Periodontal ligament (PDL) fibroblasts play an important role in preserving periodontal homeostasis and transmitting mechanical signals to alveolar bone. Connexin 43 (Cx43), a gap junction protein, is essential for bone homeostasis and regulates bone remodeling. However, the function of Cx43 in human PDL fibroblast‐regulated bone remodeling has not yet been elucidated. In this study, human PDL fibroblasts were exposed to cyclic mechanical tension with a maximum 5% elongation for different durations. We then examined the expression of signaling molecules related to osteogenesis and osteoclastogenesis at both the mRNA and protein levels as well as the activity of extracellular signal‐regulated kinase (ERK) in human PDL fibroblasts after loading. We found that mechanical tension increased Cx43, which further upregulated osteogenic (e.g., RUNX2, Osterix, and OPG) and down‐regulated osteoclastogenic (e.g., RANKL) signaling molecules. Suppressing Cx43 gene ( Gja1 ) by siRNA inhibited the increase in osteogenesis‐related molecules but enhanced RANKL expression. Similar to Cx43, activated ERK1/2 was also enhanced by mechanical tension and suppressed by Cx43 siRNA. Inhibition of ERK1/2 signaling using PD98059 reduced the tension‐regulated increase in osteogenesis‐related molecules but enhanced that of osteoclastogenesis‐related ones. These findings suggest that cyclic tension may involve into the osteogenic or osteoclastogenetic differentiation potential of human PDL fibroblasts via the Cx43‐ERK1/2 signaling pathway. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1008–1014, 2015.

Intracellular calcium oscillation and its downstream signaling in osteoclasts is believed to play critical roles in regulating bone resorption. Our previous study demonstrated that fluid shear stress (FSS) induced more calcium responsive peaks in the late differentiated osteoclasts than the early ones. In this paper, the signaling pathways of FSS-induced calcium response for the osteoclasts in different differentiation stages were studied. RAW264.7 macrophage cells were induced to differentiate into osteoclasts with the conditioned medium from MC3T3-E1 osteoblasts. Furthermore pharmacological agents were added to block the specific signaling pathways. Finally the cells were exposed to FSS at different levels (1 or 10 dyne/cm²) after being induced for 4 or 8 days. The results showed that the mechanosensitive, cation-selective channels, phospholipase C (PLC) and endoplasmic reticulum constituted the major signaling pathway for mechanical stimulation-induced calcium response in osteoclasts. Extracellular calcium or ATP involved with calcium oscillation in a FSS magnitude-dependent manner. This pathway study may help to give insight into the molecular mechanism of mechanical stimulation-regulated bone remodeling.

Focal adhesions are large macromolecular assemblies through which cells are connected with the extracellular matrix so that extracellular signals can be transmitted inside cells. Some studies have focused on the effect of cell shape on the differentiation of stem cells, but little attention has been paid to focal adhesion. In the present study, mesenchymal stem cells (MSCs) and osteoblast-like MC3T3-E1 cells were seeded onto micropatterned substrates on which circular adhesive islands with different spacing and area were created for focal adhesion. Results showed that the patterns of focal adhesion changed cell morphology but did not affect cell survival. For MSCs cultured for 3 days, patterns with small circles and large spacing promoted osteogenesis. For MSCs cultured for 7 days, patterns with large circles and spacing enhanced osteogenesis. For MC3T3-E1 cells, the patterns of focal adhesion had no effect on cell differentiation after 3 days of culture, but patterns with small circles and spacing improved osteogenic differentiation after 7 days. Moreover, the assembly of F-actin, phosphorylation of myosin, and nuclear translocation of yes-associated proteins (YAP) were consistent with the expression of differentiation markers, indicating that the pattern of focal adhesion may affect the osteogenesis of MSCs and osteoblasts through changes in cytoskeletal tension and nuclear localisation of YAP.