Study Of Ex Vivo OPG Gene Therapy In Preventing Wear Debris Induced Osteolysis

BackgroundPeriprosthetic osteolysis with subsequent aseptic loosening remains the most frequent and relevant long-term complication associated with total joint arthroplasty. Presently, there are no proven therapeutic agents for the prevention and treatment of osteolysis. Revision joint arthroplasty is the major choice for catastrophic failure of the implant from osteolysis,but consuming greater healthcare resources, having a poorer clinical result and a shorter duration of survival. In the face of these challenges, precautionary measures and noninvasive treatment alternatives are greatly needed for prevention and treatment of periprosthetic osteolysis.It is well understood that particulate wear debris such as Ti particles induce an activation of phagocytic cells at the bone-implant interface which initiates an osteolytic cascade involving peri-prosthetic granulomatous inflammation and subsequent osteoclastic bone resorption. Enormous efforts have been directed at preventing osteolysis,including development of improved bearing materials and optimizing prosthesis design,but can not eliminate wear debris at all and provide effective measures in process of osteolysis.Recently,the effects of RANKL/RANK/OPG on particle- induced osteoclastic bone resorption with aseptic loosening of total joint prostheses have been illustrated,and OPG is regarded to have the greatest potential for treatment of osteolysis.In consideration of some disadvantages of OPG protein therapy, this study aimed to explore the potential of ex vivo OPG gene therapy as a treatment for Ti particle-induced osteolysis in a murine calvarial model.ObjectiveThe encoding gene of OPG was obtained from human osteosarcoma cell line MG63.Generation of the transfer vector (pcDNA3.1-OPG-IRES-EGFP) was carried out via multiple subcloning steps.The recombinant plasmid pcDNA3.1-OPG -IRES-EGFP was transformed into C2C12 cell line and stable transfectants were selected .Implantation of stable cells inhibiting partical-induced bone loss was demonstrated, thus providing the foundation for prevention and treatment of bone resorption diseases.Materials and Methods1. Using the isolated total RNA from human osteosarcoma cell line MG63, the cDNA encoding OPG was amplified by semi-nested reverse transcription-polymerase chain reaction (RT-PCR) method. Double-stranded DNA sequencing of the OPG cDNA was performed to confirm that there were no mutations in this region of the pGEM-T Easy vector.2. Recombinant vector pcDNA3.1-OPG-IRES-EGFP was constructed by directional cloning the target gene fragments into eukaryotic expression vector pcDNA3.1(+). Extensive restriction digests and PCR were performed to confirm the authenticity of the vector.3. Using Lipofectamine?2000 mediated method, C2C12 cells were transfected by pcDNA3.1-OPG-IRES-EGFP and pcDNA3.1(+) control plasmid.Stable clones were obtained by observing the expression of EGFP after G418 screening. The expressions of EGFP and OPG were then analyzed in the cultures of the stable cells by green fluorescence microscopy and ELISA,respectively.4. Wafers were incubated in the osteoclasts and osteoblasts media with culture supernatants from stable cells. The wafers were treated for 7 days, then stained with 1 % toluiding blue and pits on the surface of the whole wafer were quantified.5. A mouse calvaria model of Ti partical-induced osteolysis was erected. 40uL Ti particles were used together with or without 107 C2C12-OPG cells. SHAM animals received surgery alone without particles, and C2C12-pcDNA3.1 control group received 107 cells without particles. The EGFP-expression C2C12 were traced in cryosections using fluorescent microscopy.The sagittal suture areas and the number of osteoclasts were quantified, and the average sagittal suture area and the average number of osteoclasts for each group of animals were determined. Significance was determined with SPSS 10.0.Results1. The PCR product of hOPG was cloned into pGEM-T Easy and sequenced, and the OPG cDNA was identical with correlative literatures.2. The authenticity of recombinant vector pcDNA3.1-OPG-IRES-EGFP was verified by extensive restriction digests and PCR.3. Stable C2C12-pcDNA3.1 and C2C12-OPG were obtained after G418 screening for four weeks . After 72h of culture the OPG from 106 stable C2C12-OPG cells peaked at 279.3pg/ml ,but stable C2C12-pcDNA3.1 cell lines produced undetectable amounts of OPG in the supernatants.4. The resulting pits showed OPG with biological activity from C2C12-OPG inhibited osteoclastic bone resorption.5. A mouse calvaria model of Ti particle-induced osteolysis is erected successfully .The EGFP-expression C2C12-OPG in cryosections were verified by fluorescent microscopy, showing live cells after transplantation.Ti-treated animals induced significant bone resorption and osteoclastogenesis and C2C12-OPG gene therapy reduced the suture area and osteoclastogenesis.Conclusions1. EGFP was beneficial to get stable cell lines and trace the transplantation cells as a reporter gene.2. The above-mentioned results demonstrated that OPG gene and EGFP gene were simultaneously expressed independently in the C2C12 cell line by the regulation of IRES sequence.3. Ti particles induced osteolysis.OPG ex vivo gene therapy was effective in blocking osteoclastogenesis and osteoclastic bone resorption, and may be useful to prevent osteolysis or treat patients for aseptic loosening.