Fabrication And Biological Assessment Of Multilayer DNA Coatings On Sandblasted-dual Acid Etched Titanium Implant Surface

Modifications of the implant surface and gene therapy have been appealing more and more attention, immobilizing functional gene onto irregular oral implant surface has become an advanced challenge. This study aims to describe the fabrication of the multilayer DNA coatings using electrostatic layer-by-layer (LBL) assembly technique, so that introduce the gene of coding green fluorescent protein (GFP) to the surface of implant.Electrostatic layer-by-layer (LBL) assembly technique was used to fabricate the multilayer DNA coatings. The titanium disk having a PEI (Linear poly (ethylene imine), PEI) layer was dipped to the HA (hyaluronic acid, HA) solution and adsorbed electrostatically HA onto the surface. The HA-loaded disk was exposed to an LDc (lipid-DNA complex (LDc), LDc) solution in Opti-MEM at room temperature to adsorb LDc onto the surface. That is to say Ti-PEI-(HA/LDc)1. These procedures were repeated sequentially to obtain a multilayer consisting of HA and LDc. So the GFP gene encoding green fluorescent protein was fabricated onto the porous titanium surface depending on the negatively charged hyaluronic acid and positively charged LDc. X-ray photoelectron spectroscopy (XPS) was performed to confirm the composition of elements on the surface of the sandblasted-dual acid etched titanium implant surface. Ti, N, O and C signals appeared in control group, while in the group of assembling 5 layers, the complete disappearance of the Ti signal combined with the presence of Na, P and S signals and the increase of N signals. The surface topography of titanium disks were determined by Field Scanning Electron Microscopy. The SEM indicated that the spherical particles appeared on the titanium surface and increased with the layer numbers. Transmission electron microscopy (TEM) was used to examine the morphology of plasmid DNA. The outcome displayed that the DNA could be released from assembled multilayered DNA coatings in phosphate buffered saline and the released buffer gradually turned into more loosely but still possess the transcriptional activity. Contact angle measurement generally indicated the wettable properties of the measured substrates. The wettabilities of multilayered titanium films were superior to the control group.In vitro biological assessment:MC3T3-el cells were inoculated on the assembled surface to observe the expression of GFP after incubation for 24 hours and 72 hours, respectively. Moreover, cell viability and morphology were also tested. The results indicated that Ti-PEI-(HA/LDc)8 possessed the highest EGFP expression efficiency when cultured for 24h. And all the EGFP expression efficiencies, whether the assembled layer numbers, displayed that at 72h was lower than 24h culture. And the higher assembled layers, the greater cytotoxicity, but compared with the control group significantly higher viability was displayed.The LBL technology can be applied to achieve the goal of loading the gene of GFP onto the roughed titanium surface. The gene of GFP could be transfected into pre-osteoblasts without any external conditions. Moreover, the GFP expression efficiency was achieved with the increasing of layer numbers. And the DNA coatings fabricated by LBL assembly technique improved the cytocompatibility of native porous titanium surface.