Natural bone repair material and preparation method thereof

Abstract

The invention provides a natural bone repair material and a preparation method thereof. The material is animal cancellous bone inorganic salt particles or animal cancellous bone inorganic salt bone slices; the invention also relates to a preparation method of the natural bone repair material. The preparation method sequentially comprises the following steps: selecting materials, crushing, cleaning, removing organic matters, efficiently cleaning and drying, calcining and sterilizing. According to the method, inorganic base heating extraction and low-temperature calcination are adopted, so that the method is non-toxic, harmless, safe, efficient, low in organic matter residue and small in damage to the structure of a natural bone material, and the structure and morphology of the natural bone are better reserved; the defects of long construction period, low efficiency, high cost and the like existing in high-temperature calcination and organic reagent removal are avoided. The size of the bone repair material can be controlled, and the obtained bone repair material has certain mechanical strength, is not easy to break after being filled, and can play a good supporting role; and the material also has the advantages of high specific surface area, high porosity, low crystallinity, good hydrophilic performance, good biocompatibility and the like, and is suitable for bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials; and more particularly to a natural bone repair material and its preparation method. Background Technology

[0002] With the rapid development of oral medicine technology, the demand for bone repair materials is increasing. These materials require excellent osteoconductivity and biodegradability, as well as high compatibility with the host bone. It has been confirmed that a crucial factor affecting the long-term outcome of implants is sufficient bone volume at the implantation site to achieve good osseointegration. Bone repair materials play a vital role in guiding bone regeneration. Clinically commonly used bone repair materials are categorized into four types based on their source: autologous bone, allogeneic bone, xenogeneic bone, and synthetic bone. Autologous bone has limitations due to issues such as a second surgical site, limited bone volume, and potential complications. Allogeneic bone has limited sources and carries a certain risk of viral infection. Xenogeneic bone generally suffers from poor compressive strength and slow degradation. Synthetic bone lacks osteoinductive properties, has low porosity, and uneven pore size, failing to mimic the complex microstructure of natural bone materials, and its degradation capacity and biological properties are far inferior to natural bone. Xenogeneic bone is a readily available and inexpensive raw material, but current processing techniques for purifying and separating hydroxyapatite from xenogeneic bone have poor bone repair effects and low efficiency.

[0003] Many researchers have tried various methods to remove antigens from xenogeneic bone, such as cryogenic freezing, gamma irradiation, high-temperature calcination, and strong oxidants, to weaken or eliminate xenogeneic bone antigens. (1) High-temperature calcination, a common method for removing organic matter from xenogeneic bone materials, can lead to significant changes in the bone mineral structure, such as increased crystallinity and changes in pore structure, which in turn affect the degradation and bone-guiding properties of bone repair materials. In addition, carbonate in xenogeneic bone materials has the function of regulating the degradation rate of materials, while high-temperature calcination can lead to the decomposition of carbonate, further reducing the degradation performance of materials. (2) The organic reagent method is a two-step method, that is, first removing some fat, and then using organic solvents to remove the remaining fat and protein. Organic solvents include ethylenediamine, triethylamine, propylamine, or propylenediamine. This method has disadvantages such as long construction time, low sample strength, low efficiency, high cost, boiling, and certain toxicity. This invention uses inorganic base to remove organic matter, avoiding the disadvantages of organic reagents and the influence of high-temperature calcination on the structure of natural bone. The obtained bone repair material has certain strength, low cost, and short construction time.

[0004] Research indicates that high-temperature extraction with alkaline reagents to remove organic matter from xenograft bone presents the challenge of removing alkaline reagent residues. Common methods involve repeated rinsing with purified water or PBS, with purified water often requiring at least 15 rinse cycles. PBS rinsing, while lengthy, introduces calcium and phosphate ions, affecting the calcium and phosphate content of hydroxyapatite, the main component of bone repair materials. This leads to inaccuracies in determining the composition of the prepared bone repair material and makes it impossible to ascertain the safety of the material for implantation into the host. Both methods have drawbacks, including long processing times, high labor costs, and safety concerns. This invention, through the introduction of ammonium salts, can quickly remove alkaline reagent residues without damaging the hydroxyapatite structure of cancellous bone.

[0005] To address the issues of poor bone repair effects of hydroxyapatite purified from xenogeneic bone, this invention employs inorganic alkali to remove organic matter, thus solving the problems of complex, inefficient, costly, and slow degradation processes in xenogeneic bone material preparation. This better preserves the multi-level porous structure and properties of natural bone, as well as its high porosity, high specific surface area, and superior hydrophilicity, thereby improving the compatibility of the material with the host bone and achieving ideal osteoconduction effects.

[0006] Therefore, improving the processing efficiency of bone repair materials while better preserving the structure of natural bone materials to achieve better bone repair results is a key challenge in enhancing the performance of bone repair materials. Summary of the Invention

[0007] The purpose of this invention is to provide a natural bone repair material and its preparation method.

[0008] This invention is achieved through the following technical solution:

[0009] This invention relates to a natural bone repair material, which is animal cancellous bone inorganic salt particles or animal cancellous bone inorganic salt bone flakes; wherein, the natural bone repair material has a bimodal porous structure and a specific surface area of ​​90-120 m² / g. 2 / g, the particle size of the inorganic salt particles of animal cancellous bone is 0.10-5.0mm; the natural bone repair material better maintains the multi-level porous three-dimensional spatial structure of natural bone, which is conducive to bone guidance; the size of the inorganic salt bone slices of animal cancellous bone is (2-5)×(5-10)×(5-20)mm.

[0010] The present invention also relates to a method for preparing the aforementioned natural bone repair material, which comprises the following steps: material selection and crushing, washing, removal of organic matter, alkali treatment, washing and drying, calcination, and sterilization, to obtain the natural bone repair material.

[0011] Preferably, the specific steps of the material selection and crushing are as follows: (1) obtaining cancellous bone of animal femur; (2) removing soft tissue attached to the cancellous bone; (3) crushing the sample and passing it through a molecular sieve of 0.10-5.0 mm to obtain irregular granular bone; or cutting the cancellous bone into sheet-like bone of (2-5)×(5-10)×(5-20) mm.

[0012] Preferably, the specific steps of the cleaning are as follows: (1) Place irregular granular bone or flaky bone into a clean container, add 0.9%-5% sodium chloride at a mass-to-volume ratio of 1:(5-20), and ultrasonically soak for 30-90 minutes; (2) Place the irregular granular bone or flaky bone into a clean container, weigh out purified water at a mass-to-volume ratio of 1:(10-30), and ultrasonically soak for 10-30 minutes.

[0013] Preferably, the specific steps for removing organic matter are as follows: ① Place the sterilized irregular granular bone or flaky bone into a clean container, and add an alcoholic organic reagent with a volume ratio of 1:(10-30) to remove some of the fat; wherein, the alcoholic organic reagent is one or more of n-butanol, isobutanol, isopropanol, or ethanol, and the partial fat removal temperature is 20-30℃, and the removal time is not less than 24h; ② Then use an inorganic alkaline reagent with a volume ratio of 1:(10-30) for heating extraction to remove the remaining fat and protein; wherein, the inorganic alkaline reagent is calcium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate, and one or more of them are selected for heating extraction, the concentration of the inorganic alkaline reagent is 0.2-2M, and the heating extraction time is not less than 12h.

[0014] The organic matter removal process of this invention involves room temperature treatment with alcohol-based organic reagents, followed by heating extraction with inorganic alkali and low-temperature calcination to remove organic components from xenogeneic bone. This avoids the impact of high-temperature calcination on the material's structural properties caused by the need to remove residual organic components. Furthermore, the inorganic alkali heating extraction and low-temperature calcination involved in this invention use non-toxic and harmless inorganic alkali reagents that do not cause boiling over during the heating extraction of organic matter. This process has a short processing cycle and is safer and more efficient. Secondly, the inorganic alkaline reagent method used in this invention removes organic matter more thoroughly, requiring only low-temperature calcination to treat the sample. This not only avoids changes in the bone mineral crystal structure caused by high-temperature calcination, such as crystallinity and porosity, but also solves the technical problems of carbonate decomposition and reduced content caused by high-temperature calcination. The carbonate in the xenogeneic bone material obtained by the method involved in this invention can improve the bioactivity and degradability of the material, resulting in bone repair materials with low immunogenicity and more complete preservation of natural bone structure. It can also control the size and specifications of natural bone repair materials, solving the problems of complex, inefficient, costly, and slow degradation in xenogeneic bone material preparation processes. It better preserves the multi-level porous structure and properties of natural bone, high porosity, high specific surface area, and better hydrophilicity, improving the compatibility of the material with the host bone and achieving ideal osteoconduction effects.

[0015] Preferably, the specific steps of the cleaning and drying are as follows: After removing organic matter, the sample is placed in a clean container and cleaned by alternating soaking and shaking with purified water and ammonium salt; Drying steps: Drying in a forced-air drying oven at 90-120℃ for 3-5 hours; wherein, the ammonium salt is ammonium chloride or ammonium sulfate; The cleaning temperature is room temperature, wherein the mass-volume ratio of the sample after alkali treatment to purified water or ammonium salt is 1:(10-30), and the alternating soaking and shaking frequency is: purified water (3-5) times / (10-30) min + ammonium salt (3-5) times / (30-60) min + purified water (3-5) times / (10-30) min; The specific steps of the drying are as follows: Drying in a forced-air drying oven at 90-120℃ for 3-5 hours.

[0016] Preferably, the specific steps of calcination are as follows: place the bone material sample in a muffle furnace and calcine at 200-400℃ for 2-8 hours.

[0017] Preferably, the sterilization conditions are: cobalt-60 irradiation sterilization.

[0018] The present invention has the following advantages:

[0019] (1) This invention provides an efficient method for removing organic matter from natural bone repair materials. It utilizes alcohol-based organic reagents, inorganic alkali heating extraction, and low-temperature calcination to remove organic components from xenogeneic bone. This avoids the impact of high-temperature calcination on the material's structural properties caused by removing residual organic components. Furthermore, it avoids the drawbacks of organic reagents, such as long processing time, low efficiency, high cost, and high temperatures. The obtained bone repair material possesses certain mechanical strength, is not easily broken after filling, and provides good support. Its degradation rate is better matched to the host bone, achieving an ideal osteoconduction effect.

[0020] (2) Existing xenogeneic bone reagent methods for removing organic matter suffer from the problem of difficult-to-handle reagent residues. Common methods involve repeated rinsing with purified water or PBS, with purified water often requiring at least a two-day rinsing cycle. PBS rinsing, while lengthy, introduces calcium and phosphorus ions, affecting the calcium and phosphorus content of hydroxyapatite, the main component of bone repair materials. This leads to inaccuracies in determining the component content of the prepared bone repair materials, making it impossible to ascertain the safety of the material for implantation into the host. Both methods suffer from long processing times, high labor costs, and safety concerns. This invention, through the introduction of ammonium salts, can quickly remove organic reagent residues without damaging the hydroxyapatite structure of cancellous bone.

[0021] (3) The natural bone repair material of the present invention has a higher specific surface area and improved hydrophilicity, which makes the material have good capillary action and can be quickly wetted by blood, thereby allowing nutrients to enter the interior of the bone repair material, promoting early vascularization, promoting bone formation, and ensuring effective bone fusion; low crystallinity, degradation rate is more matched with the growth rate of new bone; low immunogenicity, high safety, good biocompatibility and other advantages.

[0022] (4) The natural bone repair material of the present invention has a pore diameter distribution that is more similar to the structure of human bones, which can better maintain the macroscopic, microscopic pore structure and nanostructure of natural inorganic cancellous bone, and is conducive to the proliferation and vascularization of osteoblasts.

[0023] (5) Effectively removes viruses with very little organic residue.

[0024] (6) The preparation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0025] Figure 1 These are photographs of natural bone repair materials prepared by the method involved in this invention;

[0026] Figure 2 This is a SEM image of the natural bone repair material prepared by the method involved in this invention;

[0027] Figure 3This is a pore size distribution diagram of the natural bone repair material prepared by the method involved in this invention;

[0028] Figure 4 The porosity result of the natural bone repair material prepared by the method involved in this invention;

[0029] Figure 5 This is an X-ray diffraction pattern of the natural bone repair material prepared by the method involved in this invention;

[0030] Figure 6 This is a diagram showing the crystallinity of the natural bone repair material prepared by the method involved in this invention.

[0031] Figure 7 This refers to the specific surface area of ​​the natural bone repair material prepared by the method involved in this invention.

[0032] Figure 8 This is a diagram showing the results of co-culturing natural bone repair materials prepared by the method involved in this invention with osteoblasts. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are merely further illustrations of the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0034] Example 1

[0035] This embodiment relates to a method for preparing a natural bone repair material, including the following steps:

[0036] Step 1: Remove the soft tissue attached to the cancellous bone and cut it to obtain a sheet of bovine cancellous bone of (2-5)×(5-10)×(5-20);

[0037] Step 2, the specific steps of the pretreatment cleaning are as follows: (1) Put the bovine cancellous bone obtained in step 1 into a clean container, add 2% sodium chloride at a mass-volume ratio of 1:10, and ultrasonically soak for 90 minutes; (2) Put the bovine cancellous bone into a clean container again, weigh the bone and purified water at a mass-volume ratio of 1:10, and ultrasonically soak for 30 minutes.

[0038] Step 3, the specific steps of the defatting treatment are as follows: the pretreated sheet bovine cancellous bone is placed in n-butanol to remove some of the fat (the material-to-liquid mass-to-volume ratio is 1:10), and shaken and soaked at 20°C for 47 hours to obtain defatted cancellous bone.

[0039] Step 4, the specific steps of the organic removal are as follows: the defatted cancellous bone is placed in a 0.2M sodium hydroxide solution with a mass-to-volume ratio of 1:20 and refluxed at 100-120℃ for 4 hours; the solution is replaced with a 1.0M sodium bicarbonate solution with a mass-to-volume ratio of 1:20, and reflux extraction is continued at 100-120℃ for 8 hours to obtain the deproteinized cancellous bone.

[0040] Steps 3 and 4, involving defatting and deproteinization, are crucial steps in removing organic components from natural bone, mitigating the risk of immunogenicity without altering the composition and structure of the natural bone. The resulting cancellous bone exhibits virtually complete removal of organic components.

[0041] Step 5, the specific steps of the high-efficiency cleaning method are as follows: The cancellous bone after organic matter removal is cleaned by alternating soaking in purified water and ammonium salt at a mass-to-volume ratio of 1:30. The alternating soaking and shaking frequency is: purified water 3 times / 20min + ammonium salt 3 times / 30min + purified water 3 times / 30min. Drying after cleaning: Dry at 120℃ for 3 hours.

[0042] Step 6: Place the dried sample into a stainless steel tray. Calcination parameters: calcination at 260℃ in a high-temperature furnace for 6 hours; rinse with ultrapure water after calcination and dry at 120℃ for 3 hours.

[0043] Step 7: The sterilization conditions are: sterilization by cobalt-60 irradiation.

[0044] Example 2

[0045] Step 1: Remove the soft tissue attached to the cancellous bone, crush and sieve to obtain bovine cancellous bone particles;

[0046] Step 2, the specific steps of the pretreatment cleaning are as follows: (1) Put the bovine cancellous bone obtained in step 1 into a clean container, add 5% sodium chloride with a mass-volume ratio of 1:20, and ultrasonically soak for 30 minutes; (2) Put the bovine cancellous bone into a clean container again, weigh the bone and purified water with a mass-volume ratio of 1:20, and ultrasonically soak for 30 minutes.

[0047] Step 3, the specific steps of the defatting treatment are as follows: the pretreated sheet bovine cancellous bone is placed in a mixture of 80% n-butanol and 20% ethanol to remove some of the fat (the mass-volume ratio of the material to the liquid is 1:30), and shaken and soaked at 30°C for 36 hours to obtain defatted cancellous bone.

[0048] Step 4, the specific steps of the organic removal are as follows: the defatted cancellous bone is placed in a 1.0M potassium hydroxide solution with a mass-to-volume ratio of 1:15 and refluxed at 100-120℃ for 8 hours; the solution is replaced with a 0.8M potassium carbonate solution with a mass-to-volume ratio of 1:15 and refluxed at 100-120℃ for another 6 hours to obtain the deproteinized cancellous bone.

[0049] Step 5, the specific steps of the high-efficiency cleaning method are as follows: The cancellous bone after organic matter removal is cleaned by alternating soaking in purified water and ammonium salt at a mass-to-volume ratio of 1:10. The alternating soaking and shaking frequency is: purified water 5 times / 10min + ammonium salt 2 times / 10min + purified water 5 times / 10min. Drying after cleaning: Dry at 90℃ for 3 hours.

[0050] Step 6: Place the dried sample into a stainless steel tray and calcine it at 260°C for 6 hours in a high-temperature furnace. After calcine, wash with ultrapure water and dry at 90°C for 3 hours.

[0051] Step 7: The sterilization conditions are: sterilization by cobalt-60 irradiation.

[0052] The sodium bicarbonate or potassium bicarbonate involved in this invention removes organic matter and is easily decomposed during heating into sodium carbonate or potassium carbonate, carbon dioxide and water, forming a concentration gradient. This allows the reagent to penetrate more easily into the pores of cancellous bone, which is beneficial for the removal of organic matter.

[0053] Example 3

[0054] Step 1: Remove the soft tissue attached to the cancellous bone and cut it to obtain a sheet of bovine cancellous bone of (2-5)×(5-10)×(5-20);

[0055] Step 2, the specific steps of the pretreatment cleaning are as follows: (1) Put the bovine cancellous bone obtained in step 1 into a clean container, add 5% sodium chloride with a mass-volume ratio of 1:10, and ultrasonically soak for 30 minutes; (2) Put the bovine cancellous bone into a clean container again, weigh out purified water with a mass-volume ratio of 1:20 for bone and purified water, and ultrasonically soak for 30 minutes.

[0056] Step 3, the specific steps of the defatting treatment are as follows: the pretreated sheet bovine cancellous bone is placed in a mixture of 60% isobutanol-20% isopropanol-20% ethanol to remove some of the fat (the mass-volume ratio of the material to the liquid is 1:20), and shaken and soaked at 20°C for 24 hours to obtain defatted cancellous bone.

[0057] Step 4, the specific steps of the organic removal are as follows: The defatted cancellous bone is placed in a 0.2M potassium carbonate solution with a mass-to-volume ratio of 1:10, and refluxed at 100-150℃ for 16 hours to obtain deproteinized cancellous bone.

[0058] Step 5, the specific steps of the high-efficiency cleaning method are as follows: The cancellous bone after organic matter removal is alternately soaked and cleaned with purified water and ammonium salt at a mass-to-volume ratio of 1:10. The alternating soaking and shaking frequency is: purified water 5 times / 10min + ammonium salt 5 times / 10min + purified water 3 times / 10min. Drying after cleaning: dry at 90℃ for 5 hours.

[0059] Step 6: Place the dried sample into a stainless steel tray and calcine it at 350°C for 4 hours in a high-temperature furnace. After calcine, wash with ultrapure water and dry at 90°C for 5 hours.

[0060] Step 7: The sterilization conditions are: sterilization by cobalt-60 irradiation.

[0061] Natural bone is mainly composed of organic and inorganic components. When the organic components are completely removed, the inorganic components are loosely bound, resulting in reduced mechanical strength. Low-temperature calcination allows the inorganic components to recrystallize, removing residual organic matter and improving biocompatibility. Furthermore, adjusting the crystallinity avoids the negative impact of organic removal on the material's mechanical properties, thus enhancing its mechanical strength. The calcination cooling process is particularly important; it should be done slowly in a muffle furnace to avoid the stress, cracks, or brittleness caused by rapid cooling, which can affect the performance of natural bone repair materials.

[0062] The removal of organic matter by inorganic alkali and the calcination process are key control factors and essential steps in this invention. Inorganic alkali is particularly important for the removal of organic matter, while the calcination process is the recombination of inorganic components after the removal of organic matter by reagents and the further removal of residual organic matter. This ensures the biosafety of the material, improves the mechanical strength of the bone repair material, better preserves the complex three-dimensional porous structure of natural bone, and has a highly similar phase composition. This results in a natural bone repair material with high specific surface area, porosity, hydrophilicity, low immunogenicity, high safety, and good biocompatibility.

[0063] The optimal calcination temperature in the calcination step described in this invention is 250-350℃. If the calcination temperature is too low, the recombination of inorganic components is limited, mechanical properties cannot be improved, and the application of the material will be greatly limited. Lower temperatures may also result in the inability to decompose residual organic matter, potentially leading to problems such as immune rejection. Furthermore, the filling of organic matter prevents the full manifestation of the material's porous structure, reducing the specific surface area and thus decreasing the contact area between the material and host cells and body fluids, which is detrimental to cell adhesion and growth. Calcination temperatures above 350℃ will damage the nanostructure of the natural bone surface, reduce porosity, and increase crystallinity, leading to a decrease in the specific surface area and a slower degradation rate. This results in a mismatch between the degradation rate and the growth rate of new bone, failing to achieve the desired bone repair effect.

[0064] Comparative Example

[0065] This comparative example relates to a method for preparing a natural bone repair material, including the following steps:

[0066] Step 1: Remove the soft tissue attached to the cancellous bone, crush and sieve or cut it to obtain bovine cancellous bone particles or flakes.

[0067] Step 2, the specific steps of the pretreatment are as follows: (1) Put the cancellous bone obtained in step 1 into a clean container, add 5% sodium chloride at a mass-volume ratio of 1:10, and soak for 30 minutes; (2) Put the cancellous bone particles or flake bovine cancellous bone into a clean container, weigh the bone and purified water at a mass-volume ratio of 1:20, and ultrasonically soak for 30 minutes.

[0068] Step 3, the specific steps of the defatting treatment are as follows: the pretreated sheet bovine cancellous bone is placed in a mixture of 80% n-butanol and 20% ethanol to remove some of the fat (the mass-volume ratio of the material to the liquid is 1:20), and shaken and soaked at 20°C for 24 hours to obtain defatted cancellous bone.

[0069] Step 4, the specific steps of the organic removal are as follows: The defatted cancellous bone is placed in a 1.0M potassium bicarbonate solution with a mass-to-volume ratio of 1:10, and refluxed at 100-150℃ for 14 hours to obtain deproteinized cancellous bone.

[0070] Step 5, the specific steps of the high-efficiency cleaning method are as follows: The cancellous bone after organic matter removal is cleaned by alternating soaking in purified water and ammonium salt at a mass-to-volume ratio of 1:10. The alternating soaking and shaking frequency is: purified water 3 times / 10min + ammonium salt 3 times / 30min + purified water 5 times / 10min. Drying after cleaning: dry at 120℃ for 3 hours.

[0071] Step 6: Place the dried sample into a stainless steel tray and calcine it at 530°C for 4 hours in a high-temperature furnace. After calcine, wash with ultrapure water and dry at 110°C for 3 hours.

[0072] Step 7: The sterilization conditions are: sterilization by cobalt-60 irradiation.

[0073] Product Testing

[0074] The natural bone repair materials obtained in Examples 2 and 3 of the present invention were subjected to macroscopic analysis (see...). Figure 1 a) and microscopic observation (see Figure 1 See the rendering of b). Figure 1 As shown; Figure 2 The results are from SEM observations performed at 50x and 2000x magnification. Figure 1 and Figure 2 It can be seen that the natural bone repair material prepared by the method involved in this invention has a pore distribution that is more similar to the structure of human bones, and can better maintain the macroscopic and microscopic pore structure of natural inorganic cancellous bone, which is conducive to the proliferation and vascularization of osteoblasts.

[0075] Depend on Figure 3As can be seen, the natural bone repair material prepared in Example 1 or Example 2 has a bimodal pore structure, further confirming the SEM results. The bimodal pore structure facilitates the infiltration of tissue fluid and the enrichment of growth factors, while also accelerating cell ingrowth and angiogenesis, thus promoting bone growth and healing. Macropores provide space for bone tissue and blood vessel ingrowth, while micropores significantly increase the specific surface area of ​​the material, thereby facilitating protein adsorption and the infiltration of body fluids. Simultaneously, the presence of both macropores and micropores synergistically accelerates bone integration and promotes new bone growth, effectively improving bone regeneration efficiency.

[0076] Depend on Figure 4 As can be seen from the porosity data of the natural bone repair materials prepared in Examples 1, 2, 3 and the comparative examples of the present invention, the porosity of the present invention is between 72.46% and 80.46%, which is similar to that of human bone trabeculae, which is beneficial for later implantation.

[0077] Depend on Figure 5 As shown in the X-ray diffraction pattern, the characteristic diffraction peaks of the natural bone repair material prepared by this invention are consistent with those of HA. Sharp characteristic diffraction peaks appear at diffraction angles 2θ of 26°, 32°, 33°, 40°, 46°, 49°, and 53°. The phase is Ca5(PO4)3OH, and it conforms to the standard calcium phosphate card PDF: 09-0432, with no obvious impurity peaks.

[0078] Depend on Figure 6 As shown, the crystallinity detection data of the samples prepared in the embodiments and comparative examples of the present invention show that the crystallinity of the sample obtained by high-temperature calcination in the comparative example is higher than that of the sample in the embodiment. Crystallinity is closely related to the dissolution rate of the material implanted in the host. Generally, the higher the crystallinity, the slower the dissolution rate of the material. The calcium and phosphorus ions released during the dissolution process can promote the formation of new bone. While ensuring the performance of the material, the lower the crystallinity of the material within a certain range, the more conducive it is to the implantation of the material.

[0079] have Figure 7 As shown in the figures, the specific surface area of ​​the samples prepared in the embodiments and comparative examples of this invention is measured. The natural bone repair material prepared in the embodiments has a higher specific surface area. A larger specific surface area increases the contact area with body fluids, which in turn increases the adhesion space for osteoblasts, making it easier for cells to grow into the pores of the material. This facilitates the transport and exchange of cell nutrients and metabolites, provides a carrier for cell adhesion and growth, promotes cell proliferation and differentiation, and more easily promotes bone healing, thus realizing the transformation of inanimate biological materials into living matter. In contrast, the specific surface area of ​​the material in the comparative examples decreased after high-temperature calcination, which is detrimental to subsequent implantation.

[0080] Depend on Figure 8As shown in the figure, the cell diagram of the natural bone repair material shows that the prepared natural bone repair material has cells attached to its surface, spread out well, and the cells are intact and polygonal in shape, and have established a good connection with the material surface.

[0081] The porosity of this invention is between 72.46% and 80.46%, similar to that of human bone trabeculae, which is beneficial for later implantation (comparative example: 66.63%). The crystallinity of this invention is between 48.29% and 53.20% (comparative example: 75.60%), with lower crystallinity further facilitating material dissolution within the implanted body. The specific surface area of ​​this invention is 90.7 m². 2 / g-103.3m 2 Between / g, the comparative example is only 61.07m. 2 / g. In summary, the natural bone repair material prepared by this invention has superior performance compared to the material prepared in the comparative example.

[0082] The method involved in this invention utilizes inorganic alkali heating extraction and low-temperature calcination. This process is safe and efficient, with low organic residue and minimal damage to the structure of natural bone materials, thus better preserving the structure and morphology of natural bone. It avoids the drawbacks of high-temperature calcination and organic reagent removal, such as long processing time, low efficiency, and high cost. This invention allows for control over the size of the bone repair material, resulting in a material with sufficient mechanical strength, resistant to breakage after filling, and providing good support. Furthermore, it possesses advantages such as high specific surface area, high porosity, low crystallinity, excellent hydrophilicity, and good biocompatibility, making it suitable for bone defect repair.

[0083] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A natural bone repair material, characterized by, The material is animal cancellous bone inorganic salt particles or animal cancellous bone inorganic salt bone pieces. The animal cancellous bone inorganic salt particles are animal cancellous bone inorganic salts with a bimodal pore structure, and have a specific surface area of 90-120 m 2 / g and a particle size of 0.10-5.0 mm. The size of the animal cancellous bone inorganic salt bone pieces is (2-5) x (5-10) x (5-20) mm.

2. A method for preparing the natural bone repair material according to claim 1, characterized in that, The natural bone repair material is obtained through the following steps in sequence: material selection and crushing, cleaning, organic matter removal, cleaning and drying, calcination, and sterilization.

3. The method for preparing a natural bone repair material according to claim 2, wherein the step of mixing the natural bone material and the polymer is performed at a temperature of 10 to 40°C. The specific steps of the material selection and crushing are as follows: (1) obtaining animal femur cancellous bone; (2) removing soft tissues attached to the cancellous bone; (3) crushing the sample and passing through a 0.10-5.0 mm molecular sieve screen to obtain irregular granular bone; or cutting the cancellous bone into a sheet-shaped bone with a size of (2-5) x (5-10) x (5-20) mm.

4. The method for preparing a natural bone repair material according to claim 2, wherein the step of mixing the natural bone material and the polymer is performed at a temperature of 10 to 40°C. The specific steps of the cleaning are as follows: (1) placing the irregular granular bone or sheet-shaped bone into a clean container, adding 0.9%-5% sodium chloride with a mass / volume ratio of 1:(5-20), and ultrasonic soaking for 30-90 min; (2) placing the irregular granular bone or sheet-shaped bone into a clean container, weighing purified water with a mass / volume ratio of 1:(10-30), and ultrasonic soaking for 10-30 min.

5. The method for preparing the natural bone repair material as described in claim 2, characterized in that, The specific steps of the organic matter removal are as follows: placing the cleaned irregular granular bone or sheet-shaped bone into a clean container, adding an alcohol organic reagent with a volume ratio of 1:(10-30) to remove part of the fat, and then adding an inorganic alkali reagent with a volume ratio of 1:(10-30) to heat extraction to remove the remaining fat and protein; the alcohol organic reagent is one or more of n-butanol, isobutyl alcohol, isopropyl alcohol, or ethanol; the fat removal temperature is 20-30°C, and the fat removal time is not less than 24 h; the inorganic alkali reagent is one or more of calcium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate, and the inorganic alkali reagent is selected for heating extraction at a temperature of 100-150°C, a concentration of 0.2-2 M, and a heating extraction time of not less than 12 h.

6. The method for preparing the natural bone repair material as described in claim 2, characterized in that, The specific steps of the cleaning and drying are as follows: placing the sample after the organic matter removal into a clean container, alternately soaking and shaking with purified water and ammonium salt; and drying step: drying in a blast drying oven at 90-120°C for 3-5 h.

7. The method for preparing the natural bone repair material as described in claim 7, characterized in that, The ammonium salt is ammonium chloride or ammonium sulfate.

8. The method for preparing a natural bone repair material according to claim 7, wherein the step of mixing the natural bone material and the organic solvent is performed at a temperature of 10 to 40°C. The temperature of the cleaning is room temperature, the mass / volume ratio of the sample after the organic matter removal to the purified water or the ammonium salt is 1:(10-30), and the frequency of the alternate soaking and shaking is: purified water (3-5) times / (10-30) min + ammonium salt (3-5) times / (30-60) min + purified water (3-5) times / (10-30) min.

9. The method of claim 2, wherein the natural bone repair material is prepared by the steps of: a) mixing the natural bone repair material with a solvent; b) drying the natural bone repair material; and c) sterilizing the natural bone repair material. The specific steps of the calcination are as follows: placing the bone material sample in a stainless steel tray and calcining in a muffle furnace at 200-400°C for 2-8 h.

10. The method for preparing the natural bone repair material as described in claim 2, characterized in that, The sterilization condition is cobalt-60 irradiation sterilization.

Images