Osteoclastic responses on the titanium surfaces immersed in modified simulated body fluid

Abstract

Osseointegration is achieved by bone remodeling around the implant titanium (Ti) surfaces. This bone remodeling is a harmonious coupling of bone formation and bone resorption and requires a coordinated activity of osteoblasts and osteoclasts. Biomimetic deposition using modified simulated body fluid (SBF) substantially improved the surface wettability and sur-face energy of the Ti surfaces and also increased osteoblast responses on anodized Ti surfaces. We hypothesized that thin calcium phosphate (CaP) deposition onto Ti surfaces using biomimetic deposition led to not only improve osteogenesis but also suppress osteoclastogenesis in terms of high surface hydrophilicity. To understand bone remodeling on Ti surfaces with thin CaP coatings by means of immersion in modified SBF, we investigated the effect of biomimetic deposition on osteoclast differentiation activity on Ti surfaces. Ti discs with two different surface topographies were prepared: machined and anodized surfaces. The specimens of two different surfaces were immersed in modified SBF solution for two weeks at physiologic condition of 37℃, initial pH 7.4, and p(CO2) = 0.05 atm. The control groups were dry Ti (machined and anodized) discs not immersed in any solution. In this study we used murine RAW 264.7 cells as osteoclast precursor cells. To evaluate osteoclast differentiation activity on Ti surfaces, Tartrate-resistant acid phosphatase (TRAP) activity assay was performed. After 11 days of culture, cells on Ti surfaces were observed using a field emission-scanning electron microscope (FE-SEM) and confocal laser scanning microscopy (CLSM) to investigate osteoclast differentiation activity visually. The expression of nuclear factor of activated T cells 1 (NFATc1) and c-Fos, two critical transcriptional factors involved in osteoclastogenesis, were also assessed in terms of mRNA and protein levels by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, respectively. The results were as follows: 1. TRAP activities on both machined and anodized Ti surfaces immersed in modified SBF were significantly lower compared to non - immersed ones. 2. FE-SEM and CLSM observation showed that the number of differentiated osteoclasts was lower on anodized surfaces immersed in modified SBF compared to Ti surfaces not im-mersed. 3. mRNA and protein expression of two osteoclastogenesis marker, NFATc1 and c-Fos, were significantly decreased in anodized Ti surfaces immersed in modified SBF than in ano-dized dry surfaces. 4. The effects of immersion of Ti discs in modified SBF on osteoclastogenesis were higher in anodized surfaces than in machined surfaces. These results indicate that osteoclastogenesis was inhibited by biomimetic deposition using modified SBF especially on anodized Ti surfaces.