A composite bone repair scaffold, a preparation method thereof and application thereof in preparation of a product for treating radiation osteomyelitis

Abstract

The application provides a composite bone repair scaffold, a preparation method thereof and application thereof in preparation of a product for treating radiation osteomyelitis, and belongs to the technical field of biomedical materials. The composite bone repair scaffold comprises a three-dimensional porous scaffold matrix, a bacteriostatic agent and functionalized microspheres; the three-dimensional porous scaffold matrix is a gelatin-sodium alginate oxide double-network hydrogel; and the functionalized microspheres are microspheres loaded with eucommia extract and radix astragali extract. The composite bone repair scaffold provided by the application has good pore size, porosity and mechanical properties, and has good degradation performance, water absorption performance and bacteriostatic performance, and has good biocompatibility, can be used for preparing a product for treating radiation osteomyelitis, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a composite bone repair scaffold, its preparation method, and its application in the preparation of products for treating radiation-induced osteomyelitis. Background Technology

[0002] Radiation osteomyelitis is a serious complication that occurs in patients with head and neck tumors after radiotherapy. Its pathological features mainly include: local bone necrosis and defects; severely impaired tissue blood supply; susceptibility to secondary bacterial infections, especially anaerobic infections; persistent inflammatory response; and suppressed osteoblast activity. Currently, the main clinical treatments for radiation osteomyelitis are surgical debridement combined with antibiotic therapy and autologous / allogeneic bone transplantation. However, these methods have several limitations: ① Systemic or local antibiotic use is difficult to achieve effective concentrations in poorly blood-supplied necrotic bone tissue and easily leads to drug resistance. ② Autologous bone transplantation has limited sources, can cause secondary damage, and has poor healing effects in poorly blood-supplied recipient areas. ③ Allogeneic or artificial bone materials lack bioactivity, have low integration rates, and lack anti-infection capabilities, resulting in a high failure rate in contaminated or potentially infected areas of radiation osteomyelitis.

[0003] Therefore, providing a composite bone repair scaffold that simultaneously meets the multiple needs of efficient antibacterial, anti-inflammatory, and guided bone repair is key to solving the treatment challenges of radiation osteomyelitis and has significant clinical demand and market prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a composite bone repair scaffold, its preparation method, and its application in the preparation of products for treating radiation osteomyelitis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a composite bone repair scaffold, which includes a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres; the three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; and the functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract.

[0007] Preferably, the degree of oxidation of the oxidized sodium alginate is 30-40%; the mass ratio of the gelatin to the oxidized sodium alginate is 8-12:3-5;

[0008] The antibacterial agent is a nano-silver suspension; the concentration of the nano-silver suspension is 0.08~0.12 mg / mL.

[0009] Preferably, the method for preparing the functionalized microspheres includes the following steps:

[0010] (1) Liquid paraffin is mixed with a surfactant to obtain an oil phase;

[0011] (2) Under water bath conditions, gelatin and sodium alginate were mixed with water, and Eucommia ulmoides extract and Achyranthes bidentata extract were added and mixed again to obtain an aqueous phase;

[0012] (3) Add the aqueous phase obtained in step (2) to the oil phase obtained in step (1) and emulsify to obtain a W / O emulsion;

[0013] (4) The crosslinking solution is added to the W / O emulsion obtained in step (3) for crosslinking and curing. The microspheres are collected by solid-liquid separation, washed and dried to obtain functionalized microspheres.

[0014] Preferably, the volume ratio of liquid paraffin and surfactant in step (1) is 40~55:0.8~1.2; the surfactant is Span 80; the mixing rate is 300~500 rpm and the time is 10~40 min;

[0015] The temperature of the water bath in step (2) is 38~45℃; the mass-volume ratio of gelatin, sodium alginate, water, Eucommia ulmoides extract, and Achyranthes bidentata extract is 3~5g: 1.5~2.5g: 80~120mL: 0.8~1.2g: 0.8~1.2g.

[0016] Preferably, in step (3), the volume ratio of the aqueous phase to the oil phase is 450~550:80~120; the emulsification rate is 1300~1700 rpm and the time is 10~20 min;

[0017] The crosslinking solution in step (4) contains 1.5-2.5% calcium chloride and 0.8-1.2% glacial acetic acid; the volume ratio of the crosslinking solution to the W / O emulsion is 150-250:500-700; and the crosslinking curing time is 1.5-2.5 h.

[0018] This invention provides a method for preparing the composite bone repair scaffold. Under water bath conditions, gelatin, sodium alginate, and PBS buffer are mixed, functionalized microspheres and antibacterial agents are added and mixed again, and the mixture is printed using a 3D bioprinter. The printed scaffold is then crosslinked, allowed to stand, and washed to obtain the composite bone repair scaffold.

[0019] Preferably, the temperature of the water bath is 35~40℃, and the mass-to-volume ratio of gelatin, sodium alginate, PBS buffer, functionalized microspheres, and antibacterial agent is 0.8~1.2g:0.3~0.5g:6~10mL:0.15~0.25g:0.8~1.2mL.

[0020] Preferably, the printing platform temperature is 8~12℃, the nozzle diameter is 0.4~0.6mm, the pressure is 450~550kPa, the speed is 6~10mm / s, and the path is 0 / 90° alternating layering, with a total of 4~8 layers.

[0021] Preferably, the crosslinking solution used during crosslinking is a 4-6% calcium chloride solution, the crosslinking temperature is 3-5℃, and the time is 20-40 min; the standing time is 4-8 h.

[0022] This invention provides the application of the composite bone repair scaffold described above or the composite bone repair scaffold prepared by the method in the preparation of products for treating radiation osteomyelitis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a composite bone repair scaffold that combines antibacterial and anti-inflammatory properties with bone repair promotion. The scaffold uses a gelatin-sodium alginate dual-network hydrogel as its body, providing physical support and space for cell ingrowth while simultaneously carrying antibacterial agents to rapidly establish an antibacterial environment in the early stages of implantation. The composite bone repair scaffold also contains functionalized microspheres encapsulating bone-repairing active ingredients (Eucommia ulmoides extract and Achyranthes bidentata extract), preventing rapid degradation and loss of these active ingredients in the initial stages. Furthermore, as the hydrogel and the microspheres themselves degrade, they gradually release the herbal extracts, providing sustained osteogenic induction and anti-inflammatory effects, complementing the rapid antibacterial formation time of nano-silver.

[0025] The scaffold matrix provided by this invention is a gelatin-sodium oxidized alginate dual-network hydrogel. Gelatin provides excellent cell adhesion sites, while sodium oxidized alginate cross-links with gelatin and calcium ions to form a dual-network structure with superior mechanical properties and better stability, which can better simulate the physical and biochemical environment of the extracellular matrix. The functionalized microspheres use the same material system as the scaffold matrix, ensuring excellent biocompatibility and interfacial compatibility, which is conducive to the integration of microspheres and scaffolds and to achieving matching degradation rates. The Eucommia ulmoides extract and Achyranthes bidentata extract encapsulated in the functionalized microspheres work together to continuously act on the defect site, which can efficiently induce bone tissue regeneration and counteract radiation-induced tissue regeneration barriers.

[0026] This invention uses 3D bioprinting technology to prepare the composite bone repair scaffold, which has a controllable structure and can be precisely printed according to the patient's CT scan data to perfectly match the shape of the bone defect, achieving perfect implantation.

[0027] The composite bone repair scaffold provided by this invention has good pore size, porosity and mechanical properties, as well as good degradation performance, water absorption performance and antibacterial performance. It also has good biocompatibility and can be used to prepare products for the treatment of radiation osteomyelitis, showing broad application prospects. Detailed Implementation

[0028] This invention provides a composite bone repair scaffold, which includes a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres; the three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; and the functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract.

[0029] In this invention, the oxidation degree of the sodium alginate is 30-40%, preferably 32-38%, more preferably 34-36%, and even more preferably 35%; the mass ratio of the gelatin to the sodium alginate is 8-12:3-5, preferably 9-11:3.5-4.5, and even more preferably 10:4.

[0030] The antibacterial agent is a nano-silver suspension; the concentration of the nano-silver suspension is 0.08~0.12 mg / mL, preferably 0.09~0.11 mg / mL, and more preferably 0.1 mg / mL.

[0031] In this invention, the method for preparing the functionalized microspheres includes the following steps:

[0032] (1) Liquid paraffin is mixed with a surfactant to obtain an oil phase;

[0033] (2) Under water bath conditions, gelatin and sodium alginate were mixed with water, and Eucommia ulmoides extract and Achyranthes bidentata extract were added and mixed again to obtain an aqueous phase;

[0034] (3) Add the aqueous phase obtained in step (2) to the oil phase obtained in step (1) and emulsify to obtain a W / O emulsion;

[0035] (4) The crosslinking solution is added to the W / O emulsion obtained in step (3) for crosslinking and curing. The microspheres are collected by solid-liquid separation, washed and dried to obtain functionalized microspheres.

[0036] In this invention, the volume ratio of liquid paraffin and surfactant in step (1) is 40~55:0.8~1.2, preferably 45~52:0.9~1.1, and more preferably 49:1; the surfactant is Span 80; the mixing rate is 300~500 rpm, preferably 350~450 rpm, and more preferably 400 rpm, and the time is 10~40 min, preferably 15~30 min, more preferably 18~25 min, and more preferably 20 min;

[0037] The temperature of the water bath in step (2) is 38~45℃, preferably 39~42℃, and more preferably 40℃; the mass-volume ratio of gelatin, sodium alginate, water, Eucommia ulmoides extract, and Achyranthes bidentata extract is 3~5g:1.5~2.5g:80~120mL:0.8~1.2g:0.8~1.2g, preferably 3.5~4.5g:1.8~2.2g:90~110mL:0.9~1.1g:0.9~1.1g, and more preferably 4g:2g:100mL:1g:1g.

[0038] In this invention, the volume ratio of the aqueous phase to the oil phase in step (3) is 450~550:80~120, preferably 480~520:90~110, and more preferably 500:100; the emulsification rate is 1300~1700 rpm, preferably 1400~1600 rpm, and more preferably 1500 rpm; the time is 10~20 min, preferably 12~18 min, and more preferably 15 min.

[0039] The crosslinking solution in step (4) contains 1.5-2.5% calcium chloride and 0.8-1.2% glacial acetic acid, preferably 1.8-2.2% calcium chloride and 0.9-1.1% glacial acetic acid, and more preferably 2% calcium chloride and 1% glacial acetic acid; the volume ratio of the crosslinking solution to the W / O emulsion is 150-250:500-700, preferably 180-220:550-650, and more preferably 200:600; the crosslinking curing time is 1.5-2.5h, preferably 1.8-2.2h, and more preferably 2h.

[0040] This invention provides a method for preparing the composite bone repair scaffold. Under water bath conditions, gelatin, sodium alginate, and PBS buffer are mixed, functionalized microspheres and antibacterial agents are added and mixed again, and the mixture is printed using a 3D bioprinter. The printed scaffold is then crosslinked, allowed to stand, and washed to obtain the composite bone repair scaffold.

[0041] In this invention, the temperature of the water bath is 35~40℃, preferably 36~38℃, more preferably 37℃, and the mass-volume ratio of gelatin, sodium alginate, PBS buffer, functionalized microspheres, and antibacterial agent is 0.8~1.2g:0.3~0.5g:6~10mL:0.15~0.25g:0.8~1.2mL, preferably 0.9~1.1g:0.35~0.45g:7~9mL:0.18~0.22g:0.9~1.1mL, more preferably 1g:0.4g:8mL:0.2g:1mL.

[0042] In this invention, the platform temperature during printing is 8~12℃, preferably 9~11℃, more preferably 10℃; the nozzle diameter is 0.4~0.6mm, preferably 0.45~0.55mm, more preferably 0.5mm; the pressure is 450~550kPa, preferably 480~520kPa, more preferably 500kPa; the speed is 6~10mm / s, preferably 7~9mm / s, more preferably 8mm / s; the path is 0 / 90° alternating layup, with a total of 4~8 layers, preferably 5~7 layers, more preferably 6 layers.

[0043] In this invention, the crosslinking solution used during crosslinking is a 4-6% calcium chloride solution, preferably a 5% calcium chloride solution; the crosslinking temperature is 3-5°C, preferably 4°C; the crosslinking time is 20-40 min, preferably 25-35 min, and more preferably 30 min; the standing time is 4-8 h, preferably 5-7 h, and more preferably 6 h.

[0044] This invention provides the application of the composite bone repair scaffold described above or the composite bone repair scaffold prepared by the method in the preparation of products for treating radiation osteomyelitis.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The Eucommia ulmoides extract, Achyranthes bidentata extract, and Epimedium extract used below are all alcohol extracts.

[0046] Example 1

[0047] A composite bone repair scaffold is composed of a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres;

[0048] The three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; the degree of oxidation of sodium oxidized alginate is 30%, and the mass ratio of gelatin to sodium oxidized alginate is 8:3.

[0049] The antibacterial agent is a nano-silver suspension with a concentration of 0.08 mg / mL.

[0050] The functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract; the preparation method of the functionalized microspheres is as follows:

[0051] (1) Mix 400 mL of liquid paraffin with 8 mL of Span 80 at 300 rpm for 10 min to obtain the oil phase;

[0052] (2) Under the condition of 38℃ water bath, 3g gelatin and 1.5g oxidized sodium alginate (oxidation degree of 30%) were mixed with 90mL deionized water, and 0.8g Eucommia ulmoides extract and 0.8g Achyranthes bidentata extract were added and mixed again to obtain the aqueous phase;

[0053] (3) At 500 rpm, the aqueous phase obtained in step (2) is slowly added dropwise to the oil phase obtained in step (1), and emulsified at 1300 rpm for 10 min to obtain a W / O emulsion.

[0054] (4) At 500 rpm, 150 mL of a crosslinking solution consisting of 1.5% calcium chloride and 0.8% glacial acetic acid was slowly added dropwise to the W / O emulsion obtained in step (3). The crosslinking was cured for 1.5 h. The microspheres were collected by centrifugation at 3000 rpm for 10 min. The microspheres were washed three times with anhydrous ethanol and deionized water respectively and then dried to obtain functionalized microspheres. During the drying process, 3% trehalose was added to the washed microspheres as a protective agent. The microspheres were first dried at -30℃ for 1 h to allow them to crystallize completely. Then, they were dried at -20℃ and 30 Pa vacuum for 15 h to prevent the microspheres from collapsing. After that, the temperature was raised to 20℃ and the vacuum was 25 Pa. The microspheres were dried for 8 h to make the final moisture content ≤5%.

[0055] The composite bone repair scaffold was prepared as follows: 0.8 g of gelatin and 0.3 g of sodium alginate were dissolved in 6 mL of PBS buffer under a 35°C water bath. Then, 0.15 g of the functionalized microspheres prepared above and 0.8 mL of nano-silver suspension were added and mixed again. The mixture was then printed using a 3D bioprinter with the following printing parameters: platform temperature 8°C, nozzle diameter 0.4 mm, pressure 450 kPa, speed 6 mm / s, and 0 / 90° alternating layup for a total of 4 layers. The printed scaffold was then placed in a 4% calcium chloride solution at 3°C ​​for 20 min for crosslinking, and then allowed to stand for 4 h. After washing with deionized water, the composite bone repair scaffold was obtained.

[0056] Example 2

[0057] A composite bone repair scaffold is composed of a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres;

[0058] The three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; the oxidation degree of sodium oxidized alginate is 40%, and the mass ratio of gelatin to sodium oxidized alginate is 12:5.

[0059] The antibacterial agent is a nano-silver suspension with a concentration of 0.12 mg / mL.

[0060] The functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract; the preparation method of the functionalized microspheres is as follows:

[0061] (1) Mix 550 mL of liquid paraffin with 12 mL of Span 80 at 500 rpm for 40 min to obtain the oil phase;

[0062] (2) Under the conditions of 45℃ water bath, 5g of gelatin and 2.5g of oxidized sodium alginate (oxidation degree of 40%) were mixed with 110mL of deionized water, and 1.2g of Eucommia ulmoides extract and 1.2g of Achyranthes bidentata extract were added and mixed again to obtain the aqueous phase;

[0063] (3) At 500 rpm, the aqueous phase obtained in step (2) is slowly added dropwise to the oil phase obtained in step (1), and emulsified at 1700 rpm for 20 min to obtain a W / O emulsion;

[0064] (4) At 500 rpm, 250 mL of a crosslinking solution consisting of 2.5% calcium chloride and 1.2% glacial acetic acid was slowly added dropwise to the W / O emulsion obtained in step (3). The crosslinking was cured for 2.5 h. The microspheres were collected by centrifugation at 3000 rpm for 10 min. The microspheres were washed three times with anhydrous ethanol and deionized water respectively and then dried to obtain functionalized microspheres. During the drying process, 3% trehalose was added to the washed microspheres as a protective agent. The microspheres were first dried at -40℃ for 2 h to allow them to crystallize completely. Then, they were dried at -10℃ and 30 Pa vacuum for 25 h to prevent the microspheres from collapsing. After that, the temperature was raised to 40℃ and the vacuum was 25 Pa. The microspheres were dried for 12 h to make the final moisture content ≤5%.

[0065] The composite bone repair scaffold was prepared as follows: 1.2g of gelatin and 0.5g of sodium alginate were dissolved in 10mL of PBS buffer under a 40℃ water bath. Then, 0.25g of the functionalized microspheres prepared above and 1.2mL of nano-silver suspension were added and mixed again. The mixture was then printed using a 3D bioprinter with the following printing parameters: platform temperature 12℃, nozzle diameter 0.6mm, pressure 550kPa, speed 10mm / s, and 0 / 90° alternating layup for a total of 8 layers. The printed scaffold was then placed in a 6% calcium chloride solution at 5℃ for crosslinking for 40min, and then allowed to stand for 8h. After washing with deionized water, the composite bone repair scaffold was obtained.

[0066] Example 3

[0067] A composite bone repair scaffold is composed of a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres;

[0068] The three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; the oxidation degree of sodium oxidized alginate is 35%, and the mass ratio of gelatin to sodium oxidized alginate is 10:4.

[0069] The antibacterial agent is a nano-silver suspension with a concentration of 0.1 mg / mL.

[0070] The functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract; the preparation method of the functionalized microspheres is as follows:

[0071] (1) Mix 490 mL of liquid paraffin with 10 mL of Span 80 at 400 rpm for 20 min to obtain the oil phase;

[0072] (2) Under the condition of 40℃ water bath, 4g gelatin and 2g oxidized sodium alginate (oxidation degree of 35%) were mixed with 100mL deionized water, and 1g Eucommia ulmoides extract and 1g Achyranthes bidentata extract were added and mixed again to obtain the aqueous phase;

[0073] (3) At 500 rpm, the aqueous phase obtained in step (2) is slowly added dropwise to the oil phase obtained in step (1), and emulsified at 1500 rpm for 15 min to obtain a W / O emulsion.

[0074] (4) At 500 rpm, 200 mL of a crosslinking solution consisting of 2% calcium chloride and 1% glacial acetic acid was slowly added dropwise to the W / O emulsion obtained in step (3). The crosslinking was cured for 2 h, and the microspheres were collected by centrifugation at 3000 rpm for 10 min. The microspheres were washed three times with anhydrous ethanol and deionized water respectively and then dried to obtain functionalized microspheres. During the drying process, 3% trehalose was added to the washed microspheres as a protective agent. The microspheres were first dried at -35℃ for 1.5 h to allow them to crystallize completely. Then, they were dried at -15℃ and 30 Pa vacuum for 20 h to prevent the microspheres from collapsing. After that, the temperature was raised to 30℃ and the vacuum was 25 Pa. The microspheres were dried for 10 h to make the final moisture content ≤5%.

[0075] The composite bone repair scaffold was prepared as follows: 1g of gelatin and 0.4g of sodium alginate were dissolved in 8mL of PBS buffer under a 37℃ water bath. Then, 0.2g of the functionalized microspheres and 1mL of nano-silver suspension were added and mixed again. The mixture was then printed using a 3D bioprinter with the following printing parameters: platform temperature 10℃, nozzle diameter 0.5mm, pressure 500kPa, speed 8mm / s, and alternating 0 / 90° layup for a total of 6 layers. The printed scaffold was then placed in a 5% calcium chloride solution at 4℃ for 30min for crosslinking, and then allowed to stand for 6h. After washing with deionized water, the composite bone repair scaffold was obtained.

[0076] Comparative Example 1

[0077] The difference between this comparative example and Example 3 is that the scaffold matrix material of this comparative example composite bone repair scaffold is polylactic acid (PLA). The preparation method of this comparative example composite bone repair scaffold is as follows: 1 g of PLA is dissolved in 10 mL of dichloromethane and stirred at 300 rpm for 8 h to obtain a polymer solution; 0.2 g of functionalized microspheres and 1 mL of nano-silver suspension are dispersed in the above polymer solution, stirred at 400 rpm for 15 min, and then homogenized under high-speed shearing in an ice-water bath (homogenization at 10000 rpm for 3 min, with a 15 s pause every 30 s during homogenization); under 3000 rpm conditions... 9g of sodium chloride crystals with a particle size range of 200~400μm were slowly added as a pore-forming agent. The mixture was homogenized for 2 minutes at 8000rpm in an ice-water bath (with a 15s pause every 30s during homogenization) to obtain a slurry. The slurry was cast into a PTFE mold and frozen at -80℃ for 8 hours to solidify the scaffold. After initial drying at -40℃ and 10Pa vacuum for 24 hours, it was leached in deionized water for 48 hours until the sodium chloride was completely removed (the water was changed every 8 hours during this period). It was then dried again at -40℃ and 10Pa vacuum for 24 hours to obtain the composite bone repair scaffold. The functionalized microspheres and nano-silver suspension used in this comparative example are the same as in Example 3.

[0078] Comparative Example 2

[0079] The difference between this comparative example and Example 3 is that the preparation method of the functionalized microspheres is different; the remaining steps and methods are the same as in Example 3. The preparation method of the functionalized microspheres in this comparative example is as follows:

[0080] (1) Mix 490 mL of liquid paraffin with 10 mL of Span 80 at 400 rpm for 20 min to obtain the oil phase;

[0081] (2) Disperse 1g of Eucommia ulmoides extract and 1g of Achyranthes bidentata extract in 100mL of 3% sodium alginate solution and mix at 400rpm for 20min to obtain the aqueous phase;

[0082] (3) At 500 rpm, the aqueous phase obtained in step (2) is slowly added dropwise to the oil phase obtained in step (1), and emulsified at 1500 rpm for 15 min to obtain a W / O emulsion.

[0083] (4) At 500 rpm, 200 mL of a crosslinking solution consisting of 2% calcium chloride and 1% glacial acetic acid was slowly added dropwise to the W / O emulsion obtained in step (3). The crosslinking was cured for 2 h, and the microspheres were collected by centrifugation at 3000 rpm for 10 min. The microspheres were washed three times with anhydrous ethanol and deionized water respectively and then dried to obtain functionalized microspheres. During the drying process, 3% trehalose was added to the washed microspheres as a protective agent. The microspheres were first dried at -35℃ for 1.5 h to allow them to crystallize completely. Then, they were dried at -15℃ and 30 Pa vacuum for 20 h to prevent the microspheres from collapsing. After that, the temperature was raised to 30℃ and the vacuum was 25 Pa. The microspheres were dried for 10 h to make the final moisture content ≤5%.

[0084] Comparative Example 3

[0085] The difference between this comparative example and Example 3 is that the drug loaded on the functionalized microspheres is different. In this comparative example, Epimedium extract is used instead of Achyranthes bidentata extract used in Example 3. The remaining steps and methods are the same as in Example 3.

[0086] Experiment Example 1 Performance Testing

[0087] 1. Microstructure characterization

[0088] The average pore size and porosity of the composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 were analyzed by scanning electron microscopy, and the results are shown in Table 1.

[0089] Table 1. Results of average pore size and porosity measurements

[0090]

[0091] As shown in Table 1, the composite bone repair scaffolds prepared in Examples 1-3 have good pore size and porosity, which is beneficial for promoting bone tissue growth. Furthermore, compared with Comparative Examples 1-2, Examples 1-3 and Comparative Example 3 have significantly higher average pore size and greater porosity, making them more suitable for tissue attachment and growth.

[0092] 2. Mechanical property testing

[0093] The compressive and tensile properties of the composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 were tested using a universal testing machine. The composite bone repair scaffolds were tested as cylindrical specimens with a diameter of 1 cm and a height of 0.5 cm at a loading rate of 1 mm / min; and as dumbbell-shaped (dog bone type) specimens at a loading rate of 5 mm / min. All specimens were soaked in PBS buffer for 24 h before testing. The results are shown in Table 2.

[0094] Table 2 Results of compressive and tensile property tests

[0095]

[0096] As can be seen from Table 2, the composite bone repair scaffolds prepared in Examples 1-3 and Comparative Example 3 have good mechanical properties and are suitable for guiding bone repair.

[0097] 3. Degradation performance test

[0098] The composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 were weighed and then immersed in PBS buffer (10 mmol / L, pH 7.4). After 28 days of immersion, they were weighed again, and the degradation rate of the composite bone repair scaffolds [(initial mass - mass after immersion) / initial mass × 100%] was calculated. The results are shown in Table 3.

[0099] Table 3 Degradation rate determination results

[0100]

[0101] As can be seen from Table 3, the composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 all have good degradation performance, can match the growth of bone tissue well, and will not hinder the repair of bone tissue. Among them, the composite bone repair scaffolds prepared in Examples 1-3 have the best degradation performance.

[0102] 4. Water absorption performance test

[0103] The composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 were weighed and then immersed in PBS buffer (10 mmol / L, pH 7.4). After 8 hours, they were removed, the surface moisture was absorbed, and they were weighed again. The water absorption and swelling rate of the composite bone repair scaffolds was calculated [(mass after water absorption - initial mass) / initial mass × 100%]. The results are shown in Table 4.

[0104] Table 4 Results of water absorption swelling rate determination

[0105]

[0106] As shown in Table 4, all composite bone repair scaffolds have good water absorption properties, which can effectively absorb the exudate caused by bone injury, reduce tissue infiltration, and facilitate the slow release of the loaded drugs.

[0107] 5. Antibacterial ability test

[0108] Staphylococcus aureus and Escherichia coli were cultured to the logarithmic developmental stage, washed with sterile PBS buffer (10 mmol / L, pH 7.4), and prepared to a concentration of 1×10⁻⁶. 6A bacterial suspension of CFU / mL was prepared. Composite bone repair scaffolds of uniform size and weight were placed in centrifuge tubes, and an equal volume of the prepared bacterial suspension was added to each tube, ensuring complete immersion of the scaffold. A control group containing only the bacterial suspension was used as a blank control. Each centrifuge tube was incubated at 37℃ and 120 rpm for 4 h. After incubation, the bacterial suspension was diluted to the same concentration and spread onto LB solid medium, then incubated at 37℃ for 25 h. Colony counts were observed and recorded to evaluate the antibacterial effect of each composite bone repair scaffold. The results are shown in Table 5.

[0109] Table 5 Results of antibacterial rate determination (%)

[0110]

[0111] As can be seen from Table 5, the composite bone repair scaffolds prepared in Examples 1-3 and Comparative Examples 1-3 all have good antibacterial effects.

[0112] 6. Biocompatibility testing

[0113] The composite bone repair scaffold was prepared into circular samples with a diameter of 5 mm and a thickness of 2 mm using a punch. After sterilization, the samples were placed in a 24-well plate, and 5 × 10⁻⁶ μL of the solution was added to each well. 4 Bone marrow mesenchymal stem cells (BMSCs) were affixed to the sample surface and cultured in α-MEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2, with the medium being changed every 3 days. The treatment group without BMSCs served as the blank control group.

[0114] (1) Adhesion rate of BMSCs on composite bone repair scaffold

[0115] The OD values ​​of BMSCs cultured on composite bone repair scaffold samples at 3h, 6h, and 12h were measured using an ELISA reader, and the adhesion rate was calculated. Specifically, after 3, 6, and 12h of culture, the culture medium was discarded, and the cells were washed three times with PBS buffer (10mmol / L, pH 7.4) to remove non-adhered cells. The OD values ​​were then measured at 450nm using an ELISA reader, and the adhesion rate was calculated as (OD value of experimental group / OD value of blank control group × 100%).

[0116] (2) Determination of osteogenic differentiation capacity of BMSCs after culture on composite bone repair scaffold

[0117] After culturing BMSCs on composite bone repair scaffold samples for 7, 10, and 14 days, the culture medium was discarded, and the cells were washed three times with PBS buffer (10 mmol / L, pH 7.4). 200 μL of 1% NP-40 lysis buffer was added to each well to obtain cell lysates. After centrifugation, the supernatant was collected and placed in a new 24-well plate. 200 μL of alkaline phosphatase (ALP) detection working solution (components: p-nitrophenyl phosphate + 0.1 M glycine + 1 mM MgCl₂) was added to each well. ·6 After incubation at 37°C for 1 hour, the reaction was terminated with 2M NaCl. The OD value was measured at 450 nm using a microplate reader. The total protein amount used for normalization was determined using a diquinoline carboxylic acid kit, and ALP activity (OD value / total protein amount per minute) was calculated to assess the osteogenic differentiation capacity of BMSCs.

[0118] The results are shown in Table 6. It can be seen that BMSCs exhibited better adhesion rates on the composite bone repair scaffolds prepared in Examples 1-3, with adhesion rates reaching 89.5%-92.1% after 12 hours of culture. This indicates that the composite bone repair scaffolds prepared in Examples 1-3 can better stimulate the adhesion of BMSCs to their surface, thereby promoting bone tissue growth. Simultaneously, BMSCs cultured on the composite bone repair scaffolds prepared in Examples 1-3 showed higher ALP activity, significantly better than those in Comparative Examples 1-3, indicating that the composite bone repair scaffolds prepared in this invention are more conducive to promoting BMSC differentiation, thereby promoting bone repair.

[0119] Table 6. Results of Biocompatibility Tests

[0120]

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composite bone repair scaffold, characterized in that, The composite bone repair scaffold comprises a three-dimensional porous scaffold matrix, an antibacterial agent, and functionalized microspheres; the three-dimensional porous scaffold matrix is ​​a gelatin-sodium oxidized alginate dual-network hydrogel; the functionalized microspheres are microspheres loaded with Eucommia ulmoides extract and Achyranthes bidentata extract; the preparation method of the functionalized microspheres includes the following steps: (1) Liquid paraffin is mixed with a surfactant to obtain an oil phase; (2) Under water bath conditions, gelatin and sodium alginate were mixed with water, and Eucommia ulmoides extract and Achyranthes bidentata extract were added and mixed again to obtain an aqueous phase; (3) Add the aqueous phase obtained in step (2) to the oil phase obtained in step (1) and emulsify to obtain a W / O emulsion; (4) The crosslinking solution is added to the W / O emulsion obtained in step (3) for crosslinking and curing. The microspheres are collected by solid-liquid separation, washed and dried to obtain functionalized microspheres.

2. The composite bone repair scaffold as described in claim 1, characterized in that, In the gelatin-sodium oxidized alginate dual-network hydrogel, the degree of oxidation of the sodium oxidized alginate is 30-40%; the mass ratio of the gelatin to the sodium oxidized alginate is 8-12:3-5. The antibacterial agent is a nano-silver suspension; the concentration of the nano-silver suspension is 0.08~0.12 mg / mL.

3. The composite bone repair scaffold as described in claim 1, characterized in that, The volume ratio of liquid paraffin and surfactant in step (1) is 40~55:0.8~1.2; the surfactant is Span 80; the mixing rate is 300~500 rpm and the time is 10~40 min. The temperature of the water bath in step (2) is 38~45℃; the mass-volume ratio of gelatin, sodium alginate, water, Eucommia ulmoides extract, and Achyranthes bidentata extract is 3~5g: 1.5~2.5g: 80~120mL: 0.8~1.2g: 0.8~1.2g.

4. The composite bone repair scaffold as described in claim 1, characterized in that, In step (3), the volume ratio of the aqueous phase to the oil phase is 450~550:80~120; the emulsification rate is 1300~1700 rpm and the time is 10~20 min. The crosslinking solution in step (4) contains 1.5-2.5% calcium chloride and 0.8-1.2% glacial acetic acid; the volume ratio of the crosslinking solution to the W / O emulsion is 150-250:500-700; and the crosslinking curing time is 1.5-2.5 h.

5. A method for preparing a composite bone repair scaffold according to any one of claims 1 to 4, characterized in that, Under water bath conditions, gelatin, sodium alginate, and PBS buffer were mixed, functionalized microspheres and antibacterial agents were added and mixed again, and the mixture was printed using a 3D bioprinter. The printed scaffold was cross-linked, allowed to stand, and washed to obtain the composite bone repair scaffold.

6. The method as described in claim 5, characterized in that, The water bath temperature is 35~40℃, and the mass-to-volume ratio of gelatin, sodium alginate, PBS buffer, functionalized microspheres, and antibacterial agent is 0.8~1.2g:0.3~0.5g:6~10mL:0.15~0.25g:0.8~1.2mL.

7. The method as described in claim 5, characterized in that, The printing platform temperature is 8~12℃, the nozzle diameter is 0.4~0.6mm, the pressure is 450~550kPa, the speed is 6~10mm / s, the path is 0 / 90° alternating layering, and a total of 4~8 layers are used.

8. The method as described in claim 5, characterized in that, The crosslinking solution used during the crosslinking process is a 4-6% calcium chloride solution, the crosslinking temperature is 3-5℃, and the time is 20-40 min; the standing time is 4-8 h.

9. The use of a composite bone repair scaffold according to any one of claims 1 to 4 or a composite bone repair scaffold prepared by the method according to any one of claims 5 to 8 in the preparation of a product for treating radiation osteomyelitis.

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