| 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. |
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