Bisphosphonate-related osteonecrosis of the jaw (BRONJ) commonly occurs in individuals receiving bisphosphonates (BPs) with clinical manifestations of the exposed necrotic bone. Although defective wound healing of soft tissue is frequently, if not always, observed in BRONJ, the effects of BPs on oral soft tissue or cells remain unknown. To investigate the effects of BPs on cells of oral mucosal tissue, we studied the effect of pamidronate (PAM), one of the BPs most commonly administered to cancer patients, on the phenotypes of normal human oral keratinocytes (NHOK) and fibroblasts (NHOF). When exposed to PAM at 10 µM, NHOK, not NHOF, underwent senescence: NHOK overexpressed senescence-associated β-galactosidase (SA-β-Gal), p16INK4A, IL-6, and IL-8. When exposed to a higher level (50 µM) of PAM, NHOK maintained senescent phenotypes, but NHOF underwent apoptosis. PAM-induced senescence in NHOK is mediated, in part, via geranylgeranylation of the mevalonate pathway. Our in vitro 3D oral mucosal tissue construction studies further demonstrated that PAM induced senescence and impaired re-epithelialization of oral mucosa. Analysis of these data indicates that premature senescence of oral mucosal cells and subsequent defective soft-tissue wound healing might be partly responsible for the development of BRONJ in individuals receiving PAM or other BPs.

Pulp capping, or placing dental materials directly onto the vital pulp tissues of affected teeth, is a dental procedure that aims to regenerate reparative dentin. Several pulp capping materials are clinically being used, and calcium ion (Ca(2+)) released from these materials is known to mediate reparative dentin formation. ORAI1 is an essential pore subunit of store-operated Ca(2+) entry (SOCE), which is a major Ca(2+) influx pathway in most nonexcitable cells. Here, we evaluated the role of ORAI1 in mediating the odontogenic differentiation and mineralization of dental pulp stem cells (DPSCs). During the odontogenic differentiation of DPSCs, the expression of ORAI1 increased in a time-dependent manner. DPSCs knocked down with ORAI1 shRNA (DPSC/ORAI1sh) or overexpressed with dominant negative mutant ORAI1(E106Q) (DPSC/E106Q) exhibited the inhibition of Ca(2+) influx and suppression of odontogenic differentiation and mineralization as demonstrated by alkaline phosphatase (ALP) activity/staining as well as alizarin red S staining when compared with DPSCs of their respective control groups (DPSC/CTLsh and DPSC/CTL). The gene expression for odontogenic differentiation markers such as osteocalcin, bone sialoprotein, and dentin matrix protein 1 (DMP1) was also suppressed. When DPSC/CTL or DPSC/E106Q cells were subcutaneously transplanted into nude mice, DPSC/CTL cells induced mineralized tissue formation with significant increases in ALP and DMP1 staining in vivo, whereas DPSC/E106Q cells did not. Collectively, our data showed that ORAI1 plays critical roles in the odontogenic differentiation and mineralization of DPSCs by regulating Ca(2+) influx and that ORAI1 may be a therapeutic target to enhance reparative dentin formation.

No abstract