Fabrication,Characterization,and Biological Assessment Of Multilayer Laminin5γ2DNA Coatings On Titanium Surface

Backgroud:The attachment loss of soft tissues around dental implants is one of the most important reasons resulting in implant failures. It has been demonstrated that the deficient expression of Laminin-5(LN-5) in internal basal lamina (IBL) is associated with the weak attachment between PIE and titanium at the coronal and middle portion of the dental implant-PIE interface. To get long-term resistance to oral bacterial intefection and maintain the fuctional stability of the implant, some studies attempt to further improve the surface properties of titanium in order to establish a well soft tissue-implant biologic seal. Over the past decades, gene therapy has become an important technique in the field of biomedicine. To investigate the adhesion mechanism of epithelium on titanium surface at the gene level, may be a potential method to promote the healing of implant soft tissue. Our previous studies demonstrated that gene-functionalized titanium surface constructed through LBL approach could transfer plasmid DNA into cells.Objective:This work was to improve implant property via the fabrication of multilayer gene-functionalized films on titanium surfaces and investigate the effect of the gene coatings on HEK293cells. Thus provided a viable idea for the formation of the bioactive implant surface.Methods:Multilayer gene-functinalized films were fabricated by layer-by-layer (LBL) assembly technique on titanium surfaces. They were composed of chitosan (CS), hyaluronic acid (HA), cationic liposome (Lip) and pGFP-M61-LAMC2(LDc). Surfaces analysis was carried out by X-ray photoelectron specteoscopy (XPS), atomic force microscope (AFM) and surface contact angle measurements (SCA). To investigate coating degradation, LDc were labeled by IT nucleic acid labeling kits and titanium substrates were visualized with fluorescence microscope. The HEK293cell biological characteristics of the coatings were evaluted by in vitro experiments on titanium surfaces.Result:The study of XPS, AFM and CA showed polyelectrolyte multilayer films could be successfully deposited. DNA degradation behaviors in the multilayer films were dependent on the number of layers. The cells transfected with plasmids were able to express rh-y2chain. The result of ELISA showed that the concentration of LN-5on the control surface was always significantly lower than others (P<0.01). The concentation of LN-5on the surface of CS-(HA-LDc)5was higer than others at4d (P<0.05) while there was no difference at2d and6d between CS-(HA-LDc)5and CS-(HA-Lip)5. The effect of the various surfaces on cell proliferation and adhension were evaluated. The results demostrated that HEK293cells cultured on the multilayer films displayed significantly high adhension activity than control group in48h (P<0.01). After4days and6days culture, HEK293cells viability on the surface of CS-(HA-LDc)5was higher than others (P<0.05).Conclusion:The multilayer structure consisting of cationic lipid and LAMC2DNA can be fabricated via the LBL assembly process on titanium. The DNA-loaded multilayer provides the surface with good biocompatibility. Plasmid DNA is efficiently transferred to the HEK293cells cultured on the multilayer substrate surface, which show a better attachment and proliferation ability. It was concluded that the multilayer laminin γ2DNA coating was a convenient and effective approach to improve cell adhesion and proliferation on implant biomaterials.