Deer antler-DCB bone graft scaffold material and its preparation method

Abstract

This invention discloses a method for preparing antler-DCB bone graft scaffold material, specifically: S1, cutting cancellous bone from antlers into bone samples and treating them with ultrasound; S2, immersing the samples in ethanol and shaking on a shaker; S3, immersing the samples in ether and shaking on a shaker; S4, immersing the samples in sodium hydroxide solution and shaking on a shaker, then rinsing the bone samples with running water, treating them with 30% hydrogen peroxide for 24 hours, and rinsing them again; S5, placing the rinsed bone samples in a 3:1 mixture of chloroform and methanol for 1 hour, rinsing them with distilled water, and drying them at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material. This invention also discloses the antler-DCB bone graft scaffold material. The antler-DCB bone graft scaffold material and its preparation method disclosed in this invention have high porosity, large pore size, and specific surface area, which are more conducive to bone regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, specifically relating to a method for preparing antler-DCB bone graft scaffold material, and also relating to antler-DCB bone graft scaffold material. Background Technology

[0002] Bone transplantation is the second most common transplant surgery after blood transfusion, placing a significant burden on medical and social security systems. With the rapid development of modern military technology, approximately 70% of combat injuries caused by high-energy, high-explosive weapons involve fractures or bone defects. Traditional bone transplantation methods mainly include autologous bone grafting, allogeneic bone grafting, and synthetic bone materials, but each method has its limitations. Autologous bone grafting is considered the "gold standard," but its application is restricted by issues such as donor site damage, limited donor sources, and surgical complexity. Allogeneic bone grafting, while avoiding donor site damage, carries the risk of immune rejection and disease transmission. Synthetic bone materials often exhibit shortcomings in biocompatibility and bone integration. Deer antler, a naturally shed and regenerated resource annually, possesses excellent biocompatibility and mechanical properties. Through decellularization techniques, deer antler cancellous bone can effectively reduce immunogenicity while retaining its natural porous structure and bioactive components, promoting 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, possessing significant clinical application value and market prospects. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing antler-DCB bone graft scaffold material, which solves the problem of no active ingredients being retained in decellularized cancellous bone in the prior art.

[0004] Another object of the present invention is to provide a staghorn-DCB bone graft scaffold material.

[0005] The first technical solution adopted in this invention is a method for preparing antler-DCB bone graft scaffold material, which is implemented according to the following steps:

[0006] S1, cut the cancellous bone of the antler into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound.

[0007] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0008] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0009] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0010] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0011] The first technical solution of the present invention is further characterized in that,

[0012] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0013] The shaking time in ethanol in S2 is 3 hours.

[0014] The shaking time in S3 with diethyl ether is 3 hours.

[0015] The sodium hydroxide solution in S4 has a mass concentration of 20 g / L, and the shaking time in the sodium hydroxide solution is 12 hours.

[0016] The bone sample in S4 was washed with running water three times.

[0017] The specific steps for cleaning after treatment in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after another wash, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time.

[0018] Another technical solution adopted in this invention is the antler-DCB bone graft scaffold material prepared by the preparation method of antler-DCB bone graft scaffold material.

[0019] The beneficial effects of this invention are:

[0020] The antler-DCB bone graft scaffold material and its preparation method of the present invention possess the structural characteristics of natural bone. Compared with commercially available decellularized cancellous bone, it has high porosity, large pore size, and specific surface area, which is more conducive to bone regeneration. Simultaneously, its mechanical properties are similar to those of commercially available decellularized cancellous bone, effectively supporting damaged bone tissue. Furthermore, the antler-DCB bone graft scaffold material of the present invention effectively removes immune rejection components while retaining bioactive substances, thereby significantly improving its biocompatibility and bioactivity. This improvement can effectively reduce immune rejection reactions and promote endogenous bone regeneration, making it better suited for the repair of bone defects. Attached Figure Description

[0021] Figure 1 These are the manufacturing and characterization diagrams of DCBs for multiple species in Example 6;

[0022] Figure 2This is a schematic diagram of the characterization of the antler-DCB bone graft scaffold material in Example 6. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0024] This invention provides a method for preparing antler-DCB bone graft scaffold material, specifically implemented according to the following steps:

[0025] S1, cut the cancellous bone of the antler into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0026] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0027] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0028] The shaking time in ethanol in S2 is 3 hours.

[0029] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0030] The shaking time in S3 with diethyl ether is 3 hours.

[0031] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0032] The sodium hydroxide solution in S4 had a mass concentration of 20 g / L. The sample was shaken in the sodium hydroxide solution for 12 hours, and the bone sample was washed with running water 3 times.

[0033] The specific steps for cleaning after treatment in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after another wash, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time.

[0034] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0035] In the preparation method of the antler-DCB bone graft scaffold material of the present invention, the specific functions of each step are as follows:

[0036] The function of S1 is to cut the staghorn fibrous bone into 4×4mm pieces, which facilitates further decellularization. This process destroys immunogenic substances such as DNA by disrupting the cell structure. A freezing and thawing method is used, based on the principle that ice crystals are formed to disrupt cell membranes, increasing the extractability of cellular components. 40kHz ultrasound treatment is employed, which has the advantage of further disrupting cell structure and promoting the release of cellular components.

[0037] The role of S2 is that ethanol can help remove lipids and other organic substances from the sample, while also helping to fix the tissue structure and reduce the impact of components on the matrix.

[0038] The function of S3 is that ether soaking can dissolve and remove lipid components in cell membranes, and since ether is volatile, it can effectively reduce the amount of solvent remaining in the tissue.

[0039] S4's role is to dissolve organic components such as cell membranes and intracellular proteins, making it easier to remove cellular residues and helping to degrade residual cellular components in the sample, while eliminating potential microbial contamination. The purpose of the second wash is to degrade residual DNA and RNA to reduce the risk of an immune response, while also removing fat and other lipid-soluble components from the bone tissue.

[0040] The antler-DCB bone graft scaffold material of the present invention does not undergo high-temperature calcination treatment, thus effectively preserving bioactive substances and small hydroxyapatite grain size.

[0041] The present invention also provides a staghorn-DCB bone graft scaffold material, which is composed of the following components by weight percentage: hydroxyapatite 78.29%~80.63%, calcium carbonate 9.01%~11.34%, Collagen I 3.33%~4.67%, glycosaminoglycans 2.37%~3.29%, osteonectin 1.42%~2.44%, fibronectin 1.62%~2.25%, Mg 1.03%~1.44%, S 0.59%~0.61%, and Si 0.35%~0.62%.

[0042] The antler-DCB bone graft scaffold material of the present invention is composed of the following components by mass percentage according to elemental analysis:

[0043] 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%.

[0044] The roles of each component in the antler-DCB bone graft scaffold material of this invention are as follows:

[0045] Hydroxyapatite is an important mineral component of bone, providing a suitable growth environment for osteoblasts, promoting their differentiation and proliferation, and thus accelerating new bone formation. As bones develop, hydroxyapatite, as a major component of bone tissue, gradually replaces collagen fibers and cartilage, effectively increasing bone density, enhancing bone's compressive strength and toughness, thereby reducing the risk of fractures. Simultaneously, it can interfere with osteoclast function, reducing bone resorption, thus effectively inhibiting bone loss, slowing the rate of bone loss, and preventing excessive bone wastage. As the main component of the novel staghorn-DCB bone graft scaffold material, hydroxyapatite, after being transplanted into the body, can effectively exert the above-mentioned effects, playing a vital role in fracture repair and bone defect filling.

[0046] Calcium carbonate is a major component of bone. In the staghorn-DCB bone graft scaffold material, calcium carbonate exists in crystalline form, providing the material with hardness and stability. After fractures or bone defects, calcium carbonate plays a crucial role, participating in the bone repair and reconstruction process, supporting callus formation and fracture site repair.

[0047] More importantly, the decellularized scaffold of this invention retains both the supporting structure and bioactive substances such as Collagen I, GAGs, Osteonectin, Fibronectin, Mg, S, and Si. During bone defect repair, the higher proportion of Collagen I in the decellularized staghorn-DCB bone graft scaffold material helps 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 disperse stress and prevents bone fracture under external impact.

[0048] Glycosaminoglycans are a class of polysaccharide compounds widely found in human tissues, playing a crucial role in bone. They are unique components of connective tissue and intercellular matrix, acting as natural adhesives between tissue cells and providing essential mechanical support and protection for bones. Furthermore, glycosaminoglycans in materials are vital for promoting cell proliferation, osteogenic processes, mineral deposition, and bone remodeling, helping to accelerate the regeneration of bone defects.

[0049] Osteonectin is an important bone matrix protein. During bone formation, osteocalcin promotes the release of calcium from osteoblasts, maintaining proper calcium balance during bone formation and remodeling. Furthermore, osteocalcin is closely related to the mineralization process of collagen, helping to form a strong bone matrix, thereby enhancing bone strength and resilience.

[0050] Fibronectin also plays a crucial role in the immune regulation and regeneration of bone. Fibronectin is not only an important component of the extracellular matrix but also participates in cell-matrix interactions, regulating cell proliferation, migration, and differentiation. These functions are essential in bone regeneration and repair because they promote new bone formation and help maintain bone structural stability.

[0051] Mg plays a regulatory role in bone regeneration by modulating the polarization and angiogenesis of M2 macrophages. Si promotes bone formation by stimulating osteoblasts to secrete collagen fibers, supporting mineralization. Furthermore, Si enhances the osteogenic process by promoting angiogenesis. S, in its sulfate form, has the ability to activate stem cells, thereby promoting bone regeneration.

[0052] Example 1

[0053] This embodiment provides a method for preparing antler-DCB bone graft scaffold material. The hard antlers are obtained from three male sika deer, and the specific steps are as follows:

[0054] S1, cut the bone into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0055] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0056] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0057] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0058] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0059] Example 2

[0060] This embodiment provides a method for preparing antler-DCB bone graft scaffold material. The hard antlers are obtained from three male sika deer, and the specific steps are as follows:

[0061] S1, cut the bone into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0062] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0063] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0064] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0065] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0066] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0067] Example 3

[0068] This embodiment provides a method for preparing antler-DCB bone graft scaffold material. The hard antlers are obtained from three male sika deer, and the specific steps are as follows:

[0069] S1, cut the bone into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0070] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0071] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0072] The shaking time in ethanol in S2 is 3 hours, and the shaking time in ether in S3 is 3 hours.

[0073] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0074] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0075] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0076] Example 4

[0077] This embodiment provides a method for preparing antler-DCB bone graft scaffold material. The hard antlers are obtained from three male sika deer, and the specific steps are as follows:

[0078] S1, cut the bone into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound;

[0079] The ultrasonic treatment in S1 specifically involves treating the bone sample with 40kHz ultrasound for 20 minutes.

[0080] S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken.

[0081] The shaking time in ethanol in S2 is 3 hours, and the shaking time in ether in S3 is 3 hours.

[0082] S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker;

[0083] S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again.

[0084] The sodium hydroxide solution in S4 had a mass concentration of 20 g / L. The sample was shaken in the sodium hydroxide solution for 12 hours, and the bone sample was washed with running water 3 times.

[0085] The specific steps for cleaning after treatment in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after another wash, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time.

[0086] S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

[0087] Example 5

[0088] This embodiment provides a method for using the antler-DCB bone graft scaffold material, which is implemented according to the following steps:

[0089] Step 1: The experiment used 28 SD rats (approximately 300 grams, male), provided by the Experimental Animal Center of Air Force Medical University.

[0090] Step 2: First, anesthetize the rats with 3% (w / v) sodium pentobarbital.

[0091] Step 3: Make a 3 cm incision on the rat's right thigh.

[0092] Step 4: Separate the muscles and fascia to expose the femur.

[0093] Step 5: Use a trephine drill to create a 4mm diameter and 4mm depth defect in the middle of the femur.

[0094] Step 6: Clean the wound with iodine solution and allow it to cool.

[0095] Step 7: Insert a 4mm diameter spherical support into the defect and fix it to the femur with 5-0 absorbable sutures.

[0096] Step 8: Use 4-0 sutures to suture the muscles, tendons, and skin layer by layer.

[0097] Step 9: To prevent infection, all rats were injected with 25,000 units of penicillin for the first three days after surgery.

[0098] Example 6

[0099] This embodiment provides test results for the antler-DCB bone graft scaffold material. The specific results are as follows:

[0100] like Figure 1-2 As shown, the skeletal structure within the antlers provides the structural conditions that promote rapid bone growth. This is achieved through decellularization technology (antler DCB). Figure 1 A and B) obtained the natural skeletal structure of the antlers, retaining appropriate mechanical properties; such as Figure 2 As shown in the AF, porosity and pore size increased. HE, DAPI staining and quantitative analysis confirmed the effective removal of antler fragments and DNA components, thereby minimizing immunogenicity. Figure 1 C and Figure 2 (C and D). Importantly, the decellularization process of this invention preserves essential extracellular matrix (ECM) components, such as collagen I and glycosaminoglycans (GAGs), which are crucial for supporting bone mineralization. Figure 1 D and Figure 2 (G). Then, antler DCB was compared with DCB products from different species. The porous structure of antler-DCB is similar to that of spinal-DCB from deer, cattle, and humans. Figure 1 However, compared with spine-DCB, antler-DCB has significantly higher volumetric porosity (87.60±5.45%), pore size (626.5±63.9 mm), and specific surface area (199.95±16.48 m² / g). Figure 1 F and Figure 2 K, L, P < 0.05). The large porous structure may facilitate bone ingrowth and promote osseointegration between antler-DCB and surrounding bone. Furthermore, the surface roughness and hydrophilicity of antler-DCB are significantly higher than other vertebral-DCBs (K, L, P < 0.05). Figure 1 G, H and Figure 2This is beneficial for cell adhesion and fluid infiltration, which contributes to bone regeneration. Mechanical properties are a key factor supporting regenerative bone grafts, and the maximum compressive stress of deer antler DCB is 9.52±2.05MPa, that of bovine bone DCB is 9.00±0.75MPa, that of human bone DCB is 13.59±2.31MPa, and that of cancellous bone DCB is 11.00±1.24MPa. Figure 1 These values ​​are within the mechanical range of cancellous bone, indicating that its mechanical properties are suitable for bone regeneration.

[0101] Next, the ECM components in various DCBs were analyzed, including organic (proteins, proteoglycans, etc.) and inorganic ECMs, which constitute key signals for immune regulation and regeneration. Using LC-MS / MS technology, 87, 54, 53, and 76 functional proteins were identified in deer antler DCB, deer DCB, bovine DCB, and human DCB, respectively. Figure 2 Gene ontology enrichment analysis showed that the biological processes were mainly related to bone development and collagen fiber tissue. Figure 2 Specifically, collagen I is a major component of the bone ECM and is crucial for the maturation and strength of newly formed bone tissue. GAG promotes cell proliferation, osteogenic processes, mineral deposition, and bone remodeling. Osteonecrocin effectively regulates calcium release and collagen mineralization during bone formation. Compared to other DCBs, staghorn-DCB has higher levels of these specific ECM proteins, highlighting its superior ability to promote bone regeneration. Figure 1 J, K and Figure 2 P, Q). Against the background of inorganic ECM, we performed X-ray diffraction (XRD) analysis on different DCBs to determine their phase composition. The main peaks are represented by blue dots, which are assigned to the (002), (210), (211), (300), (310), (222), (213), (321) and (004) planes of hydroxyapatite (HAP) (JCPDS card number 9-432), respectively, indicating that HAP in the four DCBs all exhibits polycrystalline properties and is composed of a hexagonal system with a primitive lattice. Figure 1 Further quantitative analysis showed that the grain size of HAP in antler-DCB was smaller than that in other DCBs, indicating that antler-DCB has excellent bioactivity, biodegradability, and osteoinductive properties. Figure 2 Furthermore, FT-IR spectroscopy confirmed that DCB is primarily composed of hydroxyapatite carbonate (R). Figure 1Furthermore, proton-induced X-ray emission (PIXE) analysis revealed that the unique chemical elements in different DCBs fully met the requirements, with antler-DCB containing more magnesium (Mg, 877.2 ± 149.6 μg / g), silicon (Si, 329.8 ± 41.2 μg / g), and sulfur (S, 166.3 ± 15.7 μg / g) than other DCBs. Figure 1 In summary, the unique ECM component in antler-DCB inspires the design of high-performance bone regeneration materials, laying a solid foundation for our antler-inspired bone grafting. DCB was implanted into femoral defects in rats to further evaluate its in vivo osteogenic effect. At 12 weeks post-implantation (wpi), the antler-DCB group showed significantly higher bone mineral density (BMD), bone volume / total tissue volume (BV / TV), and trabecular bone thickness (Tb.Th.) in the local defect area. Figure 1 These findings indicate 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.

[0102] Figure 1 In this diagram, A is a schematic depiction of the manufacturing process and characterization techniques for decellularized bone marrow. 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 the antler before and after decellularization (n=4). E is a scanning electron microscope (SEM) image of the DCB surface. F is the measurement of the specific surface area (SSA) in the DCB (n=4). G is the roughness (Ra) of the DCB measured using 3D atomic force microscopy (AFM). H is the water contact angle (WCA) measured in DCB units (n=4). I is the compressive stress-strain curve of the DCB. J and K are the quantitative analysis of collagen I and GAGs content in the DCB using a detection kit, respectively. L is the X-ray diffraction (XRD) pattern of the DCB. M is the Fourier transform infrared (FT-IR) spectrum of the DCB. NP is the quantitative analysis of the Mg(N), S(O), and Si(P) content in each group (n=4). Q represents hematoxylin-eosin (H&E) and Masson staining images showing new bone formation (NB, newly formed bone tissue) at 12 weeks post-surgery. R represents 3D reconstructed and 2D micro-CT images showing regenerated bone around the defect. SU represents quantitative analysis of microstructural parameters of regenerated bone tissue, including BMD (S), BV / TV (T), and Tb.Th (U) (n=5). All statistics 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.

[0103] Figure 2In the images, A shows scanning electron microscope (SEM) images of antlers before and after decellularization. B shows porosity and pore size measurements of antlers before and after decellularization (n=4). C shows HE staining of antlers before and after decellularization. D shows DNA content of antlers before and after decellularization. E shows trabecular diameter measurements of antlers before and after decellularization (n=4). F shows elastic modulus and compressive modulus measurements of antlers before and after decellularization (n=4). G shows Sirius red staining of antlers before and after decellularization. H shows the macroscopic image of the DCB scaffold. I shows the Micro-CT image of the DCB cube reconstructed in two dimensions. J shows the 3D DAFM image of the DCB. K and L show porosity (K) and pore size (L) measurements in the DCB, respectively (n=4). M) Analysis of the amount of protein retained in the DCB during decellularization using LC / MS-MS. N) Cluster analysis of DEPs in different DCB and GO analysis results to identify key different features and biological significance. O shows Sirius red staining of isolated tissues revealing collagen deposition. P and Q represent the summation of Western blot analysis (P) and quantitative data (Q) of osteonectin, fibronectin, and elastin in DCB (n=4). R represents the grain size of DCB characterized by XRD (n=4). All statistics are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA and Bonferroni post-hoc tests. *P<0.05, **P<0.01, ***P<0.001.

Claims

1. A method for preparing antler-DCB bone graft scaffold material, characterized in that, The specific steps are as follows: S1, cut the cancellous bone of the antler into 4×4mm bone samples, repeatedly freeze and thaw, and then treat with ultrasound; The ultrasonic treatment in S1 is as follows: the bone sample is treated with 40kHz ultrasound for 20 minutes. S2, the bone sample after S1 ultrasonic treatment is immersed in ethanol and shaken. The shaking time in the ethanol described in S2 is 3 hours; S3, Immerse the bone sample soaked in S2 in ether and shake it on a shaker; The shaking time in the ether described in S3 is 3 hours; S4. Soak the bone sample after S3 in sodium hydroxide solution in a shaker. After soaking, wash the bone sample with running water. After washing, treat with 30% hydrogen peroxide for 24 hours. After treatment, wash again. The specific steps of the second cleaning after treatment described in S4 are as follows: wash the bone sample three times with running water for 0.5 hours each time, then soak the bone sample in a DNase solution containing 1 mg / mL for 48 hours, and after the second cleaning, soak the sample in a solution containing 0.5 mg / mL RNase for 48 hours, and wash it three times with running water for 0.5 hours each time. S5. After cleaning, the bone sample was placed in a mixture of chloroform and methanol at a volume ratio of 3:1 for 1 hour. The bone sample was then rinsed with distilled water and dried at 70°C for 1 day to obtain the antler-DCB bone graft scaffold material.

2. The method for preparing the antler-DCB bone graft scaffold material as described in claim 1, characterized in that, The sodium hydroxide solution in S4 has a mass concentration of 20 g / L, and the shaking time in the sodium hydroxide solution is 12 hours.

3. The method for preparing the antler-DCB bone graft scaffold material as described in claim 1, characterized in that, The bone sample was washed with running water three times as described in S4.

4. The antler-DCB bone graft scaffold material prepared by the preparation method of the antler-DCB bone graft scaffold material according to any one of claims 1-3.

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