骨科新型医用可降解植入材料JDBM镁合金的生物毒性、髓内针及植入物感染细菌生物膜的基础研究
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摘要
第一部分新型医用可吸收镁合金JDBM的生物毒性
目的：交大镁合金系列（Jiao Da bio-magnesium series, JDBM）是上海交通大学轻合金精密成型国家工程研究中心设计开发的生物相容性好、强度和塑韧性相匹配、腐蚀行为近乎均匀的新型高性能生物医用Mg-Nd-Zn-Zr (平衡-3-0.2-0.4wt.%)基合金系列，分为2类，JDBM1具有高强度和中等塑性，应用于骨科；JDBM2具有高塑性和中等强度，应用于血管支架。该合金体系添加了少量细胞毒性轻微的轻稀土元素Nd作为低合金化元素，Nd的加入可以保证镁合金具有良好的时效析出强化和固溶强化效果，并可大幅度提高合金基体的电极电位，减小基体与第二相的电偶腐蚀电位差，从而提高镁合金的耐均匀腐蚀性能。Zn是人体生理需要的微量元素，微量加入可提高合金强度及塑性加工能力；微量Zr作为晶粒细化剂可提高合金的强韧性和耐蚀性，其在镁合金中的生物相容性已经证实。JDBM的强度、柔韧性、耐蚀性能和生物相容性全面超越已经在欧洲进入临床实验的WE43镁合金。相比于商业性的WE43和AZ31镁合金，JDBM具有更好的生物相容性。JDBM在体内90天时基本完好，在180天时降解少于40%。本实验的目的，即通过研究含轻稀土Nd元素的JDBM镁合金及单纯的Nd对体外培养的小鼠胚胎成骨细胞株MC3T3-E1的毒性，分析JDBM镁合金中稀土元素Nd的体外成骨细胞毒性，体内实验部分通过研究Nd对昆明小鼠骨及周围组织的生理病理影响，以及其在小鼠各器官组织中的分布，来研究Nd的生物安全性及其动物体内分布情况。
方法：按标准方法用aMEM培养基制作Nd的浸提液，并稀释为1/2、1/4、1/8几个不同的浓度梯度，同时制作Ti合金、WE43镁合金、JDBM镁合金及CaP涂层JDBM镁合金的浸提液，以正常培养组做空白对照，以Ti合金浸提液组做阴性对照，以0.64%苯酚组做阳性对照，用含10%澳洲胎牛血清的aMEM培养基对MC3T3-E1成骨细胞进行培养，观察其不同时间的形态变化及增殖情况，并以CCK8法检测其不同时间的细胞增殖活性，以Annexin V PI双染色流式细胞术检测不同时间的细胞凋亡情况。将梯度量的Nd微粒加入正常培养的成骨细胞培养液中，最低量接近JDBM植入物释放的Nd量，观察微粒入胞情况，并制作细胞爬片，用电子目镜接普通显微镜直接动态观察细胞吞噬Nd微粒的情况，然后行HE染色观察细胞形态变化。体内实验部分，将不同量的Nd微粒置于小鼠股骨旁组织，检测1、3、5天小鼠的血液电解质、肝肾功能变化、Nd在小鼠体内的分布及局部病理切片观察Nd微粒引起的病理变化。
结果：用CCK8法检测细胞增殖活性，Nd浸提液原液组细胞毒性较大，与苯酚阳性对照组实验结果一致，其与正常对照组、Ti合金浸提液组、JDBM浸提液组、WE43浸提液组、Nd浸提液1/4、1/8浓度组相比P <0.00001，而后几者之间无明显统计学差异。Nd浸提液原液及苯酚组与Nd浸提液稀释至1/2组之间P <0.01，有明显统计学差异。从流式细胞术检测结果得知，随Nd浸提液浓度及作用时间的增加，成骨细胞凋亡明显增多。微粒入胞实验动态及静态观察发现，成骨细胞通过主动胞吞作用吞噬周围环境中的Nd微粒，微粒较少时细胞生长无明显影响，微粒量多时则可导致成骨细胞膜破裂，直至细胞崩解死亡。通过计算比较，JDBM及WE43中Nd2.5%左右的含量可能会导致周围细胞的损伤。将不同量的Nd微粒置于小鼠股骨旁组织，小鼠的血液电解质及肝肾功能较正常组无明显变化。小鼠体内各组织器官的Nd元素分布因无合适的检测合作单位而待测，病理组织切片显示大量的Nd微粒对周围组织细胞有一定的损伤。
结论： Nd浸提液的成骨细胞毒性及对成骨细胞凋亡的影响随浓度的减小而减小，JDBM中微量Nd对成骨细胞无明显毒性；成骨细胞通过主动胞吞作用摄取周围环境中的Nd微粒，当微粒数量达到一定程度时，成骨细胞崩解，JDBM中微量Nd颗粒对成骨细胞的增殖及形态无明显不良影响。相对于Nd微粒的细胞损伤，JDBM和WE43中的Nd含量可能过高。Nd微粒对小鼠的肝肾功能、电解质无明显影响，多量的Nd微粒对周围组织细胞有一定的损伤。
第二部分新型医用可吸收镁合金JDBM的髓内降解及病理生理效应
目的：生物体内可降解吸收材料是生物材料发展的重要方向，由于金属材料具有较好的强度和塑韧性，因此金属基可降解吸收材料具有重要的临床应用价值。镁是所有金属材料中生物力学性能与人体骨最接近的金属材料，具有理想的生物力学相容性，因此，镁合金作为可降解生物材料具有巨大的应用潜力。目前临床应用的生物体内可降解吸收材料主要是聚合物和某些陶瓷材料，如聚乳酸、磷酸钙等。但由于聚合物材料强度偏低、陶瓷材料的塑韧性较差限制了其广泛使用。近年来，以生物可降解镁合金(biodegradablemagnesium alloys)为主要代表的具有生物可降解(吸收)特性的新一代医用金属材料的研究受到了人们的特别关注。镁合金因具有与人体骨头接近的密度和弹性模量、高比强度和比刚度、生物可降解性以及生物相容性等优点，近10年来国内外研究人员对其应用于骨内植物、骨组织工程支架和心血管支架等领域进行了广泛的研究。然而，目前大多数研究均以现有商用镁合金为对象，如含Al元素的AZ31、AZ91以及含重稀土元素的WE43等，并未考虑到作为生物材料的安全性等问题。上海交通大学轻合金精密成型国家工程研究中心，作为我国重要的镁合金材料及精密成型工艺研究的国家级研究基地，近年来在可降解医用镁合金材料研究领域也取得了令国际同行瞩目的研究成果。该研究中心研究人员与医学研究人员合作，积极开展生物医用镁合金材料的临床应用基础研究。上海交通大学研究人员运用第一性原理计算与分子动力学模拟方法，并与实验相结合，从原子、分子水平深入系统地探索了镁的变形机制，定量评估了所优选的生物相容性好的合金元素对镁中层错能及位错滑移、孪生等变形倾向的影响，设计开发了生物相容性好、强度和塑韧性相匹配、腐蚀行为接近均匀腐蚀的新型高性能生物医用镁合金Mg-Nd-Zn-Zr基合金系列(Jiao Dabio-magnesium series，简称JDBM)，已经分别申请中国和国际专利保护。该合金体系中通过加入少量细胞毒性轻微(临床可接受)的轻稀土元素Nd作为低合金化元素，Nd的加入可以保证镁合金具有良好的时效析出强化和固溶强化效果，并可大幅度提高镁合金基体的电极电位，减小基体与第二相的电偶腐蚀电位差，从而提高镁合金的耐均匀腐蚀性能。JDBM可降解镁合金自研制成功以来，一直在进行各项临床前实验研究，以了解其各项生物学性能，以便进一步改进其各项性能并争取早日应用于临床医疗实践中。我们已经做了一些JDBM镁合金钉板系统方面的研究，取得了许多重要的第一手资料，但尚未进行JDBM镁合金髓内针方面的实验研究。本实验的目的在于检测可降解JDBM镁合金髓内针对大鼠股骨代谢的影响及其病理生理变化，并检测JDBM镁合金髓内针在大鼠股骨髓腔内的降解行为，通过动物实验研究对本材料的医用价值做出一定程度的评估。
方法：将JDBM镁合金及对照的Ti合金、WE43镁合金、CaP涂层JDBM做成1mm x30mm的髓内针，消毒后进行无菌手术将其植入雄性SD大鼠左侧股骨髓腔内，每组6只。在1、3个月进行99mTc-MDP骨三相扫描及SPECT-CT检查，通过骨显像核素扫描了解大鼠股骨植入JDBM镁合金髓内针后的代谢状况，比较与对照侧及对照组的差异。1、3个月拍摄X线片了解髓内针的位置与降解的大体情况。在15天，1、3、6个月检测大鼠的血生化电解质变化。3、6个月做骨组织病理硬切片，通过骨硬组织切片观察大鼠股骨的病理变化，并检测不同时间髓内针的降解情况，确定其降解速率。
结果：JDBM髓内针组大鼠股骨的放射性标记物摄取率明显高于对侧股骨及Ti合金对照组，P<0.01，与WE43组及CaP涂层JDBM组相比无明显差异。骨三相扫描显示镁合金髓内针植入侧血供旺盛。血电解质检测各组未见明显差异。大鼠股骨X线片摄片显示，1、3月时JDBM髓内针形态完好，密度均匀，但6、8月时影像略显模糊，X线透光程度不等，CaP涂层JDBM髓内针及WE43髓内针也出现程度不等的类似变化，可能系镁合金刺激周围骨组织增生所致。大鼠骨硬组织病理切片显示，JDBM髓内针在大鼠髓腔内缓慢降解，并促进骨组织增生，未见明显畸形细胞。与Ti合金组相比，JDBM及CaP涂层JDBM组、WE43组髓内针周围成骨明显。1个月、3个月、6个月的时间内，JDBM髓内针在大鼠骨髓腔内缓慢降解，至6个月时降解不超过20%，直径由最初的1mm至大于0.8mm，质量减少不超过20%，而CaP涂层JDBM组降解更为缓慢，WE43组降解略快于JDBM组，Ti合金组无降解。
结论： JDBM对实验动物大鼠的血清肝肾功能、电解质无明显影响。JDBM在大鼠髓腔内4个月时降解比较轻微，刺激周围骨组织增生的作用已经显现，6个月时周围骨组织明显增生，相应的JDBM髓内针影像略显模糊，应力遮挡效应减弱。JDBM等镁合金髓内针可导致大鼠局部核素摄取增多，表明局部代谢旺盛，镁合金有促进大鼠髓腔内代谢的作用。JDBM髓内针可促进骨代谢及骨形成，有利于损伤骨组织的修复。JDBM镁合金髓内针在骨髓腔内逐渐降解，后期降解趋于缓慢。JDBM及CaP涂层JDBM、WE43髓内针植入大鼠股骨后，其髓腔内组织皆未发现明显的不良反应。
第三部分新型医用可吸收镁合金JDBM与葡萄球菌粘附及生物膜模型
目的：骨科假体植入后葡萄球菌感染是世界范围内比较棘手的问题，医疗实践中通过应用各种材料改性技术和研发新材料等方法以期减少感染的发生。关节假体感染（prosthetic joint infection，PJI）和无菌性松动是人工关节置换术后的重要并发症。在美国，PJI是导致人工全膝关节置换术失败的首要原因，也是人工全髋关节置换术失败的第三大原因。微生物生物膜（biofilm，BF）作为PJI的病原基础，对感染的发生、发展和转归具有决定性作用。根据临床统计，导致关节假体感染的主要致病菌为表皮葡萄球菌。金黄色葡萄球菌是创伤感染中最常见的病原前，而且可引致败血症、疖、脓肿等。作为人兽共患病的重要病原菌，其致病性长期来为医学家所关注。但往主要对凝固酶、葡澈酶等胞外酶及肠毒素、杀白细胞毒、表皮剥脱毒素及毒性休克综合征毒素I等外毒素进行了研究，而忽视了对其牯附机制的探讨。众所周知，粘附是细菌感染的先决条件，否则细菌就无法定居和繁殖，也不能产生和释放胞外酶和外毒素=粘附虽非直接导致组织受损，但对组织的选择和疾病的严重程度却有重要的影响。因此，近年来在对金黄色葡萄球菌胞外产物研究日渐深入的研究，同时，对其粘附机制的研究亦获长足的进展，发现金黄色葡萄球菌有数种粘附素。根据临床统计，导致关节假体感染的主要致病菌为表皮葡萄球菌。表皮葡萄球菌存在于人体的体表，与其他正常菌群一道构成人体皮肤粘膜等的微生态共同抵御外来微生物的侵袭。同时由于寄生部位改变，在凝固酶阴性葡萄球菌(Coagulase Negative Staphvlococcl；CoNS)引起医院感染中最为常见。国外报道表葡占CoNS的57％和68％，特别是在病人体内留置医疗器械引起的感染中表葡是最重要的病原之一，占39％。表皮葡萄球菌通过ica fIntercellular Adhesion)操纵子表达多聚糖胞间粘附因子(Polysaccharide Intercelluar Adhesion PIA)，使之粘附于器械静脉导管、人造心瓣膜、人造关节、人造血管、心脏起搏器及隐形眼镜等)表面，形成一层菌膜，以抵抗机体免疫系统和抗生素的攻击，是表葡引起留置器械相关感染的重要环节。本院骨科假体感染分离出来的致病菌以表皮葡萄球菌为主。本实验的目的在于探索新型可降解材料JDBM镁合金对于葡萄球菌的表面粘附及生长增殖的影响，并制作JDBM镁合金植入动物体内的感染模型，以研究新材料JDBM镁合金的临床适用性。
方法：将JDBM镁合金、WE43镁合金、Ti合金制成1mm x10mm的圆形薄片，进行环氧乙烷消毒。细菌采用RP62A、ATCC12228等菌株，用细菌比浊仪，以TSB培养基调节细菌浓度至约0.5麦氏单位（菌量约1x108/ml），然后在4.5ml EP管（eppendorf管）中加入2ml调好的细菌，加入无菌合金圆片共培养1至3天，每种材料各做一个重复培养的EP管。吸去TSB培养基，用PBS冲洗金属片3次，然后加入2ml PBS，放入超声震荡仪中，中等强度超声能量分别震荡5、10分钟，将所得液体分别按102、104梯度用PBS稀释，各取100ul稀释后的PBS菌液并划到血平板，培养24小时后作细菌计数，看各组间粘附的细菌量有无差异。实验检测金属片表面不同粗糙度对细菌粘附的影响。动物实验部分，将JDBM、WE43镁合金及Ti合金做成3mm x10mm的螺钉，用环氧乙烷熏蒸法消毒，严格无菌操作下将实验螺钉植入普通级新西兰大白兔左侧股骨外上髁，手术伤口愈合后将菌株ST239、SF8300以PBS调至0.5麦氏单位，并将细菌浓度稀释至1x105/ml，分别注射各菌株0.5ml至实验兔的股骨旁组织靠近植入的螺钉，生理盐水注射作为阴性对照，于注射细菌后第1、3周时拍摄实验兔的股骨X线片，并于第3周拍摄实验兔的股骨x线片后将其处死，做局部组织病理切片检查，同时取局部分泌物涂片行姬姆萨染色，将细菌行血琼脂平板培养一日后涂片检查，最后进行细菌类别鉴定。
结果：开始所用的JDBM、WE43、Ti合金组材料光滑度相同，结果发现RP62A及ATCC12228的细菌粘附并无明显差别，其血琼脂培养板的细菌计数在一个数量级，而改变金属圆片的表面光滑度后，再次进行实验，发现粗糙度大的金属片表面粘附了更多的细菌，其粘附细菌量的多少与粗糙度成正相关。动物实验部分，细菌ST239注入兔股骨植入物旁，第1周和第三周时均可见发生严重的局部反应，有大量的乳白色乳酪样物质产生，SF8300注射后局部有少量乳白色乳酪样物质产生，直接涂片瑞氏染色及血琼脂平板培养后涂片瑞氏染色检查，皆发现大量细菌存在，经鉴定，确定感染的细菌为实验所注射的细菌，而生理盐水注射后局部无明显化脓及分泌物产生。
结论： JDBM、WE43、Ti合金材料本身对于葡萄球菌的粘附量无明显差异，但不同的材料表面粗糙度对于细菌的粘附有明显影响，材料表面粗糙度越大，细菌粘附越多，材料表面越光滑，细菌粘附越少。我们通过在实验动物新西兰大白兔的股骨植入物旁注射1x105浓度的细菌ST239很容易造成假体感染生物膜的动物模型，注射SF8300可以造成轻度感染，而注射生理盐水未造成明显的局部感染及生物膜形成。有植入的假体存在的情况下，不同种类细菌对兔股骨旁组织的感染能力及假体成膜能力不同。
第四部分JDBM镁合金、纤维蛋白原与葡萄球菌成骨细胞内化作用
目的：在骨科感染中，多种致病葡萄球菌可以进入成骨细胞，在其中生长繁殖，产生毒素，破坏细胞，并造成细胞的损伤与崩解坏死，或长期存活于细胞内，导致抗生素不能对其进行杀伤。内化是生物大分子(包括细菌、病毒等)进入细胞内的膜泡转运过程。广义地说,内化是指活细胞在生活状态下,自外环境中摄取物质分子的一种模式。小分子物质如水、氨基酸、单糖以及离子等,可借助细胞膜的通透性机制而直接进入细胞内。但生物大分子物质(如细菌、病毒等)不能直接通透细胞膜,常常需先在细胞膜的某些区域产生凹陷,将其包裹于小的膜区域内,随后凹陷进一步向细胞质内延伸,并与细胞膜脱离,在细胞质内形成独立的膜性小泡,引发一系列相关界膜小泡的生成、融合、转运及分离,上述生物大分子进入细胞内的膜泡转运全过程称之为内化,亦称为内吞作用(endocytosis)。其主要形式有吞噬作用(phagocytosis)、吞饮作用(pinocytosis)、受体介导的内化(receptor-mediated internalization)、越胞作用(d-iacytosis)和穿细胞吞吐作用(transcytosis)等。它是当今生命科学研究的重要课题之一。金黄色葡萄球菌（staphylococcus aureus, SA）能侵入成骨细胞内,其侵入能力与菌株本身特点有关。金黄色葡萄球菌侵入成骨细胞后能逃避庆大霉素的杀菌作用,这可能与临床上骨髓炎不易为抗生素治愈有关。金黄色葡萄球菌是血源性骨髓炎及骨科植入物感染的主要来源，约有65%~70%的骨髓炎由金黄色葡萄球菌引起。有研究表明金黄色葡萄球菌能侵入某些体外培养的宿主细胞，如成纤维细胞、鼠的肾细胞、内皮细胞等。金黄色葡萄球菌的这种侵入真核细胞的能力可能与其引起骨髓炎的病理机制有关。表皮葡萄球菌(Staphylococcus epidermidis,SE)是一种革兰染色阳性、凝固酶阴性的葡萄球菌,一般粘附于人体的皮肤和黏膜,是重要的条件致病菌，近年来随着留置静脉导管，植入人工心瓣膜、人工晶体、人工假体等侵袭性操作的增加,表皮葡萄球菌的感染率不断上升，并且由于抗生素的广泛使用甚至滥用，导致表皮葡萄球菌耐药菌株日益增多，出现了多重耐药。表皮葡萄球菌在生物材料表面形成的生物膜是其致病的主要因素，生物膜的形成需要许多因子的参与，这些因子又受相关基因的调控。由于表皮葡萄球菌分泌的毒性因子较金黄色葡萄球菌少，导致其致病的主要因素是表皮葡萄球菌粘附于植入体内的生物材料表面形成生物膜，它不仅使宿主体内的免疫反应呈抑制状态，促使表皮葡萄球菌增殖，而且其黏液阻碍了亲水性的抗生素进入菌体发挥杀菌作用,使感染呈慢性、持续性和反复性的特点。本实验的目的即在于研究不同的临床株表皮葡萄球菌对成骨细胞的内化作用的差异，并研究JDBM镁合金及纤维蛋白原对实验细菌内化作用的影响。
方法：将MC3T3-E1成骨细胞以每孔1ml含10%澳洲胎牛血清的DMEM培养基培养于24孔板，每孔细胞量5x104，连续两天更换培养基，至细胞生长至超过50%面积。以含胎牛血清的DMEM完全培养基通过菌量比浊仪调整23株临床株及标准对照株细菌ATCC12593的细菌浓度调至0.5麦氏单位，约1x108/ml。吸去培养孔内的培养基，每孔分别加入1ml含实验细菌的培养基，置于37℃的二氧化碳孵箱孵育2小时，然后以PBS洗三次，每孔加入含90ug/ml的万古霉素的DMEM完全培养基1ml，置于二氧化碳孵箱37℃培养2小时，以PBS洗三次去掉细胞外被万古霉素杀死的细菌，加入0.1%曲拉通作用10min，使细胞崩以解释放进入细胞内的细菌，将所得液体用PBS按102、104浓度梯度稀释后，以血琼脂平板划板，在35℃的条件下培养，次日观察平板有无细菌生长，对有细菌者比较数量级差异并计数。重复实验3次并比较结果异同。至于纤维蛋白原对细菌内化的影响部分，将MG63骨肉瘤细胞用含10%澳洲胎牛血清的DMEM培养基培养于24孔板，待细胞生长至接近铺满底面积，把9株骨科临床假体感染分离的致病菌及ATCC12228、ATCC12598、RP62A按1x108/ml细菌浓度，每孔1ml细菌液接种的于各培养孔，每株细菌按是否加纤维蛋白原做2个复孔，培养3小时后，吸去培养液，每个培养孔中各加入1ml含100ug/ml万古霉素的完全培养液，37℃条件下在二氧化碳孵箱内培养培养细胞并过夜。次日吸去各培养孔中的培养液，将各培养孔用PBS洗3遍，然后每孔加入PBS配制的0.1%曲拉通作用10min，以使细胞崩解，释放出进入细胞内的细菌，所得液体将原液及102稀释液划于血琼脂培养板，在35℃的孵箱内培养过夜，次日观察有无细菌生长，计数生长的细菌。纤维蛋白原和JDBM对细菌内化的影响方案基本同前。纤维蛋白原对细菌内化的影响部分，将MG63骨肉瘤细胞用含10%澳洲胎牛血清的DMEM培养基培养于24孔板，待细胞生长至接近铺满底面积，把9株骨科临床假体感染分离的致病菌及ATCC12228、ATCC12598、RP62A按1x108/ml细菌浓度，每孔1ml细菌液接种的于各培养孔，每株细菌按是否加纤维蛋白原做2个复孔，培养3小时后，吸去培养液，每个培养孔中各加入1ml含100ug/ml万古霉素的完全培养液，37℃条件下在二氧化碳孵箱内培养培养细胞并过夜。次日吸去各培养孔中的培养液，将各培养孔用PBS洗3遍，然后每孔加入PBS配制的0.1%曲拉通作用10min，以使细胞崩解，释放出进入细胞内的细菌，所得液体将原液连续10倍稀释后划于血琼脂培养板，在37℃的孵箱内培养过夜，次日观察有无细菌生长，计数生长的细菌。JDBM对细菌内化的影响部分，类似于纤维蛋白原的研究方案，用含10%FBS的Ti合金、WE43、JDBM浸提液按上述方案替换培养液进行实验，观察骨科植入材料浸提液对临床表皮葡萄球菌内化的影响。
结果：各个临床菌株对成骨细胞的内化作用强弱不等，有的细菌对成骨细胞的内化作用很强，有的菌株完全看不到对成骨细胞的内化。金黄色葡萄球菌的内化作用较强，毒力较大，镜下观察，成骨细胞中加入诸如ATCC12598等金黄色葡萄球菌0.5h，细胞内即可见细菌进入，作用时间稍长则可见细胞破坏或崩解。一般情况下，内化菌株加入成骨细胞培养孔中一定时间，加入万古霉素2h，然后停止万古霉素的作用，将干预后的细胞用PBS清洗3遍后，将清洗得到的液体划到羊血琼脂培养板上培养一日，有时会显示少量细菌存在。我们的实验结果显示，不管有无纤维蛋白原干预，细菌内化的量没有明显的不同。JDBM、WE43以及Ti合金的浸提液，对于葡萄球菌内化于成骨细胞没有明显的影响。
结论：一般金黄色葡萄球菌毒力比表皮葡萄球菌大，其侵袭力强，内化实验中进入成骨细胞内部的菌量多，它们在细胞内多成团生长；而表皮葡萄球菌毒力弱，侵袭力弱，内化作用也较弱，在细胞内呈散在分布。不同的临床株表皮葡萄球菌内化作用强弱也不同。在有成骨细胞存在的情况下，某些菌株在万古霉素作用2小时后，用PBS洗去万古霉素，仍可在划板培养时出现在血琼脂培养板上。这种现象的发生可能是因为细菌与成骨细胞通过某种机制发生关联，使万古霉素对细菌的杀伤作用减弱，或因为细菌有在空间内平均分布的趋势，万古霉素停止作用后细胞内存活的细菌重新移行到细胞外。我们的实验结果发现，纤维蛋白原及JDBM镁合金浸提液对实验细菌的内化无明显影响。
Part I Biotoxicity of a novel degradable medical material: JDBMmagnesium alloy
【Objective】 JDBM is shortened form Jiao Da bio-magnesium series. It is designed and developed byLight Alloy Precision Forming National Engineering Research Center of Shanghai Jiaotong University.It’s a novel biodegradable high performance Mg-Nd-Zn-Zr based magnesium alloy series, with goodbiocompatibility, excellently matched strength and toughness, and almost even corrosion behavior. Thealloy systems are added in a small quantity of light rare earth element Nd as low alloying elements, whichhas a small cell toxicity. Nd can guarantee the magnesium alloy has good precipitation strengthening andsolid solution strengthening effect, and greatly improve the electrode potential of the alloy matrix, thenthe galvanic corrosion potential difference between matrix and the second phase decreases, in results, theuniform corrosion resistance of magnesium alloy improves. Zn is a trace element of human physiologicalneeds, and trace addition can improve the strength and plastic processing capacity of the alloy. Zr couldincrease Strong toughness and corrosion resistance of alloy as a grain refiner, and its biocompatibility inmagnesium alloy has been confirmed. The purpose of this study is to research cytotoxicity of Rare earthelements Nd in JDBM magnesium alloy to osteoblast cell MC3T3in vitro. And then study itsphysiological and pathological effects to Kunming mice bone and tissues nearby in vivo.
【Methods】 Making leach liquor of Nd with aMEM culture according to the standard method, and thendilute to several different concentration gradient of1/2,1/4,1/8, as controls, making the leach liquor of Tialloy, WE43magnesium alloy, JDBM magnesium alloy and CaP coating JDBM magnesium alloy, andnormal training group as blank control, Ti alloy leach liquor group as negative control,0.64%phenolgroup as positive control. MC3T3-E1cells are cultured with aMEM culture containing10%Australianfetal bovine, observing the morphological changes and proliferation at different times. The activities of cells proliferations in different times were detected by CCK8method. Cells apoptosis in different timeswere detected by Annexin V PI double staining flow cytometry. Adding Nd particles into the culturemedium of cultured osteoblasts by quantity ladder. The minimum amount of Nd added in isapproximately equal to the amount of Nd released from JDBM implants. Observating the condition of Ndparticles into cells. Making the cell climbing glass pieces, and the morphological changes of cells wereobserved by HE staining.
【Results】CCK8method was used to detect the cell proliferation activity. The basic solution of Nd leachliquor group has a great Cytotoxicity, as same as the group of0.64%phenol positive control group.Compared to the normal control group, Ti alloy leach liquor group, JDBM leach liquor group, WE43leach liquor group,1/4and1/8of Nd leach liquor group, P<0.00001. There are no statistically significantdifferences among the after several groups. The basic solution of Nd leach liquor group and0.64%phenol group each has statistically significant difference compared to1/2of Nd leach liquor group,P<0.01. We can know from the flow cytometry results that osteoblast cells apoptosis increasedsignificantly with the increase of the solution concentration and action time of Nd leach liquor. Fromexperiment of dynamic and static observation to that Nd particles get into cells, I find that Osteoblasts getNd particles in the surrounding environment into cells through active endocytosis phagocytosis. There isno significant effect on cell growth with less amount of Nd particles. A lot amount of Nd particles canlead to bone cell membrane rupture, until cell disintegration or death.
【Conclusions】 The osteoblast cytotoxicity and effects on osteoblast cell apoptosis of Nd leach liquoreach decreases with the decrease of concentration. Trace Nd in JDBM has no obvious toxicity onosteoblast. Osteogenic cells uptake Nd particle in the surrounding environment through activeendocytosis. When the number of particles in cells arrive to a certain extent, osteogenic cells disintegrate.Trace Nd particles in JDBM have no obvious adverse effects on proliferation and morphology of bonecells.
Part II Degradation and pathophysiology research of novel biodegradablemagnesium alloy JDBM intramedullary needle with rat femur
【Objective】 The biodegradable JDBM magnesium alloy has been studied since its successfuldevelopment. The purpose of clinical front test is to demonstrate its biological properties, to improve itsperformance, and to fight for the application in clinical medical practice. We have done some research onJDBM magnesium alloy screw board system, gotten many important first-hand data, but have not donethe experimental studies about JDBM magnesium alloy intramedullary needle. The purpose of this studyis to test changes of pathophysiology and metabolism of rat’s for the first, and then to test the degradation behavior of JDBM magnesium alloy in the bone marrow cavity of the rat. So we can assess the medicalvalue of the material JDBM magnesium alloy by animal experimental study.
【Methods】 JDBM magnesium alloy and control groups of Ti alloy, WE43magnesium alloy, CaP coatedJDBM magnesium alloy were made into intramedullary needle of1mm x30mm. After disinfection,asepsis operation has been done and the magnesium alloy intramedullary needle was implanted into thethe left femoral marrow cavity of the male SD rat. Each group has6rats.99mTc-MDP3-phase bone scanand SPECT-CT examination were done at1,3month. To know about the bone metabolic status after theneedle implantation of the rat femur intramedullary JDBM magnesium alloy by bone imagingradionuclide scan, and compared differences with the control side and control groups. X ray films weretaken in order to know about the position and degradation of intramedullary nail at1,3month. To detectthe changes of biochemical and electrolyte of rat blood at half of a month and1,3month. Hard tissuepathology slice of bone was done at month3and6. Pathological changes of rat femur were observed byhard tissue slice, and detect the degradation of intramedullary needle in different time, determine itsdegradation rate.
【Results】 Rat femur radioactive marker uptake rate of JDBM intramedullary needle group wassignificantly higher than that of the contralateral femur and Ti alloy group, P <0.01. There is nodifference significant that JDBM intramedullary needle group compared with the WE43group, JDBMgroup and CaP coating JDBM group. Blood flow phase displays that magnesium alloy promotes localblood supply. No significant differences were detected in blood electrolyte test. Hard tissue pathologicalsection showed that JDBM, CaP coated JDBM group, WE43group has more new bone growth aroundintramedullary needle compared with Ti alloy. JDBM intramedullary needle corroded slowly in rat bonemarrow cavity during month1,3and6. JDBM intramedullary needle corroded less than20%of the masstill6months. While the CaP coated JDBM group was degraded more slowly. WE43group was degradedslightly faster than the JDBM group.
【Conclusions】 JDBM intramedullary needle can promote the formation and metabolism of the bone,helps to repair the damaged bone tissue. JDBM magnesium alloy intramedullary nail in bone marrowcavity has a gradual degradation, but later degradation tends to slower. We wish it could present slowdegradation when we need its supporting role, and when no longer need the support role in the healingperiod, we wish it can has a completely degradation and disappear in controllable time.
Part Novel degradable material JDBM magnesium alloy withstaphylococcal adhesion and biofilm model
【Objective】 Staphylococcal infection is a knotty problem of orthopedics in the world. We reduce the incidence of infection by the application of various materials modification technology or research anddevelopment of new materials in generally. The pathogenic bacteria isolated by the orthopedics doctors ofour hospital are mainly staphylococcus epidermidis. The purpose of this study is to explore the effect ofsurface adhesion, growth and proliferation of staphylococcus with the novel degradable JDBMmagnesium alloy. To make the animal infection model of JDBM magnesium alloy implantation, and tostudy on the clinical application of new materials JDBM magnesium alloy.
【Methods】 JDBM magnesium alloy, WE43magnesium alloy, Ti alloy each was made into1mm x10mm circular sheet, disinfecting them with ethylene oxide sterilization method. Our test using RP62A,ATCC12228strain Bacteria, etc. Turbidity instrument regulating bacterial concentration to about0.5Maxwell units (1x108/ml), using TSB medium. Take2ml bacteria TSB and put it into EP tube of4.5ml,then sterile alloy discs were added in and co-cultured for1to3days, make a repeat of cultured EP tubefor each kind of material. Absorbed TSB medium, washed metal piece with PBS for3times, then put2ml PBS and put the repeated EP tubes into ultrasonic concussion instrument and vibrated for5and10minutes with moderate power ultrasonic. The resulting liquid was diluted to102and104concentrationrespectively with PBS liquid. Take100ul of each diluted PBS liquid contained bacterium and scratchthem to blood plate, cultured for24hours and make bacterial count, observe the differences of theamount of adhesion bacteria. Thus we can study the effect of different metal sheet surface roughnesssurface on the adhesion of bacteria. As the animal test, we made JDBM, WE43alloy and Ti alloy into3mm x10mm screw, and the disinfection was use epoxy ethane fumigation method. Put the screw into thecommon New Zealand rabbit’s left femoral epicondyle with a strict aseptic operation. After operationwound healing, adjusted strain ST239, SF8300to0.5Maxwell unit (1x108/ml) with PBS liquid, andthen diluted bacterial concentration1x105/ml.0.5ml of each strain was injected into the experimentalrabbit tissues around femur near to implanted screw. Injection of saline as negative control. TakenX-ray photos at1st,3rd week after the bacteria was injected into rabbit tissue. Till the third week, X-rayphotos were taken and then the rabbits were killed, local tissue pathological examination was done. Thepartial secretion smear and Giemsa staining was done, and scratching bacteria onto blood agar cultureplate and cultured for one day and then smear examination were done after the culture ended. Bacterialcategory identification was done in the end.
【Results】 JDBM, WE43, Ti alloy material smoothness is same at first, we found that there is nosignificant difference of the bacteria adhesion between RP62A and ATCC, and the bacteria count of theblood agar plate was in an order of magnitude. When we change the metal wafer smoothness of thesurface and do the experiment again, we found that more bacteria adhered to the metal sheet surface withlarge roughness. The amount of bacteria adhered to the surface has a positive correlation to the roughnessof the metal sheet surface. As animal experiment, when bacteria ST239and SF8300were injected intothe tissues near the rabbit femoral implants for1and3weeks, we found that there is a severe localreaction, and there are a lot of milk white cheese like substance emerged. Wright’s staining examination with direct smear or smear after cultured on blood agar plate was done, and a large number of bacteriawere found. After identification, we found that the bacteria caused the infection is the bacteria injected inthe test. While the saline injection rabbit has no significant local pus and secretions produced.
【Conclusions】 There are no obvious difference about the amount of staphylococcus adhesion amongJDBM, WE43, Ti alloy material. But different surface roughness has an obvious influence on theadhesion of bacteria. Surface roughness increases, bacteria adhesion increase at the same time. Thematerial surface is smoother, the bacteria adhesion is less. We made the animal innfectionon biofilmmodel of prosthesis infection very easily by inject the bacterial ST239and SF8300of1X105concentration around the femoral implants of New Zealand white rabbits, while the injection of saline didnot cause obvious locality infection. The experimental results show that different bacterial species havedifferent infection ability to tissues near rabbit femur in the presence of a prosthesis implanted.
Part IV JDBM and fibrinogen with internalization effect ofstaphylococcus on osteoblasts
【Objective】 In orthopedics infection, a variety of pathogenic staphylococcus can enter bone cells, inwhich they grow and reproduce, produce toxins, destruct the cells, and cause cells damage, collapse andnecrosis in the end, or survive in the cells for a long time, as the result, the antibiotic cannot kill them.The purpose of this experiment is to study the difference of on the internalization role among clinicalStaphylococcus strains to the osteoblast cells, and study the effect of fibrinogen on the internalization ofthe test bacteria.
【Methods】 MC3T3-E1osteoblast cells were cultured in the24well plate with DMEM containing10%Australia fetal bovine serum medium,1ml DMEM medium each hole, about5x104cells. Medium waschanged in two consecutive days, until cells growed to more than50%area. The concentration of23clinical strains and standard strains bacteria ATCC12593was adjusted to0.5Maxwell unit with DMEMcomplete medium containing fetal bovine serum through bacteria quantity turbidimetric instrument, theamount of bacteria is about1x108/ml. Absorbed off the medium inside the culture holes, each hole arerespectively added to1ml medium containing experimental bacteria and put the culture plate into thecarbon dioxide incubator and incubated for2hours at37℃. Then we washed the culture holescontaining the bacteria for three times with PBS. Each culture hole is added in1ml DMEM completemedium containing90ug/ml vancomycin, and put the plate into the carbon dioxide incubator andcultured for2hours at37℃. The culture holes were washed three times with PBS to remove the deadbacteria killed by vancomycin outside the cultured.0.1%Triton was added in and played role for10min,so cells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. The liquid earned was diluted with PBS to102,104folds of concentration gradient dilution, and scratchedthem onto the blood agar culture plates and culture for one day at35℃, the plates were observed in thenext day if there has bacteria growth on them. If the bacteria were found on the blood agar culture plates,comparing the differences in magnitude and counting the amount of the bacteria. Repeated theexperiment for3times and compared if there were differences among the results of every times. As forthe effects of fibrinogen on bacteria internalization, MG63osteosarcoma cells were cultured with DMEMmedium containing10%Australian fetal bovine in the24well plate. When the cells developed to close tocover all of the bottom of the wells,9strains of pathogenic bacteria isolated from orthopedics clinicalinfection and ATCC12228, ATCC12598, RP62A were added to each well according to the concentrationof108/ml,1ml culture liquid containing bacteria strain every well. After3hours of culture, we absorbedthe culture liquid, and added1ml DMEM complete medium containing100ug/ml vancomycin, then weput the culture plate into carbon dioxide incubator and cultured the cells overnight at37℃. The next daywe absorbed the culture medium of every wells, and washed every culture well with PBS liquid for3times, and then added0.1%Triton prepared with PBS liquid to each well and let it play role for10min, socells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. Theliquid earned was diluted with PBS liquid to102,104folds of concentration gradient dilution, andscratched them onto the blood agar culture plates and culture for one day at35℃, the plates wereobserved in the next day if there has bacteria growth on them, and the amount of the bacteria observedwere counted. We also research the effect of JDBM extract liquid on staphylococcus internalization,replaced the medium with JDBM, Ti, and WE43extract liquid.
【Results】 The effects of internalization on osteoblast cells between every clinical bacteria strains areunequal, some bacteria have a very strong internalization effect on bone cells, and the other strains cannotbeen seen completely with the internalization effects of osteoblasts. Internalization effect ofstaphylococcus aureus is stronger in generally, and their virulence is bigger. Observed throughmicroscopic, osteoblasts with Staphylococcus aureus put in about0.5h, there are visible bacteria inosteoblasts. Let the bacteria play a role for a long time, we can see destruction or collapse of the cells. Ingenerally, internalization bacteria strains added into osteoblast cells cultured in wells plate for a certainperiod of time, added vancomycin into the wells for2h and then stopped the effect of the drug, the nextstop, washed them with PBS for3times to clear the remain of the antibacterial drug. The cleaning liquidobtained was scribed to the sheep blood agar culture plates and cultured for one day, a small amount ofbacteria may be seen sometimes. Our experimental results show that there are no obvious differenceswith the amount of internalization bacteria no matter if there is fibrinogen intervention. The extract liquidof JDBM, WE43and Ti has no obvious effects on staphylococcus internalization with osteoblasts.
【Conclusions】 In generally, the virulence of staphylococcus aureus is stronger than Staphylococcusepidermidis. The SA has a strong invasiveness. More amount of bacteria can be seen in cells with the testof internalization experiments, and they grow in cells presenting a large of groups. While the virulence of Epidermal staphylococcal is weak, their invasion force is weak, their internalization effect is also weak,this present a scattered distribution in cells. Different clinical staphylococcus epidermidis strains havedifferent degree of internalization effect. Culturing together with osteogenic cells, after vancomycin playa role for2h and stopped the drug’s effect with cleaning for3times by PBS, some strains can be culturedout on the blood agar. Because the bacteria are evenly distributed in the space of the trend. Theoccurrence of this phenomenon may be due to bacteria associated with osteogenic cells through somesort of mechanism and the killing bacteria effect of vancomycin turn weaker in the result, or because thebacteria have a evenly distributed trend in the space, when vancomycin’s killing effect is stopped, theintracellular survival bacteria shift to the outside of the cell. Our experiment results show that fibrinogenor JDBM has no obvious influence on experimental bacterial internalization effect.
目的：交大镁合金系列（Jiao Da bio-magnesium series, JDBM）是上海交通大学轻合金精密成型国家工程研究中心设计开发的生物相容性好、强度和塑韧性相匹配、腐蚀行为近乎均匀的新型高性能生物医用Mg-Nd-Zn-Zr (平衡-3-0.2-0.4wt.%)基合金系列，分为2类，JDBM1具有高强度和中等塑性，应用于骨科；JDBM2具有高塑性和中等强度，应用于血管支架。该合金体系添加了少量细胞毒性轻微的轻稀土元素Nd作为低合金化元素，Nd的加入可以保证镁合金具有良好的时效析出强化和固溶强化效果，并可大幅度提高合金基体的电极电位，减小基体与第二相的电偶腐蚀电位差，从而提高镁合金的耐均匀腐蚀性能。Zn是人体生理需要的微量元素，微量加入可提高合金强度及塑性加工能力；微量Zr作为晶粒细化剂可提高合金的强韧性和耐蚀性，其在镁合金中的生物相容性已经证实。JDBM的强度、柔韧性、耐蚀性能和生物相容性全面超越已经在欧洲进入临床实验的WE43镁合金。相比于商业性的WE43和AZ31镁合金，JDBM具有更好的生物相容性。JDBM在体内90天时基本完好，在180天时降解少于40%。本实验的目的，即通过研究含轻稀土Nd元素的JDBM镁合金及单纯的Nd对体外培养的小鼠胚胎成骨细胞株MC3T3-E1的毒性，分析JDBM镁合金中稀土元素Nd的体外成骨细胞毒性，体内实验部分通过研究Nd对昆明小鼠骨及周围组织的生理病理影响，以及其在小鼠各器官组织中的分布，来研究Nd的生物安全性及其动物体内分布情况。
方法：按标准方法用aMEM培养基制作Nd的浸提液，并稀释为1/2、1/4、1/8几个不同的浓度梯度，同时制作Ti合金、WE43镁合金、JDBM镁合金及CaP涂层JDBM镁合金的浸提液，以正常培养组做空白对照，以Ti合金浸提液组做阴性对照，以0.64%苯酚组做阳性对照，用含10%澳洲胎牛血清的aMEM培养基对MC3T3-E1成骨细胞进行培养，观察其不同时间的形态变化及增殖情况，并以CCK8法检测其不同时间的细胞增殖活性，以Annexin V PI双染色流式细胞术检测不同时间的细胞凋亡情况。将梯度量的Nd微粒加入正常培养的成骨细胞培养液中，最低量接近JDBM植入物释放的Nd量，观察微粒入胞情况，并制作细胞爬片，用电子目镜接普通显微镜直接动态观察细胞吞噬Nd微粒的情况，然后行HE染色观察细胞形态变化。体内实验部分，将不同量的Nd微粒置于小鼠股骨旁组织，检测1、3、5天小鼠的血液电解质、肝肾功能变化、Nd在小鼠体内的分布及局部病理切片观察Nd微粒引起的病理变化。
结果：用CCK8法检测细胞增殖活性，Nd浸提液原液组细胞毒性较大，与苯酚阳性对照组实验结果一致，其与正常对照组、Ti合金浸提液组、JDBM浸提液组、WE43浸提液组、Nd浸提液1/4、1/8浓度组相比P <0.00001，而后几者之间无明显统计学差异。Nd浸提液原液及苯酚组与Nd浸提液稀释至1/2组之间P <0.01，有明显统计学差异。从流式细胞术检测结果得知，随Nd浸提液浓度及作用时间的增加，成骨细胞凋亡明显增多。微粒入胞实验动态及静态观察发现，成骨细胞通过主动胞吞作用吞噬周围环境中的Nd微粒，微粒较少时细胞生长无明显影响，微粒量多时则可导致成骨细胞膜破裂，直至细胞崩解死亡。通过计算比较，JDBM及WE43中Nd2.5%左右的含量可能会导致周围细胞的损伤。将不同量的Nd微粒置于小鼠股骨旁组织，小鼠的血液电解质及肝肾功能较正常组无明显变化。小鼠体内各组织器官的Nd元素分布因无合适的检测合作单位而待测，病理组织切片显示大量的Nd微粒对周围组织细胞有一定的损伤。
结论： Nd浸提液的成骨细胞毒性及对成骨细胞凋亡的影响随浓度的减小而减小，JDBM中微量Nd对成骨细胞无明显毒性；成骨细胞通过主动胞吞作用摄取周围环境中的Nd微粒，当微粒数量达到一定程度时，成骨细胞崩解，JDBM中微量Nd颗粒对成骨细胞的增殖及形态无明显不良影响。相对于Nd微粒的细胞损伤，JDBM和WE43中的Nd含量可能过高。Nd微粒对小鼠的肝肾功能、电解质无明显影响，多量的Nd微粒对周围组织细胞有一定的损伤。
第二部分新型医用可吸收镁合金JDBM的髓内降解及病理生理效应
目的：生物体内可降解吸收材料是生物材料发展的重要方向，由于金属材料具有较好的强度和塑韧性，因此金属基可降解吸收材料具有重要的临床应用价值。镁是所有金属材料中生物力学性能与人体骨最接近的金属材料，具有理想的生物力学相容性，因此，镁合金作为可降解生物材料具有巨大的应用潜力。目前临床应用的生物体内可降解吸收材料主要是聚合物和某些陶瓷材料，如聚乳酸、磷酸钙等。但由于聚合物材料强度偏低、陶瓷材料的塑韧性较差限制了其广泛使用。近年来，以生物可降解镁合金(biodegradablemagnesium alloys)为主要代表的具有生物可降解(吸收)特性的新一代医用金属材料的研究受到了人们的特别关注。镁合金因具有与人体骨头接近的密度和弹性模量、高比强度和比刚度、生物可降解性以及生物相容性等优点，近10年来国内外研究人员对其应用于骨内植物、骨组织工程支架和心血管支架等领域进行了广泛的研究。然而，目前大多数研究均以现有商用镁合金为对象，如含Al元素的AZ31、AZ91以及含重稀土元素的WE43等，并未考虑到作为生物材料的安全性等问题。上海交通大学轻合金精密成型国家工程研究中心，作为我国重要的镁合金材料及精密成型工艺研究的国家级研究基地，近年来在可降解医用镁合金材料研究领域也取得了令国际同行瞩目的研究成果。该研究中心研究人员与医学研究人员合作，积极开展生物医用镁合金材料的临床应用基础研究。上海交通大学研究人员运用第一性原理计算与分子动力学模拟方法，并与实验相结合，从原子、分子水平深入系统地探索了镁的变形机制，定量评估了所优选的生物相容性好的合金元素对镁中层错能及位错滑移、孪生等变形倾向的影响，设计开发了生物相容性好、强度和塑韧性相匹配、腐蚀行为接近均匀腐蚀的新型高性能生物医用镁合金Mg-Nd-Zn-Zr基合金系列(Jiao Dabio-magnesium series，简称JDBM)，已经分别申请中国和国际专利保护。该合金体系中通过加入少量细胞毒性轻微(临床可接受)的轻稀土元素Nd作为低合金化元素，Nd的加入可以保证镁合金具有良好的时效析出强化和固溶强化效果，并可大幅度提高镁合金基体的电极电位，减小基体与第二相的电偶腐蚀电位差，从而提高镁合金的耐均匀腐蚀性能。JDBM可降解镁合金自研制成功以来，一直在进行各项临床前实验研究，以了解其各项生物学性能，以便进一步改进其各项性能并争取早日应用于临床医疗实践中。我们已经做了一些JDBM镁合金钉板系统方面的研究，取得了许多重要的第一手资料，但尚未进行JDBM镁合金髓内针方面的实验研究。本实验的目的在于检测可降解JDBM镁合金髓内针对大鼠股骨代谢的影响及其病理生理变化，并检测JDBM镁合金髓内针在大鼠股骨髓腔内的降解行为，通过动物实验研究对本材料的医用价值做出一定程度的评估。
方法：将JDBM镁合金及对照的Ti合金、WE43镁合金、CaP涂层JDBM做成1mm x30mm的髓内针，消毒后进行无菌手术将其植入雄性SD大鼠左侧股骨髓腔内，每组6只。在1、3个月进行99mTc-MDP骨三相扫描及SPECT-CT检查，通过骨显像核素扫描了解大鼠股骨植入JDBM镁合金髓内针后的代谢状况，比较与对照侧及对照组的差异。1、3个月拍摄X线片了解髓内针的位置与降解的大体情况。在15天，1、3、6个月检测大鼠的血生化电解质变化。3、6个月做骨组织病理硬切片，通过骨硬组织切片观察大鼠股骨的病理变化，并检测不同时间髓内针的降解情况，确定其降解速率。
结果：JDBM髓内针组大鼠股骨的放射性标记物摄取率明显高于对侧股骨及Ti合金对照组，P<0.01，与WE43组及CaP涂层JDBM组相比无明显差异。骨三相扫描显示镁合金髓内针植入侧血供旺盛。血电解质检测各组未见明显差异。大鼠股骨X线片摄片显示，1、3月时JDBM髓内针形态完好，密度均匀，但6、8月时影像略显模糊，X线透光程度不等，CaP涂层JDBM髓内针及WE43髓内针也出现程度不等的类似变化，可能系镁合金刺激周围骨组织增生所致。大鼠骨硬组织病理切片显示，JDBM髓内针在大鼠髓腔内缓慢降解，并促进骨组织增生，未见明显畸形细胞。与Ti合金组相比，JDBM及CaP涂层JDBM组、WE43组髓内针周围成骨明显。1个月、3个月、6个月的时间内，JDBM髓内针在大鼠骨髓腔内缓慢降解，至6个月时降解不超过20%，直径由最初的1mm至大于0.8mm，质量减少不超过20%，而CaP涂层JDBM组降解更为缓慢，WE43组降解略快于JDBM组，Ti合金组无降解。
结论： JDBM对实验动物大鼠的血清肝肾功能、电解质无明显影响。JDBM在大鼠髓腔内4个月时降解比较轻微，刺激周围骨组织增生的作用已经显现，6个月时周围骨组织明显增生，相应的JDBM髓内针影像略显模糊，应力遮挡效应减弱。JDBM等镁合金髓内针可导致大鼠局部核素摄取增多，表明局部代谢旺盛，镁合金有促进大鼠髓腔内代谢的作用。JDBM髓内针可促进骨代谢及骨形成，有利于损伤骨组织的修复。JDBM镁合金髓内针在骨髓腔内逐渐降解，后期降解趋于缓慢。JDBM及CaP涂层JDBM、WE43髓内针植入大鼠股骨后，其髓腔内组织皆未发现明显的不良反应。
第三部分新型医用可吸收镁合金JDBM与葡萄球菌粘附及生物膜模型
目的：骨科假体植入后葡萄球菌感染是世界范围内比较棘手的问题，医疗实践中通过应用各种材料改性技术和研发新材料等方法以期减少感染的发生。关节假体感染（prosthetic joint infection，PJI）和无菌性松动是人工关节置换术后的重要并发症。在美国，PJI是导致人工全膝关节置换术失败的首要原因，也是人工全髋关节置换术失败的第三大原因。微生物生物膜（biofilm，BF）作为PJI的病原基础，对感染的发生、发展和转归具有决定性作用。根据临床统计，导致关节假体感染的主要致病菌为表皮葡萄球菌。金黄色葡萄球菌是创伤感染中最常见的病原前，而且可引致败血症、疖、脓肿等。作为人兽共患病的重要病原菌，其致病性长期来为医学家所关注。但往主要对凝固酶、葡澈酶等胞外酶及肠毒素、杀白细胞毒、表皮剥脱毒素及毒性休克综合征毒素I等外毒素进行了研究，而忽视了对其牯附机制的探讨。众所周知，粘附是细菌感染的先决条件，否则细菌就无法定居和繁殖，也不能产生和释放胞外酶和外毒素=粘附虽非直接导致组织受损，但对组织的选择和疾病的严重程度却有重要的影响。因此，近年来在对金黄色葡萄球菌胞外产物研究日渐深入的研究，同时，对其粘附机制的研究亦获长足的进展，发现金黄色葡萄球菌有数种粘附素。根据临床统计，导致关节假体感染的主要致病菌为表皮葡萄球菌。表皮葡萄球菌存在于人体的体表，与其他正常菌群一道构成人体皮肤粘膜等的微生态共同抵御外来微生物的侵袭。同时由于寄生部位改变，在凝固酶阴性葡萄球菌(Coagulase Negative Staphvlococcl；CoNS)引起医院感染中最为常见。国外报道表葡占CoNS的57％和68％，特别是在病人体内留置医疗器械引起的感染中表葡是最重要的病原之一，占39％。表皮葡萄球菌通过ica fIntercellular Adhesion)操纵子表达多聚糖胞间粘附因子(Polysaccharide Intercelluar Adhesion PIA)，使之粘附于器械静脉导管、人造心瓣膜、人造关节、人造血管、心脏起搏器及隐形眼镜等)表面，形成一层菌膜，以抵抗机体免疫系统和抗生素的攻击，是表葡引起留置器械相关感染的重要环节。本院骨科假体感染分离出来的致病菌以表皮葡萄球菌为主。本实验的目的在于探索新型可降解材料JDBM镁合金对于葡萄球菌的表面粘附及生长增殖的影响，并制作JDBM镁合金植入动物体内的感染模型，以研究新材料JDBM镁合金的临床适用性。
方法：将JDBM镁合金、WE43镁合金、Ti合金制成1mm x10mm的圆形薄片，进行环氧乙烷消毒。细菌采用RP62A、ATCC12228等菌株，用细菌比浊仪，以TSB培养基调节细菌浓度至约0.5麦氏单位（菌量约1x108/ml），然后在4.5ml EP管（eppendorf管）中加入2ml调好的细菌，加入无菌合金圆片共培养1至3天，每种材料各做一个重复培养的EP管。吸去TSB培养基，用PBS冲洗金属片3次，然后加入2ml PBS，放入超声震荡仪中，中等强度超声能量分别震荡5、10分钟，将所得液体分别按102、104梯度用PBS稀释，各取100ul稀释后的PBS菌液并划到血平板，培养24小时后作细菌计数，看各组间粘附的细菌量有无差异。实验检测金属片表面不同粗糙度对细菌粘附的影响。动物实验部分，将JDBM、WE43镁合金及Ti合金做成3mm x10mm的螺钉，用环氧乙烷熏蒸法消毒，严格无菌操作下将实验螺钉植入普通级新西兰大白兔左侧股骨外上髁，手术伤口愈合后将菌株ST239、SF8300以PBS调至0.5麦氏单位，并将细菌浓度稀释至1x105/ml，分别注射各菌株0.5ml至实验兔的股骨旁组织靠近植入的螺钉，生理盐水注射作为阴性对照，于注射细菌后第1、3周时拍摄实验兔的股骨X线片，并于第3周拍摄实验兔的股骨x线片后将其处死，做局部组织病理切片检查，同时取局部分泌物涂片行姬姆萨染色，将细菌行血琼脂平板培养一日后涂片检查，最后进行细菌类别鉴定。
结果：开始所用的JDBM、WE43、Ti合金组材料光滑度相同，结果发现RP62A及ATCC12228的细菌粘附并无明显差别，其血琼脂培养板的细菌计数在一个数量级，而改变金属圆片的表面光滑度后，再次进行实验，发现粗糙度大的金属片表面粘附了更多的细菌，其粘附细菌量的多少与粗糙度成正相关。动物实验部分，细菌ST239注入兔股骨植入物旁，第1周和第三周时均可见发生严重的局部反应，有大量的乳白色乳酪样物质产生，SF8300注射后局部有少量乳白色乳酪样物质产生，直接涂片瑞氏染色及血琼脂平板培养后涂片瑞氏染色检查，皆发现大量细菌存在，经鉴定，确定感染的细菌为实验所注射的细菌，而生理盐水注射后局部无明显化脓及分泌物产生。
结论： JDBM、WE43、Ti合金材料本身对于葡萄球菌的粘附量无明显差异，但不同的材料表面粗糙度对于细菌的粘附有明显影响，材料表面粗糙度越大，细菌粘附越多，材料表面越光滑，细菌粘附越少。我们通过在实验动物新西兰大白兔的股骨植入物旁注射1x105浓度的细菌ST239很容易造成假体感染生物膜的动物模型，注射SF8300可以造成轻度感染，而注射生理盐水未造成明显的局部感染及生物膜形成。有植入的假体存在的情况下，不同种类细菌对兔股骨旁组织的感染能力及假体成膜能力不同。
第四部分JDBM镁合金、纤维蛋白原与葡萄球菌成骨细胞内化作用
目的：在骨科感染中，多种致病葡萄球菌可以进入成骨细胞，在其中生长繁殖，产生毒素，破坏细胞，并造成细胞的损伤与崩解坏死，或长期存活于细胞内，导致抗生素不能对其进行杀伤。内化是生物大分子(包括细菌、病毒等)进入细胞内的膜泡转运过程。广义地说,内化是指活细胞在生活状态下,自外环境中摄取物质分子的一种模式。小分子物质如水、氨基酸、单糖以及离子等,可借助细胞膜的通透性机制而直接进入细胞内。但生物大分子物质(如细菌、病毒等)不能直接通透细胞膜,常常需先在细胞膜的某些区域产生凹陷,将其包裹于小的膜区域内,随后凹陷进一步向细胞质内延伸,并与细胞膜脱离,在细胞质内形成独立的膜性小泡,引发一系列相关界膜小泡的生成、融合、转运及分离,上述生物大分子进入细胞内的膜泡转运全过程称之为内化,亦称为内吞作用(endocytosis)。其主要形式有吞噬作用(phagocytosis)、吞饮作用(pinocytosis)、受体介导的内化(receptor-mediated internalization)、越胞作用(d-iacytosis)和穿细胞吞吐作用(transcytosis)等。它是当今生命科学研究的重要课题之一。金黄色葡萄球菌（staphylococcus aureus, SA）能侵入成骨细胞内,其侵入能力与菌株本身特点有关。金黄色葡萄球菌侵入成骨细胞后能逃避庆大霉素的杀菌作用,这可能与临床上骨髓炎不易为抗生素治愈有关。金黄色葡萄球菌是血源性骨髓炎及骨科植入物感染的主要来源，约有65%~70%的骨髓炎由金黄色葡萄球菌引起。有研究表明金黄色葡萄球菌能侵入某些体外培养的宿主细胞，如成纤维细胞、鼠的肾细胞、内皮细胞等。金黄色葡萄球菌的这种侵入真核细胞的能力可能与其引起骨髓炎的病理机制有关。表皮葡萄球菌(Staphylococcus epidermidis,SE)是一种革兰染色阳性、凝固酶阴性的葡萄球菌,一般粘附于人体的皮肤和黏膜,是重要的条件致病菌，近年来随着留置静脉导管，植入人工心瓣膜、人工晶体、人工假体等侵袭性操作的增加,表皮葡萄球菌的感染率不断上升，并且由于抗生素的广泛使用甚至滥用，导致表皮葡萄球菌耐药菌株日益增多，出现了多重耐药。表皮葡萄球菌在生物材料表面形成的生物膜是其致病的主要因素，生物膜的形成需要许多因子的参与，这些因子又受相关基因的调控。由于表皮葡萄球菌分泌的毒性因子较金黄色葡萄球菌少，导致其致病的主要因素是表皮葡萄球菌粘附于植入体内的生物材料表面形成生物膜，它不仅使宿主体内的免疫反应呈抑制状态，促使表皮葡萄球菌增殖，而且其黏液阻碍了亲水性的抗生素进入菌体发挥杀菌作用,使感染呈慢性、持续性和反复性的特点。本实验的目的即在于研究不同的临床株表皮葡萄球菌对成骨细胞的内化作用的差异，并研究JDBM镁合金及纤维蛋白原对实验细菌内化作用的影响。
方法：将MC3T3-E1成骨细胞以每孔1ml含10%澳洲胎牛血清的DMEM培养基培养于24孔板，每孔细胞量5x104，连续两天更换培养基，至细胞生长至超过50%面积。以含胎牛血清的DMEM完全培养基通过菌量比浊仪调整23株临床株及标准对照株细菌ATCC12593的细菌浓度调至0.5麦氏单位，约1x108/ml。吸去培养孔内的培养基，每孔分别加入1ml含实验细菌的培养基，置于37℃的二氧化碳孵箱孵育2小时，然后以PBS洗三次，每孔加入含90ug/ml的万古霉素的DMEM完全培养基1ml，置于二氧化碳孵箱37℃培养2小时，以PBS洗三次去掉细胞外被万古霉素杀死的细菌，加入0.1%曲拉通作用10min，使细胞崩以解释放进入细胞内的细菌，将所得液体用PBS按102、104浓度梯度稀释后，以血琼脂平板划板，在35℃的条件下培养，次日观察平板有无细菌生长，对有细菌者比较数量级差异并计数。重复实验3次并比较结果异同。至于纤维蛋白原对细菌内化的影响部分，将MG63骨肉瘤细胞用含10%澳洲胎牛血清的DMEM培养基培养于24孔板，待细胞生长至接近铺满底面积，把9株骨科临床假体感染分离的致病菌及ATCC12228、ATCC12598、RP62A按1x108/ml细菌浓度，每孔1ml细菌液接种的于各培养孔，每株细菌按是否加纤维蛋白原做2个复孔，培养3小时后，吸去培养液，每个培养孔中各加入1ml含100ug/ml万古霉素的完全培养液，37℃条件下在二氧化碳孵箱内培养培养细胞并过夜。次日吸去各培养孔中的培养液，将各培养孔用PBS洗3遍，然后每孔加入PBS配制的0.1%曲拉通作用10min，以使细胞崩解，释放出进入细胞内的细菌，所得液体将原液及102稀释液划于血琼脂培养板，在35℃的孵箱内培养过夜，次日观察有无细菌生长，计数生长的细菌。纤维蛋白原和JDBM对细菌内化的影响方案基本同前。纤维蛋白原对细菌内化的影响部分，将MG63骨肉瘤细胞用含10%澳洲胎牛血清的DMEM培养基培养于24孔板，待细胞生长至接近铺满底面积，把9株骨科临床假体感染分离的致病菌及ATCC12228、ATCC12598、RP62A按1x108/ml细菌浓度，每孔1ml细菌液接种的于各培养孔，每株细菌按是否加纤维蛋白原做2个复孔，培养3小时后，吸去培养液，每个培养孔中各加入1ml含100ug/ml万古霉素的完全培养液，37℃条件下在二氧化碳孵箱内培养培养细胞并过夜。次日吸去各培养孔中的培养液，将各培养孔用PBS洗3遍，然后每孔加入PBS配制的0.1%曲拉通作用10min，以使细胞崩解，释放出进入细胞内的细菌，所得液体将原液连续10倍稀释后划于血琼脂培养板，在37℃的孵箱内培养过夜，次日观察有无细菌生长，计数生长的细菌。JDBM对细菌内化的影响部分，类似于纤维蛋白原的研究方案，用含10%FBS的Ti合金、WE43、JDBM浸提液按上述方案替换培养液进行实验，观察骨科植入材料浸提液对临床表皮葡萄球菌内化的影响。
结果：各个临床菌株对成骨细胞的内化作用强弱不等，有的细菌对成骨细胞的内化作用很强，有的菌株完全看不到对成骨细胞的内化。金黄色葡萄球菌的内化作用较强，毒力较大，镜下观察，成骨细胞中加入诸如ATCC12598等金黄色葡萄球菌0.5h，细胞内即可见细菌进入，作用时间稍长则可见细胞破坏或崩解。一般情况下，内化菌株加入成骨细胞培养孔中一定时间，加入万古霉素2h，然后停止万古霉素的作用，将干预后的细胞用PBS清洗3遍后，将清洗得到的液体划到羊血琼脂培养板上培养一日，有时会显示少量细菌存在。我们的实验结果显示，不管有无纤维蛋白原干预，细菌内化的量没有明显的不同。JDBM、WE43以及Ti合金的浸提液，对于葡萄球菌内化于成骨细胞没有明显的影响。
结论：一般金黄色葡萄球菌毒力比表皮葡萄球菌大，其侵袭力强，内化实验中进入成骨细胞内部的菌量多，它们在细胞内多成团生长；而表皮葡萄球菌毒力弱，侵袭力弱，内化作用也较弱，在细胞内呈散在分布。不同的临床株表皮葡萄球菌内化作用强弱也不同。在有成骨细胞存在的情况下，某些菌株在万古霉素作用2小时后，用PBS洗去万古霉素，仍可在划板培养时出现在血琼脂培养板上。这种现象的发生可能是因为细菌与成骨细胞通过某种机制发生关联，使万古霉素对细菌的杀伤作用减弱，或因为细菌有在空间内平均分布的趋势，万古霉素停止作用后细胞内存活的细菌重新移行到细胞外。我们的实验结果发现，纤维蛋白原及JDBM镁合金浸提液对实验细菌的内化无明显影响。
Part I Biotoxicity of a novel degradable medical material: JDBMmagnesium alloy
【Objective】 JDBM is shortened form Jiao Da bio-magnesium series. It is designed and developed byLight Alloy Precision Forming National Engineering Research Center of Shanghai Jiaotong University.It’s a novel biodegradable high performance Mg-Nd-Zn-Zr based magnesium alloy series, with goodbiocompatibility, excellently matched strength and toughness, and almost even corrosion behavior. Thealloy systems are added in a small quantity of light rare earth element Nd as low alloying elements, whichhas a small cell toxicity. Nd can guarantee the magnesium alloy has good precipitation strengthening andsolid solution strengthening effect, and greatly improve the electrode potential of the alloy matrix, thenthe galvanic corrosion potential difference between matrix and the second phase decreases, in results, theuniform corrosion resistance of magnesium alloy improves. Zn is a trace element of human physiologicalneeds, and trace addition can improve the strength and plastic processing capacity of the alloy. Zr couldincrease Strong toughness and corrosion resistance of alloy as a grain refiner, and its biocompatibility inmagnesium alloy has been confirmed. The purpose of this study is to research cytotoxicity of Rare earthelements Nd in JDBM magnesium alloy to osteoblast cell MC3T3in vitro. And then study itsphysiological and pathological effects to Kunming mice bone and tissues nearby in vivo.
【Methods】 Making leach liquor of Nd with aMEM culture according to the standard method, and thendilute to several different concentration gradient of1/2,1/4,1/8, as controls, making the leach liquor of Tialloy, WE43magnesium alloy, JDBM magnesium alloy and CaP coating JDBM magnesium alloy, andnormal training group as blank control, Ti alloy leach liquor group as negative control,0.64%phenolgroup as positive control. MC3T3-E1cells are cultured with aMEM culture containing10%Australianfetal bovine, observing the morphological changes and proliferation at different times. The activities of cells proliferations in different times were detected by CCK8method. Cells apoptosis in different timeswere detected by Annexin V PI double staining flow cytometry. Adding Nd particles into the culturemedium of cultured osteoblasts by quantity ladder. The minimum amount of Nd added in isapproximately equal to the amount of Nd released from JDBM implants. Observating the condition of Ndparticles into cells. Making the cell climbing glass pieces, and the morphological changes of cells wereobserved by HE staining.
【Results】CCK8method was used to detect the cell proliferation activity. The basic solution of Nd leachliquor group has a great Cytotoxicity, as same as the group of0.64%phenol positive control group.Compared to the normal control group, Ti alloy leach liquor group, JDBM leach liquor group, WE43leach liquor group,1/4and1/8of Nd leach liquor group, P<0.00001. There are no statistically significantdifferences among the after several groups. The basic solution of Nd leach liquor group and0.64%phenol group each has statistically significant difference compared to1/2of Nd leach liquor group,P<0.01. We can know from the flow cytometry results that osteoblast cells apoptosis increasedsignificantly with the increase of the solution concentration and action time of Nd leach liquor. Fromexperiment of dynamic and static observation to that Nd particles get into cells, I find that Osteoblasts getNd particles in the surrounding environment into cells through active endocytosis phagocytosis. There isno significant effect on cell growth with less amount of Nd particles. A lot amount of Nd particles canlead to bone cell membrane rupture, until cell disintegration or death.
【Conclusions】 The osteoblast cytotoxicity and effects on osteoblast cell apoptosis of Nd leach liquoreach decreases with the decrease of concentration. Trace Nd in JDBM has no obvious toxicity onosteoblast. Osteogenic cells uptake Nd particle in the surrounding environment through activeendocytosis. When the number of particles in cells arrive to a certain extent, osteogenic cells disintegrate.Trace Nd particles in JDBM have no obvious adverse effects on proliferation and morphology of bonecells.
Part II Degradation and pathophysiology research of novel biodegradablemagnesium alloy JDBM intramedullary needle with rat femur
【Objective】 The biodegradable JDBM magnesium alloy has been studied since its successfuldevelopment. The purpose of clinical front test is to demonstrate its biological properties, to improve itsperformance, and to fight for the application in clinical medical practice. We have done some research onJDBM magnesium alloy screw board system, gotten many important first-hand data, but have not donethe experimental studies about JDBM magnesium alloy intramedullary needle. The purpose of this studyis to test changes of pathophysiology and metabolism of rat’s for the first, and then to test the degradation behavior of JDBM magnesium alloy in the bone marrow cavity of the rat. So we can assess the medicalvalue of the material JDBM magnesium alloy by animal experimental study.
【Methods】 JDBM magnesium alloy and control groups of Ti alloy, WE43magnesium alloy, CaP coatedJDBM magnesium alloy were made into intramedullary needle of1mm x30mm. After disinfection,asepsis operation has been done and the magnesium alloy intramedullary needle was implanted into thethe left femoral marrow cavity of the male SD rat. Each group has6rats.99mTc-MDP3-phase bone scanand SPECT-CT examination were done at1,3month. To know about the bone metabolic status after theneedle implantation of the rat femur intramedullary JDBM magnesium alloy by bone imagingradionuclide scan, and compared differences with the control side and control groups. X ray films weretaken in order to know about the position and degradation of intramedullary nail at1,3month. To detectthe changes of biochemical and electrolyte of rat blood at half of a month and1,3month. Hard tissuepathology slice of bone was done at month3and6. Pathological changes of rat femur were observed byhard tissue slice, and detect the degradation of intramedullary needle in different time, determine itsdegradation rate.
【Results】 Rat femur radioactive marker uptake rate of JDBM intramedullary needle group wassignificantly higher than that of the contralateral femur and Ti alloy group, P <0.01. There is nodifference significant that JDBM intramedullary needle group compared with the WE43group, JDBMgroup and CaP coating JDBM group. Blood flow phase displays that magnesium alloy promotes localblood supply. No significant differences were detected in blood electrolyte test. Hard tissue pathologicalsection showed that JDBM, CaP coated JDBM group, WE43group has more new bone growth aroundintramedullary needle compared with Ti alloy. JDBM intramedullary needle corroded slowly in rat bonemarrow cavity during month1,3and6. JDBM intramedullary needle corroded less than20%of the masstill6months. While the CaP coated JDBM group was degraded more slowly. WE43group was degradedslightly faster than the JDBM group.
【Conclusions】 JDBM intramedullary needle can promote the formation and metabolism of the bone,helps to repair the damaged bone tissue. JDBM magnesium alloy intramedullary nail in bone marrowcavity has a gradual degradation, but later degradation tends to slower. We wish it could present slowdegradation when we need its supporting role, and when no longer need the support role in the healingperiod, we wish it can has a completely degradation and disappear in controllable time.
Part Novel degradable material JDBM magnesium alloy withstaphylococcal adhesion and biofilm model
【Objective】 Staphylococcal infection is a knotty problem of orthopedics in the world. We reduce the incidence of infection by the application of various materials modification technology or research anddevelopment of new materials in generally. The pathogenic bacteria isolated by the orthopedics doctors ofour hospital are mainly staphylococcus epidermidis. The purpose of this study is to explore the effect ofsurface adhesion, growth and proliferation of staphylococcus with the novel degradable JDBMmagnesium alloy. To make the animal infection model of JDBM magnesium alloy implantation, and tostudy on the clinical application of new materials JDBM magnesium alloy.
【Methods】 JDBM magnesium alloy, WE43magnesium alloy, Ti alloy each was made into1mm x10mm circular sheet, disinfecting them with ethylene oxide sterilization method. Our test using RP62A,ATCC12228strain Bacteria, etc. Turbidity instrument regulating bacterial concentration to about0.5Maxwell units (1x108/ml), using TSB medium. Take2ml bacteria TSB and put it into EP tube of4.5ml,then sterile alloy discs were added in and co-cultured for1to3days, make a repeat of cultured EP tubefor each kind of material. Absorbed TSB medium, washed metal piece with PBS for3times, then put2ml PBS and put the repeated EP tubes into ultrasonic concussion instrument and vibrated for5and10minutes with moderate power ultrasonic. The resulting liquid was diluted to102and104concentrationrespectively with PBS liquid. Take100ul of each diluted PBS liquid contained bacterium and scratchthem to blood plate, cultured for24hours and make bacterial count, observe the differences of theamount of adhesion bacteria. Thus we can study the effect of different metal sheet surface roughnesssurface on the adhesion of bacteria. As the animal test, we made JDBM, WE43alloy and Ti alloy into3mm x10mm screw, and the disinfection was use epoxy ethane fumigation method. Put the screw into thecommon New Zealand rabbit’s left femoral epicondyle with a strict aseptic operation. After operationwound healing, adjusted strain ST239, SF8300to0.5Maxwell unit (1x108/ml) with PBS liquid, andthen diluted bacterial concentration1x105/ml.0.5ml of each strain was injected into the experimentalrabbit tissues around femur near to implanted screw. Injection of saline as negative control. TakenX-ray photos at1st,3rd week after the bacteria was injected into rabbit tissue. Till the third week, X-rayphotos were taken and then the rabbits were killed, local tissue pathological examination was done. Thepartial secretion smear and Giemsa staining was done, and scratching bacteria onto blood agar cultureplate and cultured for one day and then smear examination were done after the culture ended. Bacterialcategory identification was done in the end.
【Results】 JDBM, WE43, Ti alloy material smoothness is same at first, we found that there is nosignificant difference of the bacteria adhesion between RP62A and ATCC, and the bacteria count of theblood agar plate was in an order of magnitude. When we change the metal wafer smoothness of thesurface and do the experiment again, we found that more bacteria adhered to the metal sheet surface withlarge roughness. The amount of bacteria adhered to the surface has a positive correlation to the roughnessof the metal sheet surface. As animal experiment, when bacteria ST239and SF8300were injected intothe tissues near the rabbit femoral implants for1and3weeks, we found that there is a severe localreaction, and there are a lot of milk white cheese like substance emerged. Wright’s staining examination with direct smear or smear after cultured on blood agar plate was done, and a large number of bacteriawere found. After identification, we found that the bacteria caused the infection is the bacteria injected inthe test. While the saline injection rabbit has no significant local pus and secretions produced.
【Conclusions】 There are no obvious difference about the amount of staphylococcus adhesion amongJDBM, WE43, Ti alloy material. But different surface roughness has an obvious influence on theadhesion of bacteria. Surface roughness increases, bacteria adhesion increase at the same time. Thematerial surface is smoother, the bacteria adhesion is less. We made the animal innfectionon biofilmmodel of prosthesis infection very easily by inject the bacterial ST239and SF8300of1X105concentration around the femoral implants of New Zealand white rabbits, while the injection of saline didnot cause obvious locality infection. The experimental results show that different bacterial species havedifferent infection ability to tissues near rabbit femur in the presence of a prosthesis implanted.
Part IV JDBM and fibrinogen with internalization effect ofstaphylococcus on osteoblasts
【Objective】 In orthopedics infection, a variety of pathogenic staphylococcus can enter bone cells, inwhich they grow and reproduce, produce toxins, destruct the cells, and cause cells damage, collapse andnecrosis in the end, or survive in the cells for a long time, as the result, the antibiotic cannot kill them.The purpose of this experiment is to study the difference of on the internalization role among clinicalStaphylococcus strains to the osteoblast cells, and study the effect of fibrinogen on the internalization ofthe test bacteria.
【Methods】 MC3T3-E1osteoblast cells were cultured in the24well plate with DMEM containing10%Australia fetal bovine serum medium,1ml DMEM medium each hole, about5x104cells. Medium waschanged in two consecutive days, until cells growed to more than50%area. The concentration of23clinical strains and standard strains bacteria ATCC12593was adjusted to0.5Maxwell unit with DMEMcomplete medium containing fetal bovine serum through bacteria quantity turbidimetric instrument, theamount of bacteria is about1x108/ml. Absorbed off the medium inside the culture holes, each hole arerespectively added to1ml medium containing experimental bacteria and put the culture plate into thecarbon dioxide incubator and incubated for2hours at37℃. Then we washed the culture holescontaining the bacteria for three times with PBS. Each culture hole is added in1ml DMEM completemedium containing90ug/ml vancomycin, and put the plate into the carbon dioxide incubator andcultured for2hours at37℃. The culture holes were washed three times with PBS to remove the deadbacteria killed by vancomycin outside the cultured.0.1%Triton was added in and played role for10min,so cells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. The liquid earned was diluted with PBS to102,104folds of concentration gradient dilution, and scratchedthem onto the blood agar culture plates and culture for one day at35℃, the plates were observed in thenext day if there has bacteria growth on them. If the bacteria were found on the blood agar culture plates,comparing the differences in magnitude and counting the amount of the bacteria. Repeated theexperiment for3times and compared if there were differences among the results of every times. As forthe effects of fibrinogen on bacteria internalization, MG63osteosarcoma cells were cultured with DMEMmedium containing10%Australian fetal bovine in the24well plate. When the cells developed to close tocover all of the bottom of the wells,9strains of pathogenic bacteria isolated from orthopedics clinicalinfection and ATCC12228, ATCC12598, RP62A were added to each well according to the concentrationof108/ml,1ml culture liquid containing bacteria strain every well. After3hours of culture, we absorbedthe culture liquid, and added1ml DMEM complete medium containing100ug/ml vancomycin, then weput the culture plate into carbon dioxide incubator and cultured the cells overnight at37℃. The next daywe absorbed the culture medium of every wells, and washed every culture well with PBS liquid for3times, and then added0.1%Triton prepared with PBS liquid to each well and let it play role for10min, socells disintegrated and the intracellular bacteria were released into the liquid of the culture wells. Theliquid earned was diluted with PBS liquid to102,104folds of concentration gradient dilution, andscratched them onto the blood agar culture plates and culture for one day at35℃, the plates wereobserved in the next day if there has bacteria growth on them, and the amount of the bacteria observedwere counted. We also research the effect of JDBM extract liquid on staphylococcus internalization,replaced the medium with JDBM, Ti, and WE43extract liquid.
【Results】 The effects of internalization on osteoblast cells between every clinical bacteria strains areunequal, some bacteria have a very strong internalization effect on bone cells, and the other strains cannotbeen seen completely with the internalization effects of osteoblasts. Internalization effect ofstaphylococcus aureus is stronger in generally, and their virulence is bigger. Observed throughmicroscopic, osteoblasts with Staphylococcus aureus put in about0.5h, there are visible bacteria inosteoblasts. Let the bacteria play a role for a long time, we can see destruction or collapse of the cells. Ingenerally, internalization bacteria strains added into osteoblast cells cultured in wells plate for a certainperiod of time, added vancomycin into the wells for2h and then stopped the effect of the drug, the nextstop, washed them with PBS for3times to clear the remain of the antibacterial drug. The cleaning liquidobtained was scribed to the sheep blood agar culture plates and cultured for one day, a small amount ofbacteria may be seen sometimes. Our experimental results show that there are no obvious differenceswith the amount of internalization bacteria no matter if there is fibrinogen intervention. The extract liquidof JDBM, WE43and Ti has no obvious effects on staphylococcus internalization with osteoblasts.
【Conclusions】 In generally, the virulence of staphylococcus aureus is stronger than Staphylococcusepidermidis. The SA has a strong invasiveness. More amount of bacteria can be seen in cells with the testof internalization experiments, and they grow in cells presenting a large of groups. While the virulence of Epidermal staphylococcal is weak, their invasion force is weak, their internalization effect is also weak,this present a scattered distribution in cells. Different clinical staphylococcus epidermidis strains havedifferent degree of internalization effect. Culturing together with osteogenic cells, after vancomycin playa role for2h and stopped the drug’s effect with cleaning for3times by PBS, some strains can be culturedout on the blood agar. Because the bacteria are evenly distributed in the space of the trend. Theoccurrence of this phenomenon may be due to bacteria associated with osteogenic cells through somesort of mechanism and the killing bacteria effect of vancomycin turn weaker in the result, or because thebacteria have a evenly distributed trend in the space, when vancomycin’s killing effect is stopped, theintracellular survival bacteria shift to the outside of the cell. Our experiment results show that fibrinogenor JDBM has no obvious influence on experimental bacterial internalization effect.
引文
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[1] Staiger MP, Pietak AM, Huadmai J, et al. Magnesium and its alloys as orthopedic biomaterials:a review. Biomaterials,2006,27:1728-1734.
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[1] Wang X, Huang J, Zhang T, et al. Cytoskeleton reorganization and FAK phosphorylation areinvolved in lanthanum (III)-promoted proliferation and differentiation in rat osteoblasts.Progress in Natural Science,2009,19:331-335.
[2] Zhang X, Yuan G, Mao L, et al. Effects of extrusion and heat treatment on the mechanicalproperties and biocorrosion behaviors of a Mg-Nd-Zn-Zr alloy. J Mech Behav Biomed Mater,2012,7:77-86.
[3] Zhang X, Yuan G, Niu J, et al. Microstructure, mechanical properties, biocorrosion behavior,and cytotoxicity of as-extruded Mg-Nd-Zn-Zr alloy with different extrusion ratios. J MechBehav Biomed Mater,2012,9:153-162.
[4] Feyerabend F, Fischer J, Holtz J, et al. Evaluation of short-term effects of rare earth and otherelements used in magnesium alloys on primary cells and cell lines. Acta Biomaterialia,2010,6:1834-1842.
[5] Wang Y, Ouyang Y, Pang X, et al. Effects of degradable MG-ND-ZN-ZR alloy on osteoblasticcell function. Int J Immunopathol Pharmacol,2012,25:597-606.
[6] Wang Y, Zhu Z, He Y, et al. In vivo degradation behavior and biocompatibility of Mg-Nd-Zn-Zralloy at early stage. Int J Mol Med,2012,29:178-184.
[7] Yang C, Yuan G, Zhang J, et al. Effects of magnesium alloys extracts on adult human bonemarrow-derived stromal cell viability and osteogenic differentiation. Biomed Mater,2010,5:045005.
[8] Hort N, Huang Y, Fechner D, et al. Magnesium alloys as implant materials–Principles ofproperty design for Mg–RE alloys. Acta Biomaterialia,2010,6:1714-1725.
[9] Witte F, Kaese V, Haferkamp H, et al. In vivo corrosion of four magnesium alloys and theassociated bone response. Biomaterials,2005,26:3557-3563.
[10] Hirano S, Suzuki KT. Exposure, metabolism, and toxicity of rare earths and related compounds.Environ Health Perspect,1996,104Suppl1:85-95.
[11] Ji YJ, Xiao B, Wang ZH, et al. The suppression effect of light rare earth elements onproliferation of two cancer cell lines. Biomed Environ Sci,2000,13:287-292.
[12] Chen Y, Yang L, Feng C, et al. Nano neodymium oxide induces massive vacuolization andautophagic cell death in non-small cell lung cancer NCI-H460cells. Biochem Biophys ResCommun,2005,337:52-60.
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[1] Staiger MP, Pietak AM, Huadmai J, et al. Magnesium and its alloys as orthopedic biomaterials:a review. Biomaterials,2006,27:1728-1734.
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[1] Wang X, Huang J, Zhang T, et al. Cytoskeleton reorganization and FAK phosphorylation areinvolved in lanthanum (III)-promoted proliferation and differentiation in rat osteoblasts.Progress in Natural Science,2009,19:331-335.
[2] Zhang X, Yuan G, Mao L, et al. Effects of extrusion and heat treatment on the mechanicalproperties and biocorrosion behaviors of a Mg-Nd-Zn-Zr alloy. J Mech Behav Biomed Mater,2012,7:77-86.
[3] Zhang X, Yuan G, Niu J, et al. Microstructure, mechanical properties, biocorrosion behavior,and cytotoxicity of as-extruded Mg-Nd-Zn-Zr alloy with different extrusion ratios. J MechBehav Biomed Mater,2012,9:153-162.
[4] Feyerabend F, Fischer J, Holtz J, et al. Evaluation of short-term effects of rare earth and otherelements used in magnesium alloys on primary cells and cell lines. Acta Biomaterialia,2010,6:1834-1842.
[5] Wang Y, Ouyang Y, Pang X, et al. Effects of degradable MG-ND-ZN-ZR alloy on osteoblasticcell function. Int J Immunopathol Pharmacol,2012,25:597-606.
[6] Wang Y, Zhu Z, He Y, et al. In vivo degradation behavior and biocompatibility of Mg-Nd-Zn-Zralloy at early stage. Int J Mol Med,2012,29:178-184.
[7] Yang C, Yuan G, Zhang J, et al. Effects of magnesium alloys extracts on adult human bonemarrow-derived stromal cell viability and osteogenic differentiation. Biomed Mater,2010,5:045005.
[8] Hort N, Huang Y, Fechner D, et al. Magnesium alloys as implant materials–Principles ofproperty design for Mg–RE alloys. Acta Biomaterialia,2010,6:1714-1725.
[9] Witte F, Kaese V, Haferkamp H, et al. In vivo corrosion of four magnesium alloys and theassociated bone response. Biomaterials,2005,26:3557-3563.
[10] Hirano S, Suzuki KT. Exposure, metabolism, and toxicity of rare earths and related compounds.Environ Health Perspect,1996,104Suppl1:85-95.
[11] Ji YJ, Xiao B, Wang ZH, et al. The suppression effect of light rare earth elements onproliferation of two cancer cell lines. Biomed Environ Sci,2000,13:287-292.
[12] Chen Y, Yang L, Feng C, et al. Nano neodymium oxide induces massive vacuolization andautophagic cell death in non-small cell lung cancer NCI-H460cells. Biochem Biophys ResCommun,2005,337:52-60.
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