Biocompatible calcium and phosphorous-doped titania nanotubes influences the osteogenic differentiation of human mesenchymal stem cells

摘要

Despite commercially pure titanium (cp-Ti)-based dental implants are widely used clinically, a significant number of failures still occur due to poor osseointegration. The modification of Ti features such as topography, morphology and chemistry seems a good strategy to achieve enhanced bone-implant integration. In particular, the decoration of Ti with titania nanotubes (NTs) has demonstrated to be a promising way to produce novel bio-functional and bio-selective dental implant surfaces. The main aim of this work is to synthesize biocompatible Calcium (Ca) and Phosphorous (P)-doped titania NTs and to study its influence on osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, Ca and P-doped NTs (Ca/P-NTs) were synthesized by two-step anodization. Firstly, NTs were grown on Ti substrates by anodizing at 60V for 30min. in an electrolyte containing ethylene glycol, 0.3wt.% NH4F and 3vol.% H2O. Secondly, NT surfaces were cathodic polarized at 20V for 30s in an electrolyte composed of 0.35M calcium acetate and 0.04M β-glycerophosphate and, immediately after, these samples were anodized at 100V for 30 min. in the same electrolyte. The NT features were characterized by Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), Energy-Dispersive X-ray Spectroscopy (EDS), X-ray Photoelectron Spectroscopy (XPS) and Water Contact Angle (WCA) measurements. In the present study, the influence of Ca/P-NTs on viability/proliferation and adhesion of osteoblasts (OB) and human mesenchymal stem cells (hMSCs) was investigated. The viability/proliferation of cells was evaluated by MTT assay and the adhesion ability was studied by fluorescence microscopy and FESEM observation. The osteogenic differentiation of hMSCs was also investigated by quantitative real-time polymerase chain reaction (PCR) assay. Real-time PCR analysis was performed on osteogenic markers of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP-2), collagen type 1 (COL-1), alkaline phosphatase (ALP) and osteopontin (OPN). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. One-Way ANOVA was used to determine statistical significance. Results showed that well-ordered Ca/P-NTs with diameter comprised between 60-85 nm and 4.9 0.5 μm length were synthesized. Chemical analysis conducted by EDS and XPS of such NTs confirmed the presence of Ca and P. After two-step anodization, the hydrophilicity of Ti was enhanced as shown by the significant decrease of WCA from 45.4 3.1o to 5.7 2.3o (TI vs. Ca/P-NTs; p＜0.001). After 6 days of incubation, OB cultured on Ca/P-NTs showed similar viability as cultured on NT surfaces. On the other hand, the viability of hMSCS on Ca/P-NTs was lower as compared to NT surfaces (p＜0.05). Both OB and hMSCs were well-adhered presenting elongated morphology with filopodia forming adhesion points with NTs. The expression of Runx2, BMP-2, COL-1, ALP and OPN was significantly higher on Ca/P-NTs than on NT and Ti surfaces. These results suggest that Ca/P-NTs induced the osteogenic differentiation of hMSCs. The major highlight of this study relies on the synthesis, for the first time, of biocompatible anodic Ca and P-doped titania NTs with osteogenesis-inducing ability, as very attractive candidates for the development of new smart implant surfaces designed to contribute for a better outcome of dental implants.