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Osteoclasts (OCs) differentiate from monocyte/macrophage-lineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)-β1 on OCP migration in vitro. Pre-osteoclastic RAW264.7 cells were cultured with and without TGF-β1 (2, 5 or 20 ng/ml), receptor activator of NF-κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF-β1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF-β1 for 3 days, and the effect was reversed by SB431542. Tartrate-resistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGF-β1 (2 ng/ml) and RANKL. When TGF-β1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF-β1 accelerates RANKL-induced OC differentiation, but does not act in a dose-dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL-induced osteoclast differentiation in the presence of TGF-β1. These results were accompanied with the increased expression of small G-proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF-β1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGF-β1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro. [ABSTRACT FROM AUTHOR]

Aging is a significant risk factor for the development of bisphosphonate-related osteonecrosis of the jaws (BRONJ). Accumulating evidence suggests that bone aging is associated with oxidative stress (OS), and OS is associated with osteonecrosis. To elucidate the mechanisms of the onset of BRONJ, the present study focused on OS and the effects of treatment with the pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO), an oxidative stressor, on healing of a surgically induced penetrating injury of the palate. Six-week-old C57BL/6J mice were randomly divided into four groups (n=5 each) and treated with or without zoledronic acid (ZOL) and with or without BSO (experimental groups: ZOL, BSO, and ZOL+BSO; control group: saline solution). A penetrating injury of the midline palate was surgically created using a root elevator. ZOL (250 µg/kg/day) was injected intraperitoneally every day from 7 days prior to the surgical treatment to 4 days following the surgical treatment. BSO (500 µg/kg/day) was administered 7 days prior to the surgical treatment as a single intraperitoneal injection. The maxillae were harvested at 5 days following the surgical treatment for histological and histochemical studies. The presence of empty osteocyte lacunae in the palatal bone was increased by ZOL and BSO treatment. The highest number of empty osteocyte lacunae was observed in the ZOL+BSO group. The number of tartrate-resistant acid phosphatase-positive cells was decreased by ZOL treatment and increased by BSO treatment. The number of canaliculi per osteocyte lacuna was significantly decreased by BSO treatment. The mineral apposition rate was significantly lower in the treatment groups than the control group. Bisphosphonates and OS suppressed bone turnover. The present study has demonstrated that BSO treatment affects osteocytes, and OS in osteocytes exacerbates impairment of the osteocytic canalicular networks. As a result, bisphosphonates and OS may induce osteonecrosis following invasive dentoalveolar surgery. OS has been identified as an additional risk factor for the development of BRONJ. [ABSTRACT FROM AUTHOR]

Background: We evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ). Materials and Methods: Male Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies. Results: In the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group. Conclusions: This study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ. [ABSTRACT FROM AUTHOR]

Aging cells not only cease growing, but also secrete various proteins such as inflammatory cytokines. This secretory phenomenon is known as the senescence-associated secretory phenotype (SASP). The aim of the present study was to elucidate the effects of senescence on the differentiation of osteoclast precursors (OCPs) and corresponding SASP. RAW264.7 cells were used as OCPs and were cultured to passage (P)5, P10 and P20. Cell proliferation assays, senescence-associated β-galactosidase staining and telomere length quantification were subsequently performed, and it was revealed that replicative senescence was induced at P20. In addition, the level of tartrate-resistant acid phosphatase activity in P20 cells treated with receptor activator of nuclear factor-κB ligand was significantly lower than that in P5 and P10 cells. The SASP factors interleukin-6, tumour necrosis factor-α and nitric oxide were significantly increased in P20 culture supernatants compared with those in P5 and P10 supernatants. Furthermore, the number of exosomes at P20 was increased compared with that at P5 and P10, and inducible nitric oxide synthase (iNOS) was expressed in exosomes at P20, but not in exosomes at P5. In conclusion, the present study revealed that senescent RAW264.7 cells exhibit increased expression of SASP factors and release iNOS in exosomes. [ABSTRACT FROM AUTHOR]