The Effect Of Adenovirus-mediated Sirna Targeting BMPR-â…¡ On UHMWPE-induced Osteoclast Formation

BackgroundTotal joint replacement is a common procedure that has provenhighly successful for the treatment of rheumatoid arthritis, ostseoarthritis,femoral head necrosis, hip fractures in the elderly and other diseases. Itreduces pain, restores joint function, and allows arthritis patients to returnto varied activities of daily living. Aseptic loosening(AL) is the single mostcommon complication of total joint arthroplasty. The long-term outcome oftotal joint replacement surgery remains compromised by weardebris-associated implant loosening. The critical factor may contribute toloosening is the adverse tissue response to wear debris. The particles arephagocytosed by macrophages, activate an inflammatory cascade, result ina bone resorption process and promote osteoclastogenesis at thebone-implant interface. A growing body of literature suggests that BMPsinfluence the formation and activity of osteoclasts, and BMP signalingplays an important role in the osteoclast formation. So we will employ anRNA interference approach by transfecting a small interfering RNA (siRNA) specific for BMPR in osteoblast and osteoclast, to inhibit BMPsignaling pathways, regulate osteoclast formation directly or indirectly, andreduce osteolysis. BMP signaling plays a two-pronged role which othersignaling pathways do not have.ObjectiveTo construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene. In order to observe the effect ofadenovirus-mediated siRNA targeting BMPR-II on UHMWPE-inducedosteoclast formation, and the effects of BMPR-II signaling on osteoclastformation are mediated directly by osteoclast itself, as well as indirectly byaltered expression of RANKL and OPG in osteoblast.Methods1. To construct the recombinant adenovirus vector containing siRNAtargeting BMPR-II gene using modified AdEasy system, the designedsiRNA oligonucleotide fragments of BMPR-II gene were cloned intoshuttle plasmid pSES-HUS to construct pSES-HUS-siBMPR-II plasmid.Afterward, the correct recombinant was linearized by Pmel, followingco-transformation with the backbone vector pAdEasy-1in Escherichia coliBJ5183to construct pAd-siBMPR-II plasmid, and then transfected into293T cell line via Lipofectamine2000to package the adenovirus. Viralsupernatants were harvested. The viral titers were determined by infecting293T cells with serial dilutions of concentrated adenoviruses. 2. In vivo studies, firstly, we should build the air pouch model inwhich the differentiation and formation of osteoclasts are induced byUHMWPE, then test the effect of adenovirus-mediated siRNA targetingBMPR-II on UHMWPE-induced osteoclast formation by real-time PCR,Western blot, TRAP staining, immunohistochemistry, immunofluorescence,electron microscopy and so on.3. In vitro studies, firstly, we should build the bone marrow cells andprimary osteoblasts culture system.(1) In the bone marrow cells culture system, to test whether theadenovirus-mediated siRNA targeting BMPR-II can inhibit the osteoclastdifferentiation by real-time PCR, Western blot, TRAP staining and so on.(2) In the primary osteoblasts culture system, to measure whether theadenovirus-mediated siRNA targeting BMPR-II can changed the gene andprotein expression of RANKL and OPG by real-time PCR, Western blot.Results1. Adenovirus-mediated siRNA targeting BMPR-II was builtsuccessfully. And adenovirus-mediated BMPR-II siRNA could successfullyinhibit BMPR-II expression in high efficiency both at mRNA and proteinlevels.2. The air pouch model with UHMWPE was built successfully. Wefound that siBMPR-II inhibited differ-entiation of osteoclasts in theUHMWPE-stimulated pouches, including reduction of gene and protein expression levels of TRAP and RANK, and TRAP positive cells, decreasein the area of absorbing lacuna.3. Furthermore, we revealed that loss of BMPR-II signaling inhibitsosteoclast differentiation directly, and acts on osteoblasts to reduceosteoclast formation through altered expression of RANKL and OPG,including down-regulation of the amount of TRAP positive cells inducedby RANKL, the gene and protein expression levels of osteoclast marker(TRAP and RANK) in the bone marrow cells culture system, and resultingin an increase of OPG mRNA and protein expression levels, a decrease ofRANKL expression in the primary osteoblasts culture system.ConclusionsIn the present study, we revealed that locally injection ofadenovirus-mediated siRNA targeting BMPR-II appears to be a feasibleand effective candidate to treat or prevent wear debris-associated osteolysis.Furthermore, we revealed that the effects of BMPR-II signaling onosteoclast formation are mediated directly by osteoclast itself, as well asindirectly by altered expression of RANKL and OPG in osteoblast.