Antler-DCB bone transplantation scaffold material and preparation method thereof

Abstract

The invention discloses a preparation method of an antler-DCB bone transplantation scaffold material, which specifically comprises the following steps: S1, cutting the cancellous bone of an antler into a bone sample, and carrying out ultrasonic treatment; s2, soaking in ethanol, and shaking with a shaking table; s3, soaking in diethyl ether, and shaking with a shaking table; s4, soaking the bone sample in a sodium hydroxide solution for shaking table oscillation, cleaning the bone sample with flowing water after soaking, treating the bone sample with hydrogen peroxide with the mass fraction of 30% for 24 hours after cleaning, and cleaning the bone sample again after treatment; and S5, putting the cleaned bone sample into a mixture of chloroform and methanol in a volume ratio of 3: 1 for 1 hour, washing the bone sample with distilled water, and drying at 70 DEG C for 1 day to prepare the antler-DCB bone transplantation scaffold material. The invention also discloses the antler-DCB bone transplantation scaffold material. According to the antler-DCB bone transplantation scaffold material and the preparation method thereof disclosed by the invention, the antler-DCB bone transplantation scaffold material has high porosity, large aperture and specific surface area, and is more beneficial to bone regeneration.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical material preparation, and in particular relates to a method for preparing a antler-DCB bone transplant scaffold material, and also relates to a antler-DCB bone transplant scaffold material. Background Art

[0002] Bone transplantation is the second largest transplantation operation after blood transfusion, which brings a huge burden to medical and social security. With the rapid development of modern military, about 70% of combat injuries caused by high-energy and high-explosive weapons have fractures or bone defects. Traditional bone transplantation methods mainly include autologous bone transplantation, allogeneic bone transplantation and synthetic bone materials, but these methods have their own limitations. Autologous bone transplantation is regarded as the "gold standard", but its application is limited by problems such as donor site damage, limited donor sources and complicated surgery. Although allogeneic bone transplantation can avoid donor site damage, there are risks of immune rejection and hidden dangers of disease transmission. Synthetic bone materials often lack biocompatibility and bone integration. Deer antlers, as a natural resource that falls off and regenerates every year, have good biocompatibility and mechanical properties. Through decellularization technology, deer antler cancellous bone can effectively reduce immunogenicity, retain its natural porous structure and bioactive components, and promote bone healing and regeneration. This material not only shows great potential in overcoming the shortcomings of traditional bone transplantation methods, but also provides new solutions for tissue engineering and regenerative medicine, with important clinical application value and market prospects. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a antler-DCB bone transplant scaffold material, which solves the problem of no active components remaining in the decellularized cancellous bone in the prior art.

[0004] Another object of the present invention is to provide antler-DCB bone transplant scaffold material.

[0005] The first technical solution adopted by the present invention is a method for preparing a antler-DCB bone transplant scaffold material, which is specifically implemented according to the following steps: S1, the cancellous bone of the antlers was cut into 4 × 4 mm bone samples, and then subjected to ultrasonic treatment after repeated freezing and thawing; S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0006] The first technical solution of the present invention is also characterized in that: The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0007] The shaking time in ethanol in S2 was 3 hours.

[0008] The shaking time of S3 in ether was 3 hours.

[0009] The mass concentration of the sodium hydroxide solution in S4 is 20 g / L, and the shaking time in the sodium hydroxide solution is 12 hours.

[0010] In S4, the bone samples were washed with running water three times.

[0011] The specific cleaning after treatment in S4 is as follows: the bone sample is cleaned 3 times with running water, each time for 0.5 hour, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After cleaning again, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and then cleaned 3 times with running water, each time for 0.5 hour.

[0012] Another technical solution adopted by the present invention is the antler-DCB bone transplant scaffold material prepared by the preparation method of the antler-DCB bone transplant scaffold material.

[0013] The beneficial effects of the present invention are: The antler-DCB bone transplant scaffold material of the present invention and its preparation method have the structural characteristics of natural bone. Compared with commercial decellularized cancellous bone, it has high porosity, large pore size and specific surface area, which is more conducive to bone regeneration. At the same time, its mechanical properties are similar to those of commercial decellularized cancellous bone, and it can effectively support defective bone tissue. At the same time, the antler-DCB bone transplant scaffold material of the present invention effectively removes immune rejection components and retains bioactive substances, thereby significantly improving its biocompatibility and bioactivity. This improvement can effectively reduce immune rejection reactions and promote endogenous bone regeneration, and can be better applied to the repair of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a diagram of the manufacture and characterization of multiple species of DCB in Example 6; Figure 2 This is a schematic diagram of the characterization of the antler-DCB bone transplant scaffold material in Example 6. DETAILED DESCRIPTION

[0015] The present invention is described in detail below in conjunction with specific implementation modes and accompanying drawings.

[0016] The present invention provides a method for preparing a antler-DCB bone transplant scaffold material, which is specifically implemented according to the following steps: S1, antler cancellous bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0017] S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; The shaking time in ethanol in S2 was 3 hours.

[0018] S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; The shaking time of S3 in ether was 3 hours.

[0019] S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; The mass concentration of the sodium hydroxide solution in S4 was 20 g / L, the shaking time in the sodium hydroxide solution was 12 hours, and the bone sample was washed 3 times with running water.

[0020] The specific cleaning after treatment in S4 is as follows: the bone sample is cleaned 3 times with running water, each time for 0.5 hour, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After cleaning again, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and then cleaned 3 times with running water, each time for 0.5 hour.

[0021] S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0022] In the method for preparing the antler-DCB bone transplant scaffold material of the present invention, the specific functions of each step are as follows: The function of S1 is to cut the antler cancellous bone into blocks of 4×4mm, which is conducive to further decellularization and destroys immunogenic substances such as DNA by destroying the cell structure. The freezing and thawing method is used, and the principle is to destroy the cell membrane by forming ice crystals to increase the extractability of cell components. The advantage of using 40kHz ultrasonic treatment is that ultrasonic treatment can further destroy the cell structure and promote the release of cell components.

[0023] The role of S2 is that ethanol can help remove lipids and other organic substances from the sample, while helping to fix the tissue structure and reduce the impact of components on the matrix.

[0024] The function of S3 is that ether soaking can dissolve and remove lipid components in the cell membrane, and ether is volatile, which can effectively reduce the amount of solvent remaining in the tissue.

[0025] The role of S4 is to dissolve organic components such as cell membranes and intracellular proteins, making cell residues easier to remove and helping to degrade residual cell components in the sample while eliminating possible microbial contamination. The purpose of the second wash is to degrade residual DNA to reduce the risk of immune response, and to degrade residual RNA to reduce the risk of immune response, while removing fat and other fat-soluble components in bone tissue again.

[0026] The antler-DCB bone transplant scaffold material of the present invention is not subjected to high-temperature calcination treatment, and thus biologically active substances and smaller hydroxyapatite grain sizes are effectively retained.

[0027] The present invention also provides a antler-DCB bone transplant scaffold material, which is composed of the following components in mass percentage: 78.29%-80.63% hydroxyapatite, 9.01%-11.34% calcium carbonate, 3.33%-4.67% collagen I, 2.37%-3.29% glycosaminoglycan, 1.42%-2.44% osteonectin, 1.62%-2.25% fibronectin, 1.03%-1.44% Mg, 0.59%-0.61% S, and 0.35%-0.62% Si.

[0028] The antler-DCB bone transplant scaffold material of the present invention is composed of the following components in mass percentage according to elemental analysis: C 33.05%~ 34.05%, O 45.45%~ 47.05%, P 3.34%~ 5.15%, Ca 12.43%~ 13.49%, Na1.12%~ 1.15%, Mg 1.06%~ 1.23%, S 0.44%~ 0.50%, Si 0.31%~0.45%.

[0029] The functions of the components in the antler-DCB bone transplant scaffold material of the present invention are as follows: Hydroxyapatite is an important mineral component in bones, providing a suitable growth environment for osteoblasts, promoting their differentiation and proliferation, and thus accelerating the formation of new bones. As bones develop, hydroxyapatite, as the main bone tissue component, gradually replaces collagen fibers and cartilage, which can effectively increase bone density, enhance bone's compressive resistance and toughness, and thus reduce the risk of fractures. At the same time, it can also interfere with the function of osteoclasts and reduce osteolysis, thereby effectively inhibiting bone absorption, slowing down the rate of bone loss, and preventing excessive bone consumption. As the main component of the new antler-DCB bone transplant scaffold material, hydroxyapatite can effectively play the above role after being transplanted into the body, making it play an important role in fracture repair and bone defect filling.

[0030] Calcium carbonate is the main component of bones. In the antler-DCB bone graft scaffold material, calcium carbonate exists in the form of crystals, which provides hardness and stability to the material. After a fracture or bone defect, calcium carbonate plays an important role in the repair and reconstruction of bones, supporting the formation of callus and the repair of fracture sites.

[0031] More importantly, the decellularized scaffold of the present invention retains both the supporting structure and the bioactive substances, such as Collagen I, GAGs, Osteonectin, Fibronectin, Mg, S, and Si. In the process of bone defect repair, a large proportion of Collagen I in the decellularized antler-DCB bone transplant scaffold material can help form a complex three-dimensional network structure at the fracture or bone defect site, which is crucial for maintaining the integrity and biomechanical properties of the bone structure. It also helps to disperse stress and prevent bones from breaking when impacted by external forces.

[0032] Glycosaminoglycans are a class of polysaccharide compounds that are widely present in human tissues. In bones, glycosaminoglycans play an important role. They are unique components of connective tissue interstitium and intercellular matrix, and are natural adhesives between tissue cells, which can provide necessary mechanical support and protection for bones. At the same time, glycosaminoglycans in materials can promote cell proliferation, osteogenesis, mineral deposition and bone remodeling, which is crucial to help accelerate the regeneration of bone defects.

[0033] Osteonectin is an important bone matrix protein. During bone formation, osteocalcin can promote the release of calcium in osteoblasts, allowing them to maintain proper calcium balance during bone formation and remodeling. In addition, osteocalcin is closely related to the mineralization process of collagen, helping to form a solid bone matrix, thereby enhancing the strength and toughness of bones.

[0034] Fibronectin also plays an important role in the immune regulation and regeneration of bones. Fibronectin is not only an important component of the extracellular matrix, but also participates in the interaction between cells and the matrix, regulating cell proliferation, migration and differentiation. These functions are crucial in the regeneration and repair of bones because they can promote the formation of new bones and help maintain the structural stability of bones.

[0035] Mg plays a regulatory role in bone regeneration by regulating the polarization of M2 macrophages and the angiogenesis process. Si promotes bone formation by stimulating osteoblasts to secrete collagen fibers and support mineralization. In addition, Si also enhances the osteogenesis process by promoting angiogenesis. S in the form of sulfate has the ability to activate stem cells, thereby promoting bone regeneration.

[0036] Example 1 This embodiment provides a method for preparing antler-DCB bone transplant scaffold material. The hard antlers come from three male sika deer. The method is specifically implemented according to the following steps: S1, the bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0037] Example 2 This embodiment provides a method for preparing antler-DCB bone transplant scaffold material. The hard antlers come from three male sika deer. The method is specifically implemented according to the following steps: S1, the bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0038] S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0039] Example 3 This embodiment provides a method for preparing antler-DCB bone transplant scaffold material. The hard antlers come from three male sika deer. The method is specifically implemented according to the following steps: S1, the bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0040] S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; The shaking time in ethanol in S2 was 3 hours, and the shaking time in ether in S3 was 3 hours.

[0041] S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0042] Example 4 This embodiment provides a method for preparing antler-DCB bone transplant scaffold material. The hard antlers come from three male sika deer. The method is specifically implemented according to the following steps: S1, the bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

[0043] S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; The shaking time in ethanol in S2 was 3 hours, and the shaking time in ether in S3 was 3 hours.

[0044] S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; The mass concentration of the sodium hydroxide solution in S4 was 20 g / L, the shaking time in the sodium hydroxide solution was 12 hours, and the bone sample was washed 3 times with running water.

[0045] The specific cleaning after treatment in S4 is as follows: the bone sample is cleaned 3 times with running water, each time for 0.5 hour, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After cleaning again, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and then cleaned 3 times with running water, each time for 0.5 hour.

[0046] S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

[0047] Example 5 This embodiment provides a method for using a antler-DCB bone transplant scaffold material, which is specifically implemented according to the following steps: Step 1: The experiment used 28 SD rats (about 300 g, male), which were provided by the Experimental Animal Center of Air Force Medical University.

[0048] Step 2: First, the rats were anesthetized with 3% (W / V) sodium pentobarbital.

[0049] Step 3: A 3 cm incision was made on the right thigh of the rat.

[0050] Step 4: Separate the muscles and fascia to expose the femur.

[0051] Step 5: A trephine is used to create a 4 mm diameter and 4 mm deep defect in the mid-femur.

[0052] Step 6: Use iodine to clean the wound and dissipate heat.

[0053] Step 7: A 4 mm diameter spherical stent was implanted into the defect and fixed to the femur with 5-0 absorbable sutures.

[0054] Step 8: Use 4-0 sutures to suture the muscle, aponeurosis, and skin layer by layer.

[0055] Step 9: To prevent infection, all rats were injected with 25,000 units of penicillin during the first three days after surgery.

[0056] Example 6 This example provides the test results of antler-DCB bone transplant scaffold material, and the specific results are as follows: like Figure 1-2 As shown, the bone structure in the antlers provides structural conditions that promote rapid bone growth. Figure 1 , A and B) obtained the natural bone structure of the antlers and retained appropriate mechanical properties; Figure 2 As shown in Figure AF, the porosity and pore size increased. HE, DAPI staining and quantitative analysis confirmed that the antler fragments and DNA components were effectively removed, thereby minimizing immunogenicity ( Figure 1 , C and Figure 2 , C and D). Importantly, the present decellularization procedure preserves essential extracellular matrix (ECM) components, such as collagen I and glycosaminoglycans (GAGs), which are critical for supporting bone mineralization ( Figure 1 , D and Figure 2 , G). Then, the antler DCB was compared with DCB products from different species. The porous structure of the antler-DCB was similar to that of the spine-DCB from deer, cattle, and humans ( Figure 1 , E), but the volume porosity (87.60 ± 5.45%), pore size (626.5 ± 63.9 mm), and specific surface area (199.95 ± 16.48 m2 / g) of antler-DCB were significantly higher than those of spine-DCB ( Figure 1 , F and Figure 2 , K, L, P < 0.05). The large porous structure may be conducive to bone ingrowth and promote the osteointegration of antler-DCB with surrounding bones. In addition, the surface roughness and hydrophilicity of antler-DCB are significantly higher than those of other spine-DCBs ( Figure 1 , G, H and Figure 2 , J). This is conducive to cell adhesion and fluid infiltration, which helps bone regeneration. Mechanical properties are key elements of bone transplantation to support regeneration, and the maximum compressive stress of deer antler DCB is 9.52±2.05MPa, the maximum compressive stress of bovine bone DCB is 9.00±0.75MPa, the maximum compressive stress of human bone DCB is 13.59±2.31MPa, and the maximum compressive stress of cancellous bone DCB is 11.00±1.24MPa ( Figure 1 , I). These values ​​are within the mechanical range of cancellous bone, indicating that its mechanical properties are suitable for bone regeneration.

[0057] Next, the ECM components in various DCBs were analyzed, including organic (proteins, proteoglycans, etc.) and inorganic ECM, which constitute key signals for immune regulation and regeneration. Using LC-MS / MS technology, 87, 54, 53, and 76 functional proteins were identified in velvet antler DCB, deer DCB, bovine DCB, and human DCB, respectively. Figure 2 , M). Gene ontology enrichment analysis showed that biological process pathways were mainly related to bone development, collagen fiber organization, etc. ( Figure 2 , N). Specifically, collagen I is a major component of bone ECM and is essential for the maturation and strength of newly formed bone tissue. GAGs promote cell proliferation, osteogenesis, mineral deposition, and bone remodeling. Osteonectin effectively regulates calcium release and collagen mineralization during bone formation. The higher levels of these specific ECM proteins in antler-DCBs compared to other DCBs highlight their superior ability to promote bone regeneration ( Figure 1 , J, K and Figure 2 , P, Q). In the context of inorganic ECM, we performed X-ray diffraction spectra (XRD) analysis on different DCBs to determine their phase compositions. The main peaks are represented by blue dots, which are attributed to the (002), (210), (211), (300), (310), (222), (213), (321), and (004) planes of hydroxyapatite (HAP) (JCPDS card number 9-432), respectively, indicating that the HAP in the four DCBs is polycrystalline and consists of a hexagonal system with a primitive lattice ( Figure 1 , L). Further quantitative analysis showed that the grain size of HAP in antler-DCB was smaller than that of other DCBs, indicating that antler-DCB has excellent bioactivity, biodegradability and osteoinductivity ( Figure 2 , R). In addition, FT-IR spectroscopy confirmed that the composition of DCB is mainly carbonated hydroxyapatite ( Figure 1 , M). In addition, proton induced X-ray emission (PIXE) detection found that the unique chemical elements in different DCBs fully met the requirements, and antler-DCB contained more magnesium (Mg, 877.2±149.6μg / g), silicon (Si, 329.8±41.2μg / g) and sulfur (S, 166.3±15.7μg / g) than other DCBs ( Figure 1 , NP). In conclusion, the unique ECM components in antler-DCB inspired the design of high-performance bone regeneration materials and laid a solid foundation for our antler-inspired bone transplantation. DCB was implanted into the femoral defect of rats to further evaluate its in vivo osteogenesis. At 12 weeks post-implantation (wpi), new bone formation in the local defect area of ​​the antler-DCB group showed significantly higher bone mineral density (BMD), bone tissue volume / total tissue volume (BV / TV), and trabecular thickness (Tb.Th.) ( Figure 1, QU). These findings suggest that antler-DCB has a stronger ability to promote new bone formation than commercially available DCB, highlighting its potential as a natural graft material for bone repair.

[0058] Figure 1 In the figure, A is a schematic description of the manufacturing process and characterization techniques of decellularized marrow bone. B is a naturally shed sika deer antler. C is DAPI staining of the antler before and after decellularization. D is the measurement of collagen I and GAGs in velvet antler before and after decellularization (n=4). E is a scanning electron microscope (SEM) image of the DCB surface. F is the specific surface area (SSA) measurement in DCB (n=4). G is the roughness (Ra) measurement of DCB using 3D atomic force microscopy (AFM). H is the water contact angle (WCA measurement) in units of DCB (n=4). I is the compressive stress-strain curve of DCB. J and K are the quantitative analysis of the collagen I and GAGs content in DCB using the detection kit, respectively. L is the X-ray diffraction (XRD) pattern of DCB. M) Fourier transform infrared (FT-IR) spectrum of DCB. NP is the quantitative analysis of the Mg (N), S (O), and Si (P) content in each group (n=4). Q is hematoxylin-eosin (H&E) and Masson staining images showing new bone formation (NB, newly formed bone tissue) 12 weeks after surgery. R is 3D reconstruction and 2D micro-CT images showing regenerated bone around the defect. SU is quantitative analysis of microstructural parameters of regenerated bone tissue, including BMD (S), BV / TV (T), and Tb.Th (U) (n=5). All statistical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.

[0059] Figure 2In the figure, A is a scanning electron microscope (SEM) image of antler before and after decellularization. B is the porosity and pore size measurement of antler before and after decellularization (n=4). C is the HE staining of antler before and after decellularization. D is the DNA content of antler before and after decellularization. E is the diameter measurement of antler trabeculae before and after decellularization (n=4). F is the elastic modulus and compression modulus measurement of antler before and after decellularization (n=4). G is the Sirius red staining of antler before and after decellularization. H is a macroscopic image DCB scaffold. I is a Micro-CT image of a two-dimensional reconstruction of a DCB cube. J is a 3D AFM image of DCB. K and L are the porosity (K) and pore size (L) measurements in DCB, respectively (n=4). M) Analysis of the number of proteins retained in DCB during decellularization by LC / MS-MS. N) Cluster analysis of DEPs in different DCBs and GO analysis results to determine key different features and biological significance. O is Sirius red staining of isolated tissues showing collagen deposition. P, Q are summary of Western blot analysis (P) and quantitative data (Q) of osteonectin, fibronectin, and elastin of DCB (n=4). R is the crystallite size of DCB characterized by XRD (n=4). All statistical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001.

Claims

1. A method for preparing a antler-DCB bone transplant scaffold material, characterized in that: Follow the steps below to implement it: S1, antler cancellous bone was cut into 4 × 4 mm bone samples and then treated with ultrasound after repeated freezing and thawing; S2, soak the bone sample treated with ultrasound in S1 in ethanol and shake it on a shaker; S3, soak the bone sample soaked in S2 in ether and shake it on a shaker; S4, soaking the bone sample soaked in S3 in a sodium hydroxide solution and shaking it on a shaker, washing the bone sample with running water after soaking, treating it with 30% hydrogen peroxide for 24 hours, and washing it again after treatment; S5, the cleaned bone sample was placed in a mixture of chloroform and methanol in a volume ratio of 3:1 for 1 hour, and then the bone sample was rinsed with distilled water and dried at 70°C for 1 day to obtain a antler-DCB bone transplant scaffold material.

2. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The specific ultrasonic treatment in S1 is: using 40kHz ultrasonic waves to treat the bone sample for 20 minutes.

3. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The shaking time in ethanol in S2 is 3 hours.

4. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The shaking time in the ether in S3 is 3 hours.

5. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The mass concentration of the sodium hydroxide solution in S4 is 20 g / L, and the shaking time in the sodium hydroxide solution is 12 hours.

6. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The bone samples were washed with running water three times as described in S4.

7. The method for preparing the antler-DCB bone transplant scaffold material according to claim 1, characterized in that: The further cleaning after the treatment described in S4 is specifically as follows: the bone sample is cleaned 3 times with running water, each time for 0.5 hour, and then the bone sample is immersed in a solution containing 1 mg / mL DNase for 48 hours. After the second cleaning, the sample is immersed in a solution containing 0.5 mg / mL RNase for 48 hours, and then cleaned 3 times with running water, each time for 0.5 hour.

8. Deer antler-DCB bone transplant scaffold material prepared by the preparation method of deer antler-DCB bone transplant scaffold material according to any one of claims 1 to 7.

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