G­CSF delays tooth extraction socket bone healing via the inhibition of bone turnover in mice.

Granulocyte colony-stimulating factor (G-CSF) regulates the survival, proliferation and differentiation of all cells in the neutrophil lineage, and is consequently used for neutropenic conditions. Upon G-CSF administration, osteoblasts and osteocytes are suppressed, and the support system allowing hematopoietic stem cells to remain in the microenvironment is diminished. The present study focused on and investigated G-CSF as a regulatory factor of bone remodeling. The aim of the present study was to investigate the effect of G-CSF administration on the bone healing of tooth extraction sockets. Significant differences in the bone volume fraction, and trabecular separation of the proximal femurs and alveolar septa were observed between the G-CSF and control (saline-treated) groups. The trabecular bone of the femur and alveolar septa was reduced in the G-CSF group compared with that in the control group. In addition, serum procollagen type 1 N-terminal propeptide levels, a marker of bone formation, were lower in the G-CSF group compared with in the control group. Fibrous connective tissues and immature bone were observed in the extraction socket, and bone healing was delayed in the G-CSF group compared with that in the control group. The bone area in the extraction socket 6 days after tooth extraction was significantly smaller in the G-CSF group (23.6%) than that in the control group (45.1%). Furthermore, G-CSF administration reduced the number of canaliculi per osteocyte and inhibited the connection of osteocyte networks. Consequently, osteoblast activation was inhibited and bone remodeling changed to a state of low bone turnover in the G-CSG group. Analysis of bone formation parameters revealed that the G-CSF group exhibited a lower mineral apposition rate compared with in the control group. In conclusion, these findings indicated that G-CSF may delay bone healing of the socket after tooth extraction.

Senescent RAW264.7 cells exhibit increased production of nitric oxide and release inducible nitric oxide synthase in exosomes.

Aging cells not only cease growing, but also secrete various proteins such as inflammatory cytokines. This secretory phenomenon is known as the senescence­associated 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, senescence­associated ß­galactosidase staining and telomere length quantification were subsequently performed, and it was revealed that replicative senescence was induced at P20. In addition, the level of tartrate­resistant acid phosphatase activity in P20 cells treated with receptor activator of nuclear factor­κB ligand was significantly lower than that in P5 and P10 cells. The SASP factors interleukin­6, tumour necrosis factor­α 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.

Effects of TGF­ß1 on the migration and morphology of RAW264.7 cells in vitro.

Osteoclasts (OCs) differentiate from monocyte/macrophage­lineage 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)­ß1 on OCP migration in vitro. Pre­osteoclastic RAW264.7 cells were cultured with and without TGF­ß1 (2, 5 or 20 ng/ml), receptor activator of NF­κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF­ß1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF­ß1 for 3 days, and the effect was reversed by SB431542. Tartrate­resistant 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 TGF­ß1 (2 ng/ml) and RANKL. When TGF­ß1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF­ß1 accelerates RANKL­induced OC differentiation, but does not act in a dose­dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL­induced osteoclast differentiation in the presence of TGF­ß1. These results were accompanied with the increased expression of small G­proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF­ß1 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 TGF­ß1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.

Osteonecrosis of the jaws caused by bisphosphonate treatment and oxidative stress in mice.

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.

Treatment with teriparatide for advanced bisphosphonate-related osteonecrosis of the jaw around dental implants: a case report.

We report a case of a 66-year-old severely osteoporotic woman with bisphosphonate-related osteonecrosis of the jaw (BRONJ) around her dental implants, who was treated successfully with teriparatide and sequestrectomy of the mandible. After 5 months of teriparatide therapy, the sequestrum separation had progressed and a sequestrectomy was performed under general anesthesia. Five months after the operation, new bone formation was observed around the bone defect in the region of the sequestrectomy. A repeat computed tomographic image revealed improvement in the bone defect in the mandible. These results suggest that teriparatide provides beneficial effects in the treatment of advanced BRONJ around dental implants.