【作者】 宋德业；

【导师】 王万春；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 目的无菌性松动是关节假体置换术后失败的主要原因,目前无特别有效的防治办法。二磷酸盐是一类人工合成的焦磷酸类似物,它可作用无菌性松动的多个环节如提高假体周围骨密度、抑制术后骨量丢失等,有望成为防治无菌性松动的理想药物。它的给药方式主要是口服,存在生物利用度低、服用时间长、治疗费用昂贵和上消化道溃疡等不足。局部用药是一个比较理想的给药途径。骨水泥是多种药物的良好载体,把阿伦磷酸钠加入骨水泥中是否安全、有效地防治无菌性松动,必须进行骨水泥生物力学、洗提特性、细胞生物学的研究,本文就从这几个方面初步探讨复合阿伦磷酸钠骨水泥的可行性并确定骨水泥中阿伦磷酸钠合适的加入量。方法1.生物力学研究（1）在50g骨水泥粉末中分别加入0 mg、10 mg、50 mg、100mg、500mg和1000mg制备骨水泥压缩、弯曲（模量）强度和疲劳实验标本。用INSTRON 8032型万用测试仪测定各组骨水泥浸提前和浸提4W后的压缩强度、弯曲（模量）强度和张力性疲劳寿命。四点弯曲试验标本断面和疲劳实验标本孔隙率分别用电镜和MCT检测。（2）用推出实验检测6组不同阿伦磷酸钠加入量的骨水泥-金属界面浸提前和浸提4W后的界面剪切强度,实验后骨水泥标本表面用电镜扫描。在大白兔股骨远端制备骨水泥-骨界面,在术后1天和60天用推出实验检测界面剪切强度并扫描界面周围骨密度。2.洗提实验在50g骨水泥粉末中分别加入10mg、50mg、100mg、500mg和1000mg阿伦磷酸钠制备浸提标本,每组1个标本放在30ml生理盐水中,在37℃恒温箱里浸提24周,在1、2、3、4、5、6、7、9、11、14、21、28、35、42、49、56、84、112、140、168d取样品5ml,在高效液相色谱仪串联质谱仪上测定阿伦磷酸钠的浓度,计算各时点的释放速率和释放总量百分比。3.细胞生物学研究（1）破骨前体细胞Raw264.7与不同浓度的阿伦磷酸钠(0M、10^-9M、10^-8M、10^-7M、10^-6M和10^-5M)在RANKL存在条件下培养8天,细胞计数检测破骨前体细胞向破骨细胞分化的功能;同条件下培养14天后取出培养板中的骨磨片行电镜扫描,观察骨吸收陷窝的数目并用Image-Pro（R）Plus软件分析骨吸收陷窝面积检测骨吸收功能。（2）在50g骨水泥粉末中分别加入0mg、10mg、50mg、100mg、500mg和1000mg阿伦磷酸钠制备浸提标本和粘附标本。各组骨水泥标本浸提液或未浸提培养液与成骨样细胞MG-63共培养,检测成骨样细胞MG-63增殖、细胞毒性、凋亡、碱性磷酸酶活性、蛋白合成及骨矿化能力。用电镜扫描与成骨样细胞MG-63共培养的粘附标本,观察细胞表面粘附情况。结果1.在50g骨水泥粉末中分别加入10 mg、50 mg、100 mg、500 mg和1000 mg阿伦磷酸钠对浸提前和浸提4W后的压缩强度、张力性疲劳寿命、疲劳标本和骨水泥-金属界面孔隙率,浸提前的弯曲模量和骨水泥-金属界面剪切强度,术后第1天骨-骨水泥界面的剪切强度和骨密度无明显影响;当分别加入50 mg、100 mg、500 mg、1000mg阿伦磷酸钠时可显著增大术后第60天骨-骨水泥界面的剪切强度和骨密度;当加入1000mg阿伦磷酸钠时浸提前和浸提4W后的弯曲强度、浸提后的弯曲模量、浸提后的骨水泥-金属界面剪切强度显著性减少。2.各组骨水泥中阿伦磷酸钠释放速率先快后慢,在同一时间点随加入量增加而增大。各组阿伦磷酸钠释放总量百分比在快速释放期相似,约11%;在缓慢释放期不同,但在24周时均不超过25%。释放总量百分比斜率在快速释放期相似,在缓慢释放期也不同:500mg组斜率最小,10mg组最大。3.阿伦磷酸钠随浓度(0M-10^-5M)增加呈剂量依赖性抑制破骨细胞骨吸收功能;高浓度(10^-6M和10^-5M)时明显抑制破骨细胞分化,而低浓度(10^-9M、10^-8M和10^-7M)时对细胞分化无影响。在50g骨水泥粉末中分别加入10 mg、50 mg、100 mg、500 mg和1000mg阿伦磷酸钠对成骨样细胞MG-63无毒性,对特异性碱性磷酸酶活性、胞体外钙化结节形成和增殖无显著性影响;前4组显著抑制细胞凋亡,而1000mg组使细胞凋亡加剧。结论1.各组骨水泥浸提前和浸提4W后的静态机械性能均符合骨水泥ISO5833（2002）标准。在50g骨水泥粉末中阿伦磷酸钠不超过500mg时不会降低浸提前和浸提4W后的压缩强度、弯曲（模量）强度、张力性疲劳寿命、骨水泥-金属界面剪切强度和术后1、60天骨-骨水泥界面的剪切强度。2.骨水泥是一种良好的阿伦磷酸钠的载体和缓释体,阿伦磷酸钠在骨水泥固化时未被破坏且可从骨水泥中缓慢释放,释放浓度随加入量增加而增加。3.各组复合阿伦磷酸钠骨水泥无成骨细胞毒性,其药物释放浓度在较长时间（至少24周）高于抑制破骨细胞的最小有效浓度。更多还原

【Abstract】 OBJECTIVEAseptic loosening is the main failure reason after prosthetic replacement for joints and there are no effective methods for its prevention and cure.Bisphosphonate is a class of compounds whose chemical constitution is similar with pyrophosphate.It can act on lots of steps during development of aseptic loosening such as periprosthetic bone density after operation et.al,and may become a ideal drug.Oral administration is the main way for alendronate and it have lots of shortcomings such as low bioavailability,long time’s drug administration, expensive treatment cost,upper disgestive tract ulcer,et,al.Local application is a better route of administration.Bone cement had been proved as a good drug carrier.It is unknown that alendronate -impregnated bone cement is effective or not for aseptic loosening.In order to solve it,there are three questions to study which are mechanical properties of bone cement,drug-releasing law of alendronate in bone cement and effects on osteoblasts and osteoclasts.Therefore,this experiment studied these three aspects and according to these results, feasibility of alendronate-impregnated bone cement and the adapted addition of alendronate in bone cement were determined.METHODS1.Study of biomechanics （1）Samples of compression strength,bending strength,bending modulus and fatigue life were prepared with bone cements added 10mg,or 50mg、or 100mg、or 500mg,or 1000mg alendronate in 50g bone cement powders respectively.Compression strength,bending strength, bending modulus and tensive fatigue life of bone cements were examinated by INSTRON 8032 tester before elution and after elution for 4 weeks.Broken samples of four-point bending test were examinated by SEM and porosity rates of fatigue samples were determinated by MCT.（2）Cement-metal interface’s shear strength of six groups with different alendronate in 50g bone cement powders were examinated before elution and after elution for 4 weeks.Surfaces of bone cement samples after test were observed by SEM.Cement-bone interfaces of six groups with different alendronate in 50g bone cement powders were prepared in distal femur of rabbits and their shear strength was examinated on 1d and 60d after operation.Bone densities around bone cement were evaluated.2.Elution experimentsSamples Lixiviated were prepared with bone cement added 10mg,or 50mg、or 100mg、or 500mg,or 1000mg alendronate in 50g bone cement powders respectively.One sample of each group was dipped into 30ml 0.9%physiological saline and Lixiviated for 24 weeks.5 ml leaching liquor of each group was collected at 1、2、3、4、5、6、7、9、11、14、 21、28、35、42、49、56、84、112、140、168d,and concentrations of alendronate were detected by mass spectrometer combined with high performance liquid chromatograph.Alendronate’s release percent of total amount and its releasing velocity of each group were determined at different time points.3.Cells bioresearch（1）Osteoclast precursor Raw264.7 were co-cultured with different concentrations of alendronate(0M、or 10^-9M、or10^-8M、or10^-7M、or10^-6M, or10^-5M)for 8 days in presence of RANKL and osteoclast differentiation was detected by cells account.After 14 days under the forementioned conditions,bone slices of each group were taken out of culture plates and examinated by SEM.Numbers and morphous of absorption lacuna were observed and their absorption areas were evaluated by image-Pro（?）Plus software.（2）Lixiviating and adhesive samples were prepared with bone cement added 10mg,or50mg、or 100mg、or 500mg,or 1000mg alendronate in 50g bone cement powders respectively.Osteoblast-like cells MG-63 were co-cultured with leaching liquor of samples from different groups and their proliferation,apoptosis,alkaline phosphatase activities,protein synthesis ability and bone mineralization capability were evaluated. Adhesive samples cultured with osteoblast-like cells MG-63 were examinated by SEM and cell adhesion and morphous on samples’ surfaces were observed.RESULTS1.10mg,or 50mg、or 100mg、or 500mg,or 1000mg alendronate added in 50g bone cement powders had no effects on compression strength,tensive fatigue life,porosity rates of fatigue samples and cement-metal interfaces before elution and after elution for 4 weeks, flexural modulus and shear strength of cement-metal interfaces before elution,shear strength and bone density in cement-bone interface of 1d after operation.When 50mg、or 100mg、or 500mg,or 1000mg alendronate was added,shear strength and bone density in cement-bone interface of 60d after operation increased remarkedly.When addition of alendronate was 1000mg,significant reduction was found in flexural strength before elution and after elution for 4 weeks,flexural modulus and shear strength of cement-metal interfaces after elution for 4 weeks.2.Alendronate in bone cement of all groups released quickly in early stage and then slowly in late stage,and its releasing velocity at the same time point increased step by step with more addition of alendronate.All groups had similar releasing percent of total amount and its slope in quick-release stage.However,in slow-release stage they became different. Maximal and minimal slope were found in 500mg group and 10mg group respectively.3.Alendronate with concentration range from 0M to 10^-5M inhibited bone absorption in dose-dependent way.However,alendronate inhibited osteoclast differentiation in high concentrations(10^-5M and 10^-6M)and there are no effects in low concentrations(10^-7M、10^-8M and 10^-9M). Alendronate whose addition was 10 mg,or 50 mg,or 100 mg,0r 500 mg,or 1000mg in 50g acrylic bone cement powders,had no cytotoxicity on osteoblast-like cells MG-63.Four groups with less amount of alendronate inhibited their apoptosis in dose-dependent way and largest amount group showed contrary effect on osteoblast-like cells MG-63.10mg,or 50mg、or 100mg、or 500mg,or 1000mg alendronate added in 50g bone cement powders had no effects on specific AKP activities,proliferation and formation of extracellular calcified tubercles.CONCLUSIONS1.Static mechanical properties of bone cement in all groups accorded to standard of ISO 5833（2002）for acrylic bone cement.Less than 500mg alendronate added in 50g bone cement powders didn’t decrease compressive strength、flexural（modulus）strength,tensive fatigue life and shear strength of cement-metal interface before elution and after elution for 4 weeks,shear strength of cement-bone interface on 1d and 60d after operation significantly.2.Bone cement was a good slow-release carrier of alendronate.It wasn’t destructed and released from cement slowly.When more alendronate was added,its releasing concentrations increased gradually. 3.All alendronate-impregnated bone cement had no cytotoxicity on osteoblasts and their releasing concentration lasted for a long time（more than 24 weeks）above minimum effective drug concentration for osteoclasts.更多还原