Prevention And Treatment Effect Of Zoledronic Acid Combined With Teriparatide On Prosthetic Aseptic Loosening In Knee Model Of Rabbit

Objectives 1)To successfully establish titanium particles induced aseptic loosening model of knee in rabbit.2)To explore the prevention and treatment effect of Zoledronic Acid combined with Teriparatide on aseptic loosening and to verify the underlying mechanism,in order to establish the foundation for further study of non-surgery therapy of aseptic loosening.Methods 1)A total of 16 New Zealand white rabbits,average weight 2.85 kg,were randomly divided into two groups.All animals were operated on the right knee after 1 weeks adaptive feeding.The anterior middle incision was adopted to expose the knee joint.Titanium rod prosthesis,length 2.0cm and 0.5cm in diameter,was implanted into the femoral medullary cavity along the longitudinal axis from just above the femoral endpoint of posterior cruciate ligment.Titanium particles,weight 50 ? g and 20 ? m in diameter,were coated evenly on the surface of titanium rod prosthesis inthe study group while nothing was coated in the control group.Each rabbit of both groups was treated with the same protocol perioperatively and was sacrificed 12 weeks after surgery.Datas were collected as below:X-ray photos of the ipislateral knee 2 days before sacrificing;Hematoxylin and Eosin(H-E)staining of the synovial membrane,undecalcificated slicing of bones including titanium rod were performed with Goldner's trichrome staining and Von Kossa staining;The interface membrane tissues were performed with Real-time PCR and Western Blot to detect the m RNA and protein content of OCN,OPG,RANKL and TRAP5 b.Statistic analysis between the two groups was performed to confirm whether the model was successfully eatablished and to verify the mechanism.2)Another 24 of New Zealand white rabbits were involved and were operated on the right knee with the same method as step 1.Titanium rod prosthesis coated evenly with titanium particles on the surface,weight 50?g and 20?m in diameter,were implanted into the right femoral medullary cavity of each animal.Three groups,8 in each group,were divided according todifferent drugs received postoperatively.Zoledronic Acid(ZL)or Teriparatide(TP)or Zoledronic Acid combined with Teriparatide(ZL+TP)were given to the animals of different group from the first day after surgery to the sacrificed day of 12 weeks postoperatively.Datas were collected as below:X-ray photos of the ipislateral knee 2 days before sacrificing;undecalcificated slicing of bones including titanium rod were performed with Goldner's trichrome staining and Von Kossa staining;the interface membrane tissues were performed with Real-time PCR and Western Blot detect the m RNA and protein content of OCN,OPG,RANKL and TRAP5 b.The study group of the first part was involved as the control group(CG)of this part for statistic analyse with the other three groups,to confirm the prevention and treatment effect of Zoledronic Acid combined Teriparatide and mechanism on prosthetic aseptic loosening.Results 1)Obvious signs of periprosthetic osteolysis were detected in the study group upon the X-ray examination before sacrificing.The result of H&E staining of synovial membrane showed that obvious inflammatory cell infiltration and synovial were observed in the synovial membrane of the sdudy group.Goldner's trichrome staining of bony hard sections showed poor combination at bone-prosthesis interface of the study group and the gap around prosthesis was mainly filled with fibrous connective tissue.While the integration of bone and prosthesis in the control group was better.The bone tissue was more evenly distributed around the prosthesis without no significant fibrous connective tissue proliferation.The Bone-Prosthesis contact ratio(BPCR)and Bone Volume Percentage(BVP)in the study group were lower than those in the control group.Von Kossa staining of bone hard tissue sections showed that calcium salt deposition islands(CSDI)around the prostheses of the study group were mainly linear,with a larger number but a smaller area.However,The CSDIs of the control group were mostly star-shaped with the larger single area but the number was less.The results of Real-time PCR and Western Blot of interface membrane tissue around prosthesis showed that the m RNA and protein content of OCN and OPG increased in the study group,while m RNA and protein content of RANKL and TRAP5 b decreased,and the OPG/RANKL ratio of study group was significantly lower than the control group.2)Although obvious osteolysis signs could be detected in all groups,there was no significant difference according to the X-ray examination results.Goldner's trichrome staining of undecalcificated bones slicing showed that The bone-implant contact rate and the bonevolume percentage of ZL,TP and ZL+TP group were higeher than the CG group.The VonKossa staining showed that the numbers of calcium salt deposition island of ZL,TP andZL+TP group were less than CG group,however,the average areas of calcium saltdeposition island were larger than the control group.The results of Real-time PCR andWestern Blot of interface membrane tissues around prosthesis showed that the m RNAand protein content of OCN increased in ZL,TP and ZL+TP group,however,the amount ofincrease was easpecially greater in TP and ZL+TP group.Meanwhile,the m RNA andprotein content of TRAP5 b decreased in ZL,TP and ZL+TP group,and the amount ofdecrease was easpecially greater in ZL and ZL+TP group.Compared with the CG group,the m RNA and protein content of OPG were higher,but RANKL were lower in ZL,TPand ZL+TP groups.So the OPG/RANKL ratio of the three groups was obviously higherthan CG group and the ratio of ZL+TP group was especially higher.Conclusions 1)The method of implanting titanium rod prosthesis coating with 20?m titanium particles into the femoral medullary cavity of rabbits could make continuous titanium particles stimulation around the prosthesis,which could effectively induce osteolysis and prosthetic loosening.The main mechanism was that the microm titanium particles could induce macrophages to produce multiple inflammative factors,which could stimulate osteoclast differentiation through OPG/RANKL/RANK signaling pathway,resulting in osteoelastic osteolysis.Meanwhile,the particles could also suppress the function of osteoblasts and reduce new bone formarion around the prosthesis.Both of the two aspects contributed to the result of aseptic loosening.This rabbit model was ideal in etiology to modulate the whole process of prosthetic aseptic loosening.The model had the advantages of being economical,effective,relatively fast and repeatable,which could be used either in etiology and or in biomechanics research.It could also be apllied in a variety of ways of prevention and treatment research on aseptic loosening.2)Zoledronic acid and teriparatide could inhibit the activation of osteoclasts induced by titanium particles through OPG/RANKL/RANK pathway,and upregulate the function of osteoblasts,which played a key role in prevention and treatment of prosthetic aseptic loosening through inhibiting osteolysis and promoting bone formation around prosthesis.Zoledronic acid was more potent in inhibiting osteoclast activation and reducing periprosthetic osteolysis,while teriparatide mainly activated osteoblast function and was more powerful in promoting osseointegration around the prosthesis.The combination ofthese two drugs played a synergistic effect on inhibition of osteolysis and more powerful to promot bone formation.Overall,zoledronic acid combined with teriparatide may be a potential effective choice in the prevention and treatment of prosthetic aseptic loosening.