Previously, we discovered that periodontal ligament (PDL) cells not only support osteoclastogenesis through cell-to-cell contact, but also inhibit the formation of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells by a producing soluble factor(s). Furthermore, PDL cells express both receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) messenger RNA (mRNA). Clinically, “ankylosed teeth,” which lack periodontal ligament, cannot be moved with orthodontic tooth treatment. From this, we hypothesized that PDL cells under mechanical stress should play a pivotal role in osteoclast formation during orthodontic tooth movement. This study examined how mechanical stress affects the osteoclastogenesis-supporting activity of PDL cells. PDL cells were compressed continuously and then cocultured with peripheral blood mononuclear cells (PBMCs) for 4 weeks. PDL cells under mechanical stress up-regulated osteoclastogenesis from PBMCs. Furthermore, the expression of RANKL mRNA and protein in PDL cells increased with compressive force in parallel with the change in the number of osteoclasts. In addition, cyclo-oxygenase 2 (COX-2) mRNA expression was induced by compressive force, and indomethacin inhibited the RANKL up-regulation resulting from compressive force. PDL cells under compressive force exhibited significantly increased prostaglandin E2 (PGE2) production in comparison with control PDL cells. Exogenous PGE2 treatment increased RANKL mRNA expression in PDL cells. Interestingly, OPG expression remained constant throughout compressive force or PGE2 treatment. In conclusion, compressive force up-regulated RANKL expression in PDL cells. Furthermore, RANKL up-regulation in mechanically stressed PDL cells was dependent on PGE2.

Reactive oxygen species (ROS) act as intracellular signaling molecules in the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclast differentiation, but they also have cytotoxic effects that include peroxidation of lipids and oxidative damage to proteins and DNA. Cellular protective mechanisms against oxidative stress include transcriptional control of cytoprotective enzymes by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). This study investigated the relationship between Nrf2 and osteoclastogenesis. Stimulation of osteoclast precursors (mouse primary peritoneal macrophages and RAW 264.7 cells) with RANKL resulted in the up-regulation of kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2. It also decreased the Nrf2/Keap1 ratio, and it down-regulated cytoprotective enzymes (heme oxygenase-1, γ-glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase). Nrf2 overexpression up-regulated the expression of cytoprotective enzymes, decreased ROS levels, decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells, reduced marker genes for osteoclast differentiation, and attenuated bone destruction in both in vitro and in vivo models. Overexpression of Keap1 or RNAi knockdown of Nrf2 exerted the opposite actions. In addition, in vivo local Nrf2 overexpression attenuated lipopolysaccharide-mediated RANKL-dependent cranial bone destruction in vivo. This is the first study to show that the Keap1/Nrf2 axis regulates RANKL-dependent osteoclastogenesis through modulation of intracellular ROS signaling via expression of cytoprotective enzymes. This raises the exciting possibility that the Keap1-Nrf2 axis may be a therapeutic target for the treatment of bone destructive disease.

Periodontitis, an inflammatory disease that affects the tissues surrounding the teeth, is a common disease worldwide. It is caused by a dysregulation of the host inflammatory response to bacterial infection, which leads to soft and hard tissue destruction. In particular, it is the excessive inflammation in response to bacterial plaque that leads to the release of reactive oxygen species (ROS) from neutrophils, which, then play a critical role in the destruction of periodontal tissue. Generally, ROS produced from immune cells exhibit an anti-bacterial effect and play a role in host defense and immune regulation. Excessive ROS, however, can exert cytotoxic effects, cause oxidative damage to proteins and DNA, can interfere with cell growth and cell cycle progression, and induce apoptosis of gingival fibroblasts. Collectively, these effects enable ROS to directly induce periodontal tissue damage. Some ROS also act as intracellular signaling molecules during osteoclastogenesis, and can thus also play an indirect role in bone destruction. Cells have several protective mechanisms to manage such oxidative stress, most of which involve production of cytoprotective enzymes that scavenge ROS. These enzymes are transcriptionally regulated via NRF2, Sirtuin, and FOXO. Some reports indicate an association between periodontitis and these cytoprotective enzymes’ regulatory axes, with superoxide dismutase (SOD) the most extensively investigated. In this review article, we discuss the role of oxidative stress in the tissue destruction manifest in periodontitis, and the mechanisms that protect against this oxidative stress.

Periodontal ligament (PDL) cells play an important role in maintaining the homeostasis of periodontal tissues. However, it is not known how PDL cells contribute to osteoclastogenesis. In this study, we examined the consequences of cell-to-cell interactions between peripheral blood mononuclear cells (PBMCs) and PDL cells during osteoclastogenesis. PBMCs were co-cultured directly or indirectly with PDL cells for two to four weeks. PBMCs that were directly co-cultured with PDL cells formed significantly more resorption pits on dentin slices than did PBMCs that were cultured alone. However, soluble factor(s) produced from PDL cells inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Furthermore, PDL cells expressed both receptor activator nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) mRNA. In conclusion, PDL cells support osteoclastogenesis through cell-to-cell contact. PDL cells might regulate osteoclastogenesis by opposing mechanisms-stimulation of resorptive activity by RANKL and inhibition by OPG-thus affecting processes such as periodontitis and orthodontic tooth movement.

Previously, we discovered that RANKL expression is induced in compressed periodontal ligament cells, and that this promotes osteoclastogenesis on the compression side in orthodontic tooth movement. We hypothesized that local OPG gene transfer to the periodontium would neutralize the RANKL activity induced by mechanical compressive force, thereby inhibiting osteoclastogenesis and diminishing tooth movement. The upper first molars of six-week-old male Wistar rats were moved palatally by means of a fixed-orthodontic wire. A mouse OPG expression plasmid [pcDNA3.1(+)-mOPG] was constructed, and the production of functional OPG protein was confirmed in vitro. The inactivated HVJ envelope vector containing pcDNA3.1(+)-mOPG or PBS was injected periodically into the palatal periodontal tissue of upper first molars. When this local OPG gene transfer was performed, OPG production was induced, and osteoclastogenesis was inhibited. Local OPG gene transfer significantly diminished tooth movement. In this study, we report that OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and inhibited experimental tooth movement.