An injectable bone repair material and its preparation method

Abstract

The present invention relates to the field of biomedical materials and biomedical engineering. Specifically, an injectable composite bone repair material and its preparation method are provided. For the first time, nano-hydroxyapatite, bone morphogenetic protein-loaded, and microbubbles are combined. After the surface of nano-hydroxyapatite is modified, it is coupled with bone morphogenetic protein-loaded microbubbles to obtain a hydroxyapatite-BMP-loaded microbubble complex, which is applied to promote bone defect repair. The preparation method has simple process, good stability, and high reproducibility, providing a new idea for the treatment of bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical materials and biomedical engineering. It relates to an injectable bone repair material and a preparation method thereof. Background Art

[0002] Bone defect healing is a complex wound repair process that requires the regeneration of bone tissue and the restoration of biological properties, and its treatment is a huge challenge faced by the medical community. Currently, the treatment of bone defects mainly focuses on supplementing bone materials to promote osteogenic healing. Existing bone repair techniques mainly include autologous cancellous bone grafting technique, vascularized fibula transplantation technique, bone transport technique, Masquelet technique, etc. However, autologous bone transplantation may cause more serious injuries to patients due to large surgical trauma, resulting in persistent pain, numbness and other sensations; allogeneic bone transplantation has risks such as immune response and virus infection. And artificial material repair has become an important alternative method, but its tissue compatibility and osteogenic healing have become the key to determining bone defect repair. Therefore, finding an effective method to promote osteogenic healing has become an urgent problem to be solved in bone defect repair treatment.

[0003] Bone morphogenetic protein (BMP) is a hydrophobic acidic polypeptide with a high affinity for hydroxyapatite. It is the only growth factor that can alone induce heterotopic osteogenesis of bone tissue among many factors. This active protein can chemotactically attract, aggregate, and directionally differentiate undifferentiated mesenchymal cells into osteoblasts, synthesize collagen to promote the formation of bone matrix, and form calcified bone tissue.

[0004] Hydroxyapatite (HAP) is the main inorganic component of bone tissue and has good biological activity. It is currently a research hotspot for tissue engineering bone scaffold materials. Compared with traditional metal (stainless steel, titanium alloy) and ceramic (aluminum oxide, silicon nitride) bone substitute materials, hydroxyapatite not only has strong corrosion resistance and strong osteoinductive ability, but also endows bone with compressive strength and is the main load-bearing part of bone tissue. If the hydroxyapatite structure can be increased at the bone defect repair site, it will have a positive effect on accelerating repair and healing and increasing bone stress.

[0005] Ultrasound contrast agent microbubbles are microcapsules composed of an outer shell layer and internal gas, which can be used as drug carriers, and specific ligands or nanoparticles can be connected to the surface of the microbubbles. Under the action of ultrasonic waves with a certain intensity, cavitation occurs, opening the vascular endothelial barrier, and the drug can be targeted into the target bone defect tissue through the directional physical action of ultrasonic waves. Summary of the Invention

[0006] The present invention provides an injectable composite bone repair material and a preparation method thereof. For the first time, nano-hydroxyapatite, bone morphogenetic protein, and microbubbles are combined to prepare a hydroxyapatite-loaded BMP microbubble composite, which is applied to bone defect repair. The preparation method has simple process, good stability, and high repeatability, providing a new idea for the treatment of bone defect repair.

[0007] An injectable composite bone repair material, characterized by comprising nano-hydroxyapatite, bone morphogenetic protein-loaded, and microbubbles. After surface modification of the nano-hydroxyapatite, it is coupled with the bone morphogenetic protein-loaded microbubbles to obtain a hydroxyapatite-loaded BMP microbubble composite, which is applied to promote bone defect repair.

[0008] Step (1): Prepare nano-hydroxyapatite with amino groups on the surface

[0009] Dissolve the nano-hydroxyapatite and (3-aminopropyl)triethoxysilane in an organic solvent, reflux at 90°C for 5 h, centrifuge, collect the white amino-functionalized hydroxyapatite (HAP), wash it several times with absolute ethanol and deionized water, and freeze-dry for 24 h to obtain amino-functionalized hydroxyapatite (NH2@HAP);

[0010] Step (2): Prepare the freeze-dried powder of recombinant bone morphogenetic protein (rhBMP)-loaded microbubbles by the thin-film dispersion method combined with the freeze-drying method

[0011] Dissolve the phospholipid material and cholesterol in a certain mass ratio in 100 ml of an organic solvent, distill under reduced pressure to remove the organic solvent, form a thin film on the flask wall, dry it overnight in a vacuum drying oven, add pH 7.4 PBS buffer solution to hydrate for 20 min until the thin film peels off from the flask wall, then add the diluted rhBMP, incubate at room temperature or 4°C, centrifuge to remove the unreacted rhBMP protein completely, collect the precipitate, resuspend it with an appropriate amount of water, and freeze-dry to obtain the white freeze-dried powder of rhBMP-loaded microbubbles;

[0012] Step (3): Prepare the hydroxyapatite-loaded rhBMP microbubble composite

[0013] Add NH2@HAP to the freeze-dried powder of rhBMP microbubbles, then add N,N'-carbonyldiimidazole, react at room temperature for 5 h, quickly dialyze the reaction solution with 1000 ml of grade III water for 5 h, and then freeze-dry to obtain the white solid hydroxyapatite-loaded rhBMP microbubble composite. Pack the microbubble composite into a sealed container, displace the air, and fill it with an inert gas for standby. When in use, inject a certain amount of normal saline or PBS into the sealed container, and obtain the final product, the hydroxyapatite-loaded rhBMP microbubble composite, after mechanical oscillation.

[0014] In the specific preparation process, the molar ratio of nano-hydroxyapatite and (3-aminopropyl)triethoxysilane in step (1) is (1:1) to (1:5), the organic solvent is anhydrous ethanol or toluene, the reaction temperature is 85-100 °C, and the reaction time is 5-24 h.

[0015] In the specific preparation process, the phospholipid material in step (2) is any one of DSPE-mPEG2000, DSEP-mPEG2000-NH2, DSPE-mPEG2000-COOH, DPPC, DPPE, DSPE, and DMPC, a phospholipid modified with polyethylene glycol, and the molecular weight of polyethylene glycol is 1000-5000.

[0016] In the specific preparation process, for any one of DPPC, DPPE, DSPE, and DMPC in step (2): cholesterol: DSPE-mPEG2000: DSEP-mPEG2000-NH2: DSPE-mPEG2000-COOH, the mass ratio is 10-40: 1-15: 3-10: 3-10: 3-10, and the organic solvent is any one or several of chloroform, anhydrous ethanol, and acetone.

[0017] In the specific preparation process, for any one of DPPC, DPPE, DSPE, and DMPC in step (2): cholesterol: DSPE-mPEG2000: DSEP-mPEG2000-NH2: DSPE-mPEG2000-COOH, the mass ratio is 20: 10: 3: 3: 3.

[0018] In the specific preparation process, the hydration time in step (2) is 20 min-2 h, the incubation temperature with rhBMP-2 is 4-25 °C, the time is 4-12 h, and the buffer solution is any one or several of HEPES, BES, PBS, PIPES, and Tris.

[0019] In the specific preparation process, the centrifugation speed in step (2) is 15000-20000 rpm / min, and the time is 20-40 min.

[0020] In the specific preparation process, the room temperature reaction time in step (3) is 5 h-24 h, the dialysis time is 5 h-48 h, the inert gas is at least one of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, and nitrogen, the frequency of mechanical oscillation is 4000 times / minute-5000 times / minute, and the mechanical oscillation time is 30 seconds-60 seconds.

[0021] The recombinant bone morphogenetic protein BMP can be any one of recombinant BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, and BMP-8.

[0022] The results of the above hydroxyapatite - BMP - loaded microbubble complex indicate that the injectable bone defect repair material constructed in the present invention has the advantages of simple preparation process, good stability and safety, and bone repair. Compared with traditional bone repair materials, it has injectability, good tissue compatibility and promotes osteogenic healing efficiency, and has good clinical application prospects. Description of the Drawings

[0023] Figure 1A It is a bright - field photograph of a laser confocal image of microbubbles loaded with recombinant human bone morphogenetic protein - 2 (rhBMP - 2), and round microbubbles can be seen;

[0024] Figure 1B It is a laser confocal image of rhBMP - 2 - loaded microbubbles, and rhBMP - 2 labeled with FITC emits green fluorescence;

[0025] Figure 1C It is a laser confocal co - localization image of rhBMP - 2 - loaded microbubbles, indicating that rhBMP - 2 is successfully conjugated to round microbubbles;

[0026] Figure 2 It is an inverted microscope image of the rhBMP - 2 - loaded microbubble complex;

[0027] Figure 3 It is a particle size diagram of the hydroxyapatite - rhBMP - 2 - loaded microbubble complex;

[0028] Figure 4A It is a scanning electron microscope image of rhBMP - 2 - loaded microbubbles not conjugated with nano - hydroxyapatite;

[0029] Figure 4B It is a scanning electron microscope image of the hydroxyapatite - rhBMP - 2 - loaded microbubble complex;

[0030] Figure 5 It is an alizarin red staining image of BMSCs cells induced by the hydroxyapatite - rhBMP - 2 - loaded microbubble complex (HAP - BMP - 2@MB). Detailed Embodiments

[0031] The following further describes the specific embodiments of the present invention through examples. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0032] Example 1

[0033] (1) Preparation of nano - hydroxyapatite with amino groups on the surface

[0034] Dissolve nano-hydroxyapatite with an average particle size of 20 nm and (3-aminopropyl)triethoxysilane in anhydrous ethanol, reflux at 90 °C for 5 h, centrifuge, collect the white amino-functionalized HAP, wash it several times with anhydrous ethanol and deionized water, and freeze-dry for 24 h to obtain amino-functionalized hydroxyapatite.

[0035] Bone recombinant morphogenetic protein BMP can be any one of recombinant BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, and BMP-8. In this example, only BMP-2 is used to illustrate the preparation process.

[0036] Example 2

[0037] (2) Preparation and characterization of recombinant bone morphogenetic protein-2 (rhBMP-2) microbubbles

[0038] The phospholipid material can be any one of DSPE-mPEG2000, DSEP-mPEG2000-NH2, DSPE-mPEG2000-COOH, DPPC, DPPE, DSPE, and DMPC, which are phospholipids modified with polyethylene glycol, and the molecular weight of polyethylene glycol is 1000 - 5000. In this example, only DPPC is used to illustrate the preparation process.

[0039] ① Preparation of fluorescently labeled rhBMP-2: Take 20 μg of rhBMP-2, add distilled water for dilution (0.1 M, 700 μL, pH 9.0), react with FITC solution (0.24 mg, 0.1 M, 300 ml, pH 9.0) at room temperature for 2 hours, and dialyze with a dialysis bag with a molecular weight of 3500 Da for 24 hours to obtain FITC-labeled rhBMP-2. ② Prepare microbubbles by the thin film dispersion method combined with freeze-drying method. Dissolve DPPC:cholesterol:DSPE-mPEG2000:DSEP-mPEG2000-NH2:DSPE-mPEG2000-COOH in a mass ratio of 20:10:3:3:3 in 100 ml of chloroform, distill under reduced pressure to remove the organic solvent, form a thin film on the flask wall, dry overnight in a vacuum drying oven, add pH 7.4 PBS buffer solution to hydrate for 20 min until the thin film peels off from the bottle wall, then add the diluted FITC-labeled rhBMP-2, incubate at 4 °C for 12 hours, centrifuge at 15000 rpm for 30 minutes to remove the unreacted rhBMP-2 protein, collect the precipitate, resuspend with an appropriate amount of water, and freeze-dry to obtain the freeze-dried powder of rhBMP-2-loaded microbubbles.

[0040] ③ Structural identification: To prove that in this embodiment, rhBMP-2 is adsorbed on the surface of microbubbles, an appropriate amount of freeze-dried powder of FITC-labeled rhBMP-2 microbubbles is dissolved in distilled water. If rhBMP-2 is adsorbed on the surface layer of microbubbles, green fluorescence can be seen. Observed under a laser confocal microscope, microbubbles can be seen in the bright field ( Figure 1A ), FITC-labeled rhBMP-2, exciting green fluorescence ( Figure 1B ). Through co-localization, it can be seen that rhBMP-2 is connected to the surface of microbubbles. As shown in Figure 1C , it can be seen from the figure that the microbubbles prepared in this embodiment are well combined with rhBMP-2. ② Determination of the coupling rate of rhBMP-2 and microbubbles: A standard curve was established through a rhBMP-2 ELISA kit, and the content of rhBMP-2 in microbubbles was determined. The coupling rate of rhBMP-2 and microbubbles was 95.9%.

[0041] ③ Morphological observation of rhBMP-2-loaded microbubbles: Take a certain amount of freeze-dried powder of rhBMP-2-loaded microbubbles, displace the air, and fill it with perfluoropropane. Inject a certain amount of normal saline into a sealed container and mechanically oscillate for 50 s to obtain rhBMP-2 microbubbles. Observe the morphology of the microbubbles under an inverted microscope. As shown in Figure 2 , the particle sizes are uniform and the distribution is uniform, and they are circular.

[0042] Example 3

[0043] (3) Preparation of hydroxyapatite-rhBMP-2-loaded microbubble composite

[0044] ① Add NH2@HAP to rhBMP-2 microbubbles, then add N,N'-hydroxydicarbodiimide, and react at room temperature for 5 hours. The reaction solution is quickly dialyzed with 1000 ml of grade III water for 5 h and then freeze-dried to obtain a white solid hydroxyapatite-rhBMP-2-loaded microbubble composite. The solid composite is dispensed into a sealed container, the air is displaced, and an inert gas is filled for standby. When in use, inject a certain amount of PBS into the sealed container and mechanically oscillate for 50 s to obtain a hydroxyapatite-rhBMP-2 microbubble composite.

[0045] ② Structural characterization (measurement of composite particle size): Take 2 ml of hydroxyapatite-rhBMP-2 microbubble composite and measure the particle size with a laser particle size analyzer to be 2265.8 nm ( Figure 3 ).

[0046] ③ Structural characterization (measurement by scanning electron microscope) Take a small amount of freeze-dried powder of hydroxyapatite-rhBMP-2-loaded microbubble composite and place it on a silicon wafer. After sputtering with gold, observe it under a scanning electron microscope. It can be seen that smaller granular nano-hydroxyapatite is attached to the surface of the microbubbles ( Figure 4B), as a control, the surface of the rhBMP-2 microbubbles not conjugated with nano-hydroxyapatite was relatively smooth ( Figure 4A ), indicating that hydroxyapatite was successfully attached to the surface of the microbubbles.

[0047] Example 4

[0048] (1) Preparation of nano-hydroxyapatite with amino groups on the surface

[0049] Dissolve nano-hydroxyapatite with an average particle size of 20 nm and (3-aminopropyl)triethoxysilane in toluene, reflux at 85 °C for 5 h, centrifuge, collect the white amino-functionalized HAP, wash it several times with absolute ethanol and deionized water, and freeze-dry for 24 h to obtain amino-functionalized hydroxyapatite.

[0050] Example 5

[0051] (2) Preparation and characterization of recombinant bone morphogenetic protein-2 (rhBMP-2) microbubbles Prepare microbubbles by the thin film dispersion method combined with freeze-drying method. Dissolve DPPC:cholesterol:DSPE-mPEG2000:DSEP-mPEG2000-NH2:DSPE-mPEG2000-COOH in a mass ratio of 30:10:3:3:3 in 100 ml of absolute ethanol, distill under reduced pressure to remove the organic solvent, form a thin film on the wall of the flask, dry overnight in a vacuum drying oven, add pH 7.4 PBS buffer solution to hydrate for 20 min until the thin film detaches from the bottle wall, then add the diluted rhBMP-2, incubate at room temperature for 4 hours, centrifuge at 15000 rpm for 30 minutes to remove the unreacted rhBMP-2 protein, collect the precipitate, resuspend it with an appropriate amount of water, and freeze-dry to obtain the freeze-dried powder of rhBMP-2-loaded microbubbles.

[0052] Example 6

[0053] (3) Preparation of hydroxyapatite-rhBMP-2 microbubble complex

[0054] Add NH2@HAP to the rhBMP-2 microbubbles, then add N,N'-carbonyldiimidazole, react at room temperature for 24 hours, quickly dialyze the reaction solution with 1000 ml of grade III water for 5 h, and then freeze-dry to obtain a white solid hydroxyapatite-rhBMP-2 microbubble complex. Divide the solid complex into sealed containers, displace the air, and fill with perfluoropropane gas for standby. When in use, inject a certain amount of normal saline into the sealed container and mechanically oscillate for 60 s to obtain the hydroxyapatite-rhBMP-2 microbubble complex.

[0055] Example 7

[0056] Calcium deposition in the extracellular matrix (ECM) of bone cells can be regarded as a phenotypic marker in the late stage of osteoblast differentiation and can be used to detect the osteoinductive and formation abilities of matrix materials. Alizarin Red S staining is used to evaluate the formation of mineralized nodules in osteoblasts. The specific steps are as follows: Bone marrow mesenchymal stem cells (BMSCs) are induced for 7 days and 10 days with different culture media containing HAP, BMP-2 microbubbles, and HAP-BMP-2 microbubble complexes. Then, the culture medium is removed and the cells are washed three times with PBS. The cells are fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and 1% Alizarin Red (Solarbio, China) solution is added and cultured at 37°C for 30 minutes. After removing the staining solution, the stained cells are rinsed 3 times with PBS (pH 4.2), and then observed under an inverted optical microscope.

[0057] Mineralization occurs through cell-mediated deposition of ECM components. When anionic matrix molecules absorb Ca 2+ , such as Figure 5 shown, at 7 days of culture, almost no stained nodules were observed in the PBS group control. For the HAP and BMP-2@MB groups, there were a small number of red nodule stains, and for the HAP-BMP-2@MB group, there were a large number of red nodule stains. This indicates that both HAP and BMP-2@MB have a certain ability to promote osteogenic induction. In particular, the HAP-BMP-2@MB complex exhibits stronger calcium deposition ability and osteogenic promotion ability. At 10 days of culture, the change trend was the same as that at 7 days.

[0058] The prepared hydroxyapatite-BMP microbubble complex is injected into rats via the caudal vein. Under the contrast-enhanced ultrasound mode of an ultrasound diagnostic instrument, an obvious imaging effect can be seen at the bone defect site. The microbubbles burst at the bone defect site, releasing BMP to promote bone repair and healing, and causing nano-hydroxyapatite particles to settle locally, strengthening the formation of osteogenic stress, exploring new biomaterials and treatment ideas for bone defect repair.

Claims

1. A preparation method of an injectable composite bone repair material, characterized in that It includes nano-hydroxyapatite and recombinant bone morphogenetic protein (rhBMP) microbubbles. After surface modification of the nano-hydroxyapatite, it is coupled with the recombinant bone morphogenetic protein microbubbles to obtain a hydroxyapatite-BMP-loaded microbubble complex; Specifically, it includes the following steps: Step (1) Preparation of nano-hydroxyapatite with amino groups on the surface Dissolve nano-hydroxyapatite and (3-aminopropyl)triethoxysilane in an organic solvent, reflux at 90 °C for 5 h, centrifuge, collect the white amino-functionalized hydroxyapatite, wash it several times with absolute ethanol and deionized water, and freeze-dry for 24 h to obtain amino-functionalized hydroxyapatite (NH2@HAP); Step (2) Preparation of recombinant bone morphogenetic protein (rhBMP) microbubble freeze-dried powder by thin film dispersion method combined with freeze-drying method Dissolve the phospholipid material and cholesterol in a certain mass ratio in 100 ml of an organic solvent, distill under reduced pressure to remove the organic solvent, form a thin film on the flask wall, dry overnight in a vacuum drying oven, add pH 7.4 PBS buffer solution to hydrate for 20 min until the thin film peels off from the bottle wall, then add the diluted rhBMP, incubate at room temperature or 4 °C, centrifuge to remove the unreacted rhBMP protein completely, collect the precipitate, resuspend it with an appropriate amount of water, and freeze-dry to obtain a white rhBMP-loaded microbubble freeze-dried powder; Step (3) Preparation of hydroxyapatite-rhBMP-loaded microbubble complex Add NH2@HAP to the rhBMP microbubble freeze-dried powder, then add N,N'-hydroxydicarbodiimide, react at room temperature for 5 hours, quickly dialyze the reaction solution with 1000 ml of grade III water for 5 h, and then freeze-dry to obtain a white solid hydroxyapatite-rhBMP-loaded microbubble complex. Divide the microbubble complex into sealed containers, displace the air, and fill with an inert gas for standby. When in use, inject a certain amount of normal saline or PBS into the sealed container, and obtain the final product, the hydroxyapatite-rhBMP-loaded microbubble complex, after mechanical oscillation; In step (2), the phospholipid material is any one of DSPE-mPEG2000, DSEP-mPEG2000-NH2, DSPE-mPEG2000-COOH, DPPC, DPPE, DSPE, and DMPC, a phospholipid modified with polyethylene glycol, and the average molecular weight of polyethylene glycol is 2000; In step (2), for any one of DPPC, DPPE, DSPE, and DMPC:cholesterol:DSPE-mPEG2000:DSEP-mPEG2000-NH2:DSPE-mPEG2000-COOH, the mass ratio is 10~40:1~15:3~10:3~10:3~10, and the organic solvent is any one or several of chloroform, absolute ethanol, and acetone; The injectable bone defect repair material prepared by the above method has injectability.

2. The preparation method of an injectable composite bone repair material according to claim 1, characterized in that, Step (1) The molar ratio of nano-hydroxyapatite to (3-aminopropyl)triethoxysilane is (1:1) to (1:5), the organic solvent is absolute ethanol or toluene, the reaction temperature is 85 to 100 °C, and the reaction time is 5 to 24 h.

3. The preparation method of an injectable composite bone repair material as described in claim 1, characterized in that, Step (2) Any one of DPPC, DPPE, DSPE, and DMPC: cholesterol: DSPE-mPEG2000: DSEP-mPEG2000-NH2: DSPE-mPEG2000-COOH has a mass ratio of 20:10:3:3:

3.

4. The preparation method of an injectable composite bone repair material as described in claim 1, characterized in that, Step (2) The hydration time is 20 min to 2 h, the incubation temperature with rhBMP is 4 to 25 °C, the time is 4 to 12 h, and the buffer is any one or several of HEPES, BES, PBS, PIPES, and Tris.

5. The preparation method of an injectable composite bone repair material as described in claim 1, characterized in that, Step (2) The centrifugation speed is 15000 to 20000 rpm / min, and the time is 20 - 40 min.

6. The preparation method of an injectable composite bone repair material as described in claim 1, characterized in that, Step (3) The reaction time at room temperature is 5 h - 24 h, the dialysis time is 5 h - 48 h, the inert gas is at least one of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, and nitrogen, the frequency of mechanical oscillation is 4000 times / minute - 5000 times / minute, and the mechanical oscillation time is 30 seconds - 60 seconds.

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