【作者】 王鹏；

【导师】 孙剑飞；

【作者基本信息】 东南大学 ， 生物医学工程（专业学位）， 2025， 博士

【摘要】 退行性骨科疾病发病率高、危害大,是非外伤致残率最高的疾病,其中骨关节炎、骨质疏松、周围神经损伤等疾病在中老年人群中患病率超过50%,且患病人群成有年轻化趋势,严重影响国民生命健康。神经网络是退行性骨科疾病的生理病理信号交互感知与调控的重要环节,在骨关节发育、代谢、重塑及修复等过程中发挥关键作用。作为一名在医院骨科工作的非全日制博士研究生,希望寻求一种对退行性骨科疾病有普适作用的治疗方法。基于此,本论文开发了一种精准的神经刺激系统,该系统由磁性氧化铁纳米颗粒(Iron oxide nanoparticles,IONPs)与壳聚糖/β-甘油磷酸钠(Chitosan/β-glycerophosphate sodium,CS/GP)组成的磁性可注射凝胶(Magnetic hydrogel,M-Gels)结合温和脉冲磁场构建而成。IONPs已获得中国国家药品监督管理局的批准,而CS/GP温敏水凝胶的组分亦为美国食品药品监督管理局批准的无毒材料,具备良好的生物安全性。在此基础上,进一步探索通过磁性可注射凝胶包裹单根靶神经,在温和脉冲磁场作用下,利用磁性纳米材料介导的非聚焦磁场,实现具有选择性的、精准的磁作用。研究重点包括磁性可注射水凝胶在靶神经周围的驻留规律,揭示其介导的精准神经刺激在神经功能活化中的机制,进一步通过调节治疗靶区及磁场遥控系统的参数,为实现退行性骨科疾病的精准治疗提供了新的方式和思路。主要研究内容包括如下几个部分:(1)磁性可注射凝胶及磁场遥控装置的构建和表征。将IONPs负载至CS/GP温敏水凝胶中,构建M-Gels,通过调节IONPs的负载量构建不同成分的M-Gels(M-Gels-1,M-Gels-2,M-Gels-3)。利用透射电子显微镜、Zeta电位仪和纳米粒度分析仪对IONPs的材料学特性进行表征,并通过扫描电子显微镜、傅里叶变换红外光谱、旋转流变仪等技术对M-Gels进行全面分析,进一步通过活/死细胞染色和鬼笔环肽染色对M-Gels的生物相容性进行评估。最后,成功构建了用于小动物和大动物的温和脉冲磁场,用于后续精准神经磁刺激对退行性骨科疾病的改善效果评估。(2)精准迷走神经磁刺激(Magnetic vagus nerve stimulation,m VNS)改善骨质疏松的效果及机制研究。通过基于人工智能的知识挖掘技术发现迷走神经可能是m VNS改善骨质疏松的新靶点。构建大鼠骨质疏松模型,并将M-Gels直接注射至骨质疏松大鼠颈部以包裹单根迷走神经。结合磁共振成像(Magnetic resonance imaging,MRI)与人工智能辅助的定量磁化率成像(Quantitative susceptibility mapping,QSM)技术,发现M-Gels显著延长了纳米氧化铁在外周组织中的安全、稳定驻留时间(目前最长可达20周),从而实现了100 m T温和脉冲磁场对迷走神经的精准磁刺激。神经电生理结果显示,M-Gels与磁场的协同作用能够有效激活迷走神经,并具有良好的实时响应特性。研究表明,经过16周的精准神经磁刺激(20 Hz,每天2次,每次15分钟),大鼠因雌激素缺乏导致的骨质疏松症状得到显著改善。进一步通过知识挖掘技术发现肠道微生物可能是m VNS改善骨质疏松的潜在途径。结合大鼠盲肠内容物16S r RNA测序分析,发现m VNS不但显著增加了骨质疏松大鼠肠道菌群的α多样性,而且可以有效逆转因雌激素缺乏导致的特定菌群的改变,提示肠道微生物的变化可能是m VNS改善骨质疏松的重要机制之一。(3)精准迷走神经刺激改善骨关节炎的效果及机制研究。通过改良Hulth手术构建大鼠骨关节炎模型,并将M-Gels直接注射至大鼠颈部以包裹单根迷走神经。通过血清炎症指标、血清软骨代谢相关指标、MRI扫描及组织学分析发现,m VNS能够有效抑制炎症,缓解氧化应激,同时正向调控软骨代谢,从而发挥软骨保护作用。通过步态分析和疼痛阈值检测发现,m VNS能够显著恢复骨关节功能。进一步通过对大鼠滑膜组织单细胞RNA测序进行细胞和分子层面的作用机制分析,发现m VNS可能通过改变M1巨噬细胞的衰老状态来发挥作用,m VNS通过上调M1巨噬细胞中的高迁移率蛋白B2(High mobility group box 2,Hmgb2)表达水平,显著抑制衰老细胞的抗凋亡能力以实现OA中衰老M1巨噬细胞的自我清除。最后,通过MRI、Micro-CT和组织学分析等手段,在巴马小型猪模型上验证了m VNS对骨关节炎的改善效果,发现m VNS不但具有良好的生物安全性,而且有助于减缓骨关节炎过程中软骨的退变进程,改善关节结构。(4)精准坐骨神经磁刺激(Magnetic sciatic nerve stimulation,m SNS)改善坐骨神经损伤的效果及机制研究。构建大鼠坐骨神经卡压模型,并将M-Gels直接注射至大鼠腿部以包裹单根坐骨神经。通过神经电生理测试发现,m SNS可以明显促进坐骨神经的功能恢复。通过步态分析、坐骨神经功能指数(Sciatic functional index,SFI)、机械痛阈值检测、BBB(Basso,Beattie and Bresnahan)评分、旷场实验及腓肠肌指数研究,表明m SNS可以显著加速大鼠坐骨神经损伤后的运动功能和神经恢复。通过对神经生化标志物NF200和S100组织免疫荧光染色,蛋白质印迹法(Western blot,WB)及半定量分析发现,m SNS能够有效促进轴突形成,加速损伤神经的再髓鞘化过程,进而有效加快坐骨神经卡压损伤的修复进程。进一步进行机制探索,结果表明m SNS可能通过促进细胞粘附和细胞迁移来发挥促进轴突的再生和神经功能的恢复。另外,I-kappa B激酶/NF-kappa B信号传导、JAK-STAT信号通路以及PI3K-AKT信号通路也可能是m SNS促进神经损伤修复过程发挥作用的分子机制。更多还原

【Abstract】 Degenerative orthopedic diseases are marked by their high prevalence and significant harm,being the leading cause of disability due to non-traumatic factors.Diseases such as osteoarthritis,osteoporosis,and peripheral nerve injury have a prevalence rate exceeding 50%among the elderly populations,and the incidence is increasingly affecting younger individuals,severely impacting the health of the nation.The neural network plays a crucial role in the physiological and pathological signal interaction and regulation of degenerative orthopedic diseases,and is key to processes such as bone joint development,metabolism,remodeling,and repair.As a part-time Ph D student working in the orthopedic department of a hospital,I aim to explore a universally effective treatment for degenerative orthopedic diseases.Based on this,this dissertation develops a precise neural stimulation system consisting of magnetic iron oxide nanoparticles(IONPs)and chitosan/β-glycerophosphate sodium(CS/GP)to form a magnetic injectable hydrogel(Magnetic hydrogel,M-Gels),combined with mild pulsed magnetic fields.IONPs have been approved by the National Medical Products Administration(NMPA)of China,and the components of CS/GP thermosensitive hydrogel are non-toxic materials approved by the U.S.Food and Drug Administration(FDA),ensuring excellent biocompatibility and biosafety.Building on this foundation,the study further explores the use of magnetic injectable hydrogels encapsulating single target nerves,where mild pulsed magnetic fields mediate non-focused magnetic fields through magnetic nanoparticles,achieving selective and precise magnetic effects.The research focuses on the residence patterns of magnetic injectable hydrogels around the target nerve,elucidating the mechanisms of precise neural stimulation in nerve function activation.Moreover,by adjusting treatment target areas and the parameters of the magnetic field control system,this approach offers a novel method and strategy for the precise treatment of degenerative orthopedic diseases.The main research content includes the following sections:(1)Construction and Characterization of Magnetic Injectable Hydrogel and Magnetic Field Remote Control Device.IONPs were loaded into CS/GP thermosensitive hydrogels to form M-Gels.Different compositions of M-Gels(M-Gels-1,M-Gels-2,M-Gels-3)were created by adjusting the loading amounts of IONPs.The material characteristics of IONPs were characterized using Transmission Electron Microscopy and nanoparticle size analysis,and M-Gels were comprehensively analyzed using Scanning Electron Microscopy,Fourier Transform Infrared Spectroscopy,and rotational rheometer.Furthermore,biocompatibility of M-Gels was evaluated through live/dead cell staining and calcein-AM staining.Finally,a mild pulsed magnetic field was constructed for both small and large animals to assess the therapeutic effects of precise neural magnetic stimulation on degenerative orthopedic diseases.(2)Effect and Mechanism of Magnetic Vagus Nerve Stimulation(m VNS)in Improving Osteoporosis.Based on artificial intelligence-driven knowledge mining,vagus nerve was identified as a new target for m VNS in improving osteoporosis.An ovariectomy-induced osteoporosis rat model was established,and M-Gels were directly injected into the necks of the osteoporosis rats to encapsulate the single vagus nerve.Using Magnetic Resonance Imaging(MRI)combined with Artificial Intelligence-assisted Quantitative Susceptibility Mapping(QSM),it was found that M-Gels significantly extended the safe and stable retention time of nano-iron oxide in peripheral tissues(up to 20 weeks),achieving precise vagus nerve magnetic stimulation with a100 m T mild pulsed magnetic field.Neurophysiological results showed that the synergistic effect of M-Gels and magnetic field could effectively activate the vagus nerve and demonstrate excellent real-time responsiveness.After 16 weeks of precise neural magnetic stimulation(20 Hz,twice daily,15 minutes each time),the osteoporosis symptoms caused by estrogen deficiency were significantly improved.Further knowledge mining identified gut microbiota as a potential pathway for m VNS to improve osteoporosis.16S r RNA sequencing analysis of the rat cecal contents revealed that m VNS significantly increased theα-diversity of the gut microbiota in the osteoporosis rats and could effectively reverse the changes in specific microbial populations caused by estrogen deficiency.These findings suggest that changes in gut microbiota may be one of the important mechanisms through which m VNS improves osteoporosis.(3)Effect and Mechanism of Magnetic Vagus Nerve Stimulation in Improving Osteoarthritis.A modified Hulth surgery was used to establish an osteoarthritis rat model,and M-Gels were directly injected into the rats’necks to encapsulate the single vagus nerve.Serum inflammatory markers,cartilage metabolism-related biomarkers,MRI scans,and histological analysis showed that m VNS effectively suppressed inflammation,alleviated oxidative stress,and positively regulated cartilage metabolism,thereby providing cartilage protection.Gait analysis and pain threshold testing indicated that m VNS significantly restored joint function.Further cellular and molecular mechanism analysis through single-cell RNA sequencing of rat synovial tissue revealed that m VNS might exert its effects by altering the senescence state of M1macrophages,with high mobility group box 2(Hmgb2)playing a key role in the regulation of M1 macrophages.Finally,MRI,Micro-CT,and histological analyses in a Bama mini-pig model validated the therapeutic effects of m VNS on osteoarthritis,showing that m VNS not only demonstrated excellent biocompatibility but also helped slow down the cartilage degeneration process during osteoarthritis and improved joint structure.(4)Effect and Mechanism of Magnetic Sciatic Nerve Stimulation(m SNS)in Improving Sciatic Nerve Injury.A rat model of sciatic nerve compression was established,and M-Gels were directly injected into the rats’legs to encapsulate the sciatic nerve.Electrophysiological testing revealed that m SNS significantly promoted the functional recovery of the sciatic nerve.Gait analysis,Sciatic functional index(SFI),mechanical pain threshold assessments,Basso,Beattie and Bresnahan(BBB)scoring,open-field tests,and gastrocnemius muscle index studies demonstrated that m SNS notably accelerated the recovery of motor function and neural regeneration following sciatic nerve injury.Immunofluorescence staining for the neural biochemical markers NF200 and S100,along with Western blot(WB)and semi-quantitative analysis,showed that m SNS effectively enhanced axonal regeneration and accelerated the remyelination of damaged nerves,thereby facilitating the repair process of sciatic nerve compression injury.Further mechanistic investigation suggested that m SNS may promote axon regeneration and the recovery of nerve function by enhancing cell adhesion and migration.Additionally,the IκB kinase/NF-κB signaling pathway,JAK-STAT signaling pathway,and PI3K-AKT signaling pathway may be involved in the molecular mechanisms by which m SNS promotes the repair of nerve injury.更多还原