Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways.

Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low- (10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low- and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-κB, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment.

Histochemical examination of systemic administration of eldecalcitol combined with guided bone regeneration for bone defect restoration in rats.

The aim of this experiment was to elucidate the histological alterations after systemic administration of eldecalcitol (ELD) combined with guided bone regeneration during the restoration of bone defect healing in rats. The femurs of 8-week-old Wister rats were used to generate bone defect models. The defect was covered with a collagen membrane, and ELD group was administrated with eldecalcitol (50 ng/kg body weight) intragastrically once every other day. Femora were harvested at 1, 2, 4 and 8 weeks post-surgery. Decalcify tissue slices were made and used for histological and immunohistochemical examination. Bone biomarkers of RANKL, OPG and osteocalcin (OCN) were detected by western blot. The results revealed that the system administration of ELD could improve new bone formation demonstrated by the increased bone volume/tissue volume ratio and accelerated mineralization. ELD suppressed osteoclastic bone resorption by reducing the number of osteoclasts, decreasing the expression of cathepsin-K and the ratio of RANKL/OPG at the early stage of bone defect restoration (1 and 2 weeks) and upregulating OCN expression at the later stage of bone defect healing (4 and 8 weeks). These data suggested that systemic administration of eldecalcitol accelerated bone formation and promoted bone maturation by decreasing bone resorption and promoting bone mineralization during bone defect restoration.

Adipocytes enhance expression of osteoclast adhesion-related molecules through the CXCL12/CXCR4 signalling pathway.

OBJECTIVES: The purpose of this study was to investigate effects of adipocytes on osteoclast adhesion-related molecules. MATERIALS AND METHODS: ST2 cells, a cloned stromal cell line from mouse bone marrow, able to differentiate into adipocytes, were cultured in serum-free α-MEM which was then collected to be used as adipocyte-conditioned medium (ADIPO CM). RAW264.7 cells were cultured in ADIPO CM in the presence of RANKL, and bone marrow-derived macrophages were cultured in ADIPO CM in the presence of RANKL and macrophage-colony stimulating factor to induce osteoclast differentiation. TRAP staining, resorption pit assay, qRT-PCR and western blotting assays were performed. RESULTS: ELISAs revealed that CXCL12 was abundant in ADIPO CM and CCK-8 assay revealed no proliferation of RAW264.7 cells after exogenous CXCL12 treatment. ADIPO CM enhanced osteoclast formation and resorption, both by RAW264.7 cells and BMMs. In addition, exogenous CXCL12 efficiently potentiated formation of TRAP-positive osteoclast and resorption by RAW264.7 cells. Western blotting and qRT-PCR suggested that ADIPO CM or combined treatment with exogenous CXCL12 caused significant increase in expression of NFAT2, src and osteoclast adhesion-related molecules, including ß3 integrin, CD44 and osteopontin. However, these promotional effects were largely abrogated on treatment of AMD3100, a CXCR4 antagonist. CONCLUSIONS: Adipocytes promoted osteoclast differentiation, function and expression of adhesion-related molecules through the CXCL12/CXCR4 signalling pathway.

Histochemical examination of the effects of high-dose 1,25(OH)2D3 on bone remodeling in young growing rats.

Vitamin D has an anabolic effect on bone developmental processes and is involved in maintaining skeletal integrity. In recent years, pediatric cases of vitamin D intoxication have attracted attention. Therefore, the aim of this study was to investigate the influence of long-term administration of physiologically-high-dose calcitriol (1,25(OH)2D3) on bone remodeling in young developing rats. Neonatal rats received once-daily subcutaneous injection of calcitriol (250 ng/kg body weight), or PBS only as a control, for 3 weeks. At 1, 2 and 4 weeks' post-administration, rats were sacrificed and fixed by transcardial perfusion with 4 % paraformaldehyde, following which tibiae were extracted for histochemical analysis. Compared with the control group, the number of tartrate-resistant acid phosphatase- and Cathepsin K-positive osteoclasts were significantly increased, and the expression of alkaline phosphatase in osteoblasts was decreased in trabecular bone of rats administered high-dose 1,25(OH)2D3, leading to decreased trabecular bone volume. In addition, the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) was increased, while that of osteoprotegerin was weaker in osteoblasts in the experimental group compared with the control group. Moreover, there was weaker immunoreactivity for EphrinB2 in osteoclasts and EphB4 in osteoblasts of trabecular bone in the experimental group compared with the control group. These findings suggest that long-term use of physiologically-high dose calcitriol may result in bone loss through RANKL/RANK/osteoprotegerin and EphrinB2-EphB4 signaling pathways, and that these negative effects could continue after drug withdrawal. Therefore, optimal limits for vitamin D administration need to be established for children and adolescents.

Immunolocalization of MMP9 and MMP2 in osteolytic metastasis originating from MDA-MB-231 human breast cancer cells.

The aim of the present study was to investigate the expression of matrix metalloproteinase (MMP)9 and MMP2, and their potential roles in bone metastasis nests using a well-standardized model of breast cancer bone metastasis in nude mice. BALB/c nu/nu mice (5-week-old; n=10) were subjected to intracardiac injection of MDA-MB-231 human breast cancer cells. After 4 weeks, the mice exhibiting radiolucent lesions in tibiae were sacrificed, and the tibiae were removed for histochemical analysis. The gene expression of MMP2 and MMP9 in the tumor cells, metaphysis and diaphysis of normal BALB/c nu/nu mice were determined using reverse transcription-polymerase chain reaction analysis. The metastatic tumor tissue occupied almost the entire bone marrow cavity. Numerous tartrate-resistant acid phosphatase-positive osteoclasts were found in the metastasized lesions. The invaded tumor cells positive for mammaglobin 1 exhibited different proliferation activities and apoptosis between the metaphysis and diaphysis. Proliferating cell nuclear antigen was expressed at high levels in the metaphyseal area, whereas TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells were more evident in the diaphysis area. Of note, MMP9 was expressed predominantly in the proliferating cell nuclear antigen­positive area, whereas the expression of MMP2 was observed predominantly in the diaphysis, which had more TUNEL­positive cells. Taken together, the results suggested that MMP9 and MMP2 may have their own importance in extracellular matrix degradation and trabecular bone damage in different zones of bone metastasis, including the metaphysis and diaphysis.

The effect of calcitriol on high mobility group box 1 expression in periodontal ligament cells during orthodontic tooth movement in rats.

High mobility group box 1 (HMGB1) is a late inflammatory cytokine that plays an important role in periodontal tissue remodeling during orthodontic tooth movement. Calcitriol (1,25-dihydroxyvitamin D3 [1α,25 (OH)2D3]) is a systemic calcium-regulating hormone shown to downregulate expression of multiple proinflammatory cytokines in human periodontal ligament cells in response to orthodontic force. The purpose of this study was to investigate the effect of 1α,25(OH)2D3 on the expression of HMGB1 in periodontal ligament (PDL) cells during orthodontic tooth movement. Seven-week-old male Wistar rats were used for experimentation. Tooth movement was assessed using a nickel-titanium coil spring to apply mechanical loading to the tooth for 5 days. This was followed by administration of either 1α,25(OH)2D3 or normal saline by gavage every other day for up to 28 days. Immunohistochemistry was used to analyze the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and HMGB1. After discontinuation of orthodontic force, expression of the early inflammatory cytokines IL-6 and TNF-α were time-dependently reduced in the 1α,25(OH)2D3 group compared with the control group at each time point. Similarly, expression of HMGB1 was decreased over time in both the 1α,25(OH)2D3 and normal saline groups, and 1α,25(OH)2D3 administration enhanced this decline. These findings indicate that administration of 1α,25(OH)2D3 might provide a favorable microenvironment for orthodontic tooth movement by downregulating expression of HMGB1 in PDL cells.

Altered distribution of Ghrelin protein in mice molar development.

OBJECTIVE: Ghrelin, an appetite-stimulating hormone, plays diverse regulatory functions in cell growth, proliferation, differentiation and apoptosis during mammalian development. There is limited information currently available regarding Ghrelin expression during mammalian tooth development, thus we aimed to establish the spatiotemporal expression of Ghrelin during murine molar odontogenesis. DESIGN: Immunohistochemistry was performed to detect the expression pattern of Ghrelin in mandible molar from E15.5 to PN7 during murine tooth development. RESULTS: The results showed that Ghrelin initially expressed in the inner enamel epithelium and the adjacent mesenchymal cells below, further with persistent expression in the ameloblasts and odontoblasts throughout the following developmental stages. In addition, Ghrelin was also present in Hertwig's epithelial root sheath at the beginning of tooth root formation. CONCLUSIONS: These results suggest that Ghrelin was present in tooth organs throughout the stages of tooth development, especially in ameloblasts and odontoblasts with little spatiotemporal expression differences. However, the potential regulatory roles of this hormone in tooth development still need to be validated by functional studies.

Long-Term Administration of High-Fat Diet Corrects Abnormal Bone Remodeling in the Tibiae of Interleukin-6-Deficient Mice.

In this study, we aimed to evaluate the influence of diet-induced obesity on IL-6 deficiency-induced bone remodeling abnormality. Seven-week-old IL-6(-/-) mice and their wild type (WT) littermates were fed a standard diet (SD) or high-fat diet (HFD) for 25 weeks. Lipid formation and bone metabolism in mice tibiae were investigated by histochemical analysis. Both IL-6(-/-) and WT mice fed the HFD showed notable body weight gain, thickened cortical bones, and adipose accumulation in the bone marrow. Notably, the HFD normalized the bone phenotype of IL-6(-/-) mice to that of their WT counterpart, as characterized by a decrease in bone mass and the presence of an obliquely arranged, plate-like morphology in the trabecular bone. Alkaline phosphatase and osteocalcin expressions were attenuated in both genotypes after HFD feeding, especially for the IL-6(-/-) mice. Meanwhile, tartrate-resistant acid phosphatase staining was inhibited, osteoclast apoptosis rate down-regulated (revealed by TUNEL assay), and the proportion of cathepsin K (CK)-positive osteoclasts significantly increased in IL-6(-/-) mice on a HFD as compared with IL-6(-/-) mice on standard chow. Our results demonstrate that HFD-induced obesity reverses IL-6 deficiency-associated bone metabolic disorders by suppressing osteoblast activity, upregulating osteoclastic activity, and inhibiting osteoclast apoptosis.

Immunolocalization of MMP 2, 9 and 13 in prednisolone induced osteoporosis in mice.

Long-term use of glucocorticoids (GC) causes rapid bone loss and increases the risk of osteoporotic fractures. Matrix metalloproteinase (MMPs), the most prominent kind of proteases implicated in the proteolytic degradation of the extracellular matrix (ECM), have been reported to be involved in pathological process of GC induced osteoporosis. However, the underlining mechanisms are still unclear. The aim of this study was to investigate the spatial expression and the potential function of MMP 2, 9 and 13 in osteoporosis induced by prednisolone in the tibiae of mice. In this experiment, mice were given prednisolone (15 mg/kg body weight) in PBS intragastrically every other day, or only PBS as control. Two weeks later, mice were fixed with transcardial perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and tibiae were extracted for histochemical analysis. Compared with control group, the number of TRAP-positive osteoclasts and the immunoreactivity of MMP 2, 9 and 13 were significantly increased in the trabecular bone of mice administered with prednisolone, leading to the decrease of trabecular bone volume. On the other hand, lighter eosin staining areas containing numerous empty lacunae of osteocytes and crevices were seen in the narrowing cortical bone. Furthermore, intense immunoreaction of MMP 2 and MMP 13 were found in the enlarged lacunae and the crevices, respectively. Taken together, we concluded that prednisolone administration induced the increase of MMP 2, 9 and 13 expressions, while MMP 2 and MMP 13 played essential roles in the osteocytic osteolysis and the early impaired areas in the cortical bone. Therefore, MMPs might be new potential therapeutic targets for prevention and treatment of glucocorticoid induced osteoporosis, especially osteocytic osteolysis.

Histochemical examination of adipose derived stem cells combined with ß-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3.

The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (ß-TCP) in the restoration of bone defects under intraperitoneal administration of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with ß-TCP for 21 days under osteogenic induction. Then the ADSC-ß-TCP complexes were implanted into bone defects in the femora of rats. 1α,25(OH)2D3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: ß-TCP-NS group; ß-TCP-ADSC-NS group; ß-TCP-VD group and ß-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the ß-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded ß-TCP under the administration of 1α,25(OH)2D3 made a promising therapy for bone defects restoration.

Altered distribution of HMGB1 in the periodontal ligament of periostin-deficient mice subjected to Waldo's orthodontic tooth movement.

Periostin is essential for the integrity and function of the periodontal ligament (PDL), and periostin knockout is related to an enhanced inflammatory status in PDL. High mobility group box 1 (HMGB1), a late inflammatory cytokine, is up-regulated in PDL cells in response to mechanical stress. This study aimed to investigate the effect of periostin deficiency (Pn-/-) on HMGB1 expression in PDL during orthodontic tooth movement. We used 8-week-old male mice homozygous for the disrupted periostin gene and their wild-type (WT) littermates. Tooth movement was performed according to Waldo's method, in which 0.5-mm-thick elastic bands were inserted between the first and second upper molars of anesthetized mice. After 3 days of mechanical loading, mice were fixed by transcardial perfusion of 4% paraformaldehyde in phosphate buffer, and the maxilla was extracted for histochemical analyses. Compared with the WT group, Pn-/- mice showed higher basal expression of HMGB1 in the absence of mechanical loading. Following 3 days of orthodontic tooth movement, the PDL in the compression side of both groups was almost replaced by cell-free hyaline zones, and Pn-/- mice showed a much wider residual PDL than WT mice. In the tension side, the number of HMGB1-positive cells in PDL in both Pn-/- and WT groups increased remarkably without a significant difference between the two groups. Our findings suggest an inhibitory effect of periostin on HMGB1 production by PDL and confirmed the critical role of periostin in integrity of PDL collagen fibrils during orthodontic tooth movement, although mechanical loading is the predominant stimulant of HMGB1 expression relative to periostin deficiency.

Histochemical evidence of zoledronate inhibiting c-src expression and interfering with CD44/OPN-mediated osteoclast adhesion in the tibiae of mice.

The purpose of this study was to investigate the effect of zoledronate (ZA) on osteoclast functions and viability in the tibiae of 8-week-old male mice. After weekly intravenous administration of ZA (125 µg/kg body weight) for 8 weeks, the mice were fixed by transcardial perfusion of 4% paraformaldehyde under anesthesia, and their tibiae were extracted for histochemical analysis. Compared with the control group, many tartrate-resistant acidic phosphatase-positive osteoclasts were found on the surface of the trabecular bone, but cartilage cores were obviously increased in the metaphysis of the ZA group. Osteoclasts of both groups showed similar expression of cathepsin K and matrix metalloproteinase-9. However, hardly any expression of c-src, a gene necessary for ruffled border formation and bone resorption, was found in osteoclasts of the ZA group. Moreover, no expression of CD44 or osteopontin (OPN) was observed in osteoclasts of the ZA group. Taken together, our findings suggest that ZA administration decreases the bone resorption ability of osteoclasts by inhibiting c-src expression and suppressing osteoclast adhesion by interfering with CD44/OPN binding.

Local administration of calcitriol positively influences bone remodeling and maturation during restoration of mandibular bone defects in rats.

The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects.

Expression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice.

Recent studies indicate that high mobility group box protein 1 (HMGB1) originating from periodontal ligament (PDL) cells can be a potential regulator in the process of orthodontic tooth movement and periodontal tissue remodeling. The aim of this study is to investigate HMGB1 expression in periodontal tissue during orthodontic tooth movement in mice according to Waldo's method. Six 7-week-old C57BL6 mice were used in these experiments. The elastic band was inserted into the teeth space between the right first and second maxillary molars. After 3 days of mechanical loading, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the maxillary was extracted for histochemical analyses. The histological examination revealed local PDL tear at the tension side and the formation of extensive cell-free hyaline zones at the compression side. The immunolocalization of HMGB1 was significantly presented at tension side of PDL, apical area and dental pulp, whereas at the compression side of PDL, the labeling of HMGB1 was almost undetectable as the presence of hyaline zone. Taken together, we concluded that the orthodontic tooth movement by Waldo's method leads to histological changes and HMGB1 expression pattern that differ from those of coil spring method, including PDL tear and extensive hyaline zone which may severely destroy periodontal tissue and in turn impede tooth movement.