Implant abutment microgrooves affect soft tissue cells response via connexin 43 pathway

Abstract

Background The down-regulation of Connexin 43 (CX43) hemichannels (HCs) function in soft tissue cells promotes the up-regulation of numerous pro-healing genes, as the down-regulation of pro-fibrotic ones, leading to its identification as a potential target for the modulation of wound healing. On the other hand, titanium is commonly used as a transmucosal material, both for dental implants and abutments, and its surface micro-topography is known to affect cell responses. Aim/Hypothesis To investigate whether threaded titanium surfaces with different groove characteristics modulate CX43 expression and its organization in HCs rather than in gap junction (GJs) in an immortalized mouse myoblast cell line. In addition, to verify if the growth pattern and arrangement of cells on the grooves. Material and Methods Murine myoblasts C2C12 were seeded on discs with grooved titanium surfaces with different patterns- UTM (Ultrathin Threaded Microsurface) and XA microgrooved surface. A machined surface was used as control (MA). Titanium surface specimens were kindly provided by Sweden & Martina (Padova, Italy). Cells were cultured up to 7 days and daily monitored for viability through chemiluminescence (cell proliferation). Gene expression analysis for CX43, alkaline phosphatase (ALP) and collagen 1 (COL1a) was performed+ after 3 days of culture total RNA was purified, retro-transcribed to cDNA and amplified through quantitative RT-PCR. To localize the expression of CX43 and thus to identify its organization as HCs or as GJs, cells were observed under fluorescence after actin, CX43 and nuclei labeling. Differences among the groups were evaluated through Two-Way ANOVA statistical test. Results Very weak signal for CX43 was observed on UTM surface (mRNA 6.12-fold less than XA and 2.58-fold less than MA). CX43 expression was extremely up-regulated on XA surface after 3 days, when compared to UTM and MA surface (P < 0.0001). Conversely, cell proliferation was significantly reduced (P < 0.0001). On the XA surface, CX43 expression was labelled in the peri-nuclear area, indicating its organization in HCs. MA showed a slightly enhanced level of CX43 (2.37-fold less than XA) and, where cells were confluent, CX43 signal was marked around cell nuclei (HCs) and at the edges of cell profile, indicating GJs with other cells. Noteworthy, the disposition of cells on XA and UTM surfaces followed the grid of the grooved surface topography, with cells laying on the grid hollows and elongated to colonize the surface. Furthermore, gene expression analysis revealed that MA control surface induced a commitment of C2C12 towards an osteoblastic phenotype, showing greater levels of ALP and COL1a. Conclusions and Clinical Implications The microgrooved titanium surfaces showed a modulatory role on myoblasts, promoting an evident cell arrangement along the surface grooves on UTM and XA surfaces. This may lead in vivo to the deposition of an oriented extracellular matrix. Additionally, UTM surface caused CX43 down-regulation, leading to the hypothesis of a more favorable in vivo soft tissues wound healing.