Antibacterial degradable bone fixation support and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical engineering, and discloses an antibacterial degradable bone fixing bracket and a preparation method thereof. The chitosan, the nHAP, the rGO and the nano MgO are compounded to form a multi-component composite material system, so that the mechanical property of the stent is improved, and the regulation and control of the degradation rate are realized; and nano MgO is added, so that the degradation rate of the stent is matched with the regeneration rate of bone tissues, and the problem of support failure caused by premature degradation of a traditional stent is avoided. Moreover, a PDA coating and AgNPs loaded bifunctional surface modification process is adopted, so that the problem of low loading capacity of an antibacterial agent caused by smooth surface of chitosan is solved, and drug resistance possibly caused by a single antibacterial agent is avoided. The stent is prepared through low-temperature 3D printing, the biological activity of chitosan is effectively kept, slurry performance and printing parameters are optimized, precise forming of a complex structure is achieved, and a foundation is provided for a personalized bone fixing stent.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to an antibacterial and biodegradable bone fixation scaffold and its preparation method. Background Technology

[0002] Bone defect repair is a significant challenge in orthopedic clinicians, especially in cases of large bone defects caused by trauma, tumor resection, or infection. Effective repair methods are needed to restore the structural integrity and function of the bone. While traditional autologous bone grafting better meets the repair needs, its source is limited, and it can cause secondary damage to the donor site and potential complications. Allogeneic bone grafting, on the other hand, presents challenges such as immune rejection, disease transmission risks, and insufficient bioactivity. Therefore, developing artificial bone repair materials with good biocompatibility, biodegradability, and suitable mechanical properties has become a research hotspot in the field of bone tissue engineering.

[0003] Among numerous biomaterials, chitosan has attracted widespread attention due to its natural origin, good biocompatibility, biodegradability, and certain antibacterial properties, and is considered a promising scaffold substrate for bone tissue engineering. However, pure chitosan materials still have significant limitations in practical applications for bone defect repair: First, its mechanical strength is relatively low, especially in humid environments where it is difficult to provide the mechanical support required for repairing bone defects in load-bearing areas; second, its degradation rate is often relatively fast, which may not match the rate of new bone formation, causing the scaffold to lose its structural integrity prematurely before tissue ingrowth; third, its strong hydrophilicity easily causes the material to swell, affecting the dimensional stability and pore structure of the scaffold; fourth, its inherent antibacterial spectrum is relatively limited, with insufficient inhibitory effects on some common orthopedic pathogens (such as Staphylococcus aureus and Escherichia coli), and it is difficult to achieve long-term antibacterial effects.

[0004] To overcome the aforementioned shortcomings, existing technologies typically combine chitosan with other materials. While existing composite strategies have improved certain properties of the materials to some extent, they still face a series of common problems in practical preparation and application: First, the rheological properties of composite slurries are often difficult to optimize, resulting in insufficient flowability, printability, and molding accuracy in precision molding processes such as 3D printing, affecting the fabrication of complex structural scaffolds; second, the proportions of each component in the composite material are difficult to precisely control, making it difficult to achieve an ideal balance among multiple properties such as mechanical strength, degradation rate, bioactivity, and antibacterial properties; third, the antibacterial mechanisms of existing composite materials are relatively simple, with insufficient antibacterial durability, and limited inhibitory effects on synergistic infections of multiple bacterial species. Summary of the Invention

[0005] The purpose of this invention is to provide an antibacterial and biodegradable bone fixation scaffold and its preparation method, and to provide a chitosan-based composite bone scaffold material that has excellent printability, suitable mechanical strength and degradation rate, good structural stability and broad-spectrum long-lasting antibacterial properties.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, comprising the following steps: (1) Chitosan, nano-hydroxyapatite, reduced graphene oxide, nano-MgO, dispersant and water are mixed and the pH is adjusted to 4.5~5.5 to obtain composite slurry; (2) According to the designed scaffold model, the composite slurry described in step (1) is 3D printed and freeze-dried to obtain a bone fixation scaffold; (3) The bone fixation scaffold described in step (2) is immersed in a buffer solution containing dopamine, and the dopamine undergoes a self-polymerization reaction to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine described in step (3) is immersed in a silver salt solution, a reducing agent is added, and an in-situ reduction reaction is carried out to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0007] Furthermore, in the preparation method, the degree of deacetylation of the chitosan in step (1) is 85-95%; The particle size of the nano-hydroxyapatite in step (1) is ≤100nm; The particle size of the nano-MgO in step (1) is 50~100nm; The dispersant in step (1) includes polyethylene glycol and / or sodium alginate.

[0008] Furthermore, in the preparation method, the mass ratio of the nano-hydroxyapatite to the chitosan in step (1) is 2~4:4~6; In step (1), the mass ratio of reduced graphene oxide to chitosan is 0.02~0.12:4~6; In step (1), the mass ratio of nano-MgO to chitosan is 0.5~1.5:4~6; In step (1), the mass ratio of the dispersant to the chitosan is 0.05~0.12:4~6.

[0009] Furthermore, in the preparation method, the scaffold model in step (2) includes: a porosity of 45~55% and a pore size of 300~500μm; The 3D printing conditions in step (2) include: printing temperature of 0~5℃, printing speed of 3~5mm / s, and printing layer thickness of 0.2~0.4mm.

[0010] Furthermore, in the preparation method, the pH value of the dopamine-containing buffer solution in step (3) is 7.5~9.0.

[0011] Furthermore, in the preparation method, the concentration of dopamine in the dopamine-containing buffer solution in step (3) is 1~4 mg / mL.

[0012] Furthermore, in the preparation method, the conditions for the self-polymerization reaction in step (3) include: a reaction temperature of 15~30℃ and a reaction time of 12~36h.

[0013] Furthermore, in the preparation method, the reducing agent in step (4) includes one or more of ascorbic acid, dopamine, glucose, and sodium citrate.

[0014] Furthermore, in the preparation method, the loading of AgNPs in step (4) is 0.1~0.6wt%.

[0015] The present invention also provides an antibacterial and biodegradable bone fixation scaffold.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) The mechanical strength and toughness of the scaffold are effectively improved by combining nano-hydroxyapatite (nHAP) with reduced graphene oxide (rGO). nHAP is uniformly dispersed in the chitosan matrix as a rigid reinforcing phase, and rGO forms physical cross-linking points and transmits stress through its two-dimensional sheet structure, so that the compressive strength of the final scaffold reaches 12~15MPa. Its strength range matches the mechanical properties of human cancellous bone, which can provide necessary initial mechanical support for bone defect sites and meet the requirements of bone fixation scaffolds in non-load-bearing or low-load-bearing sites.

[0017] (2) The introduced nano-MgO can release magnesium ions and generate a weakly alkaline microenvironment during the degradation process, which not only helps to regulate the local pH, but also actively regulates the hydrolysis rate of the chitosan matrix. By adjusting the amount of nano-MgO added, the degradation rate of the scaffold can be controlled within a range of several months, so that its degradation cycle is synchronized with the natural process of bone tissue regeneration (usually the initial healing period of 3 to 6 months), avoiding mechanical support failure caused by premature degradation of the scaffold or hindering new bone ingrowth due to delayed degradation.

[0018] (3) By utilizing the strong adhesion and abundant active groups of the polydopamine (PDA) coating, uniform and stable loading of silver nanoparticles (AgNPs) on the surface and pores of the scaffold was achieved. The PDA layer itself has certain antibacterial properties, which form a synergistic antibacterial system with AgNPs: AgNPs can continuously release silver ions, interfering with bacterial cell membranes and enzyme systems; the PDA layer can capture bacteria and enhance the contact sterilization effect. The dual mechanism enables the scaffold to achieve an antibacterial rate of over 99% against common orthopedic infectious pathogens (such as Staphylococcus aureus and Escherichia coli), and the antibacterial activity is long-lasting, which can effectively cover the high-risk infection period after surgery and prevent implantation-related infections.

[0019] (4) After being modified with dopamine, the surface hydrophilicity and bioactivity of the scaffold are significantly improved, and the moderate surface roughness is more conducive to osteoblast adhesion, spreading and proliferation. The pore size (300~500μm) and porosity (45~55%) precisely controlled by 3D printing form an interconnected porous network, which not only provides channels for nutrient delivery and metabolic waste removal, but also provides an ideal three-dimensional space for the ingrowth of new bone tissue and angiogenesis, effectively promoting bone integration.

[0020] (5) Based on the patient’s CT image data, a personalized scaffold that closely matches the shape of the bone defect can be directly formed by 3D printing, ensuring the best anatomical fit between the scaffold and the host bone, reducing intraoperative trimming, shortening the operation time, and better restoring the original anatomical structure, providing a precise solution for the repair of complex bone defects. Detailed Implementation

[0021] This invention provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, comprising the following steps: (1) Chitosan, nano-hydroxyapatite, reduced graphene oxide, nano-MgO, dispersant and water are mixed and the pH is adjusted to 4.5~5.5 (preferably 4.8~5.2, more preferably 4.98~5.10, more preferably 5.0) to obtain a composite slurry; (2) According to the designed scaffold model, the composite slurry described in step (1) is 3D printed and freeze-dried to obtain a bone fixation scaffold; (3) The bone fixation scaffold described in step (2) is immersed in a buffer solution containing dopamine, and the dopamine undergoes a self-polymerization reaction to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine described in step (3) is immersed in a silver salt solution, a reducing agent is added, and an in-situ reduction reaction is carried out to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0022] In this invention, the preferred method for mixing chitosan, nano-hydroxyapatite, reduced graphene oxide, nano-MgO, dispersant, and water in step (1) is as follows: Chitosan is dissolved in an aqueous acetic acid solution to obtain a chitosan solution. Nano-hydroxyapatite, reduced graphene oxide, and nano-MgO are sequentially added to water and ultrasonically dispersed separately to obtain a mixed dispersion. The mixed dispersion is then mixed with the chitosan solution, and a dispersant is added for further mixing. The concentration of the aqueous acetic acid solution and the mixing conditions in each step are not limited; any method well known to those skilled in the art can be used.

[0023] In this invention, the degree of deacetylation of the chitosan in step (1) is preferably 85-95%, more preferably 88-92%, and even more preferably 90%.

[0024] In this invention, the particle size of the nano-hydroxyapatite in step (1) is preferably ≤100nm, more preferably ≤60nm, and even more preferably 20nm.

[0025] In this invention, the reduced graphene oxide in step (1) is preferably vitamin C-reduced graphene oxide.

[0026] In this invention, the particle size of the nano-MgO in step (1) is preferably 50~100nm.

[0027] In this invention, the dispersant in step (1) preferably includes polyethylene glycol and / or sodium alginate, more preferably polyethylene glycol or sodium alginate, and even more preferably polyethylene glycol.

[0028] In this invention, the number average molecular weight of the polyethylene glycol is preferably 4,000 to 8,000, more preferably 5,000 to 7,000, and even more preferably 6,000.

[0029] In this invention, the sources of chitosan, nano-hydroxyapatite, reduced graphene oxide, nano-MgO and dispersant in step (1) are not limited, and commercially available products known to those skilled in the art can be used.

[0030] In this invention, the mass ratio of nano-hydroxyapatite to chitosan in step (1) is preferably 2~4:4~6, more preferably 2.5~3.8:4.5~5.5, and even more preferably 3.5:4.5.

[0031] In this invention, the mass ratio of the reduced graphene oxide to the chitosan in step (1) is preferably 0.02~0.12:4~6, more preferably 0.04~0.10:4.5~5.5, and even more preferably 0.08:4.5.

[0032] In this invention, the mass ratio of nano-MgO to chitosan in step (1) is preferably 0.5~1.5:4~6, more preferably 0.7~1.0:4.5~5.5, and even more preferably 0.8:4.5.

[0033] In this invention, the mass ratio of the dispersant to the chitosan in step (1) is preferably 0.05~0.12:4~6, more preferably 0.07~0.1:4.5~5.5, and even more preferably 0.08:4.5.

[0034] In this invention, the role of chitosan is to ensure that the material has good biocompatibility and antibacterial properties. Chitosan has positively charged amino groups, which can interact with negatively charged microbial cell membranes, disrupting their structure and inhibiting microbial growth.

[0035] In this invention, the role of nano-hydroxyapatite (nHAP) is as follows: nHAP has a chemical composition highly similar to human bone tissue, exhibiting excellent osteoconductivity and biocompatibility. Furthermore, nHAP can significantly improve the mechanical strength and bone regeneration induction capacity of the scaffold.

[0036] In this invention, the reduced graphene oxide serves to improve the conductivity and mechanical strength of the scaffold, while simultaneously promoting cell adhesion and proliferation.

[0037] In this invention, the role of the nano-MgO is to: release Mg slowly. 2+ Ion regulation of scaffold degradation rate, while Mg 2+ The ions themselves have biological activity that promotes bone regeneration.

[0038] In this invention, the scaffold model in step (2) includes: a porosity preferably of 45-55%, more preferably of 48-52%, and even more preferably of 50%; and a pore size preferably of 300-500μm, more preferably of 350-450μm, and even more preferably of 400μm.

[0039] In this invention, the 3D printing conditions in step (2) include: the printing temperature is preferably 0~5℃, more preferably 1~3℃, and more preferably 2℃; the printing speed is preferably 3~5mm / s, more preferably 3.5~4.5mm / s, and more preferably 4mm / s; the printing layer thickness is preferably 0.2~0.4mm, more preferably 0.25~0.35mm, and more preferably 0.3mm.

[0040] In this invention, by adding a dispersant and adjusting the pH value of the slurry, a balance between fluidity and stability is ensured during the 3D low-temperature printing process, which is beneficial for forming a uniform and stable printed structure. Setting the printing temperature to 0~5℃ effectively maintains the bioactivity of chitosan and avoids material degradation caused by high temperatures; printing speed and layer thickness ensure printing accuracy and structural integrity. Furthermore, the use of a cubic close-packed pore structure, controlling the pore size and porosity, ensures both the mechanical strength of the scaffold and provides sufficient space to promote bone tissue ingrowth and angiogenesis.

[0041] In this invention, the freeze-drying method in step (2) is preferably: sequential pre-freezing and sublimation. The conditions for pre-freezing and sublimation are not limited, and any method well-known to those skilled in the art can be used. Specifically, in the embodiments, the pre-freezing conditions include: a temperature of -20°C and a time of 12 hours; the sublimation conditions include: a temperature of 0°C and a time of 48 hours.

[0042] In this invention, the pH value of the dopamine-containing buffer solution in step (3) is preferably 7.5 to 9.0, more preferably 8.0 to 8.8, and even more preferably 8.5.

[0043] In this invention, the concentration of dopamine in the buffer solution containing dopamine in step (3) is preferably 1~4 mg / mL, more preferably 2~3 mg / mL, and even more preferably 2 mg / mL.

[0044] In this invention, the buffer solution in the dopamine-containing buffer solution in step (3) preferably includes Tris-HCl buffer, borate buffer, carbonate buffer or phosphate buffer, more preferably Tris-HCl buffer or phosphate buffer, and more preferably Tris-HCl buffer.

[0045] In this invention, the conditions for the self-polymerization reaction in step (3) include: the reaction temperature is preferably 15~30℃, more preferably 20~30℃, and even more preferably 25℃; the reaction time is preferably 12~36h, more preferably 20~30h, and even more preferably 24h.

[0046] In this invention, the PDA coating can improve the surface roughness of the stent, enhance the loading capacity of antibacterial agents, and improve the biocompatibility of the stent.

[0047] In this invention, the silver salt solution in step (4) is preferably a silver nitrate solution.

[0048] In this invention, the reducing agent in step (4) preferably includes one or more of ascorbic acid, dopamine, glucose, and sodium citrate, more preferably ascorbic acid or sodium citrate, and even more preferably ascorbic acid.

[0049] In this invention, the conditions for the in-situ reduction reaction in step (4) include: the reaction temperature is preferably 15~30℃, more preferably 20~30℃, and even more preferably 25℃; the reaction time is preferably 0.5~4h, more preferably 1~3h, and even more preferably 2h.

[0050] In this invention, the loading of AgNPs in step (4) is preferably 0.1 to 0.6 wt%, more preferably 0.2 to 0.4 wt%, and even more preferably 0.35 wt%.

[0051] In this invention, the concentration of the silver salt solution in step (4) and the amount of the reducing agent are not limited, and those skilled in the art can obtain the required AgNPs loading.

[0052] In this invention, the function of AgNPs is as follows: AgNPs impart long-lasting antibacterial ability to the stent, while Ag... + The sustained release of ions can maintain the antibacterial effect.

[0053] In this invention, step (4) preferably includes, after the in-situ reduction reaction, the following steps: sequentially washing and freeze-drying the support.

[0054] In this invention, the washing process preferably uses deionized water.

[0055] In this invention, the freeze-drying method is preferably to perform pre-freezing and sublimation sequentially. The conditions for pre-freezing and sublimation are not limited, and any method well-known to those skilled in the art can be used. Specifically, in the embodiments, the pre-freezing conditions include: a temperature of -20°C and a time of 12 hours; the sublimation conditions include: a temperature of 0°C and a time of 48 hours.

[0056] The present invention also provides an antibacterial and biodegradable bone fixation scaffold.

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] In the examples and comparative examples, unless otherwise specified, the chitosan used was food-grade chitosan produced by Henan Wokas Biotechnology Co., Ltd., with a degree of deacetylation of 90%; the nHAP used was Junzhuo HAP04-20 (20nm), needle-shaped, with a purity of 98% produced by Nanjing Junzhuo Biotechnology Co., Ltd.; the rGO used was vitamin C-reduced graphene oxide produced by Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.; the particle size of nano MgO was 50~100nm; and the polyethylene glycol used was PEG 6000.

[0059] Example 1

[0060] This embodiment provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, including the following steps: (1) Dissolve 5g of chitosan in 250mL of 2wt% acetic acid aqueous solution and stir until completely dissolved; take 3g of nHAP, 0.06g of rGO and 0.7g of nano MgO and ultrasonically disperse them in 50mL of deionized water, pour them into the chitosan solution, add 0.08g of PEG6000, adjust the pH to 5.0, and stir for 3h to form a uniform composite slurry; (2) Set the printing temperature to 2℃, the printing speed to 4mm / s, and the layer thickness to 0.3mm; design a cubic close-packed hole structure with a pore diameter of 400μm and a porosity of 50% using CAD; after printing, pre-freeze at -20℃ for 12h and sublimate at 0℃ for 48h to obtain a bone fixation scaffold. (3) Place the bone fixation scaffold from step (2) in a Tris-HCl buffer solution with pH=8.5, add dopamine solution, the concentration of dopamine in the mixture is 2mg / mL, react at 25℃ for 24h to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine was immersed in 50 mL of silver nitrate solution, and an equimolar amount of ascorbic acid containing silver ions was added. The scaffold was reduced in situ at 25 °C for 2 h, and the AgNPs loading was controlled to be 0.3 wt%. After washing with deionized water, the scaffold was pre-frozen at -20 °C for 12 h and then sublimated and dried at 0 °C for 48 h to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0061] Performance testing: (1) Mechanical properties: Tested in accordance with YY / T 1782-2021 "Mechanical Properties Test Method for Orthopedic External Fixation Frames" standard; (2) Antibacterial properties: The antibacterial properties of the stent were evaluated by the inhibition zone test and the viable bacteria count method.

[0062] (3) Degradation rate: The stent was immersed in modified SBF simulated body fluid (Condis Chemical (Hubei) Co., Ltd.) and the mass loss of the stent was detected; (4) Biocompatibility: MC3T3-E1 osteoblasts (Shanghai Pituo Biotechnology Co., Ltd.) were used for culture experiments, and the biocompatibility of the scaffold was evaluated by CCK-8 method and live-dead staining method.

[0063] The antibacterial and biodegradable bone fixation scaffold prepared in Example 1 was tested as follows: Mechanical properties: compressive strength 13.5MPa, compression modulus 100MPa, meeting the standards; Antibacterial properties: 99.2% inhibition rate against Staphylococcus aureus and 99.1% inhibition rate against Escherichia coli, with antibacterial effect lasting for more than 28 days; Degradation rate: 45% mass loss after 12 weeks of SBF immersion, with a support period of up to 15 weeks; Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 32% compared with the blank control group, and the cell adhesion rate reached 85%.

[0064] Example 2

[0065] This embodiment provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, including the following steps: (1) Dissolve 4.5g of chitosan in 225mL of 2wt% acetic acid aqueous solution and stir until completely dissolved; take 3.5gnHAP, 0.08g rGO and 0.8g nano MgO and ultrasonically disperse them in 50mL of deionized water, pour them into the chitosan solution, add 0.1g PEG6000, adjust the pH to 5.05, and stir for 4h to form a uniform composite slurry; (2) Set the printing temperature to 3℃, the printing speed to 3.5mm / s, and the layer thickness to 0.35mm; design a cubic close-packed hole structure with a pore diameter of 350μm and a porosity of 48% using CAD; after printing, pre-freeze at -20℃ for 12h and sublimate at 0℃ for 48h to obtain a bone fixation scaffold. (3) Place the bone fixation scaffold from step (2) in a Tris-HCl buffer solution with pH=8.5, add dopamine solution, the concentration of dopamine in the mixture is 2mg / mL, react at 25℃ for 30h to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine was immersed in 50 mL of silver nitrate solution, and an equimolar amount of ascorbic acid containing silver ions was added. The scaffold was reduced in situ at 25 °C for 2 h, and the AgNPs loading was controlled to be 0.35 wt%. After washing with deionized water, the scaffold was pre-frozen at -20 °C for 12 h and then sublimated and dried at 0 °C for 48 h to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0066] The antibacterial and biodegradable bone fixation scaffold prepared in Example 2 was tested as follows: Mechanical properties: compressive strength 14.8MPa, compressive modulus 115MPa, close to the upper limit of human cancellous bone; Antibacterial properties: 99.3% inhibition rate against Staphylococcus aureus and 99.0% inhibition rate against Escherichia coli, with antibacterial effect lasting for more than 28 days; Degradation rate: 38% mass loss after 12 weeks of SBF immersion, with a support period of up to 16 weeks; Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 35% compared with the blank control group, and the cell adhesion rate reached 86%.

[0067] Example 3

[0068] This embodiment provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, including the following steps: (1) Dissolve 5.5g of chitosan in 275mL of 2wt% acetic acid aqueous solution and stir until completely dissolved; take 2.5gnHAP, 0.04g rGO and 0.6g nano MgO and ultrasonically disperse them in 50mL of deionized water, pour them into the chitosan solution, add 0.05g PEG6000, adjust the pH to 4.98, and stir for 3h to form a uniform composite slurry; (2) Set the printing temperature to 1℃, the printing speed to 4.5mm / s, and the layer thickness to 0.25mm; design a cubic close-packed hole structure with a pore diameter of 450μm and a porosity of 55% using CAD; after printing, pre-freeze at -20℃ for 12h and sublimate at 0℃ for 48h to obtain a bone fixation scaffold. (3) Place the bone fixation scaffold from step (2) in a Tris-HCl buffer solution with pH=8.5, add dopamine solution, the concentration of dopamine in the mixture is 2mg / mL, react at 25℃ for 24h to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine was immersed in 50 mL of silver nitrate solution, and an equimolar amount of ascorbic acid containing silver ions was added. The scaffold was reduced in situ at 25 °C for 2 h, and the AgNPs loading was controlled to be 0.4 wt%. After washing with deionized water, the scaffold was pre-frozen at -20 °C for 12 h and then sublimated and dried at 0 °C for 48 h to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0069] The antibacterial and biodegradable bone fixation scaffold prepared in Example 3 was tested as follows: Mechanical properties: compressive strength 12.2MPa, compression modulus 85MPa, meeting the requirements for non-load-bearing bone fixation; Antibacterial properties: 99.6% inhibition rate against Staphylococcus aureus and 99.4% inhibition rate against Escherichia coli, with antibacterial effect lasting for more than 32 days; Degradation rate: 52% mass loss after 12 weeks of SBF immersion, with a support period of up to 13 weeks; Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 29% compared with the blank control group, and the cell adhesion rate reached 83%.

[0070] Example 4

[0071] This embodiment provides a method for preparing an antibacterial and biodegradable bone fixation scaffold, including the following steps: (1) Dissolve 4g of chitosan in 200mL of 2wt% acetic acid aqueous solution and stir until completely dissolved; take 3.8g of nHAP, 0.1g of rGO and 1.0g of nano MgO and ultrasonically disperse them in 50mL of deionized water, pour them into the chitosan solution, add 0.07g of PEG 6000, adjust the pH to 5.10, and stir for 5h to form a uniform composite slurry; (2) Set the printing temperature to 0℃, the printing speed to 3mm / s, and the layer thickness to 0.4mm; design a cubic close-packed hole structure with a pore diameter of 300μm and a porosity of 45% using CAD; after printing, pre-freeze at -20℃ for 12h and sublimate at 0℃ for 48h to obtain a bone fixation scaffold. (3) Place the bone fixation scaffold from step (2) in a Tris-HCl buffer solution with pH=8.5, add dopamine solution, the concentration of dopamine in the mixture is 2mg / mL, react at 25℃ for 20h to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine was immersed in 50 mL of silver nitrate solution, and an equimolar amount of ascorbic acid containing silver ions was added. The scaffold was reduced in situ at 25 °C for 2 h, and the AgNPs loading was controlled to be 0.2 wt%. After washing with deionized water, the scaffold was pre-frozen at -20 °C for 12 h and then sublimated and dried at 0 °C for 48 h to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

[0072] The antibacterial and biodegradable bone fixation scaffold prepared in Example 4 was tested as follows: Mechanical properties: compressive strength 14.2 MPa, compression modulus 110 MPa, residual compressive strength still reaches 8.5 MPa after 24 weeks; Antibacterial properties: 99.2% inhibition rate against Staphylococcus aureus and 99.1% inhibition rate against Escherichia coli, with antibacterial effect lasting for 28 days; Degradation rate: 30% mass loss after 12 weeks of SBF immersion and 68% mass loss after 24 weeks, matching the regeneration cycle of large bone segments (12~24 weeks); Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 36% compared with the blank control group, and the cell adhesion rate reached 87%.

[0073] Comparative Example 1 (without rGO)

[0074] The difference between Comparative Example 1 and Example 1 is that rGO in step (1) is deleted, while other parameters and conditions are the same as in Example 1.

[0075] The bone fixation scaffold prepared in Comparative Example 1 was tested: Mechanical properties: The compressive strength is only 6.8 MPa and the compression modulus is 55 MPa. The mechanical strength is significantly reduced and cannot meet the basic requirements for fixation and support of cancellous bone. Antibacterial properties: Staphylococcus aureus inhibition rate of 98.5%, Escherichia coli inhibition rate of 98.3%, and antibacterial properties were not significantly affected; Degradation rate: SBF immersion for 12 weeks resulted in a 60% mass loss, indicating excessively rapid degradation, with a support period of only about 10 weeks. Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts was increased by 15% compared with the blank control group, and the cell adhesion rate was 70%, which was significantly lower than that of the example.

[0076] Comparative Example 2 (without nano-MgO)

[0077] The difference between Comparative Example 2 and Example 1 is that nano MgO in step (1) is removed, while other parameters and conditions are the same as in Example 1.

[0078] The bone fixation scaffold prepared in Comparative Example 2 was tested: Mechanical properties: compressive strength 12.1 MPa, compression modulus 95 MPa, mechanical properties are acceptable; Antibacterial properties: Staphylococcus aureus inhibition rate of 98.8%, Escherichia coli inhibition rate of 98.5%, antibacterial properties decreased slightly, indicating that MgO has a certain auxiliary antibacterial effect; Degradation rate: SBF immersion for 12 weeks resulted in a mass loss of up to 70%, and the support period was less than 9 weeks. The degradation was too fast and could not match the bone regeneration rate, which could easily lead to premature failure of the scaffold. Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts was increased by 20% compared with the blank control group, the cell adhesion rate was 75%, and the improvement in cell behavior was weaker than that in Example 1, indicating that Mg 2+ The release of [something] promotes osteogenic formation.

[0079] Comparative Example 3 (without surface antibacterial modification)

[0080] The difference between Comparative Example 3 and Example 1 is that steps (3) and (4) are deleted, while other parameters and conditions are the same as in Example 1.

[0081] The bone fixation scaffold prepared in Comparative Example 3 was tested: Mechanical properties: compressive strength 13.0 MPa, compressive modulus 98 MPa, similar to Example 1; Antibacterial properties: No inhibition zone is produced, and the inhibition rate of Staphylococcus aureus and Escherichia coli is less than 10%. The stent has virtually no antibacterial ability and the risk of infection is extremely high. Degradation rate: 50% mass loss after 12 weeks of SBF immersion, comparable to Example 1; Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 25% compared with the blank control group, and the cell adhesion rate was 78%, indicating that the PDA coating has a certain effect on improving cell affinity.

[0082] Comparative Example 4 (using micron-level nHAP)

[0083] The difference between Comparative Example 4 and Example 1 is that nHAP in step (1) is replaced with Junzhuo HAP07-M, 45~150μm, while other parameters and conditions are the same as in Example 1.

[0084] The bone fixation scaffold prepared in Comparative Example 4 was tested: Slurry stability: Micron-sized HA particles settle more easily, resulting in poor slurry uniformity and printing continuity; Mechanical properties: compressive strength 10.2 MPa; due to the weak bonding between micron particles and the matrix interface, the reinforcing effect is limited. Antibacterial and degradation properties: No significant difference from Example 1; Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 18% compared with the blank control group, but their biological activity was significantly reduced. The cells spread poorly on the material surface, indicating that the nano-effect of nHAP is more effective in simulating the extracellular matrix and promoting osteoogenesis.

[0085] Comparative Example 5 (without PDA coating)

[0086] The difference between Comparative Example 5 and Example 1 is that step (3) is deleted, and step (4) is loaded with AgNPs under the same conditions, while other parameters and conditions are the same as in Example 1.

[0087] The bone fixation scaffold prepared in Comparative Example 5 was tested: The actual loading of AgNPs was only 0.18 wt%, and the loading efficiency was significantly reduced. Due to the lack of the chelating and reducing effect of PDA's adhesive phenolic hydroxyl and quinone groups on silver ions, AgNPs mainly adhered through physical adsorption and were largely detached during the cleaning process. Mechanical properties: compressive strength 13.1 MPa, compressive modulus 98 MPa; PDA coating and AgNPs have a weak impact on the mechanical properties of the stent body. Antibacterial properties: Staphylococcus aureus inhibition rate of 92.5%, Escherichia coli inhibition rate of 91.8%, antibacterial effect lasts only 14 days, antibacterial efficacy and durability are greatly reduced, this is due to low AgNP loading and weak binding, resulting in insufficient effective antibacterial concentration, lack of PDA contact antibacterial synergistic effect, AgNP is easily depleted quickly. Degradation rate: 47% mass loss after 12 weeks of SBF immersion; the surface modification layer had little impact on the overall degradation behavior. Biocompatibility: The proliferation rate of MC3T3-E1 osteoblasts increased by 22% compared with the blank control group, and the cell adhesion rate reached 76%. The cell behavior was significantly worse than that of Example 1. This is due to the lack of the cell adhesion-promoting properties inherent in the PDA coating. The poorly bound AgNPs caused slight toxicity to cells due to local burst release, which affected proliferation.

[0088] In summary, the antibacterial and biodegradable bone fixation scaffold prepared using the aforementioned method of this invention combines chitosan, nHAP, rGO, and nano-MgO to form a multi-component composite material system. This not only improves the mechanical properties of the scaffold but also enables the regulation of the degradation rate. nHAP provides osteoconductivity and mechanical support, rGO enhances conductivity and cell adhesion, and nano-MgO regulates the degradation rate and provides bone regeneration-promoting ions. The synergistic effect of these three components significantly improves the overall performance of the scaffold.

[0089] This invention achieves a match between the scaffold degradation rate and the bone tissue regeneration rate by adding nano-MgO. Mg 2+ The sustained release of ions can regulate the degradation rate of the scaffold, while Mg 2+ The ions themselves have bioactivity that promotes bone regeneration, forming a virtuous cycle of degradation-regeneration, thus avoiding the problem of premature degradation leading to support failure in traditional scaffolds.

[0090] Furthermore, this invention employs a dual-functional surface modification process combining PDA coating and AgNPs loading. The PDA coating improves the surface roughness of the scaffold, enhancing its antibacterial loading capacity; AgNPs impart long-lasting antibacterial properties to the scaffold, while Ag... + The sustained release of ions can maintain the antibacterial effect, which not only solves the problem of low antibacterial agent loading caused by the smooth surface of chitosan, but also avoids the drug resistance that may be caused by a single antibacterial agent.

[0091] This invention utilizes low-temperature 3D printing technology to fabricate scaffolds. Printing at a low temperature of 0-5℃ effectively preserves the bioactivity of chitosan and avoids material degradation caused by high temperatures. Simultaneously, by optimizing slurry properties and printing parameters, precise molding of complex structures is achieved, providing a foundation for personalized bone fixation scaffolds.

[0092] 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 method for preparing an antibacterial and biodegradable bone fixation scaffold, characterized in that, Includes the following steps: (1) Chitosan, nano-hydroxyapatite, reduced graphene oxide, nano-MgO, dispersant and water are mixed and the pH is adjusted to 4.5~5.5 to obtain composite slurry; (2) According to the designed scaffold model, the composite slurry described in step (1) is 3D printed and freeze-dried to obtain a bone fixation scaffold; (3) The bone fixation scaffold described in step (2) is immersed in a buffer solution containing dopamine, and the dopamine undergoes a self-polymerization reaction to obtain a bone fixation scaffold containing polydopamine. (4) The bone fixation scaffold containing polydopamine described in step (3) is immersed in a silver salt solution, a reducing agent is added, and an in-situ reduction reaction is carried out to obtain an antibacterial and biodegradable bone fixation scaffold loaded with AgNPs.

2. The preparation method according to claim 1, characterized in that, The degree of deacetylation of the chitosan in step (1) is 85-95%; The particle size of the nano-hydroxyapatite in step (1) is ≤100nm; The particle size of the nano-MgO in step (1) is 50~100nm; The dispersant in step (1) includes polyethylene glycol and / or sodium alginate.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the mass ratio of the nano-hydroxyapatite to the chitosan is 2~4:4~6; In step (1), the mass ratio of reduced graphene oxide to chitosan is 0.02~0.12:4~6; In step (1), the mass ratio of nano-MgO to chitosan is 0.5~1.5:4~6; In step (1), the mass ratio of the dispersant to the chitosan is 0.05~0.12:4~6.

4. The preparation method according to claim 1, characterized in that, The scaffold model in step (2) includes: a porosity of 45-55% and a pore size of 300-500 μm; The 3D printing conditions in step (2) include: printing temperature of 0~5℃, printing speed of 3~5mm / s, and printing layer thickness of 0.2~0.4mm.

5. The preparation method according to claim 1, characterized in that, The pH value of the dopamine-containing buffer solution in step (3) is 7.5~9.

0.

6. The preparation method according to claim 1 or 5, characterized in that, The concentration of dopamine in the buffer solution containing dopamine in step (3) is 1~4 mg / mL.

7. The preparation method according to claim 6, characterized in that, The conditions for the self-polymerization reaction in step (3) include: a reaction temperature of 15~30℃ and a reaction time of 12~36h.

8. The preparation method according to claim 1, characterized in that, The reducing agent in step (4) includes one or more of ascorbic acid, dopamine, glucose, and sodium citrate.

9. The preparation method according to claim 1 or 8, characterized in that, The loading amount of AgNPs in step (4) is 0.1~0.6wt%.

10. An antibacterial and biodegradable bone fixation scaffold prepared by the preparation method according to any one of claims 1 to 9.