Fabrication, Characterization, And Biological Assessment Of Multilayer RhBMP-2DNA Coatings On Sandblasted Dual Acid Etched Titanium Surface

Achieving osseointegration (stable fixation by bone formation directly onto the implant surface) is a major goal of implantology. A new strategy has been developed which coat titanium surfaces with multilayer gene coatings to achieve osseointegration. The objective of this study was to evaluate the effect of the BMP-2gene coating surface on cell viability, proliferation, ALP activity, OC secretion and calcium deposition in vitro, and also peri-implant bone formation and osseointegration at titanium implants coated with BMP-2cDNA placed into canine models in vivo.First titanium discs were sandblasted and dual acid etched, multilayer gene coatings were generated using the layer by layer assembly technique. The roughened titanium disks were initially immersed in5mg/ml chitosan solution (PH=2.8) for20min, thus obtaining a precursor layer with a stable positive charge to initiate the layer-by-layer assembly process. The titanium disks having a chitosan layer were dipped into the0.5mg/ml hyaluronan solution (PH=7.2) and kept for5min at room temperature to adsorb hyaluronan electrostatically onto the surfaces. Next, the disks were exposed to the cationic liposome/pEGFP-C1-BMP-2DNA complexes solution for5min at room temperature to adsorb cationic liposome/DNA complexes onto the surfaces. These procedures were repeated sequentially to obtain a multilayer consisting of hyaluronan and cationic liposome/DNA complexes. The XPS survey indicated that the N element was found increasing from the coating. The wettabilities of the multilayered titanium coating were different from those of native porous titanium surfaces, owing to the hydrophilic natures of hyaluronan and cationic liposome/DNA complexes. Fluorescence microscope of labeled plasmids showed each additional bilayer resulted in an increase in the amount of plasmid DNA present on the titatanium discs. All these results demonstrated that BMP-2gene DNA were successfully fabricated on the titanium surfaces, and cell transfection expriments showed MC3T3-E1could successfully be transfected and could express EGFP.Then MC3T3-E1cells were cultured on the roughend titanium surfaces coated with multilayer of pcDNA3.1+rhBMP-2plasmid, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2secreted into the mediums by the MC3T3-E1cells and the effect of the various surfaces on cell viability, proliferation, ALP activity, OC secretion and calcium deposition were evaluated. Messenger mRNA levels of OC, ALP, Runx2and Osterix were also investigated. The results demonstrated that rhBMP-2protein secreted into culture medium at3days was significantly higher than control groups. MC3T3-E1cells cultured on the rhBMP-2gene coating displayed significantly higher ALP activity and OC secretion at7days and14days culture, respectively. MC3T3-E1cells cultured on rhBMP-2gene coating upregulated expression of mRNA level of the osteoblast differentiation markers, especially on days12for OC and on days6and12for ALP and Osx.After that rhBMP-2plasmids coated implants were installed into the edentulated posterior mandible in twelve adult beagle dogs. After4,8, and12weeks in situ, mandibles were retrieved and prepared for removal torque testing and histomorphometric evaluation. Histomorphometric analysis showed that the intra-thread bone area was slightly higher for test implants than for control implants after4and8weeks of healing, respectively, but no statistically significant differences between rhBMP-2plasmid coated and control implants both in bone area and bone-implant contact rate at any time points. Mechanical tests showed that mean removal torque values of rhBMP-2plasmids coated implants revealed greater than that of control implants after8weeks of healing.In conclusion, rhBMP-2plasmid coated implants slightly enhanced/accelerated peri-implant bone formation and osseointegration, and may be a new method for implant surface modification.