Effect of titanium with nanotopography on the interaction between osteoblasts and osteoclasts

Author(s)

Rayana Longo Bighetti Trevisan

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-07-22

Examining board members

Márcio Mateus Beloti; Emanuela Prado Ferraz; Gustavo Mendonça; Tarcilia Aparecida da Silva

• Advisor: Márcio Mateus Beloti

Abstract

The bone remodeling process, essential for the maintenance of osseointegration of titanium (Ti) implants, comprises the balance between the resorption of bone tissue by osteoclasts and the formation by osteoblasts. Several studies developed by our research group demonstrated the role of the Ti surface with nanotopography, obtained by chemical conditioning with a H2SO4/H2O2 solution, in osteoblast differentiation under different conditions. However, the effects of this surface on osteoclast differentiation and activity, and their interaction with osteoblasts have not been investigated yet. In this context, our hypothesis is that nanotopography, in addition to favor osteoblast differentiation, is capable of modulating osteoclast differentiation and the osteoblast/osteoclast interaction. Thus, the aim of this study was to determine the influence of the Ti surface with nanotopography (Ti Nano), compared to the machined Ti surface (Ti Control), on the interaction between osteoblasts and osteoclasts. Pre-osteoblastic cells (MC3T3-E1) and macrophages (RAW 264.7) were plated on both Ti surfaces and on inserts at 10,000 cells/disc or insert and cultured under osteogenic and osteoclastogenic conditions for 5 days. On day 5, inserts containing osteoblasts were placed in osteoclastic cultures and inserts containing osteoclasts were placed in osteoblastic cultures, thus establishing indirect co-cultures, which were maintained for 2 days. Controls were non-co-cultured cells grown on both Ti surfaces. Assays with conditioned media by osteoblasts grown on Ti Nano and Ti Control, used in osteoclast cultures grown on polystyrene were also performed, with the same cell density and time points. Regarding the effect of osteoclasts on osteoblasts grown on Ti surfaces, the RNASeq (DESeq2: FC>1.7; p≤0.05) evidenced that osteoclasts downregulated the expression of genes related to osteogenesis and upregulated genes related to histone modification and chromatin organization in osteoblasts cultured on both Ti Nano and Ti Control. Osteoclasts also inhibited (p≤0.05) gene and protein expression of osteogenic markers, and this effect was reduced by Ti Nano. Regarding the mechanism involved, an increase in the expression of H3K9me2, H3K27me3 and EZH2 proteins was observed on both Ti surfaces under co-culture conditions (p≤0.05). The ChIP assay revealed accumulation of H3K9me2 repressing the promoter region of Alpl and H3K27me3 repressing Runx2, Alpl, Ibsp and Opg in osteoblasts in the presence of osteoclasts, which was attenuated by Ti Nano. Immunofluorescence corroborated the ChIP assay, exhibiting less RUNX2 and ALP and more H3K27me3 staining in osteoblasts under co-culture conditions in a less pronounced way on Ti Nano. The results of the effect of osteoblasts on osteoclasts grown on Ti Nano and Ti Control and in the presence of conditioned medium showed, through gene expression analysis, that nanotopography favored osteoclast differentiation (p≤0.05), which was potentiated by the presence of osteoblasts, with an increased expression of markers such as Rank, Ctsk, Tnf-α and genes related to Rank/Rankl signaling pathway such as cFos, Mitf, Nfatc1, and Traf6 (p≤0.05); however, osteoclasts showed lower activity, as indicated by reduced TRAP staining and protein expression of CTSK and TRAP, mainly on Ti Nano in the presence of osteoblasts. In conclusion, Ti Nano may favor clinical results in terms of implants osseointegration, since nanotopography favors osteoblast differentiation, protects osteoblasts from the deleterious effect of osteoclasts and modulates osteoclast differentiation, evidencing a possible role in the bone remodeling process. Through these findings, new therapeutic approaches can be developed using strategies capable of modulating the activities of osteoblasts and osteoclasts by different nanotopographies, in order to favor the interaction between biomaterials and bone tissue. (AU)

• FAPESP's process: 17/23888-8 - Effect of titanium surface with nanotopography on the interaction between osteoblasts and osteoclasts
• Grantee: Rayana Longo Bighetti Trevisan
• Support Opportunities: Scholarships in Brazil - Doctorate