Xenogeneic bone repair scaffold and preparation method thereof
By using high-temperature calcination and negative pressure vacuum perfusion technology to prepare xenogeneic bone repair scaffolds, the problems of immune rejection and performance degradation of xenogeneic bone materials have been solved, and the biocompatibility and osseointegration performance of xenogeneic bone materials have been improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGZHOU BONE-RENEWAL MEDICAL TECHNOLOGIES LLC
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-26
Smart Images
Figure CN122272910A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an xenogeneic bone repair scaffold and its preparation method. Background Technology
[0002] Bone defects are typically caused by trauma, infection, tumor resection, or congenital factors, requiring the use of bone repair materials for filling and repair. Among bone repair materials, autologous bone, with its superior osteogenic potential, bone guiding ability, and osteoinductive activity, has long been considered the "gold standard" for bone repair and reconstruction. However, the amount of autologous bone available is often insufficient to meet the repair needs of large-scale defects, and secondary surgery is often required. Therefore, allogeneic bone, with similar osteogenic activity, is widely used for bone repair. However, the application of allogeneic bone still faces multiple challenges: on the one hand, there are ethical controversies regarding donor sources and potential immune rejection; on the other hand, due to differences in donor age and sex, the physicochemical properties and absorption rates of the material itself vary significantly, leading to room for improvement in the stability and availability of its clinical efficacy.
[0003] Animal-derived bone materials have been widely used in bone tissue engineering due to their wide availability, convenient access, and low processing costs. However, the implantation of xenogeneic bone materials into the human body as a foreign component can induce immune rejection, which is one of the key issues restricting its clinical translation and application. Studies have shown that the antigenicity of xenogeneic bone mainly originates from the proteins, lipids, and cellular components in its organic matter. When xenogeneic bone that has not been completely processed for antigens is implanted into the human body, it can activate the host's innate and adaptive immune systems, manifested as macrophage infiltration, upregulation of inflammatory cytokines (such as TNF-α, IL-1β, and IL-6), and T lymphocyte-mediated immune responses (see reference 1: Rani K, Al-Rawi A, Al Qabbani A, et al. A comparative study on immune responses to demineralized and decellularized bone substitute following intraperitoneal implantation in mouse model. PLoS One, 2025, 20(5): e0323666.). Severe immune rejection not only interferes with implant integration and vascularization but may also lead to increased bone resorption, ultimately affecting the clinical outcome of bone defect repair (see reference 2: Supronowicz P, Zhukauskas R, York-Ely A, et al. Immunologic analyses of bovine bone treated with a novel tissue sterilization process. Xenotransplantation, 2008, 15(6): 398-406.). Therefore, effectively removing immunogenic substances from organic matter is a key factor in optimizing xenogeneic bone applications. In existing deantigenation processes, high-temperature heat treatment to remove organic components is currently the main technical approach for preparing immunogenic-free basic scaffolds.
[0004] However, while high-temperature calcination removes organic matter and antigenic components from xenogeneic bone, it also significantly alters the physicochemical properties of the material, leading to a marked decrease in its water absorption and wettability, which is detrimental to the early attachment and proliferation of osteoblasts. This decrease in wettability is mainly due to the complete removal of organic components and the rearrangement and densification of the inorganic hydroxyapatite crystal structure during high-temperature calcination. At the same time, high-temperature calcination also removes the active components originally present in the organic bone, making it difficult for the material to promote new bone regeneration after implantation in the human body (see reference 3: Li Mao, Bai Yulong, et al. Performance evaluation of two types of deantigenated xenogeneic bone and experimental study on repair of rat skull defects. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(10): 1303-1310.).
[0005] The information disclosed in the background section is only intended to enhance the understanding of the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention
[0006] The purpose of this invention is to provide an xenogeneic bone repair scaffold and its preparation method. The xenogeneic bone repair scaffold completely removes immunogenic substances from xenogeneic bone materials while maintaining excellent water absorption and infiltration properties to promote the regeneration of new bone and blood vessels.
[0007] The present invention adopts the following technical solution: A method for preparing an xenogeneic bone repair scaffold, comprising: Pretreatment steps: Obtain bone blocks cut from animal bones, decellularize, wash, and calcine to remove organic components to obtain the basic skeleton; Injection molding step: A mixed slurry of collagen, bioactive tricalcium phosphate and cross-linking agent is provided and injected into the basic skeleton through negative pressure vacuum. After freeze drying, a preliminary composite xenogeneic bone is obtained. Secondary crosslinking step: The composite xenogeneic bone is immersed in a crosslinking agent for secondary crosslinking, and then freeze-dried to obtain a xenogeneic bone repair scaffold.
[0008] In some preferred embodiments, the basic skeleton is generally cubic in shape with dimensions of (3~50)×(3~50)×(3~100) mm; or, the basic skeleton is generally cylindrical in shape with dimensions of φ(3~50) × (3~100) mm.
[0009] In some preferred embodiments, the bone blocks are animal bones cut into blocks of a specified size by a cutting machine, and the animal bones include pig bones, cow bones, or sheep bones.
[0010] In some preferred embodiments, the preprocessing step is specifically implemented as follows: The bone block was soaked and cleaned alternately with a mixture of hydrogen peroxide and methanol-chloroform, repeated 2 to 5 times. Then, the residual solvent inside the bone block was cleaned with anhydrous ethanol, and the bone block was rinsed again with running water. The bone fragments were immersed in a surfactant solution, then sequentially immersed in a hydrogen peroxide solution, a methanol-chloroform mixture, and anhydrous ethanol, then thoroughly washed with running purified water, and finally dried; the surfactants included one or more of Triton X-100, sodium dodecyl sulfate, and ethylenediaminetetraacetate. The bone blocks are calcined at 600-1000 ℃ for 2-24 hours.
[0011] In some preferred embodiments, in the infusion molding step, collagen is dissolved in an acidic solution, bioactive tricalcium phosphate is added to the collagen acidic solution, stirred for 5-20 minutes, and then a cross-linking agent is added; wherein, the concentration of collagen is 5-30 mg / mL, the concentration of the cross-linking agent is 10-500 ppm, and the mass ratio of collagen to bioactive tricalcium phosphate is 0.1-1; The bioactive tricalcium phosphate microspheres contain 0.1-3 wt.% of active elements, including one or more combinations of zinc, manganese, and magnesium. The microspheres have a particle size of 500 nm-50 μm. The micron-sized particles are more conducive to effectively filling the microcracks and defects in calcined xenogeneic bone along with collagen. The crosslinking agent includes one or more of genipin and glutaraldehyde.
[0012] In some preferred embodiments, during the injection molding step, the base skeleton is immersed in the mixed slurry, and the mixed slurry is injected into the base skeleton by vacuuming, so that the internal pores of the base skeleton are filled by the mixed slurry.
[0013] In some preferred embodiments, in the secondary crosslinking step, the preliminarily formed composite xenogeneic bone is immersed in a crosslinking agent solution for 6-48 hours, and after crosslinking, it is placed in purified water and stirred and washed at 200 rpm-600 rpm for 6-24 hours, and then freeze-dried to form the shape. The crosslinking agent is one or more of genipin, glutaraldehyde, and carbodiimide, and the concentration of the crosslinking agent is 0.05-5 wt.%.
[0014] In some preferred embodiments, the density of the xenogeneic bone repair scaffold is 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.
[0015] The present invention also adopts the following technical solution: An xenogeneic bone repair scaffold obtained by the above preparation method includes a basic framework and a filling material filling the internal pores of the basic framework, the filling material including collagen and bioactive tricalcium phosphate.
[0016] In some preferred embodiments, the basic skeleton is generally cubic in shape with dimensions of (3~50)×(3~50)×(3~100) mm; or, the basic skeleton is generally cylindrical in shape with dimensions of φ(3~50) × (3~100) mm.
[0017] In some preferred embodiments, the xenogeneic bone repair scaffold has a compressive strength of 0.6-1.2 MPa, a water absorption rate of 120-200%, and a density of 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.
[0018] The technical solution of the present invention has the following advantages: The xenogeneic bone repair scaffold obtained by this invention uses xenogeneic bone as a basic supporting framework. Collagen is infused into the internal pores of the bone, giving the scaffold excellent hydrophilicity and blood adsorption capacity, significantly improving bone integration performance. Bioactive tricalcium phosphate particles are introduced into the collagen slurry and infused to fill the scaffold. This not only provides calcium and phosphorus ions as active substances and raw materials for bone regeneration, but also the bioactive ions (zinc, magnesium, manganese, etc.) in the bioactive tricalcium phosphate can directly promote osteogenic and angiogenesis. Simultaneously, the bioactive tricalcium phosphate micron-sized particles are effectively filled into the microcracks and defects of the calcined xenogeneic bone along with the collagen, continuously maintaining the overall strength of the implant during degradation, forming a gradient composite structure that simulates the natural bone microenvironment. In particular, this invention uses a collagen solution as a carrier to uniformly infuse the bioactive tricalcium phosphate particles into all the pore systems of the xenogeneic bone, while simultaneously repairing the micro-damage caused by calcination, achieving precise spatial distribution of active ingredients and in-situ reinforcement of mechanical defects. Attached Figure Description
[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a photograph of the xenograft bone repair scaffold of Embodiment 1 of the present invention.
[0021] Figure 2These are dynamic infiltration experimental photos of the xenograft bone repair scaffold of Embodiment 1 of the present invention.
[0022] Figure 3 Photographs showing the dynamic infiltration experiment of calcined xenogeneic bone as a comparative example.
[0023] Figure 4 This is a comparison chart of the water absorption rates of the bone repair materials in Example 1 and the comparative example.
[0024] Figure 5 This is a comparison diagram of the compressive strength of the bone repair materials in Example 1 and the comparative example. Detailed Implementation
[0025] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is for the purpose of aiding understanding the present invention, but does not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0026] This invention aims to prepare a xenogeneic bone repair scaffold, specifically a composite xenogeneic bone repair scaffold of bioactive tricalcium phosphate and collagen. This embodiment employs a combination of chemical degreasing, high-temperature calcination, and vacuum perfusion technology to prepare a modified basic framework. First, animal bones are cut into shapes, washed with pure water, and then alternately immersed in hydrogen peroxide, a methanol-chloroform mixture, and Triton X-100, repeatedly washing to remove fat and protein. Subsequently, the bones are freeze-dried and then calcined at high temperatures to obtain an inorganic basic framework. Finally, a mixture of collagen and bioactive tricalcium phosphate is perfused into the basic framework using a negative pressure vacuum method. Compared to traditional calcined bone, this embodiment completely removes the immunogenicity of organic matter through a higher calcination temperature and provides bone regeneration activity through the combination of bioactive tricalcium phosphate and collagen. The degradation of tricalcium phosphate and the bone regeneration process repair the microstructure and mechanical support properties of the calcined bone.
[0027] The mainstream technologies for xenograft bone fabrication in the industry are organic solvent treatment and high-temperature calcination. The main problem with organic solvent treatment is that the organic components in clinically used xenograft bone may be a direct trigger for immune recognition and rejection responses in the host. If antigen treatment is incomplete, residual xenograft proteins will become a major risk factor for inducing an immune response. While conventional high-temperature calcination can effectively remove organic matter, it also leads to significant degradation of the material's surface properties: decreased hydrophilicity and reduced liquid wetting ability make it difficult for blood to fully penetrate the material, thus affecting its ability to adsorb functional proteins from plasma, ultimately limiting the early osseointegration effect of the implant and subsequent degradation behavior. Furthermore, the chemical composition of calcined bone scaffolds tends to be homogeneous, retaining only the inorganic hydroxyapatite phase and lacking endogenous growth factors or signaling molecules with inducible activity. This lack of biological activity makes it difficult for the material to actively mediate cell function or promote angiogenesis; it only serves as a physical support framework during bone defect repair and cannot construct a functional microenvironment conducive to tissue regeneration.
[0028] To address these issues, this embodiment employs high-temperature calcination of xenogeneic bone, which effectively removes all organic matter and immunogenic substances from the material, fundamentally eliminating immune rejection and giving the scaffold excellent biocompatibility and safety. Collagen is loaded into the internal pores of the calcined xenogeneic bone scaffold, significantly increasing the material's water absorption and wettability, allowing it to fully absorb effective components from the blood, thereby greatly improving bone integration performance and promoting early healing and tissue remodeling. Tricalcium phosphate microparticles doped with bioactive ions such as zinc, magnesium, and manganese are thoroughly filled into the submicron pores of the xenogeneic bone and into defects appearing during sintering, along with a collagen solution. During bone regeneration, these active ions are continuously released to synergistically promote osteoogenesis and angiogenesis, while simultaneously providing calcium and phosphate ions as raw materials for bone regeneration. Furthermore, the effective filling of defects by the microparticles stabilizes the mechanical strength of the implant during degradation, thus comprehensively solving the dual problems of insufficient activity and rapid strength reduction inherent in traditional calcined xenogeneic bone treatment.
[0029] The method for preparing the xenogeneic bone repair scaffold in this embodiment includes: Pretreatment steps: Obtain bone blocks cut from animal bones, decellularize, wash, and calcine to remove organic components to obtain the basic skeleton; Injection molding step: A mixed slurry of collagen, bioactive tricalcium phosphate and cross-linking agent is provided and injected into the basic skeleton through negative pressure vacuum. After freeze drying, a preliminary composite xenogeneic bone is obtained. Secondary crosslinking step: The composite xenogeneic bone is immersed in a crosslinking agent for secondary crosslinking, and then freeze-dried to obtain a xenogeneic bone repair scaffold.
[0030] Furthermore, the basic skeleton is generally cubic in shape, with dimensions of (3~50)×(3~50)×(3~100) mm; or, the basic skeleton is generally cylindrical in shape, with dimensions of φ(3~50) × (3~100) mm. The bone blocks are animal bones cut into blocks of specified specifications by a cutting machine, and the animal bones include pig bones, cow bones, or sheep bones.
[0031] The preprocessing steps specifically include: The bone block was soaked and cleaned alternately with a mixture of hydrogen peroxide and methanol-chloroform, repeated 2 to 5 times. Then, the residual solvent inside the bone block was cleaned with anhydrous ethanol, and the bone block was rinsed again with running water. The bone fragments were immersed in a surfactant solution, then sequentially immersed in a hydrogen peroxide solution, a methanol-chloroform mixture, and anhydrous ethanol, then thoroughly washed with running purified water, and finally dried; the surfactants included one or more of Triton X-100, sodium dodecyl sulfate, and ethylenediaminetetraacetate. The bone blocks are calcined at 600-1000 ℃ for 2-24 hours.
[0032] In some specific embodiments, the preprocessing step is as follows: (1) Cut the animal bones into pieces of the specified size using a cutting machine and clean the surface impurities with 10-50 times the mass of purified water or an alkaline solution. Soak the bone pieces alternately in a 5-20 times mass of hydrogen peroxide solution and a methanol-chloroform mixture for 6-24 hours, cleaning 3-5 times. Then clean the remaining organic solvent inside the bones with 5-20 times the mass of anhydrous ethanol, and rinse the bone pieces again with running water. The alkaline solution includes one or more of sodium hydroxide, sodium bicarbonate, and sodium carbonate with water, at a concentration of 0.5-8 wt.%. The hydrogen peroxide solution has a concentration of 5-30 wt.%, and the volume ratio of chloroform to methanol in the methanol-chloroform mixture is 0.5-5.
[0033] (2) Soak the bone fragments in 5-20 times their weight of a 0.5-2 wt.% surfactant solution for 12-48 hours, then sequentially soak them in a 37°C hydrogen peroxide solution, a methanol-chloroform mixture, anhydrous ethanol, and flowing purified water, each step lasting 6-24 hours. Dry the bone fragments in an oven at 60-150°C to obtain organic xenogeneic bone. The surfactant includes one or more of Triton X-100, sodium dodecyl sulfate, and ethylenediaminetetraacetic acid. The hydrogen peroxide solution concentration is 5-30 wt.%, and the volume ratio of chloroform to methanol in the methanol-chloroform mixture is 0.5-5.
[0034] (3) The organic heterogeneous bone is placed in a muffle furnace and calcined at high temperature to remove the organic components, thereby obtaining the above-mentioned basic skeleton; the calcination temperature is 600-1000 ℃ and the calcination time is 2-24 hours.
[0035] In the injection molding step, collagen is dissolved in an acidic solution, and bioactive tricalcium phosphate is added to the collagen acidic solution. The mixture is stirred for 5-20 minutes, and then a cross-linking agent is added. The concentration of collagen is 5-30 mg / mL, the concentration of the cross-linking agent is 10-500 ppm, and the mass ratio of collagen to bioactive tricalcium phosphate is 0.1-1. Collagen includes, but is not limited to, type I collagen, type II collagen, and recombinant collagen, with a molecular weight of 20,000-400,000 Da. The acidic solution includes one or more of hydrochloric acid and acetic acid solutions. The bioactive tricalcium phosphate microspheres contain 0.1-3 wt.% of active elements, including one or more combinations of zinc, manganese, and magnesium. The particle size of the bioactive tricalcium phosphate microspheres is 500 nm-50 μm; micron-sized particles are more conducive to effectively filling the microcracks and defects in calcined xenogeneic bone along with collagen. The cross-linking agent includes one or more of genipin and glutaraldehyde.
[0036] Furthermore, in the injection molding step, the base skeleton is immersed in the mixed slurry, and the mixed slurry is injected into the base skeleton by vacuuming, so that the internal pores of the base skeleton are filled by the mixed slurry.
[0037] In some specific embodiments, the injection molding step is as follows: Weigh out collagen and dissolve it in an acidic solution, stirring for 10-30 minutes until completely dissolved. Add bioactive tricalcium phosphate to the collagen solution and continue stirring for 5-20 minutes. Then, add a chemical cross-linking agent to the mixture and continue stirring for 1-10 minutes until homogeneous. Immerse the basic skeleton in the mixture, and use vacuum to completely fill the pores inside the basic skeleton with the mixture. After freeze-drying, a preliminary composite xenogeneic bone is obtained.
[0038] In the secondary crosslinking step, the preliminarily formed composite xenogeneic bone is immersed in a crosslinking agent solution for 6-48 hours. After crosslinking, it is placed in purified water and stirred and washed at 200-600 rpm for 6-24 hours, and then freeze-dried to form the final product. The crosslinking agent is one or more of genipin, glutaraldehyde, and carbodiimide, and the concentration of the crosslinking agent is 0.05-5 wt.%.
[0039] In some specific embodiments, the secondary crosslinking step is as follows: The pre-formed composite xenogeneic bone was immersed in a cross-linking agent solution for 6-48 hours, and then placed in purified water and stirred and washed at 200-600 rpm for 6-24 hours. Finally, it was freeze-dried to form a composite bioactive tricalcium phosphate and collagen xenogeneic bone repair scaffold.
[0040] The crosslinking agents used in the injection molding step and the secondary crosslinking step can be the same or different.
[0041] The density of the prepared xenogeneic bone repair scaffold was 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.
[0042] The xenogeneic bone repair scaffold obtained by the above preparation method includes a basic framework and a filling material filling the internal pores of the basic framework, wherein the filling material includes collagen and bioactive tricalcium phosphate.
[0043] The basic framework is block-shaped, with dimensions of (3~50)×(3~50)×(3~100) mm. Alternatively, the basic framework is cylindrical, with dimensions of φ(3~50) × (3~100) mm.
[0044] The xenogeneic bone repair scaffold has a compressive strength of 0.6-1.2 MPa, a water absorption rate of 120-200%, and a density of 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.
[0045] This method employs high-temperature calcination to treat xenograft bone, effectively removing all organic matter and immunogenic substances from the bone tissue. This fundamentally eliminates the immune rejection response induced by residual protein or DNA in traditional xenograft treatments. Compared to conventional decellularization, high-temperature calcination is more thorough, resulting in a base scaffold material with excellent biocompatibility and safety. This provides a non-immunogenic, naturally porous scaffold for subsequent bone repair, avoiding the risk of implantation failure due to inflammatory reactions.
[0046] This embodiment loads collagen into the internal pores of the calcined xenogeneic bone scaffold, significantly increasing the material's water absorption and wettability. This allows the xenogeneic bone repair scaffold to rapidly absorb growth factors, cytokines, and other effective components from the blood, promoting host cell adhesion, migration, and ingrowth. This characteristic not only improves the poor osteointegration performance of traditional xenogeneic bone due to its hydrophobicity but also accelerates early blood clot formation and tissue healing, thereby greatly enhancing the bonding strength and long-term stability between the implant and the host bone.
[0047] In this embodiment, tricalcium phosphate micron-sized particles doped with bioactive ions such as zinc, magnesium, and manganese are uniformly dispersed in a collagen solution and fully fill the micron-sized pores of the xenogeneic bone scaffold and the microcracks and structural defects caused by calcination. During bone regeneration, these bioactive ions are continuously released, synergistically promoting osteoblast differentiation and angiogenesis. Simultaneously, tricalcium phosphate provides calcium and phosphate ions as raw materials for bone regeneration. Furthermore, the micron-sized particles, effectively filling defects along with the collagen, maintain the overall mechanical strength of the implant during material degradation, avoiding the sudden drop in strength caused by rapid degradation of traditional simple bone scaffolds. Thus, a multiple synergistic effect is achieved: bioactive ion-induced bone regeneration, raw material supply, and mechanical stability assurance.
[0048] The present invention will be further illustrated below through specific embodiments.
[0049] Example 1 Pretreatment: Select the cancellous bone portion of the pig femoral condyle and cut it into pairs of 10×10×20 mm bone blocks. Weigh 20 g of the bone blocks and soak them in 400 mL of 1 wt.% sodium hydroxide solution for 1 hour. Then, soak them in 400 mL of purified water for 10 minutes.
[0050] Organic solvent treatment: The bone blocks were alternately immersed in 200 mL of 20% hydrogen peroxide solution and 200 mL of a 1:1 methanol-chloroform mixture for 12 hours, repeated 3 times. After treatment, the bone blocks were rinsed in 200 mL of anhydrous ethanol to remove residual organic reagents, and then rinsed in running water.
[0051] Decellularization: The bone fragments were soaked in 200 mL of 1% Triton X-100 for 24 hours. Then, they were soaked in 200 mL of 20% hydrogen peroxide solution at 37 °C for 12 hours, and then in 200 mL of a 1:1 methanol-chloroform mixture for 12 hours. The bone fragments were then washed sequentially with 200 mL of anhydrous ethanol and 200 mL of purified water for 12 hours each.
[0052] Drying and calcination: The bone blocks were placed in an oven at 105 ℃ for 6 hours to dry, and then calcined at 800 ℃ for 4 hours to obtain the calcined xenogeneic bone basic skeleton.
[0053] Preparation of the mixed slurry: Weigh 100 g of 1 wt.% acetic acid solution using an analytical balance, add 1.2 g of type I collagen sponge (molecular weight approximately 300,000 Da), and stir at 400 rpm for 25 minutes until the collagen is completely dissolved. Add 6 g of magnesium tricalcium phosphate powder and stir at 400 rpm for 10 minutes until homogeneous. Then, add 1 mL of 1% glutaraldehyde solution and stir at 400 rpm for 2 minutes.
[0054] Casting molding: The base skeleton is completely immersed in the mixed slurry, vacuumed to 0.05 MPa and then released. This process is repeated 5 times to ensure that the internal pores of the base skeleton are completely filled with the mixed solution. The material is then transferred to a -40 ℃ freezer and frozen for 6 hours. After freeze-drying, a preliminary composite xenogeneic bone is obtained.
[0055] Secondary cross-linking and cleaning: The preliminarily formed composite heterogeneous bone was immersed in 1 wt.% glutaraldehyde solution for 24 hours, and then placed in pure water and stirred and cleaned at 400 rpm for 12 hours.
[0056] Freeze-drying molding: The composite xenogeneic bone was frozen in a -40 ℃ freezer for 6 hours, and then freeze-dried for 36 hours to finally obtain a xenogeneic bone repair scaffold composed of composite bioactive tricalcium phosphate and collagen (see Figure 1).
[0057] Performance Testing: The infiltrative properties of the composite bioactive tricalcium phosphate and collagen xenogeneic bone repair scaffold (composite xenogeneic bone) were tested using a dynamic infiltration test. After 60 seconds, the test solution (red ink) filled the interior of the composite xenogeneic bone repair scaffold (see [link]). Figure 2 The xenograft bone repair scaffold had a water absorption rate of 159.9% of its own weight (see Figure 4) and a strength of 0.81 MPa (see Figure 5). The density of the xenograft bone repair scaffold was 0.38 g / cm³. 3 The collagen content is 5.3 wt.%, the bioactive tricalcium phosphate content is 26.1 wt.%, and the porosity is 65%.
[0058] Example 2 Pretreatment: Select the cancellous bone portion of the bovine femoral condyle and cut it into pairs of 15×15×15 mm bone blocks. Weigh 10 g of the bone block and soak it in 300 mL of 2 wt.% sodium bicarbonate solution for 1 hour, and then soak it in 200 mL of purified water for 10 minutes.
[0059] Organic solvent treatment: The bone blocks were alternately immersed in 100 mL of 10% hydrogen peroxide solution and 100 mL of 2:1 methanol-chloroform mixture for 12 hours, repeated 5 times. After treatment, the bone blocks were rinsed in 100 mL of anhydrous ethanol to remove residual organic reagents, and then rinsed in running water.
[0060] Decellularization: The bone fragments were soaked in 100 mL of 1% Triton X-100 for 12 hours. Then, they were soaked in 100 mL of 10% hydrogen peroxide solution at 37 °C for 12 hours, and then in 100 mL of a 2:1 methanol-chloroform mixture for 12 hours. The bone fragments were then washed sequentially with 100 mL of anhydrous ethanol and 100 mL of purified water for 12 hours each.
[0061] Drying and calcination: The bone blocks were placed in an oven at 105 ℃ for 6 hours and then calcined at 1000 ℃ for 3 hours to obtain the calcined xenogeneic bone basic skeleton.
[0062] Preparation of the mixed slurry: Weigh 100 g of 1 wt.% acetic acid solution using an analytical balance, add 1 g of type I collagen sponge (molecular weight approximately 300,000 Da), and stir at 400 rpm for 25 minutes until the collagen is completely dissolved. Add 4 g of zinc-containing tricalcium phosphate powder and stir at 400 rpm for 10 minutes until well mixed. Then, add 1 mL of 1% glutaraldehyde solution and stir at 400 rpm for 2 minutes.
[0063] Casting molding: The base skeleton is completely immersed in the mixed slurry, vacuumed to 0.05 MPa and then released. This process is repeated 5 times to ensure that the internal pores of the base skeleton are completely filled with the mixed solution. The material is then transferred to a -40 ℃ freezer and frozen for 6 hours. After freeze-drying, a preliminary composite xenogeneic bone is obtained.
[0064] Secondary cross-linking and cleaning: The preliminarily formed composite heterogeneous bone was immersed in 1 wt.% glutaraldehyde solution for 24 hours, and then placed in pure water and stirred and cleaned at 400 rpm for 12 hours.
[0065] Freeze-drying molding: The composite xenogeneic bone was frozen in a -40 ℃ freezer for 6 hours, and then freeze-dried for 36 hours to finally obtain a xenogeneic bone repair scaffold composed of composite bioactive tricalcium phosphate and collagen.
[0066] Performance testing: The density of the xenogeneic bone repair scaffold composed of bioactive tricalcium phosphate and collagen was 0.33 g / cm³. 3 The collagen content is 4.5 wt.%, the bioactive tricalcium phosphate content is 18.2 wt.%, and the porosity is 68%.
[0067] Comparative Example Pretreatment: Select the cancellous bone portion of the pig femoral condyle and cut it into pairs of 10×10×20 mm bone blocks. Weigh 20 g of the bone blocks and soak them in 400 mL of 1 wt.% sodium hydroxide solution for 1 hour. Then, soak them in 400 mL of purified water for 10 minutes.
[0068] Organic solvent treatment: The bone blocks were alternately immersed in 200 mL of 20% hydrogen peroxide solution and 200 mL of a 1:1 methanol-chloroform mixture for 12 hours, repeated 3 times. After treatment, the bone blocks were rinsed in 200 mL of anhydrous ethanol to remove residual organic reagents, and then rinsed in running water.
[0069] Decellularization: The bone fragments were soaked in 200 mL of 1% Triton X-100 for 24 hours. Then, they were soaked in 200 mL of 20% hydrogen peroxide solution at 37 °C for 12 hours, and then in 200 mL of a 1:1 methanol-chloroform mixture for 12 hours. The bone fragments were then washed sequentially with 200 mL of anhydrous ethanol and 200 mL of purified water for 12 hours each.
[0070] Drying and calcination: The bone blocks were placed in an oven at 105 ℃ for 6 hours and then calcined at 800 ℃ for 4 hours to obtain calcined xenogeneic bone.
[0071] Performance testing: The wettability of the calcined xenograft was tested using a dynamic infiltration test. After 60 seconds, the test solution (red ink) failed to completely infiltrate the calcined xenograft (see [link]). Figure 3 The water absorption rate of calcined xenogeneic bone is 104.7% of its own weight (see [reference needed]). Figure 4 The strength was 0.58 MPa (see Figure 5). The density of the calcined xenogeneic bone was 0.25 g / cm³. 3 The porosity is 78%.
[0072] The improvements in the above embodiments are as follows: high-temperature calcination is used to thoroughly remove immunogenic substances, which is more effective than conventional decellularization treatment; traditional xenogeneic bone has poor water absorption and infiltrative properties, affecting bone integration and degradation performance; by perfusing collagen into the internal pores of the basic scaffold, the scaffold is endowed with excellent hydrophilicity and blood adsorption capacity, significantly improving bone integration performance; traditional xenogeneic bone has a single component and insufficient activity, and cannot provide active substances during bone regeneration. In this embodiment, bioactive tricalcium phosphate microparticles are introduced into the collagen solution and perfused to fill it, which not only provides calcium and phosphorus ions as active substances and raw materials for bone regeneration, but also the zinc, magnesium, manganese and other ions in the bioactive tricalcium phosphate can directly promote osteoogenesis and angiogenesis. At the same time, the microparticles are effectively filled into the microcracks and defects of the calcined xenogeneic bone along with the collagen, and continuously maintain the overall strength of the implant during the degradation process, forming a gradient composite structure that simulates the natural bone microenvironment. In particular, compared to solutions where tricalcium phosphate is used only as a coating or mixed with bone scaffold particles, which have the problem of tricalcium phosphate being difficult to penetrate deep micropores and fill structural defects, this embodiment uses collagen solution as a carrier to uniformly infuse bioactive tricalcium phosphate micron particles into all the pore systems of xenograft bone, while repairing micro-damage caused by calcination. This achieves precise spatial distribution of active ingredients and in-situ reinforcement of mechanical defects, which is significantly different from conventional mixing-forming or surface coating techniques.
[0073] As indicated in this specification and claims, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, and these steps and elements do not constitute an exclusive list; the method or apparatus may also include other steps or elements. The term "and / or" as used herein includes any combination of one or more of the associated listed items.
[0074] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In the event of any contradiction or inconsistency between the definitions used herein and those contained in other published documents, the definitions used herein shall prevail.
[0076] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are preferred embodiments. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made according to the principles of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing an xenogeneic bone repair scaffold, characterized in that, include: Pretreatment steps: Obtain bone blocks cut from animal bones, decellularize, wash, and calcine to remove organic components to obtain the basic skeleton; Injection molding step: A mixed slurry of collagen, bioactive tricalcium phosphate and cross-linking agent is provided and injected into the basic skeleton through negative pressure vacuum. After freeze drying, a preliminary composite xenogeneic bone is obtained. Secondary crosslinking step: The composite xenogeneic bone is immersed in a crosslinking agent for secondary crosslinking, and then freeze-dried to obtain the xenogeneic bone repair scaffold.
2. The preparation method according to claim 1, characterized in that, The basic framework is cubic in shape with dimensions of (3~50)×(3~50)×(3~100) mm; or, the basic framework is cylindrical in shape with dimensions of φ(3~50) ×(3~100) mm.
3. The preparation method according to claim 1, characterized in that, The bone blocks are animal bones cut into blocks of a specified size by a cutting machine. The animal bones include pig bones, cow bones, or sheep bones.
4. The preparation method according to claim 1, characterized in that, The preprocessing steps are implemented as follows: The bone block was soaked and cleaned alternately with a mixture of hydrogen peroxide and methanol-chloroform, repeated 2 to 5 times. Then, the residual solvent inside the bone block was cleaned with anhydrous ethanol, and the bone block was rinsed again with running water. The bone fragments were immersed in a surfactant solution, then sequentially immersed in a hydrogen peroxide solution, a methanol-chloroform mixture, and anhydrous ethanol, then thoroughly washed with running purified water, and finally dried; the surfactants included one or more of Triton X-100, sodium dodecyl sulfate, and ethylenediaminetetraacetate. The bone blocks are calcined at 600-1000 ℃ for 2-24 hours.
5. The preparation method according to claim 1, characterized in that, In the injection molding step, collagen is dissolved in an acidic solution, bioactive tricalcium phosphate is added to the collagen acidic solution, stirred for 5-20 minutes, and then a cross-linking agent is added; wherein, the concentration of collagen is 5-30 mg / mL, the concentration of cross-linking agent is 10-500 ppm, and the mass ratio of collagen to bioactive tricalcium phosphate is 0.1-1; The bioactive tricalcium phosphate microspheres contain 0.1-3 wt.% of active elements, including one or more combinations of zinc, manganese, and magnesium, and the particle size of the bioactive tricalcium phosphate microspheres is 500 nm-50 μm; the crosslinking agent includes one or more of genipin and glutaraldehyde.
6. The preparation method according to claim 1 or 5, characterized in that, In the injection molding step, the base skeleton is immersed in the mixed slurry, and the mixed slurry is injected into the base skeleton by vacuuming, so that the internal pores of the base skeleton are filled by the mixed slurry.
7. The preparation method according to claim 1, characterized in that, In the secondary crosslinking step, the preliminarily formed composite xenogeneic bone is immersed in a crosslinking agent solution for 6-48 hours, and after crosslinking, it is placed in purified water and stirred and washed at 200 rpm-600 rpm for 6-24 hours, and then freeze-dried to form the shape. The crosslinking agent is one or more of genipin, glutaraldehyde, and carbodiimide, and the concentration of the crosslinking agent is 0.05-5 wt.%.
8. The preparation method according to claim 1, characterized in that, The density of the xenogeneic bone repair scaffold is 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.
9. A xenogeneic bone repair scaffold obtained by the preparation method according to any one of claims 1 to 8, comprising a basic framework and a filling material filling the pores inside the basic framework, the filling material comprising collagen and bioactive tricalcium phosphate.
10. The xenograft bone repair scaffold according to claim 9, characterized in that, The basic framework is cubic in shape with dimensions of (3~50)×(3~50)×(3~100) mm; or, the basic framework is cylindrical in shape with dimensions of φ(3~50) × (3~100) mm. The xenogeneic bone repair scaffold has a compressive strength of 0.6-1.2 MPa, a water absorption rate of 120-200%, and a density of 0.3-0.4 g / cm³. 3 The porosity is 60-80%, with collagen content of 2-10 wt.% and bioactive tricalcium phosphate content of 10-30 wt.%.