sample is placed in a vacuum drying oven and vacuum dried to obtain collagen skeleton material.

[0043] In this embodiment of the invention, the crosslinking agent in the crosslinking agent solution is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or glutaraldehyde. Preferably, when different types of crosslinking agents are used, the concentration of the crosslinking agent solution is different. When the crosslinking agent is glutaraldehyde, the concentration of the crosslinking agent solution is 0.02%~0.5%, preferably 0.1% (w / w); when the crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or glutaraldehyde, the concentration of the crosslinking agent solution is 0.4%~4%, preferably 2% (w / w). This helps to ensure sufficient crosslinking while controlling the degree of crosslinking appropriately, avoiding increased material brittleness due to excessive crosslinking or poor structural stability due to insufficient crosslinking, thereby facilitating the acquisition of collagen matrix materials with both good mechanical properties and biocompatibility.

[0044] According to some preferred embodiments, the crosslinking agent solution also contains a functional component; wherein the functional component is arginyl glycyl aspartic peptide (RGD), and the concentration of the functional component is 0.1~5.0 mmol / L; in the embodiments of the present invention, it was found that adding a certain amount of arginyl glycyl aspartic peptide to the crosslinking agent can further increase the hydrophilicity of the collagen skeleton while ensuring the mechanical properties of the collagen skeleton, and can enhance the adhesion of osteoblasts to the subsequent composite scaffold material.

[0045] According to some preferred embodiments, in step (3), the solute in the biodegradable polymer solution is polylactic acid or polycaprolactone, and the solvent is 1,4-dioxane, chloroform or dichloromethane; the mass concentration of the biodegradable polymer solution is 5~15% (for example, it can be 5%, 8%, 10%, 12% or 15%); the mass ratio of the solute to the inorganic material in the biodegradable polymer solution is 1:(0.1~3) (for example, it can be 1:0.1, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3).

[0046] According to some preferred embodiments, the collagen skeleton material is immersed in a biodegradable polymer solution under vacuum; wherein the vacuum degree is 0.04~0.06MPa (for example, it can be 0.04MPa, 0.05MPa or 0.06MPa), and the immersion time is 2~5min (for example, it can be 2min, 3min, 4min or 5min).

[0047] In this embodiment of the invention, a polymer material (polylactic acid or polycaprolactone) is first added to a solvent and mixed to obtain a biodegradable polymer solution of a certain concentration. Then, the collagen skeleton material is immersed in the polymer solution and a vacuum is applied to remove air from the pores of the collagen skeleton material. This allows the polymer solution to fully penetrate the pores of the collagen skeleton and uniformly coat the pore walls of the collagen skeleton, forming a three-dimensional network composite structure. This not only enhances the mechanical strength of the scaffold but also regulates the degradation rate and local acid-base environment through the barrier effect of the polymer layer.

[0048] Simultaneously, by controlling the concentration of the biodegradable polymer solution and the mass ratio of biopolymer to inorganic materials, effective control of the local pH during the degradation of the composite scaffold can be achieved. Lactic acid produced during the degradation of polylactic acid can neutralize the alkaline ions released from the degradation of inorganic materials (such as hydroxyapatite, β-tricalcium phosphate, or bioactive glass), thereby maintaining the pH stability of the local microenvironment, preventing inflammatory reactions caused by the accumulation of acidic products, and ensuring normal bone healing. Furthermore, the collagen scaffold maintains good structural integrity in the early and middle stages of degradation, ensuring that the scaffold does not mechanically disintegrate before new tissue ingrowth, providing continuous and stable support for bone repair.

[0049] It should be noted that, in this embodiment of the invention, after the impregnation is completed, the steps of freeze-drying and cleaning the impregnated sample are also included; specifically, the freeze-drying process can use the same parameters as the freeze-drying process in step (2), and the cleaning process can use anhydrous ethanol for cleaning and perform ultrasound to remove the residual organic solvent in the composite scaffold, avoid adverse effects on cell activity and tissue compatibility, and ensure the safety and biomedical performance of the composite scaffold material.

[0050] This invention also provides a biodegradable bone scaffold composite material, prepared using any of the above-described preparation methods.

[0051] The bone scaffold composite material prepared in this invention possesses both excellent mechanical and biological properties. Experimental measurements show that its median pore size is 30-100 μm, its porosity is 55-85%, and its compressive stress at 40% wet strain is 1-5 MPa. This achieves a balance between good pore connectivity, suitable mechanical support strength, and lightweight structure, effectively meeting the multiple requirements for cell ingrowth, material transport, and mechanical matching during bone repair. Furthermore, this composite material can regulate the local pH during scaffold degradation, effectively avoiding inflammatory reactions caused by acidic environments, thereby ensuring normal bone tissue healing.

[0052] To more clearly illustrate the technical solution and advantages of the present invention, the following describes in detail a biodegradable bone scaffold composite material and its preparation method through several embodiments; the particle size of the inorganic material (hydroxyapatite) is <0.5mm, and the molecular weight of the polymer material (polylactic acid) is 50,000~100,000 Da.

[0053] Example 1: (1) 60g of animal tissue derivative (pig dermal material) was placed in a 0.3wt% peracetic acid solution for virus inactivation treatment for 2h. After washing twice with purified water, it was placed in a 1mol / L potassium hydroxide solution for decellularization treatment for 3h. After washing twice with purified water, it was placed in methanol solvent and defatted at 35℃ and under ultrasonic conditions for 2h to obtain pretreated animal tissue derivative. Subsequently, it was added to purified water at 1℃ and pulverized 3 times. After centrifugation at 3000r / min for 3min, the supernatant was removed to obtain wet collagen fibers with a solid content of 15% (length 0.05~2mm, diameter 10~200μm). The above-mentioned wet collagen fibers and purified water were stirred, and hydrochloric acid was added to mix and swell, so that the concentration of hydrochloric acid in the collagen swelling solution was 0.05 mol / L and the dry basis of collagen fibers was 10%; then inorganic material (hydroxyapatite) was added and stirred to obtain a mixed viscous solution; wherein, the mass ratio of dry basis of collagen fibers to inorganic material was 1:2. (2) The mixed viscous liquid was put into a mold and placed in a freeze dryer. First, it was pre-frozen at -20℃ for 4 hours, then vacuumed to 4Pa and freeze-dried at -3℃ for 24 hours. Then, the temperature was gradually increased to room temperature and placed in a container containing a crosslinking agent solution (0.1% glutaraldehyde solution). The container was then placed in a vacuum chamber and vacuumed to 4Pa. The crosslinking reaction was carried out at room temperature for 24 hours. After the crosslinking was completed, it was soaked in purified water for 2 hours, then centrifuged at 2000r / min for 5 minutes. This process was repeated 10 times. Finally, it was vacuum dried at 40℃ to obtain the collagen skeleton material. (3) Add the polymer material (polylactic acid) to the solvent (dichloromethane) and stir at 50°C for 10 h to obtain a biodegradable polymer solution with a concentration of 10 wt%; immerse the collagen skeleton material in the biodegradable polymer solution and evacuate to 0.05 MPa for 2 min, then put it in a freeze dryer, first pre-freeze at -20°C for 4 h, then evacuate to 4 Pa ​​and freeze dry at -3°C for 24 h, then gradually increase the temperature to room temperature; ultrasonically clean it 3 times in anhydrous ethanol to obtain a biodegradable bone scaffold composite material; wherein the mass ratio of inorganic material to polymer material is 2.5:1.

[0054] Example 2: Example 2 is basically the same as Example 1, except that in step (1), the mass ratio of collagen fiber dry base to inorganic material (hydroxyapatite) is 1:3.

[0055] In this embodiment, the prepared mixed viscous liquid is not easy to form during mold molding, the collagen skeleton material has low toughness and porosity, and the wet mechanical properties of the final scaffold decrease.

[0056] Example 3: Example 3 is basically the same as Example 1, except that in step (3), the polymer material (polylactic acid) is added to the solvent (dichloromethane) and stirred at 50°C for 10 hours to obtain a biodegradable polymer solution with a concentration of 3wt%.

[0057] In this embodiment, experiments have confirmed that the prepared stent product has poor strength under wet conditions.

[0058] Example 4: Example 4 is basically the same as Example 1, except that in step (3), the polymer material (polylactic acid) is added to the solvent (dichloromethane) and stirred at 50°C for 10 hours to obtain a biodegradable polymer solution with a concentration of 20wt%.

[0059] In this embodiment, the viscosity of the biodegradable polymer solution is too high. During the process of immersing the collagen skeleton material in the biodegradable polymer solution, the polymer solution is not easily adsorbed into the collagen skeleton, resulting in uneven distribution of the polymer solution on the surface and inside the final scaffold material.

[0060] Example 5: Example 5 is basically the same as Example 1, except that in step (2), the crosslinking agent solution is a glutaraldehyde ethanol solution, and the crosslinking agent solution also contains a functional component (arginyl glycyl aspartic peptide RGD). The final mass concentration of glutaraldehyde in the mixed liquid is 0.1 wt%, and the concentration of the functional component is 2.0 mmol / L.

[0061] Comparative Example 1 (1) A polylactic acid scaffold was prepared by 3D printing at 150°C using polylactic acid as raw material; wherein the pore size of the polylactic acid scaffold was 200 μm and the porosity was 69%; (2) A mixed slurry was prepared by mixing collagen and inorganic material (hydroxyapatite, particle size 50 nm) in a mass ratio of 1:2 using purified water; wherein the total concentration of collagen and inorganic material in the mixed slurry was 10%; (3) The polylactic acid scaffold was immersed in the vacuum-degassed mixed slurry and impregnated for 5 minutes under a vacuum of 0.04 MPa to obtain the composite scaffold; (4) The composite scaffold was placed in a container containing a crosslinking agent solution (0.1% glutaraldehyde solution), and the container was placed in a vacuum chamber and evacuated to 4 Pa. The crosslinking reaction was carried out at room temperature for 24 h. After the crosslinking was completed, it was soaked in purified water for 2 h, then centrifuged at 2000 r / min for 5 min, repeated 10 times, and then vacuum dried at 40 °C to obtain the bone scaffold composite material.

[0062] Experiments have shown that the composite material prepared in this comparative example has a low recovery rate after wet bending.

[0063] Comparative Example 2 (1) Type I collagen was freeze-dried according to a predetermined procedure: the temperature of the freeze-drying tray was -20℃, and after reaching -20℃, it was kept at the temperature for 4 hours. Then, the temperature was increased to 20℃ at a rate of 10℃ / 4 hours, and then frozen-dried at 25℃ for 8 hours to obtain freeze-dried collagen. (2) Cut the freeze-dried collagen into pieces with a particle size of no more than 1 mm. Mix 1.5 g of type I collagen with 7.5 g of water and add hydrochloric acid to mix and swell, so that the concentration of hydrochloric acid in the collagen swelling solution is 0.05 mol / L. Then add 1 g of inorganic material (hydroxyapatite) and mix to obtain a mixture. Add 0.1 g of polymer material (polylactic acid) to 10 mL of solvent (dichloromethane) and mix. Then mix it with the mixture to obtain a mixed slurry. (3) The mixed slurry was injected into the mold, compacted, and pre-formed into a film; then it was pre-frozen at -20℃ for 6 hours, then vacuumed to 4Pa, and freeze-dried at -3℃ for 24 hours to obtain the bone scaffold composite material.

[0064] Experiments have shown that the mixed liquid in this comparative example is not uniform and cannot form an orderly spatial structure, which is not conducive to osteogenic formation.

[0065] Comparative Example 3 Comparative Example 3 is basically the same as Example 1, except that in step (1), collagen fibers are replaced with type I collagen with a solid content of 6wt%.

[0066] Experiments have shown that the composite scaffold formed in this comparative example has low pore size and porosity, low adsorption of polymer materials, and poor mechanical properties in the final wet scaffold.

[0067] The performance of the biodegradable bone scaffold composite materials (hereinafter referred to as samples) provided in the examples and comparative examples was tested, and the test results are shown in Tables 1 and 2: Porosity and pore size: Median pore size and porosity were measured using the mercury intrusion porosimetry method. Compressive strength: Immerse the sample in physiological saline for 5 minutes, and measure the compressive stress at 40% strain according to the method specified in GB / T 1041-2008 "Determination of compressive properties of plastics".

[0068] Recovery rate after wet bending: The wet sample was bent at 30° and the recovery was observed in physiological saline after the force was released. pH value: 1g of sample was placed in tris(hydroxymethyl)aminomethane (TRIS) buffer at (37±1)℃ and pH=7.3±0.1, and placed on a shaker at 200r / min. After 24h, the pH value was measured.

[0069] pH during degradation: The sample was placed in a tris(hydroxymethyl)aminomethane (TRIS) buffer solution at (37±1)℃ and pH=7.3±0.1, and loaded on a shaker at 200 r / min. The pH value was measured on day 1, day 5, day 10, day 15 and day 30.

[0070] Contact angle: The sample is fixed flat on a glass slide, and deionized water is slowly added to the sample surface. After 5 seconds, a photograph is taken and the contact angle is calculated using a contact angle meter. The hydrophilicity of the scaffold is evaluated by the contact angle.

[0071] Table 1 Table 2 As shown in Tables 1 and 2, the bone scaffold composite materials prepared in Examples 1 and 5 of this invention possess both excellent mechanical and physical properties, effectively meeting the multiple requirements for cell ingrowth, material transport, and mechanical matching during bone repair. At the same time, the composite material can regulate the local pH during scaffold degradation, effectively avoiding inflammatory reactions caused by acidic environments, thereby ensuring normal healing of bone tissue.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of preparing a degradable bone scaffold composite material, characterized by, The preparation method includes the following steps: (1) After the wet collagen fibers are swollen in an acid solution, inorganic materials are added and stirred to obtain a mixed liquid; wherein the wet collagen fibers are obtained by mixing pretreated animal tissue derivatives and water, followed by crushing and centrifugation; (2) The mixed liquid is freeze-dried and then mixed with a crosslinking agent solution to obtain a collagen skeleton material; (3) The collagen skeleton material is immersed in a biodegradable polymer solution and then freeze-dried to obtain the biodegradable bone scaffold composite material.

2. The production method according to claim 1, characterized by, In step (1), the animal tissue derivative is dermal material, peritoneal tissue, or bladder basement membrane material; and / or The pretreatment process includes virus inactivation, decellularization, and defatting.

3. The production method according to claim 2, characterized by, In step (1), the animal tissue derivatives are inactivated with peracetic acid solution; the virus treatment time is 1-3 hours, and the mass concentration of the peracetic acid solution is 0.1-0.5%. Animal tissue derivatives are decellularized using a strongly alkaline solution; wherein the strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution with a concentration of 0.1~2 mol / L, and the treatment time is 2~5 h; Animal tissue derivatives are defatted using an organic solvent under ultrasonic conditions; wherein the organic solvent is at least one of methanol, chloroform, acetone or isopropanol; the treatment temperature is 30~40℃ and the treatment time is 1~3h.

4. The preparation method according to claim 1, characterized in that, In step (1), the pulverization temperature is 1~10℃, and the pulverization is performed 2~3 times; The wet collagen fibers have a length of 0.05~2mm and a diameter of 10~200μm; Preferably, the solid content of the wet collagen fiber is 10-25%.

5. The preparation method according to claim 1, characterized in that, In step (1), the inorganic material is hydroxyapatite, bioactive glass, or β-tricalcium phosphate; and / or The mass ratio of the collagen fiber dry base to the inorganic material is 1:(1~2).

6. The preparation method according to claim 1, characterized in that, In step (1), an acid solution and water are added to the wet collagen fibers, and a collagen swelling solution is obtained after stirring; wherein, the acid solution is a hydrochloric acid solution or an acetic acid solution; Preferably, in the collagen swelling solution, the dry weight of collagen fibers is 5-15 wt%, and the concentration of the acid solution is 0.01-0.1 mol / L.

7. The preparation method according to claim 1, characterized in that, In step (2), the solute of the crosslinking agent solution is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or glutaraldehyde, and the solvent is anhydrous ethanol; preferably, the mass concentration of the crosslinking agent solution is 0.02~4%; The reaction is carried out at a temperature of 25-30°C for a time of 24-48 hours; and / or The freeze-drying temperature is -4℃ to -2℃, the vacuum degree is less than 5Pa, and the time is 24 to 30 hours.

8. The preparation method according to claim 1, characterized in that, In step (3), the solute in the biodegradable polymer solution is polylactic acid or polycaprolactone, and the solvent is 1,4-dioxane, chloroform or dichloromethane; Preferably, the mass concentration of the biodegradable polymer solution is 5-15%.

9. The preparation method according to claim 1, characterized in that, In step (3), the collagen skeleton material is immersed in a biodegradable polymer solution under vacuum; wherein the vacuum degree is 0.04~0.06MPa and the immersion time is 2~5min.

10. A biodegradable bone scaffold composite material, characterized in that, It is prepared by any one of the preparation methods according to claims 1 to 9.

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