Titanium with nanotopography induces osteoblast and inhibits osteoclast differentiation Lopes HB, Bighetti-Trevisan RL; Poker BC; Castro-Raucci LMS; Ferraz EP; Souza ATP; Freitas GP; Rosa AL; Beloti MM. Research Lab, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

Statement of Purpose Titanium (Ti) topographic modifications at the nanoscale level generate surfaces that are able to modulate several cellular functions and signaling pathways.1,2 The development of nanomaterials that are able to control osteoblast and osteoclast activities and consequently the bone remodeling process is of relevance to improve the phenomenon of osseointegration. In this context, the aim of this study was evaluated the effect of Ti with nanotopography (Ti-Nano) on osteoblast and osteoclast differentiation. Methods: Mesenchymal stem cells (MSCs) from rat bone marrow obtained under the approval of the Committee of Ethics in Animal Research (#2014.1.796.58.7) and RAW 264.7 cells were cultured on Ti-Nano or machined surface (Ti-Machined). The MSCs were cultured in growth medium for up to 7 days and the RAW 264.7 cells were cultured in medium to stimulate osteoclast differentiation for up to 10 days. To investigate the osteogenic potential of the nanotopography, the expression the osteoblast markers, runt-related transcription factor 2 (Runx2), Osterix (Sp7), alkaline phosphatase (Alp), (Oc) and bone sialoprotein (Bsp) was evaluated by real-time PCR on days 3, 5 and 7 and the protein expression of RUNX2 was evaluated by Western blot on days 3, 5 and 7. To evaluate the osteoclast differentiation, the gene expression of Rank was evaluated by real-time PCR on day Figure 1. Gene expression of Runx2 (A), Osx (B), Alp (C), 7, and the osteoclast activity was evaluated by staining for Oc (D), Bsp (E), Opn (F) and RUNX2 protein expression tartrate-resistant acid phosphatase (TRAP) on days 3, 7 and (G,H) of MSCs cultured on Ti-Nano or Ti-Machined (n=3). 10. The real-time PCR and Western blot experiments were * indicate statistical significant differences between Ti- done in triplicate (n=3) and the TRAP staining was done in Nano and Ti-Machined in the same time-point (p≤0.05). quintuplicate (n=5). The data were analyzed by t-test (p≤0.05) to compare cells grown on Ti-Machined or Ti- Nano at the same time-point. Results: The gene expression of Runx2 was higher on days 3 and 7 (p<0.05) and was lower on day 5 (p>0.05) in cells grown on Ti-Nano compared with Ti-Machined (Fig. 1A). The gene expression of Osx, Alp and Bsp was higher in all time-points in cells grown on Ti-Nano compared with Ti- Machined (p<0.05, Fig. 1B, C and E, respectively). The Figure 2. Gene expression of Rank (A, n=3) on day 7, and gene expression of Oc did not show differences on day 3 staining for TRAP (B, n=5) on days 3, 7 and 10 in RAW (p>0.05) and was higher on days 5 and 7 (p<0.05) in cells 264.7 cells cultured on Ti-Nano or Ti-Machined. * indicate grown on Ti-Nano compared with Ti-Machined (Fig. 1D). statistical significant differences between Ti-Nano and Ti- The gene expression of Opn was higher on days 3 and 5 Machined in the same time-point (p≤0.05). (p<0.05) and did not show difference on day 7 (p>0.05) in cells grown on Ti-Nano compared with Ti-Machined (Fig. Conclusions: Our results showed that Ti-Nano induces 1F). The protein expression of RUNX2 was higher on days osteoblast differentiation of MSCs, even in non-osteogenic 3 and 5 (p<0.05) and did not show difference on day 7 conditions, concomitantly with the inhibition of osteoclast (p>0.05) in cells grown on Ti-Nano compared with Ti- differentiation of RAW 264.7 cells. Thus, these findings Machined. The gene expression of Rank was lower in cells open windows for the development of smart surfaces with grown on Ti-Nano compared with Ti-Machined on day 7 ability to regulate the bone remodelling process and (Fig. 2A). The TRAP staining did not show differences on consequently the implant osseointegration. day 3 (p>0.05) and was lower on days 5 and 7 (p<0.05) in References: 1. Variola F. Nanoscale. 2011;3:335-353. cells grown on Ti-Nano compared with Ti-Machined (Fig. 2. Rosa AL. J. Cell Biochem. 2014;115:540-548. 2B). Grants: FAPESP (# 2016/14171-0 and 2014/08443-1).