Basic Research Of Zoledronic Acid-loaded Bone Cement As Drug Delivery Carrier

Part I The Solidification Time and Biomechanies of Zoledronic acid-loaded Bone CementObjective polymethylmethacrylate (PMMA) bone cement has played such an important role and has been in widespread use in orthopedics in the last 60 years. With the coming of ageing society, End-stage joint diseases happen more and more frequently in clinic.Artificial joint replacement which needs bone cement as a filler is a effective therapeutic method for such diseases and is widely used in clinical.In the meantime,vertebral diseases which are caused by metastatic bone tumor and osteoporosis appeared to be an increasing trend.And percutaneous vertebroplasty is a feasible and rational way to relieve symptoms.Therefore, PMMA bone cement has an irreplaceable role in the clinic.And it seem to have no additional benefit for these diseases,just as a biological material.Bisphosphonates are a kind of synthetic pyrophosphate analogues,which are used in the treatment of osteoporosis,metastatic bone disease,hypercalcemia,Paget disease and some other metabolic bone diseases.It aslo can effectively preventing aseptic loosening through inhibiting osteolysis caused by particulate wear debris.The purpose of this part is to explore the influence on solidification time and biomechanies,which has changed after we add zoledronic to PMMA bone cement,to lay the foundation for the clinic application of the Zoledronic acid-loaded Bone Cement.Methods Zoledronic acid mixed with bone cement in five different mass fractions:A(0mg), B(4mg), C(20mg), D(40mg), E(80mg).Materials preheat in a 23±1℃ condition for two hours, then we make cylindrical bone cement with aseptic operation in a surgical environment. Measured cylindrical samples’solidification time by the Vicat apparatus every once in a while.The needle of the Vicat apparatus has a diameter of lmm,a wight of 400g.Drop Vicat apparatus’needle to the sample surface carefully with 5 seconds and record solidification time until the indentation don’t appear.Under the same conditions,wo make compressive specimens and bending specimens with reference to the ISO5833:2002 standard.Then test their compressive and flexural strength with electronic universal material testing machine.Results The addition of zoledronic has no effect on the bone cement solidification time. 4mg、20mg、40mg and 80mg zoledronic addition group do not have significantly different solidification time when compared with controls (P>0.05).Mechanical properties of zoledronic acid-loaded done cement show that,the compressive strength、flexural strength and flexural modulus are decreased with the increasing amount of zoledronic.The reduction of 40mg and 80mg zoledronic addition group were statistical differences in three mechanical properties compared to the controls (P<0.05).Leaching after 4 weeks,the compressive strength of all groups are on the rise except E group.For the flexural strength and flexural modulus,E group has a decreased flexural strength after 4 weeks extraction and other groups are unchanged.Conclusion The additional zoledronic acid has a effect on compressive strength、flexural strength and flexural modulus of bone cement.And these indicators show a trend of decrease with the increase amount of zoledronic acid,but they all up to the ISO5833:2002 《Implants for surgery:acrylic resin cements》 standard. Therefore 44g bone cement which is mixed with less than 80mg zoledronic acid will have no influence on the physical and mechanical property.Part Ⅱ The Release property of Zoledronic acid-loaded Bone CementObjective Zoledronic acid is a representative drug of third generation bisphosphonates,and know sa the strongest bisphosphonates on clinic. The drug excretes quickly through the kidney by intravenous infusion,therefore it has low bioavailability and nephrotoxicity.The rest of the drug slow-release from bone tissue and high concentrations of zoledronic acid cannot form in the specified site.Local releasing which has attracted increasing attention can be a useful way to solve these problems Zoledronic acid-loaded Bone Cement as a local delivery system can form a slow-release and high-concentration environment to enhance the drug effect on osteoclast、osteoblast and tumor cells.The release property of composite bone cement is very important for us,by which wo dicide whether it can be used on the clinic and acknowledge its microstructure indirectly.The purpose of this part is to explore the release property of composite bone cement by HPLC, summarize its characteristics and provide the pharmacokinetic foundation for clinic use.Methods Zoledronic acid mixed with bone cement in five different mass fractions:A(0mg), B(4mg), C(20mg), D(40mg), E(80mg).The samples used in leaching experiment is same sa compressive specimens Materials preheat in a 23±1℃ condition for two hours, then we make cylindrical bone cement with aseptic operation in a surgical environment following ISO5833:2002《Implants for surgery:acrylic resin cements》.Then select qualified specimen that has the right size、flat surface and uniform density by Vernier caliper and X-ray apparatus Five groups with 4 in each group.Place each simple inside 5ml sterile deionized water and elute in a 37℃ condition.We collect eluents every 24 hours and put in 5ml new sterile deionized water after washing three times. 1、2、3、4、5、6、7、9、11、14、28d samples in each group will send to our test center for concentration measurement by HPLC.Results The detectiom signal appears at 11th minute according to our HPLC detecting condition.Every group has the biggest release concentration in the first day, and with the increase amount of Zoledronic acid the release concentration increases in the same test point.The release concentration of each group decreases quickly in the previous three days. When it come to the fifth day, the concentration of Zoledronic acid in lixiviating sample of Team B is exceed the detection range. On the eleventh day, Each team from C to E do not detest the concentration of Zoledronic acid. The curve of release is matched with the index curve model:the R-value from team B to E was 0.994,0.950,0.973,0.990 in sequence. The release percentage of Team B at every time is the maximum, which reached 40.36% on forth day. By the seventh day, the release percentage of Team C to E, in turn,28.34%,15.90%, 12.91%。Conclusions Zoledronic acid can release from compound bone cement. The total release amount of each team is increasing and the total release percentage of every time is decreasing, when adding more and more Zoledronic acid. The Zoledronic acid-loaded Bone Cement which presenting index figure has a obvious releasing in first three days.Part Ⅲ The Cells bioresearch of lixiviating samples from Zoledronic acid-loaded Bone CementObjective The polymerization of methyl methacrylate monoer caused rapidly when the bone cement liquid phase was mixed with its solid phase,and has a high reaction temperature between 70-120 ℃.Whether this high temperature will affect Zoledronic acid pharmaceutical properties is a concerned problem which determines its clinical applications.The root cause of aseptic loosening is a destabilizing performance of ossification and osteolysis around the prosthesis. Zoledronic acid can significantly inhibite osteoclast activity and proliferation when in 10’9M concentration.Although there do not have many researchs about the relation between Zoledronic acid and osteoblast.The incidence of malignant tumors are rising in recent years,and bone metastasis is one of the metastasis path ways.Breast cancer is more common tumor that is in the habit of metastasizing,and Zoledronic acid can inhibit the growth of breast cancer cell by reducing FPPS activity and finally activating caspase-apoptosis pathway.The lixiviating samples from Zoledronic acid-loaded bone cement are acted on osteoblast and breast cancer cell to probe whether the polymerization exotherm has a effect on Zoledronic acid pharmaceutical properties by MTT experiment,and provide reference for clinic use.Methods Elute the compound bone cements which respectively contain 0mg、4mg、20mg、 40mg,80mg Zoledronic acid 24 hours with 15ml L-15 mediums to get processed mediums,and which will be used to breast cancer cell on 96-well plate. Primary rat calvaria osteoblasts were isolated from neonatal SD rats,and purified by centrifuge and in vitro culture.The 1、9、14、21d lixiviating samples of E group were selected as a part of experimental groups,and the DMEM mediums which contain 10-5M、10-7M、10-9M、10-11M Zoledronic acid as another part of experimental groups.Apply these processed DMEM mediums to rat calvaria osteoblasts on 96-well plate,and set a unprocessed mediums as blank control. To study the effect of Zoledronic acid on rat osteoblasts and breast cancer cell, cell count and MTT analysis were used in observation of cell proliferation to study Zoledronic acid pharmaceutical properties which is released from the loaded bone cement.Results The Zoledronic acid which is released from the loaded bone cement has cytotoxic effects on breast cancer cell.The inhibiting effect increases with increase the concentration of Zoledronic acid and reached the maxium value for 60.4%.For rat osteoblast, the Zoledronic acid has a obvious inhibitory effect at 10-5M and a anxoaction at 10-11M.The 10-7M、10-9M groups have no statistical difference with blank group in four MTT tests.The group that contains 21 d lixiviating sample of E group has significant difference with blank group starting from the fifth day, and show up a proliferation promotion effect to osteoblast. The rest lixiviating sample groups have no statistical difference with blank group in four MTT tests.Conclusions The Zoledronic acid released from the bone cement still has pharmacological activity,and has a proliferation promotion effect to osteoblast when at a low concentration, a promotion effect at a high concentration.With the increase of Zoledronic acid concentration, breast cancer cell bear a growing proliferation inhibition effect..