Construction And Expression Of The Adenovirus-mediated BMP-2and TNF-α-SiRNA In Cellular Level

Objective To examine whether the constructed recombinantadenovirus(ad-TNF-α-siRNA) efficaciously down regulation TNF-α release from activatedmocrophages in response to titanium particles, We also observed the inhibition effects ofad-TNF-α-siRNA on titanium particle induced osteoclast formation and bone resorption in thepresence of RANKL,and investigated impacts of the conditioned medium of macrophageschallenged with titanium particles (Ti CM) and ad-TNF-ɑ-siRNA (Ti-ad CM) onosteoblasts(MC3T3E-1).Methods Recombination Adenovirus(ad) vector carrying both target genes(TNF-a-siRNA and BMP-2)was synthesized, which transfected into osteoclastic RAW264.7and osteoblastic MC3T3-E1cells, respectively, detection of the target gene BMP-2expressionand silencing of TNF-α gene in cells by real-time PCR and westernblot. Mouse RAW264.7cell line was induced to osteoclast by RANKL and titanium,which was treatmented by ad-BMP-2-TNF-α-siRNA to detect osteoclast specific gene and identify with tartrate resistantacid phosphatase (TRAP) staining and Measuring osteoclasts absorb bone area. mouseMC3T3-E1cell line was induced to osteoblast which was identified with alkalinephosphatase(ALP).Results Recombinant adenovirus Ad-BMP-2-TNF-α-siRNA could down-regulationtitanium particles-induced the expression of TNF-α on RAW264.7cells.Ad-BMP-2-TNF-α-siRNA inhibit titanium particles-induced osteoclastogenesis and boneresorption. It has been confirmed that Recombination Adenovirus (Ad-BMP-2-TNF-α-siRNA)can induce osteoblast differentiation treat with conditioned medium of macrophagesRAW264.7challenged with a combination of titanium (Ti) particles and Ad-siTNFα-BMP2(Ti-ad CM) is assessed by alkaline phosphatase (ALP) activity. Thereceptor activator of nuclear factor (NF)-κB ligand (RANKL) was down regulation onpro-osteoblastic MC3T3-E1cells treated with Ti-ad CM comparison to with conditionedmedium of macrophages RAW264.7challenged with titanium (Ti) particles(Ti CM).Conclusion Recombination Adenovirus can inhibit the formation of osteoclasts, whilepromoting the differentiation of osteoblast cells, which may reduce or prevent theperiprosthetic bone loss. Our findings suggest that ad-TNF-a-siRNA-BMP-2effects and maybe useful for the prevention and/or and treatment of osteolysis and loosening after TJA.