【作者】 张磊；

【导师】 袁广银； 裴佳；

【作者基本信息】 上海交通大学 ， 材料科学与工程， 2020， 博士

【摘要】 镁合金由于其优异的力学性能、生物降解性和生物相容性而在骨科领域引起了极大的关注。镁合金的密度和弹性模量与人骨相近,可有效避免应力遮挡效应,是一种非常有应用潜力的可降解骨修复材料。然而,镁合金应用到临床上尚存在关键技术难题有待解决,主要是降解过快和局部腐蚀严重等问题;同时,骨肌系统疾病及骨修复材料相关的骨感染也对镁合金材料提出了新的生物功能需求。对于骨修复用镁合金材料,除了保证可控降解和提供足够的力学性能外,还希望其在生物学上以适当的方式发挥生物功能作用,包括提高骨整合性、抗菌能力或局部递送治疗剂。本课题组在前期工作中开发了一种新型生物可降解镁合金Mg-Nd-Zn-Zr系列（Jiao Da Bio-Mg,简称JDBM）。研究表明,JDBM镁合金具有优良的均匀腐蚀行为;然而其降解速度依然偏快,在体内会产生氢气,并且过高的渗透压和碱性环境对细胞也具有一定的毒性。因此,为了进一步降低JDBM镁合金的腐蚀速度,改善其生物相容性和生物活性,并赋予特定的生物学功能,本论文基于JDBM镁合金设计开发了三种不同的功能复合涂层,以满足不同的骨科临床应用需求。主要研究内容和成果如下:1.设计并合成了一种cRGD/PEG功能化接枝聚合物,可在水溶液中通过自组装方式与JDBM基羟基磷灰石涂层（JDBM/HA）表面结合,形成超薄涂层。由于功能化接枝聚合物中聚丙烯酸主链接枝的PEG基团赋予了涂层抗菌性能,接枝的RGD多肽又可特异性结合成骨细胞,实现了兼具抗菌和促成骨细胞粘附的功能。（1）探索得到适合JDBM/HA基体的己二胺（NH₂）和硝基多巴胺（ND）表面锚固基团组合,揭示了锚固基团与基体的结合机制。通过NH₂的静电结合和ND的共价结合的协同作用,实现聚合物在JDBM/HA表面牢固结合,具有很强的吸附稳定性和优异的抗牛血清白蛋白吸附性能。（2）对接枝锚固基团和PEG基团的聚合物进一步接枝修饰不同密度的cRGD多肽,研究了cRGD/PEG功能化接枝聚合物与基体的结合动力学、成骨细胞相容性及抗菌性能。XPS和QCM-D研究结果表明,cRGD/PEG功能化接枝聚合物可在JDBM/HA基体表面牢固结合,在4-羟乙基哌嗪乙磺酸和氯化钠缓冲溶液中保持稳定,基本无脱附。体外成骨细胞和细菌实验结果表明,相比于未修饰的HA基体,修饰cRGD/PEG功能化接枝聚合物的HA显著促进了成骨细胞的粘附,同时抑制了金黄色葡萄球菌在其表面的粘附。2.通过简便的两步化学液相沉积法在JDBM骨组织工程支架表面设计制备了一种抗菌促成骨DCPD/Ag₃PO₄复合涂层。制备的JDBM基复合涂层支架具有分级结构的涂层表面形貌,纳米尺度的立方体Ag₃PO₄颗粒均匀分布在微米尺度的透钙磷石晶粒表面。复合涂层支架保持了JDBM支架利于细胞进入生长的开孔多孔结构。与氟化JDBM支架相比,复合涂层支架降解速率降低了～81%。与氟化JDBM和裸Ti支架相比,复合涂层支架的成骨细胞相容性和ALP活性显著升高,这主要是由于JDBM基体的可控降解产生的Mg²⁺和接近生理条件的微环境导致。体外抗菌结果表明,通过JDBM的碱性降解产物和最外层涂层中Ag⁺的协同抗菌作用,复合涂层支架对革兰氏阳性菌和革兰氏阴性菌均表现出优异的抗菌性能。3.在JDBM镁合金表面设计并制备了一种具有高界面结合强度的聚乳酸/钙磷（PLA/CaP）复合涂层,由作为药物载体的聚乳酸内层和透钙磷石外层构成,研究并阐明了PLA/CaP复合涂层的形成机制。（1）通过UV改性聚乳酸膜层,增加其表面亲水性,显著促进了透钙磷石在聚乳酸膜层表面的异相形核和沉积。（2）由于外层致密的透钙磷石涂层阻碍水分子和侵蚀性阴离子向内扩散侵蚀镁合金,PLA/CaP复合涂层显著降低了JDBM基体的降解速率。（3）生物活性透钙磷石涂层及显著降低的降解速率为成骨细胞提供了更有利的界面和微环境,因此PLA/CaP复合涂层对MC3T3-E1成骨细胞无毒性,并可促进成骨细胞的粘附、增殖和成骨分化,显示出优异的生物相容性和骨诱导潜力。（4）将模型药物紫杉醇负载到PLA/CaP复合涂层,体外药物释放曲线显示载药复合涂层初期药物突释低,具有持续可控的药物释放特征。4.在上述PLA/CaP复合涂层中负载抗肿瘤模型药物紫杉醇,系统研究了载紫杉醇复合涂层递药系统（简称JDBM/PLAT/CaP）的体外降解行为、体外成骨细胞相容性和抑制肿瘤细胞增殖能力及其体内抑制肿瘤效率和成骨能力。（1）体外长期浸泡实验结果表明,JDBM/PLAT/CaP显著改善了JDBM基体的耐蚀性,能够持续可控地释放镁降解产物和紫杉醇药物。（2）体外细胞实验结果表明,由于镁降解产物和紫杉醇药物的协同作用,JDBM/PLAT/CaP可有效抑制骨肉瘤细胞增殖,杀灭骨肉瘤细胞;同时,鉴于外层透钙磷石和JDBM降解产物Mg²⁺的生物活性,递药系统对成骨细胞没有明显毒性,可促进成骨细胞的粘附、增殖和成骨分化,显示出优异的成骨细胞相容性和生物活性。（3）Balb/c裸鼠和Sprague-Dawley大鼠体内植入实验显示,JDBM/PLAT/CaP局部缓释紫杉醇药物和镁降解产物可显著抑制裸鼠骨肉瘤的生长,对骨肉瘤的抑制作用具有药物剂量依赖性;在大鼠体内表现出均匀缓慢的降解模式,显著促进了股骨髁缺损部位的骨重塑,比Ti对照组具有更优异的促成骨能力。（4）提出了JDBM/PLAT/CaP递药系统在骨肿瘤切除后骨缺损部位局部递送镁降解产物和化疗药物,杀灭残余骨肿瘤细胞和诱导骨再生的协同作用机理,具有促进骨再生和减轻骨肿瘤复发的双重功能。更多还原

【Abstract】 Mg alloys have attracted intense attention in the field of orthopedics due to their unique biodegradability,excellent mechanical properties,and good biosafety.The density and elastic modulus of Mg alloys are similar to those of human bone,which can effectively mitigate the stress shielding effect.Thus,Mg alloys present great potential as implant materials in orthopedic applications.However,the excessive degradation and severe local corrosion of Mg has posed the foremost obstacle on its way towards clinical acceptance.Meanwhile,musculoskeletal diseases and infections associated with implants present new biological functional requirements for Mg alloy.In addition to ensuring controlled degradation and providing sufficient mechanical properties for Mg alloy implants,it is also desirable to function biologically in an appropriate manner,such as to achieve enhanced osteointegration,antibacterial activity and even to provide local delivery of therapeutic agents.Previously,a patented biodegradable Mg alloy Mg-Nd-Zn-Zr（Jiao Da Bio-Mg,denoted as JDBM）has been developed in our laboratory.It has been verified that JDBM alloy exhibits uniform degradation characteristics.However,the relatively fast degradation,produced hydrogen in vivo,certain toxicity to cells in high osmolality and alkaline environment need further improvement to meet clinical requirements.To modulate the degradation,improve biocompatibility,and simultaneously impart specific biological functions to JDBM alloy,we developed three different functional composite coating strategies to meet the requirements of a variety of applications.The main research contents and results are shown as follows:1.The cRGD/PEG functionalized graft polymer was designed and synthesized,which can be firmly bonded to the surface of hydroxyapatite coated JDBM alloy（JDBM/HA）by self-assembly in solution.The grafted PEG functional group of the polymer exhibited antifouling property,while the grafted RGD peptide could specifically bond to osteoblasts.（i）Due to the synergistic effect of the electrostatic bonding of NH2 and the covalent bonding of the ND group,the graft polymer could strongly bond to the surface of JDBM/HA,showing strong adsorption stability,and excellent antifouling property to bovine serum albumin.（ii）The optimized graft polymer was further functionalized with cRGD peptide.The binding kinetics,cytocompatibility and antibacterial property of the functionalized polymers were evaluated.The results of XPS and QCM-D assays indicated that the polymer could be firmly bonded to the surface of JDBM/HA and remained stable in HEPES buffer solution with bare desorption.In vitro osteoblast adhension assay and bacterial experiment verified that HA modified with cRGD/PEG functionalized graft polymer significantly promoted osteoblast adhesion compared to bare HA,while inhibiting the adhesion of S.aureus to the surface.2.The DCPD/Ag3PO4 coating with enhanced osteogenesis and antimicrobial properties was designed and synthesized on JDBM tissue engineering scaffold by two-step chemical solution deposition.The prepared scaffold exhibited a hierarchical structured coating surface with nanoscale cubic-shaped Ag3PO4 particles uniformly distributed on the microscale brushite grains.The coated JDBM scaffold retains the advantageous open-cell structure of the initial JDBM scaffold to facilitate cell growth.Immersion test reveals the initial degradation rate of the coated JDBM scaffold could be reduced by as much as～81%compared to JDBM scaffold.The osteoblast cytocompatibility and ALP activity of the coated JDBM scaffold were remarkably elevated as compared with Ti and JDBM scaffolds,mainly due to the released Mg²⁺and the microenvironment closing to physiological condition generated by controlled Mg biodegradation.In addition,the coated JDBM scaffold demonstrated potent antimicrobial capability via the synergistic actions of alkaline degradation products of Mg and the Ag specie in the coating,achieving desirable antibacterial property against both gram-positive and gram-negative bacterial.3.A composite coating of PLA/CaP with high interfacial bonding strength was designed and developed on JDBM alloy,consisted of an inner layer of PLA served as drug carrier and an outer layer composed of brushite.The formation mechanism of PLA/CaP coating was investigated and clarified.（i）A facile pretreatment of UV irradiation was applied to PLA coating to increase the surface hydrophilicity and significantly facilitate heterogeneous nucleation and deposition of brushite on the surface of PLA.（ii）The PLA/CaP coating considerably reduced the degradation rate of JDBM substrate,mainly due to the impeded inward diffusion of water molecules and aggressive anions of the outermost compact layer of brushite coating.（iii）The PLA/CaP coating induced no toxicity to MC3T3-E1 osteoblastic cells but rather fostered cell attachment and proliferation,and promoted osteogenic differentiation,which is attributed to the deposition of bioactive brushite coating and greatly reduced corrosion rate that provide a more favorable interface and microenvironment for osteoblasts,revealing excellent biocompatibility and enhanced osteoinductive potential.（iv）After incorporating taxol into PLA/CaP coating,the in vitro drug release profile showed alleviated initial burst release and a sustained and controlled release feature.4.The anti-tumor model drug,taxol,was loaded to the aforementioned PLA/CaP composite coating.The in vitro degradation behavior,osteoblast compatibility and antineoplastic activity,as well as in vivo inhibitory efficiency of tumor growth and osteogenesis activity of the taxol-loaded composite coating delivery system（denoted as JDBM/PLAT/CaP）were systematically investgated.（i）In vitro long-term immersion test showed that JDBM/PLAT/CaP significantly improved the corrosion resistance of JDBM substrate,exhibiting a controllable,sustained release of Mg degradation products and taxol.（ii）In vitro cell response revealed that JDBM/PLAT/CaP can effectively inhibit the proliferation of osteosarcoma cells and kill them;meanwhile,it has no obvious toxicity to osteoblasts,but promotes the adhesion,proliferation and differentiation of osteoblasts,showing excellent osteoblast compatibility and bioactivity.This was attributed to the synergistic effect of the released Mg degradation products and taxol.（iii）The animal experiments indicated that local sustained-release of taxol and Mg degradation products from JDBM/PLAT/CaP significantly inhibited the growth of osteosarcoma in Balb/c nude mice.The inhibitory effect on osteosarcoma is dose-dependent.JDBM/PLAT/CaP exhibited a slow and uniform degradation,and presented advantages in osteogenesis of femoral condyle defect of SD rats compared to the conventional titanium in vivo.（iv）The synergistic mechanism of JDBM/PLAT/CaP delivery system for local delivery of chemotherapeutic drug and Mg degradation products in bone tumor-induced defect site to kill residual bone tumor cells and promote bone regeneration was proposed,exhibiting dual functions of promoting bone regeneration and alleviating bone tumor recurrence.更多还原