The effect of osteoclasts on epigenetic regulation by long non-coding RNAs in osteoblasts grown on titanium with nanotopography.

Titanium (Ti) implant osseointegration is regulated by the crosstalk among bone cells that are affected by epigenetic machinery, including the regulation of long non-coding RNAs (lncRNAs). Nanotopography Ti (Ti Nano) induces the differentiation of osteoblasts that are inhibited by osteoclasts through epigenetic mechanisms. Thus, we hypothesize that osteoclasts affect lncRNA expression in Ti Nano-cultivated osteoblasts. Osteoblasts were grown on Ti Nano and Ti Control that were then co-cultured with osteoclasts for 48 h. Using RNAseq, we identified 252 modulated lncRNAs in osteoblasts regulated by both surfaces of Ti, but mainly in Ti Nano-cultivated osteoblasts. A negative correlation was observed between Kcnq1ot1 and the mRNAs of Alpl, Bglap, Bmp8a, Col1a1, and Vim in Ti Nano-cultivated osteoblasts with osteoclasts. The pull-down indicated that Bglap mRNA is a direct target of Kcnq1ot1, with enhanced physical interaction in Ti Nano-cultivated osteoblasts, and greater osteoclast inhibition than the Ti Control. The bone marker expression at the levels of mRNA and protein were downregulated by the Kcnq1ot1 silencing, indicating its pivotal role in osteoblast differentiation. These results showed that nanostructured Ti surface modulates the osteoblast-osteoclast crosstalk, at least in part, through the regulation of lncRNA expression in osteoblasts. We demonstrate that the lncRNA Kcnq1ot1 directly interacts with Bglap mRNA, and this interaction is enhanced by nanotopography and reduced by osteoclasts with greater intensity in Ti Nano-cultivated osteoblasts. These findings confirm the molecular mechanisms associated with the high osteogenic potential of nanotopography and can potentially support osteointegration of dental and skeletal prostheses.