Study Of Pure Titanium Surface Activated By Amino-group Plasma On Osteoblast Adhesion And Its Mechanism

Objective and background:Osseointegration is a direct bone integration with no fibrous tissue between dental implant surface and alveolar bone. To achieve this gold standard, recently researchers have focused on dental implant surface modification methods to change surface morphology.Excellent osseointegration requires a bio-interface with stable adhesion to the titanium surface. In addition, conventional problems such as the lack of stable surface modification, lower surface modification efficiency, and difficulty controlling the modification effect, etc., intensify the need to find a high efficient method which does not require heat treatment or interface stress and is easy to operate and easy to control the surface modification effect.We used the PECVD method to obtain a novel bio-interface. Biologically active amino functional groups were introduced to the surfaces of titanium discs. Our preliminary studies demonstrated the modified surface stability and biocompatibility of pure titanium surface were increased obviously.New bone growth at the implant-bone interface occurs in two ways, i.e., distal and through contacting new bone growth and formation. It is generally believed that the theory new bone growth via contact osteogenesis would be a better explanation for the ideal status of the osseointegration. There are multiple types of cells responsible for bone formation. Osteoblasts are the major functional cells for bone formation. Osteoblasts secreted osteoid (organic compounds of bone matrix mineralization). As the deposition and mineralization of osteoid occur, osteoblasts become a lining cellular layer of bone or apoptosis. As the anchor-dependent cells, adhesion and clustering of osteoblasts on the surface of the dental implant is essential for contact osteogenesis. Cell adhesion is mainly mediated by integrin. Integrin α2is recognized as an important adhesion-related integrin subunit of the osteoblast. Therefore, in-depth understanding of the adhesive function of cells on the biological materials, especially on the surface of the bioactive material, is the key for optimizing the implant-bone interface.Our work has been aimed at investigating the effect and mechanism of adhesion of the osteoblast to different treated titanium surfaces based on our preliminary studies. It is believed that our work can provide an experimental basis for clinical applications.Methods:The experiment used RF plasma method to introduce biologically active amino functional groups to titanium dental implants’surfaces. According to the relatively optimum parameters of the plasma discharge experiment, we selected five different modes of discharge plasma activation on the surfaces of the titanium discs:CW, P(10%), P(30%), P(60%) and CW+P(30%). We used untreated surfaces of pure titanium as control. Among the modes, CW stands for continuous wave, P stands for pulsed (percentage means duty cycle) and CW+P stands for continuous wave plus pulsed.The adhesion and spreading of the osteoblasts on different surfaces of modified pure titanium were studied in vitro using a series of methodologies, including:CCK-8cell toxicity assay, absolute cell counting by flow cytometry, DAPI staining, assay of adhesion-relevant proteins, including integrin α2, FAK and p-FAK, with fluorescent immunocytochemistry, and western blot analysis.Results:1. CCK-8cells cytotoxicity assay results showed that the amino-group plasma modified surfaces were suitable for the growth of osteoblasts and exhibited no cytotoxicity.2. Detection of adhesion rate of the osteoblast with absolute cell counting by flow cytometry:The study of the effect of modified amino-group plasma surface on cellular behavior demonstrated that the surface modification promoted adhesion of osteoblast Mc3T3-E1. It was found that cell adhesion rate of osteoblasts on the surface of titanium discs in CW+P(30%) group significantly increased after co-culture for5min,30min,120min(p<0.01). Furthermore, the increase of adhesion rate could be observed in groups CW and P(30%) at30min,120min and5min,120min(p<0.05).3. DAPI staining results showed that osteoblasts on the titanium surface with treated groups like groups P(30%) and CW+P(30%) at5min after co-culture significantly increased than those on the untreated titanium surface, and cell density also increased. There were more adhered cells in groups CW, P(30%) and CW+P(30%) than those of controls.SEM observation showed that osteoblasts in untreated groups displayed shrinking shaped clusters with smaller adhesion areas and loose adhesion; cells appeared tilting. The osteoblasts in groups CW, P (30%) and CW+P(30%) showed stronger adhesion with larger adhesion areas; no tilting appeared. The osteoblasts in groups P(30%) and CW+P(30%) showed multiple pseudopodia enhancing the adhesion, especially those in group CW+P(30%). These cells fully adhered on the surface of the materials with large stretch areas and tight adhesion.4. Immunofluorescence staining:The two target markers α2and p-FAK protein expression in the treated groups CW, P(30%) and CW+P(30%) were significantly higher than those in the untreated groups, especially group CW+P(30%) exhibited a best efficiency. The yellow area was the co-expression zone of α2and p-FAK co-expression zone, which showed the yellow area in groups P(30%) and CW+P(30%) with relatively higher fluorescence photometric value.5. Western blot further confirmed the α2and p-FAK protein expression in groups CW, P(30%) and CW+P(30%) were significantly higher than those in the untreated groups, especially group CW+P(30%) exhibited a best efficiency.Conclusion:1. Pure titanium surface activated by amino-group plasma showed no cytotoxicity, so it was suitable for cell growth.2. Significant increases in early MC3T3-E1attachment, adhesion, and spreading were observed on the amino-group plasma modified surface.3. Increased α2integrin expression and phosphorylated focal adhesion kinase (p-FAK) expression indicated possible activation of the integrin signaling pathways.4. Results of this study suggest that an amino-group plasma modified surface has the potential to safely enhance bone formation and promote early osseointegration of titanium dental implants.