纳米银骨水泥预防关节置换术后感染的实验研究
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摘要
研究背景
关节感染是关节置换术后的一个灾难性并发症，对此采取了多种措施，其中抗生素骨水泥能有效预防感染，但同时却出现了越来越多感染对抗生素耐药，对这些病例，骨水泥中的抗生素不能起到保护作用。因此，需要考虑新的抗菌剂。
银作为抗菌剂自远古以来就开始使用，现在已用于医学的不同领域，如心血管植入物，隐形眼镜，尿管和创口护理。纳米技术把银变成纳米大小从而有更高的比表面积，与微生物有更好的接触，具有良好的抗菌、抗病毒性能。
骨水泥是固定人工关节的金标准，如果纳米银加入骨水泥中同样具有抗菌性，那么使用纳米银骨水泥对预防关节置换术后感染、提高关节感染翻修成功率的临床应用提供初步依据。
第一部分纳米银骨水泥机械性能的检测
目的:检测骨水泥中加入少剂量的纳米银对骨水泥的机械性能会否产生不良影响。方法:纳米银粉末和骨水泥粉末混合后再与骨水泥单体混合真空搅拌，制成浓度为0.1％、0.5％、1.0％的纳米银骨水泥，以普通骨水泥作为对照组，根据机械性能测量要求制成相应形状的试件，用万能材料试验机和气动疲劳试验机测量各种骨水泥的抗压性能、抗弯曲强度、抗疲劳性能。结果:0.1％、0.5％、1.0％纳米银骨水泥的抗压性能、抗弯曲强度、抗疲劳性能与普通骨水泥相比无显著性差异（P>0.05）。结论:骨水泥中加入少剂量纳米银制成浓度低于1.0％对骨水泥的机械性能无不良影响。
第二部分纳米银骨水泥的体外抗菌性能及细胞毒性
目的:评估纳米银骨水泥有无抗菌活动及细胞毒性。
方法:以普通骨水泥和2%庆大霉素骨水泥分别作为阴性对照组和阳性对照组，浓度分别为0.1%、0.5%、1%的纳米银骨水泥作为实验组，采用体外杀菌实验的活菌计数法测试各组骨水泥对表皮葡萄球机、耐甲氧西林表皮葡萄球菌的抗菌活性。以有毒的1%聚乙二醇辛基苯基醚（Triton X-100）和无毒的细胞培养液作为对照组，1%纳米银骨水泥培养后的提取液作为实验组，各组加入小鼠成纤维细胞并培养，检测乳酸脱氢酶释放量和蛋白质含量以测试体外细胞毒性。
结果:1%纳米银骨水泥和2%庆大霉素骨水泥完全抑制了表皮葡萄球菌的增殖，有较强的杀菌作用。1%纳米银骨水泥完全抑制了耐甲氧西林表皮葡萄球菌的增殖，有较强的杀菌作用，而庆大霉素骨水泥对耐甲氧西林表皮葡萄球菌没有抑菌作用。纳米银骨水泥组和无毒对照组间乳酸脱氢酶释放无显著性差异（P＞0.05），而有毒Triton组和银骨水泥组间乳酸脱氢酶释放有显著性差异（P＜0.01），有毒的Triton组和无毒的细胞培养组间也有显著性差异（P＜0.01）。纳米银骨水泥提取液中总蛋白含量和无毒的细胞培养液中总蛋白含量无显著性差异（P＞0.05）；有毒的对照组和纳米银银骨水泥之间及有毒对照组和无毒的细胞培养液间有显著性差异（P＜0.01）。
结论:1%纳米银骨水泥对表皮葡萄球菌和耐甲氧西林表皮葡萄球菌都有良好的抗菌活性。2%庆大霉素骨水泥对表皮葡萄球菌有良好的抗菌活性，但对耐甲氧西林表皮葡萄球菌没有抗菌活性。1%纳米银骨水泥体外实验没有细胞毒性。
第三部分纳米银骨水泥预防膝关节置换术后感染的实验研究
目的:检验纳米银骨水泥对生物膜形成的影响及对感染是否有预防作用。
方法:在兔子左股骨髁间凹内沿股骨髓腔方向植入一长约3cm克氏针，外露1mm并用普通骨水泥固定模拟关节置换术。于皮肤缝合好后往关节腔内注射0.5ml不同浓度的表皮葡萄球菌，术前、术后检测血沉、C反应蛋白，术后两周取关节内组织作细菌培养，确定合适浓度的表皮葡萄球菌制作感染模型。
然后把36只兔子随机分为三组，每组12只，以普通骨水泥作为阴性对照组，2%庆大霉素骨水泥作为阳性对照组，以1%纳米银骨水泥作为实验组。根据分组，用不同的骨水泥固定假体，然后关节腔内注射合适浓度的表皮葡萄球菌0.5ml，术后定期检测各组血沉、C反应蛋白，术后2周对假体表面行扫描电镜观察及取关节腔内组织作细菌培养。
结果:0.5ml1×10~6CFU/ml和1×10~8CFU/ml的表皮葡萄球菌均能制成感染模型。选择1×10~6CFU/ml作为预防感染时接种菌浓度。在预防感染的实验中，1%纳米银骨水泥组和2%庆大霉素骨水泥组的血沉和C反应蛋白较普通骨水泥组明显低（P＜0.01），纳米银骨水泥组和庆大霉素骨水泥组间血沉和C反应蛋白无明显差异(P>0．05)；细菌培养阳性率示普通骨水泥组10~0%(12／12)，庆大霉素骨水泥组0％(0／12)，纳米银骨水泥组0％(0／12)，纳米银骨水泥、庆大霉素骨水泥组与普通骨水泥组之间有显著性差异(P<0．01)，庆大霉素骨水泥组与纳米银骨水泥组间无显著性差异(P>0．05)；扫描电镜观察到普通骨水泥组假体表面细菌聚集并成簇，形成生物膜，纳米银组和庆大霉素组假体表面细菌孤立，没有簇形成。
结论:1%纳米银骨水泥能抑制兔膝关节置换术后生物膜的形成，能起到有效预防感染作用。
Background
Infection after joint replacement is a catastrophic complication. Many measures havebeen taken to prevent infection. Among them, antibiotics impregnated in bone cement is aneffective one, but at the same time it has given rise to an increasing number ofmicroorganisms resistant to antibiotics. Antibiotics in the bone cement can not play apreventive role for these microorganisms. There is a need to find a new antimicrobialagent.
Silver has a strong antimicrobial potential, which has been used since the ancient times.Now it is used in different areas of medicine, such as cardiovascular implants, contactlenses, catheter and wound care. Nanotechnology changes silver into the nano-size andthus it has a better contact with the microorganisms, owing to high surface-area-to-volumeratio..Nano-silver has proved to have good antibacterial and antiviral properties.
The gold standard for fixation of artificial joints is bone cement. If nanoparticles silveradded to the bone cement has antibacterial properties, it will provide a preliminary basisfor prevention arthroplasty infections and improvement of success rate for infectedarthroplasty.
Part One
Objective: To detect wether the addition of a small dose of.nano-silver to bone cementwill have adversely effect on the mechanical properties of bone cement.
Methods: Nano-silver powder and bone cement polymer powder were mixed, then thebone cement monomer was added and stirred under vacuum.0.1%,0.5%,1.0%nano-silverbone cement were measured as study groups, while polymethylmetacrylate(PMMA) ascontrol group. Compressive properties, bending strength and fatigue properties were testedfor every group.
Results: There was no significant difference between0.1%,0.5%,1.0%nano-silver bonecement and ordinary bone cemen when mechanical properties such as compressiveproperties, bending strength and fatigue properties were compared (P>0.05).Conclusion: The addition of a dose less than1.0%nano-silver to bone cement had noadverse effects.on the mechanical properties of bone cement.
Part Two
Objective: To assess whether nano-silver bone cement have antimicrobial activity andcytotoxicity.in vitro.
Methods: Experiments were divided into five groups: ordinary bone cement was used asnegative control group and2%gentamicin bone cement was used as positive control group,0.1%,0.5%,1%nano-silver bone cement were studied as experimental group. Viable cellscount of in vitro bactericidal test was tested to measure the antibacterial activity of everygroup of bone cement to Staphylococcus epidermidis and methicillin-resistantStaphylococcus epidermidis. In vitro cytotoxicity testing was measured by release oflactate dehydrogenase and protein content of the incubated mouse fibroblasts. Experimentsfor in vitro cytotoxicity testing were divided into three groups: extraction medium after1%nano-silver bone incubated was studied as experimental group,while1%toxic TritonX-100as postive control group and non-toxic cell culture medium as a negative controlgroup.
Results:1%nano-silver bone cement and gentamicin bone cement completely inhibittedthe proliferation of Staphylococcus epidermidis, showing strong bactericidal effect.1%nano silver bone cement completely inhibitted the proliferation of methicillin-resistantStaphylococcus epidermidis (MRSE), while2%gentamicin bone cement had no inhibitoryeffect on MRSE. There was no significant difference in LDH release of1%nano-silverbone cement when compared with that of non-toxic control group (P＞0.05). There wassignificant difference in LDH release of1%nano-silver bone cement when compared withthat of toxic Triton X-100(P＜0.01). There was no significant difference in the amount of
total protein between extraction liquid from nano-silver bone cement and that of non-toxiccell culture medium (P＞0.05). There was significant difference in the amount of totalprotein between extraction liquid from nano-silver bone cement and that of toxic TritonX-100(P＜0.01). There was significant difference in the amount of total protein betweennon-toxic cell culture medium and that of toxic Triton X-100(P＜0.01).
Conclusion:1%nano-silver bone cement had strong antibacterial activity againstStaphylococcus epidermidis and MRSE. Gentamicin bone cement had strong antibacterialactivity against Staphylococcus epidermidis, but had no effect on MRSE.1%nano-silverbone cement was free of in vitro cytotoxicity.
Part Three
Objective: To evaluate the effect of1%nano-silver bone cement on the formation ofbiofilm and preventing infection.
Methods: Implant of a Kirschne about3cm long in concave of rabbit femoral condylesimulated arthroplasty, with1mm expsosed in articular space. The Kirschne was fixed withordinary bone cement. After skin closure,0.5ml different concentrations of Staphylococcusepidermidis were injected into knee. Erythrocyte sedimentation rate and C-reactive proteinwere measured preoperatively and postoperatively. Tissues in the knee were cultured forbacterial two weeks postoperatively. Then appropriate concentration of Staphylococcusepidermidis was choosed to establish infection model.
36rabbits were randomly divided into three groups, each group consisted of12rabbits:ordinary bone cement was treated as a negative control group,2%gentamicin bone cementwas treated as a positive control group, and1%nano-silver bone cement was treated asexperimental group. According to group, different bone cement was used to fixe prosthesis.Then0.5ml suitable concentration of Staphylococcus epidermidis was injected into knee.Erythrocyte sedimentation rate and C-reactive protein were measured regularly aftersurgery. Biofilm on the surface of prosthesis was detected by scanning electron microscope(SEM) and tissue in the knee were cultured for bacterial two weekspostoperatively.
Results:0.5ml1×10~6and1×10~8S. epidermidis could successfully lead to infection and1×10~6was choosed for next step experiment. In experiment of preventing infection,erythrocyte sedimentation rate and C-reactive protein was significantly lower innano-silver bone cement group and gentamicin bone cement group than that of ordinarybone cement group (P <0.01). There was no significant difference between nano-silverbone cement group and gentamicin bone group in erythrocyte sedimentation rate andC-reactive protein (P>0.05). Positive rate of bacterial culture for ordinary bone cementgroup, gentamicin bone cement group and nano-silver bone cement group was10~0%(12/12),0%(0/12) and0%(0/12) respectively; there was significant difference betweennano-silver bone cement group and ordinary bone cement group (P <0.01), there wassignificant difference between gentamicin bone cement group and ordinary bone cementgroup (P <0.01), no significant difference existed betwwen nano-silver bone cement groupand gentamicin bone cement group (P>0.05). SEM showed that bacteria on the surface ofthe prosthesis gathered and clusters，forming biofilm in ordinary bone cement group;andthat the bacteria isolated, no cluster formation in nano-silver group and gentamicin group.Conclusion:1%nano-silver bone cement inhibitted biofilm formation and preventedinfection in knee replacement of rabbit.
关节感染是关节置换术后的一个灾难性并发症，对此采取了多种措施，其中抗生素骨水泥能有效预防感染，但同时却出现了越来越多感染对抗生素耐药，对这些病例，骨水泥中的抗生素不能起到保护作用。因此，需要考虑新的抗菌剂。
银作为抗菌剂自远古以来就开始使用，现在已用于医学的不同领域，如心血管植入物，隐形眼镜，尿管和创口护理。纳米技术把银变成纳米大小从而有更高的比表面积，与微生物有更好的接触，具有良好的抗菌、抗病毒性能。
骨水泥是固定人工关节的金标准，如果纳米银加入骨水泥中同样具有抗菌性，那么使用纳米银骨水泥对预防关节置换术后感染、提高关节感染翻修成功率的临床应用提供初步依据。
第一部分纳米银骨水泥机械性能的检测
目的:检测骨水泥中加入少剂量的纳米银对骨水泥的机械性能会否产生不良影响。方法:纳米银粉末和骨水泥粉末混合后再与骨水泥单体混合真空搅拌，制成浓度为0.1％、0.5％、1.0％的纳米银骨水泥，以普通骨水泥作为对照组，根据机械性能测量要求制成相应形状的试件，用万能材料试验机和气动疲劳试验机测量各种骨水泥的抗压性能、抗弯曲强度、抗疲劳性能。结果:0.1％、0.5％、1.0％纳米银骨水泥的抗压性能、抗弯曲强度、抗疲劳性能与普通骨水泥相比无显著性差异（P>0.05）。结论:骨水泥中加入少剂量纳米银制成浓度低于1.0％对骨水泥的机械性能无不良影响。
第二部分纳米银骨水泥的体外抗菌性能及细胞毒性
目的:评估纳米银骨水泥有无抗菌活动及细胞毒性。
方法:以普通骨水泥和2%庆大霉素骨水泥分别作为阴性对照组和阳性对照组，浓度分别为0.1%、0.5%、1%的纳米银骨水泥作为实验组，采用体外杀菌实验的活菌计数法测试各组骨水泥对表皮葡萄球机、耐甲氧西林表皮葡萄球菌的抗菌活性。以有毒的1%聚乙二醇辛基苯基醚（Triton X-100）和无毒的细胞培养液作为对照组，1%纳米银骨水泥培养后的提取液作为实验组，各组加入小鼠成纤维细胞并培养，检测乳酸脱氢酶释放量和蛋白质含量以测试体外细胞毒性。
结果:1%纳米银骨水泥和2%庆大霉素骨水泥完全抑制了表皮葡萄球菌的增殖，有较强的杀菌作用。1%纳米银骨水泥完全抑制了耐甲氧西林表皮葡萄球菌的增殖，有较强的杀菌作用，而庆大霉素骨水泥对耐甲氧西林表皮葡萄球菌没有抑菌作用。纳米银骨水泥组和无毒对照组间乳酸脱氢酶释放无显著性差异（P＞0.05），而有毒Triton组和银骨水泥组间乳酸脱氢酶释放有显著性差异（P＜0.01），有毒的Triton组和无毒的细胞培养组间也有显著性差异（P＜0.01）。纳米银骨水泥提取液中总蛋白含量和无毒的细胞培养液中总蛋白含量无显著性差异（P＞0.05）；有毒的对照组和纳米银银骨水泥之间及有毒对照组和无毒的细胞培养液间有显著性差异（P＜0.01）。
结论:1%纳米银骨水泥对表皮葡萄球菌和耐甲氧西林表皮葡萄球菌都有良好的抗菌活性。2%庆大霉素骨水泥对表皮葡萄球菌有良好的抗菌活性，但对耐甲氧西林表皮葡萄球菌没有抗菌活性。1%纳米银骨水泥体外实验没有细胞毒性。
第三部分纳米银骨水泥预防膝关节置换术后感染的实验研究
目的:检验纳米银骨水泥对生物膜形成的影响及对感染是否有预防作用。
方法:在兔子左股骨髁间凹内沿股骨髓腔方向植入一长约3cm克氏针，外露1mm并用普通骨水泥固定模拟关节置换术。于皮肤缝合好后往关节腔内注射0.5ml不同浓度的表皮葡萄球菌，术前、术后检测血沉、C反应蛋白，术后两周取关节内组织作细菌培养，确定合适浓度的表皮葡萄球菌制作感染模型。
然后把36只兔子随机分为三组，每组12只，以普通骨水泥作为阴性对照组，2%庆大霉素骨水泥作为阳性对照组，以1%纳米银骨水泥作为实验组。根据分组，用不同的骨水泥固定假体，然后关节腔内注射合适浓度的表皮葡萄球菌0.5ml，术后定期检测各组血沉、C反应蛋白，术后2周对假体表面行扫描电镜观察及取关节腔内组织作细菌培养。
结果:0.5ml1×10~6CFU/ml和1×10~8CFU/ml的表皮葡萄球菌均能制成感染模型。选择1×10~6CFU/ml作为预防感染时接种菌浓度。在预防感染的实验中，1%纳米银骨水泥组和2%庆大霉素骨水泥组的血沉和C反应蛋白较普通骨水泥组明显低（P＜0.01），纳米银骨水泥组和庆大霉素骨水泥组间血沉和C反应蛋白无明显差异(P>0．05)；细菌培养阳性率示普通骨水泥组10~0%(12／12)，庆大霉素骨水泥组0％(0／12)，纳米银骨水泥组0％(0／12)，纳米银骨水泥、庆大霉素骨水泥组与普通骨水泥组之间有显著性差异(P<0．01)，庆大霉素骨水泥组与纳米银骨水泥组间无显著性差异(P>0．05)；扫描电镜观察到普通骨水泥组假体表面细菌聚集并成簇，形成生物膜，纳米银组和庆大霉素组假体表面细菌孤立，没有簇形成。
结论:1%纳米银骨水泥能抑制兔膝关节置换术后生物膜的形成，能起到有效预防感染作用。
Background
Infection after joint replacement is a catastrophic complication. Many measures havebeen taken to prevent infection. Among them, antibiotics impregnated in bone cement is aneffective one, but at the same time it has given rise to an increasing number ofmicroorganisms resistant to antibiotics. Antibiotics in the bone cement can not play apreventive role for these microorganisms. There is a need to find a new antimicrobialagent.
Silver has a strong antimicrobial potential, which has been used since the ancient times.Now it is used in different areas of medicine, such as cardiovascular implants, contactlenses, catheter and wound care. Nanotechnology changes silver into the nano-size andthus it has a better contact with the microorganisms, owing to high surface-area-to-volumeratio..Nano-silver has proved to have good antibacterial and antiviral properties.
The gold standard for fixation of artificial joints is bone cement. If nanoparticles silveradded to the bone cement has antibacterial properties, it will provide a preliminary basisfor prevention arthroplasty infections and improvement of success rate for infectedarthroplasty.
Part One
Objective: To detect wether the addition of a small dose of.nano-silver to bone cementwill have adversely effect on the mechanical properties of bone cement.
Methods: Nano-silver powder and bone cement polymer powder were mixed, then thebone cement monomer was added and stirred under vacuum.0.1%,0.5%,1.0%nano-silverbone cement were measured as study groups, while polymethylmetacrylate(PMMA) ascontrol group. Compressive properties, bending strength and fatigue properties were testedfor every group.
Results: There was no significant difference between0.1%,0.5%,1.0%nano-silver bonecement and ordinary bone cemen when mechanical properties such as compressiveproperties, bending strength and fatigue properties were compared (P>0.05).Conclusion: The addition of a dose less than1.0%nano-silver to bone cement had noadverse effects.on the mechanical properties of bone cement.
Part Two
Objective: To assess whether nano-silver bone cement have antimicrobial activity andcytotoxicity.in vitro.
Methods: Experiments were divided into five groups: ordinary bone cement was used asnegative control group and2%gentamicin bone cement was used as positive control group,0.1%,0.5%,1%nano-silver bone cement were studied as experimental group. Viable cellscount of in vitro bactericidal test was tested to measure the antibacterial activity of everygroup of bone cement to Staphylococcus epidermidis and methicillin-resistantStaphylococcus epidermidis. In vitro cytotoxicity testing was measured by release oflactate dehydrogenase and protein content of the incubated mouse fibroblasts. Experimentsfor in vitro cytotoxicity testing were divided into three groups: extraction medium after1%nano-silver bone incubated was studied as experimental group,while1%toxic TritonX-100as postive control group and non-toxic cell culture medium as a negative controlgroup.
Results:1%nano-silver bone cement and gentamicin bone cement completely inhibittedthe proliferation of Staphylococcus epidermidis, showing strong bactericidal effect.1%nano silver bone cement completely inhibitted the proliferation of methicillin-resistantStaphylococcus epidermidis (MRSE), while2%gentamicin bone cement had no inhibitoryeffect on MRSE. There was no significant difference in LDH release of1%nano-silverbone cement when compared with that of non-toxic control group (P＞0.05). There wassignificant difference in LDH release of1%nano-silver bone cement when compared withthat of toxic Triton X-100(P＜0.01). There was no significant difference in the amount of
total protein between extraction liquid from nano-silver bone cement and that of non-toxiccell culture medium (P＞0.05). There was significant difference in the amount of totalprotein between extraction liquid from nano-silver bone cement and that of toxic TritonX-100(P＜0.01). There was significant difference in the amount of total protein betweennon-toxic cell culture medium and that of toxic Triton X-100(P＜0.01).
Conclusion:1%nano-silver bone cement had strong antibacterial activity againstStaphylococcus epidermidis and MRSE. Gentamicin bone cement had strong antibacterialactivity against Staphylococcus epidermidis, but had no effect on MRSE.1%nano-silverbone cement was free of in vitro cytotoxicity.
Part Three
Objective: To evaluate the effect of1%nano-silver bone cement on the formation ofbiofilm and preventing infection.
Methods: Implant of a Kirschne about3cm long in concave of rabbit femoral condylesimulated arthroplasty, with1mm expsosed in articular space. The Kirschne was fixed withordinary bone cement. After skin closure,0.5ml different concentrations of Staphylococcusepidermidis were injected into knee. Erythrocyte sedimentation rate and C-reactive proteinwere measured preoperatively and postoperatively. Tissues in the knee were cultured forbacterial two weeks postoperatively. Then appropriate concentration of Staphylococcusepidermidis was choosed to establish infection model.
36rabbits were randomly divided into three groups, each group consisted of12rabbits:ordinary bone cement was treated as a negative control group,2%gentamicin bone cementwas treated as a positive control group, and1%nano-silver bone cement was treated asexperimental group. According to group, different bone cement was used to fixe prosthesis.Then0.5ml suitable concentration of Staphylococcus epidermidis was injected into knee.Erythrocyte sedimentation rate and C-reactive protein were measured regularly aftersurgery. Biofilm on the surface of prosthesis was detected by scanning electron microscope(SEM) and tissue in the knee were cultured for bacterial two weekspostoperatively.
Results:0.5ml1×10~6and1×10~8S. epidermidis could successfully lead to infection and1×10~6was choosed for next step experiment. In experiment of preventing infection,erythrocyte sedimentation rate and C-reactive protein was significantly lower innano-silver bone cement group and gentamicin bone cement group than that of ordinarybone cement group (P <0.01). There was no significant difference between nano-silverbone cement group and gentamicin bone group in erythrocyte sedimentation rate andC-reactive protein (P>0.05). Positive rate of bacterial culture for ordinary bone cementgroup, gentamicin bone cement group and nano-silver bone cement group was10~0%(12/12),0%(0/12) and0%(0/12) respectively; there was significant difference betweennano-silver bone cement group and ordinary bone cement group (P <0.01), there wassignificant difference between gentamicin bone cement group and ordinary bone cementgroup (P <0.01), no significant difference existed betwwen nano-silver bone cement groupand gentamicin bone cement group (P>0.05). SEM showed that bacteria on the surface ofthe prosthesis gathered and clusters，forming biofilm in ordinary bone cement group;andthat the bacteria isolated, no cluster formation in nano-silver group and gentamicin group.Conclusion:1%nano-silver bone cement inhibitted biofilm formation and preventedinfection in knee replacement of rabbit.
引文
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