[Show abstract][Hide abstract] ABSTRACT: Grafted macromolecules often induce granuloma formation with foreign body giant cell (FBGC) infiltration, and this is the main reason for graft failure. Diacylglycerol kinase (DAGK) is an important intracellular mediator of FBGC formation in macrophages. In this study, 4-hexylresorcinol (4HR) inhibited DAGKδ in a macrophage cell line (RAW264.7 cells). As a result of DAGK-δ inhibition by 4HR, FBGC formation was significantly inhibited in RAW264.7 cells. Silk fibroin is a well-known natural macromolecule, and when it is grafted into bone defects, it results in granuloma formation with massive FBGC formation. 4HR-incorporating silk graft materials displayed significant reduction of granuloma formation and increases in the extent of new bone formation in a rabbit calvarial defect model. In conclusion, 4HR could inhibit foreign body reaction via a DAGK-mediated pathway.
[Show abstract][Hide abstract] ABSTRACT: Circulating osteoclast precursor cells highly express CX3C chemokine receptor 1 (CX3CR1), which is the only receptor for the unique CX3C membrane-anchored chemokine, fractalkine (CX3CL1). An irradiated murine model was used to evaluate the role of the CX3CL1-CX3CR1 axis in osteoclast recruitment and osteoclastogenesis. Ionizing radiation (IR) promoted the migration of circulating CD11b+cells to irradiated bones and dose-dependently increased the number of differentiated osteoclasts in irradiated bones. Notably, CX3CL1 was dramatically upregulated in the vascular endothelium after IR. IR-induced production of CX3CL1 by skeletal vascular endothelium promoted chemoattraction of circulating CX3CR1+/CD11b+cells and triggered homing of these osteoclast precursor cells toward the bone remodeling surface, a specific site for osteoclast differentiation. CX3CL1 also increased the endothelium-derived expression of other chemokines including stromal cell-derived factor-1 (CXCL12) and macrophage inflammatory protein-2 (CXCL2) by activating the hypoxia-inducible factor-1 α pathway. These effects may further enhance osteoclastogenesis. A series of in vivo experiments confirmed that knockout of CX3CR1 in bone marrow-derived cells and functional inhibition of CX3CL1 using a specific neutralizing antibody significantly ameliorated osteoclastogenesis and prevented bone loss after IR. These results demonstrate that the de novo CX3CL1-CX3CR1 axis plays a pivotal role in osteoclast recruitment and subsequent bone resorption, and verify its therapeutic potential as a new target for anti-resorptive treatment.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to compare peri-implant bone formation among uncoated (UC), hydroxyapatite (HA), collagen plus HA (CH), and collagen, HA, plus bone morphogenetic protein-2 (BMP-2) implant groups.
Implants in the UC group had acid-etched surfaces. The surface coating was applied using the aerosol deposition method. The coated surfaces were examined by scanning electron microscopy, x-ray diffraction (XRD), and Fourier-transformed infrared absorption analysis. Subsequently, 6 implants from each group (total, 24 implants) were installed in the tibias of rabbits. The animals were sacrificed at 6 weeks after implant installation. Peri-implant bone formation and bone-to-implant contact (BIC) were measured in histologic sections. Significant differences among groups were evaluated using analysis of variance.
Based on the measured XRD patterns, there was a characteristic HA phase (International Centre for Diffraction Data [ICDD], 086-0740) coated on the titanium (ICDD, 089-3725). Subsequent coating processes for collagen and BMP-2 did not display additional diffraction peaks, but maintained the diffraction patterns of the HA-coated titanium. The presence of collagen was verified by infrared absorption analysis. When comparing these modifications with UC surfaces, only the CH coating displayed significantly greater peri-implant bone formation and BIC (P = .003 and P < .001, respectively). Adding BMP-2 to the implant surface did not produce any advantage compared with the CH coating.
In this study, the CH group displayed significantly greater new bone formation and BIC than the other groups. There was no significant difference among the other groups.
Journal of oral and maxillofacial surgery: official journal of the American Association of Oral and Maxillofacial Surgeons 01/2014; 72(1):53-60. · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis.
[Show abstract][Hide abstract] ABSTRACT: Objective
Interleukin-10 (IL-10) is a pleiotropic immunoregulatory cytokine with a chondroprotective effect that is elevated in cartilage and synovium in patients with osteoarthritis. However, the role of IL-10 during endochondral bone formation and its mechanism of action have not been elucidated. MethodsIL-10–/– mice and IL-10–treated tibial organ cultures were used to study loss and gain of IL-10 functions, respectively, during endochondral bone formation. Primary chondrocytes from the long bones of mouse embryos were cultured with and without IL-10. To assess the role of IL-10 in chondrogenic differentiation, we conducted mesenchymal cell micromass cultures. ResultsThe lengths of whole skeletons from IL-10–/– mice were similar to those of their wild-type littermates, although their skull diameters were smaller. The tibial growth plates of IL-10–/– mice showed shortening of the proliferating zone. Treatment with IL-10 significantly increased tibial lengths in organ culture. IL-10 also induced chondrocyte proliferation and hypertrophic differentiation in primary chondrocytes in vitro. Mechanistically, IL-10 activated STAT-3 and the Smad1/5/8 and ERK-1/2 MAP kinase pathways and induced the expression of bone morphogenetic protein 2 (BMP-2) and BMP-6 in primary chondrocytes. Furthermore, the blocking of BMP signaling attenuated the IL-10–mediated induction of cyclin D1 and RUNX-2 in primary chondrocytes and suppressed Alcian blue and alkaline phosphatase staining in mesenchymal cell micromass cultures. Conclusion
These results indicate that IL-10 acts as a stimulator of chondrocyte proliferation and chondrogenic or hypertrophic differentiation via activation of the BMP signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are polykaryons that have the unique capacity to degrade bone. Modulation of osteoclast formation and function is a promising strategy for the treatment of bone-destructive diseases. Here, we report that obovatol, a natural compound isolated from Magnolia obovata, inhibits receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation in vitro and inflammatory bone loss in vivo. We found that obovatol strongly inhibited osteoclast formation from bone marrow-derived macrophages in a dose-dependent manner without cytotoxicity. Obovatol significantly suppressed RANKL-induced activation of NF-κB, c-Jun-N-terminal kinase, and extracellular signal-regulated kinase signaling pathways. Obovatol also inhibited RANKL-induced expression of the genes c-Fos and nuclear factor of activated T cells c1, which are transcription factors important for osteoclastogenesis. In addition to osteoclast differentiation, obovatol blocked cytoskeletal organization and abrogated the bone resorbing activity of mature osteoclast. Obovatol also accelerated osteoclast apoptosis through the induction of caspase-3 activation. Consistent with its in vitro anti-resorptive effect, obovatol prevented bone loss induced by lipopolysaccharide in vivo. Together, our data suggest that obovatol may be a useful therapeutic agent for the treatment of pathological bone disorders characterized by excessive osteoclastic bone resorption.
European journal of pharmacology 10/2013; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GNF-2, a tyrosine kinase inhibitor, was developed to overcome imatinib-resistant mutations found in CML patients. Osteoclasts are the principal bone-resorbing cells that are responsible for bone diseases, such as osteoporosis, tumor-induced osteolysis, and metastatic cancers. In this study, we investigated the effect of GNF-2 on osteoclast development induced by RANKL and M-CSF. We found that GNF-2 inhibited osteoclast differentiation from BMMs. GNF-2 suppressed RANKL-induced NF-κB transcriptional activity and the induction of c-Fos and NFATc1, which are two key transcription factors in osteoclastogenesis. We also observed that GNF-2 dose-dependently inhibited the proliferation of osteoclast precursors through the suppression of the M-CSFR c-Fms. In addition, GNF-2 accelerated osteoclast apoptosis by inducing caspase-3 and Bim expression. Furthermore, GNF-2 interfered with actin cytoskeletal organization and subsequently blocked the bone-resorbing activity of mature osteoclasts. In agreement with its in vitro effects, GNF-2 reduced osteoclast number and bone loss in a mouse model of LPS-induced bone destruction. Taken together, our data reveal that GNF-2 possesses anti-bone-resorptive properties, suggesting that GNF-2 may have therapeutic value for the treatment of bone-destructive disorders that can occur as a result of excessive osteoclastic bone resorption.
Journal of leukocyte biology 10/2013; · 4.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an antimetastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolin-mediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512].
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare a carbon plate (CP) and a titanium mandibular reconstruction plate (TMRP) in finite element analysis and an animal model.
Twelve rabbits were used for this experiment. After a mandible continuity defect was created, either a CP or a TMRP was used for mandibular reconstruction. Postoperatively, daily feed intake amount (DFIA) was measured for 4 weeks. Radiographic images were also acquired to evaluate screw loosening. For the analysis of the stress distribution, a simple continuity defect model was used, and finite element analysis was performed.
The CP group had 0.80 ± 0.45 lost screws in an animal during the 4 weeks postoperative observation; however, the TMRP group had 1.86 ± 0.69 lost screws (p = 0.014). Overall, the 5 out of 5 of rabbits in the CP group and 3 out of 7 in the TMRP group exhibited preoperative levels of DFIA during the 4 week observation (p = 0.038). The finite element analysis showed that the stress was more evenly distributed in the CP than in the TMRP model.
The CP group showed decreased screw loosening and increased recovery of preoperative DFIA compared to the TMRP group in a rabbit model of mandibular continuity defects. Perfect adaptation of CP during the operation could not be achieved in spite of reshaping to the mandibular curvature. This disadvantage of the CP system can be overcome by the prefabricated technique using a prototype model.
Journal of cranio-maxillo-facial surgery: official publication of the European Association for Cranio-Maxillo-Facial Surgery 09/2013; · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material. [BMB Reports 2013; 46(6): 328-333].
[Show abstract][Hide abstract] ABSTRACT: Runt-related transcription factor 2 (Runx2) plays an important role in bone formation and de novo synthesis of proteins, including type 1 collagen. Runx2 has a potent effect on signaling of transforming growth factor (TGF)-β and vice versa, implicating its significant role in fibrosis. Chronic renal failure comprises fibrosis, characterized as an increase in TGF-β signaling, and expression of α-smooth muscle actin (α-SMA), and extracellular matrix proteins. Here, we evaluated the role of Runx2 in ureteral obstruction (UO)-induced kidney fibrosis using mice whose Runx2 gene expression is genetically down-regulated. UO caused tubular atrophy and dilation, expansion of interstitium, and increased expression of collagens and α-SMA with a concomitant decrease in expression of Runx2. Deficiency of Runx2 gene (Runx2(+/-) mice) showed higher expression of collagens and α-SMA in the kidney following UO compared to wild type (Runx2(+/+)) mice. UO-induced activation of TGF-β signaling was higher in the Runx2(+/-) kidney than Runx2(+/+) kidney, suggesting an inhibitory effect of Runx2 on TGF-β signaling in kidney fibrosis. Besides, overexpression of the Runx2 gene using an adenoviral vector in kidney tubule cells resulted in attenuated TGF-β-induced Smad3 phosphorylation and expressions of α-SMA and collagen I. Furthermore, Runx2 gene deficient mouse embryonic fibroblasts induced greater activation of Smad3 and expression of α-SMA in response to TGF-β. Collectively, Runx2 plays a protective role in UO-induced kidney fibrosis by inhibition of TGF-β signaling, suggesting Runx2 as a novel target for protection against fibrosis-related diseases such as chronic renal failure.
Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bacterial dormancy-inducing factor 4-hexylresorcinol (4-HR) has been shown to have synergistic antitumor effects when used in combination with cisplatin. In the present study, 4-HR was used as a single agent in the squamous carcinoma cell line SCC-9. The results demonstrated that 4-HR suppressed SCC-9 cell proliferation compared to primary cultured gingival fibroblasts. 4-HR dose-dependently induced SCC-9 cell apoptosis as determined by caspase-3 activity, annexin V expression, as well as by scanning and transmission electron microscopy. 4-HR inhibited intracellular calcium oscillation in both SCC-9 cells and normal human dermal fibroblasts. 4-HR-induced apoptosis was partly reversed by calcium channel blockers. Of note, 4-HR reduced the tumor mass formed by SSC-9 cell implantation in BALB/cAnNCrj-nu/nu mice and mass size reduction was also partly reversed by the concomitant application of calcium channel blockers. Collectively, our results suggest that 4-HR has strong antitumor effects by inhibiting calcium channel oscillation and inducing apoptosis.
[Show abstract][Hide abstract] ABSTRACT: AIMS: Dual Ig domain Containing Adhesion Molecule (DICAM), a protein with homology to the junctional adhesion molecule family, has been demonstrated to interact with integrin αVβ3 that plays a critical role in angiogenesis. Here, we determined the role of DICAM during angiogenesis and the molecular mechanisms involved in the inhibition of angiogenesis.Methods and ResultsDICAM was expressed on the endothelial cells of large vessels to small capillaries. In human umbilical vein endothelial cells (HUVECs), DICAM was up-regulated by vascular endothelial growth factor (VEGF) through the MEK/ERK and PI3 K/AKT pathways. Furthermore, the exogenous expression of DICAM in HUVECs suppressed angiogenesis in vitro Matrigel and in vivo plug assays, and conversely, DICAM knockdown enhanced angiogenesis. In addition, DICAM inhibited HUVEC migration and accelerated apoptosis via down-regulation of Bcl-2, but did not affect viability or proliferation of HUVEC. Mechanistically, the exogenous expression of DICAM suppressed VEGF-induced phosphorylarion of AKT and p38 MAP kinase. When integrin signaling was activated by vitronectin, a forced expression of DICAM attenuated integrin β3/FAK signaling and downstream AKT and p38 MAP kinase signaling in HUVECs. CONCLUSION: Collectively, DICAM suppressed angiogenesis by attenuating AKT and p38 MAP kinase signaling, which suggests that DICAM may be a novel negative regulator of angiogenesis.
Cardiovascular research 02/2013; · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr (-/-) ) and Runx2 carboxy-terminus truncated heterozygous (Runx2 (+/ΔC) ) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr (-/-) or Runx2 (+/ΔC) mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.
PLoS ONE 01/2013; 8(12):e83584. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Epiphyseal stapling has been widely used to correct angular deformity. The mechanism, however, has not been well determined. To determine the effect of temporary hemiepiphyseal stapling on the cellular layers of the physis, a histomorphometric study was performed using immature rabbits. METHODS: Distal lateral epiphyseal stapling of the right femur was performed on 6-week-old New Zealand white rabbits. Thirty rabbits were randomly assigned to five groups, and six rabbits in each group were analyzed weekly for up to 5 weeks. RESULTS: The distal femur was deformed into the valgus, and the anatomical lateral distal femoral angle decreased with the passage of time. In the sequential histomorphometry of the operated physeal plate, the area ratio of each layer, compared to the control side, decreased every week. The total area of the physeal plate had decreased up to 60 % at the 5th week compared to the area of the 1st week, and the area of the proliferative layer decreased by the greatest amount among the three layers. CONCLUSIONS: Our findings suggest that the proliferation of chondrocytes seemed to be more suppressed by the compression of the stapling, thereby slowing the growth rate, although hypertrophy of the chondrocytes was also suppressed.
Journal of Orthopaedic Science 11/2012; · 0.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Early growth response 2 (Egr2) is a zinc finger transcription factor that acts as an important modulator of various physiological processes. In this study, we show that Egr2 negatively regulates receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of Egr2 in bone marrow-derived macrophages (BMMs) suppresses the formation of multinuclear osteoclasts and the expression of osteoclastogenic markers, including nuclear factor of activated T cells c1 (NFATc1). On the other hand, Egr2 overexpression does not impact the phagocytic activity of osteoclast precursors or the expression of macrophage-specific markers in the presence of the osteoclastogenic stimuli, RANKL and M-CSF. We further demonstrate that Egr2 induces the expression of the inhibitors of differentiation/DNA binding (Ids) helix-loop-helix (HLH) transcription factors, which are important repressors in RANKL-mediated osteoclastogenesis. Egr2 transactivates the Id2 promoter and increases its recruitment to the Id2 promoter region. In addition, Egr2-dependent induction of Id2 promoter activity, and its binding to the Id2 promoter is abrogated by the overexpression of the Egr2 repressor, NGFI-A binding protein 2 (Nab2). Accordingly, coexpression with Nab2 restores Egr2-mediated suppression of osteoclast differentiation. Furthermore, knockdown of Egr2 using shRNA enhances osteoclastogenesis and decreases Id2 gene expression. Ectopic expression of Id2 reverses the phenotype mediated by Egr2 silencing. Taken together, our results identify Egr2 as an important modulator of RANKL-induced osteoclast differentiation and provide the link between RANKL, Egr2 and Id proteins in osteoclast-lineage cells.
[Show abstract][Hide abstract] ABSTRACT: The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: The objective of this study was to evaluate the effects of silver nanoparticles (AgNPs) on the Toll-like receptor 2 (TLR-2) pathway in cultured cells. STUDY DESIGN: Human chondrocytes, periodontal ligament (PDL) cells, and SCC-9 cells (squamous cell carcinoma from the tongue) were cultured and subjected to cytotoxicity assays. To evaluate the effects of AgNPs on the TLR-2 pathway, TLR-2 small interfering (si) RNA or TLR-2 antibodies were applied to the chondrocytes, followed by the application of AgNPs. RESULTS: AgNPs induced dose-dependent effects on the examined cell types in terms of both cytotoxicity and TLR-2 expression levels. AgNP-mediated apoptosis was reduced after treatment with TLR-2 siRNA in both PDL cells and chondrocytes. Furthermore, functional blocking of TLR-2 with anti-TLR2 antibodies inhibited AgNP-mediated cytotoxicity. AgNPs increased c-Jun phosphorylation, an effect that was reversed after treatment with TLR-2 siRNA. CONCLUSIONS: The results indicate that AgNP-mediated apoptosis most likely occurs via the TLR-2 pathway.