[Show abstract][Hide abstract] ABSTRACT: Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.
PLoS ONE 11/2015; 10(11):e0142201. DOI:10.1371/journal.pone.0142201 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE.
[Show abstract][Hide abstract] ABSTRACT: After palatal fusion, the dorsal and ventral epithelia of the palatal shelf differentiate into the nasal and oral mucosa, respectively. The tissue-specific differentiation of palatal epithelia along the dorsal–ventral axis is regulated by the signaling molecules expressed in the underlying mesenchyme. Thus, as in many other epithelial organs, differentiation relies on epithelial–mesenchymal interactions. To screen for region-specific mesenchymal signaling molecules that determine the fate of the palatal epithelia, we employed a laser microdissection (LMD) method. LMD allowed us to collect region-specific mesenchymal tissues at E13, prior to palatal fusion and the development of distinct dorsal and ventral epithelial morphology. Genome-wide screening was performed on the tissues collected using LMD to identify candidate mesenchymal signaling molecules. The microarray results were validated using real-time quantitative (qPCR) and in situ hybridization methods. The developmental role and interactions of the candidate genes were evaluated in in vitro-cultivated E13 palates using an anti-sense oligodeoxynucleotide (AS-ODN)-based loss-of-function approach. Apparent changes in the expression patterns of Runt-related transcription factor 2 (Runx2) and LIM homeobox 8 (Lhx8) were observed after knocking down each gene. Knock-down of Runx2 and Lhx8 also altered the immunolocalization pattern of cytokeratin18 (CK18), an established marker for nasal epithelium. These results were confirmed using Runx2 heterozygote mice. The mesenchymal signaling molecules Runx2 and Lhx8, which possess region-specific expression patterns along the dorsoventral axis, functionally interact to regulate the cellular and molecular characteristics of dorsal and ventral epithelia, suggesting that mesenchymal signaling molecules determine the dorsoventral fate of epithelial structures in the developing palate.
Cell and Tissue Research 06/2015; DOI:10.1007/s00441-015-2222-8 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the effects of triptolide on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and on titanium (Ti) particle-induced osteolysis.
To examine the effect of triptolide on osteoclast differentiation, bone marrow macrophages (BMMs) were treated with 100 ng/mL of RANKL and 30 ng/mL of macrophage-colony stimulating factor, or co-cultured with osteoblasts stimulated with 10 nM vitamin D3 and 1 μM prostaglandin E2 in the presence or absence of triptolide (2.8-14 nM). Osteoclast differentiation and activation were assessed using tartrate-resistant acid phosphatase staining, reverse transcriptase-polymerase chain reaction analysis to determine differentiation marker gene expression and pit formation assays. To examine the effect of triptolide on wear debris-induced osteolysis, titanium (Ti) particles were injected into the calvaria of ICR mice. Then, the mice were divided into three groups and were orally administered vehicle, or 16 or 32 μg/kg/day triptolide for ten days, followed by histomorphometric analysis.
Triptolide suppressed RANKL-mediated osteoclast differentiation of BMMs in a dose-dependent manner. In a co-culture system, osteoblasts treated with triptolide could not induce osteoclast differentiation of BMMs, which was accompanied by down-regulation of RANKL and up-regulation of osteoprotegrin. Moreover, triptolide significantly inhibited bone resorption, and expression of the bone resorption marker genes. RANKL-induced activation of p38, ERK, and JNK was substantially inhibited by triptolide. Further, in a Ti-induced mouse calvarial erosion model, mice perorally administrated with triptolide showed significant attenuation of Ti-mediated osteolysis.
Our data indicated that triptolide had an anti-osteoclastic effect and significantly suppressed wear debris-induced osteolysis in mice.
International Orthopaedics 11/2014; 39(1). DOI:10.1007/s00264-014-2596-3 · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SPARC related modular calcium binding 1 (SMOC1) has been implicated in the regulation of the osteoblast differentiation of human bone marrow mesenchymal stem cells (BMSCs); however, it is not clear how it regulates this process. In this study, we analyzed molecular networks during the SMOC1-induced osteoblast differentiation of BMSCs. SMOC1 overexpression induced the deposition of minerals and the expression of osteoblast marker genes. During osteoblast differentiation induced by SMOC1 overexpression, gene profiling was performed using an Affymetrix GeneChip. The expression of 191 genes was increased by SMOC1 overexpression, and these genes were classified into 17 groups based on gene ontology: developmental process (23%), cellular component organization (23%), cell death (10.5%), cell cycle (9.9%), cell adhesion (7.9%), and cytoskeleton organization (5.2%). Protein interaction analysis also showed the strong interaction of proteins involved in development, cell death, and cell cycle. Consistently, proliferation of BMSCs overexpressing SMOC1 was significantly increased compared to BMSCs expressing the control vector. These results suggest that SMOC1 induces the osteoblast differentiation of BMSCs by inducing the expression of an array of genes involved in cell growth and development.
Tissue Engineering and Regenerative Medicine 08/2014; 11(4):304-316. DOI:10.1007/s13770-014-0021-z · 1.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives
This study contributes three well-documented cases of multiple simple bone cysts (SBCs) of the jaws and reviews previously published cases.
A comprehensive literature search of multiple SBCs was conducted using PubMed database. Synonyms of SBC were used as search key words in combination with “mandible or jaw”, “bilateral, multiple, multifocal, atypical, and unusual”.
A total of 34 cases of multiple SBCs (including 2 asynchronous cases) were identified, including the three new cases reported here. Multiple SBCs primarily occurred in the second decade (52.9 %) and bilaterally in the posterior mandible. Lesions showed female predominance (1.8:1) and were frequently accompanied by bony expansion (44.1 %) and a multilocular radiolucent appearance (20.6 %). Recurrence was reported in 3 patients (mean age: 39.3 years old).
Knowledge of the clinical and radiographic features of multiple SBCs is important in the diagnosis and management of this entity.
[Show abstract][Hide abstract] ABSTRACT: Background
Transcription factors have been implicated in regulating the differentiation of odontoblasts from dental pulp stem cells/progenitors (DPSCs/progenitors), but their regulatory network is not completely understood.
New transcription factors that control the odontoblast differentiation of human DPSCs/progenitors were analyzed using a microarray. The result revealed bobby sox homolog (BBX) to be expressed most strongly during odontoblast differentiation. Validation using RT-PCR also revealed the strong expression of BBX during the odontoblast differentiation of DPSCs/progenitors. BBX expression was also detected in adult molar odontoblasts and other tissues, including the heart, kidney, testis, and bone marrow. To understand the role of BBX in odontoblast differentiation, BBX variant 1 and 2 cDNA were cloned and overexpressed in DPSCs/progenitors. The results showed that the overexpression of BBX cDNA in DPSCs/progenitors induced substantial mineralization and expression of the odontoblast marker genes, such as ALP, OPN, BSP, DMP1, and DSPP. The knockdown of BBX using shRNA, however, did not affect mineralization, but the expression of ALP and DSPP was decreased substantially. Meanwhile overexpression or knockdown of BBX did not modulate proliferation of DPSCs/progenitors.
Our results suggest that BBX plays an important role during the odontoblast differentiation of human DPSCs/progenitors.
Cell Communication and Signaling 05/2014; 12(1):35. DOI:10.1186/1478-811X-12-35 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.
Cell and Tissue Research 05/2014; 358(1). DOI:10.1007/s00441-014-1908-7 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)2D3-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)2D3-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.
The Journal of Cell Biology 03/2014; 204(6). DOI:10.1083/jcb.201308102 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Without exploitation of possibly immunogenic and carcinogenic bone morphogenetic protein, we developed simple but clinically feasible artificial bone graft using osteoconductive hyaluronate (HA) hydrogels and bioactive MegaGen synthetic bone (MGSB).
HA hydrogels were synthesized by the crosslinking reaction between carboxyl groups of HA and amine groups of gelatin (GEL). Then, artificial bone grafts were prepared by mixing MGSB with HA-GEL hydrogels. The bone regeneration by the MGSB/HA-GEL hydrogel complex was assessed in the skull of New Zealand white male rabbits in 4 and 8 weeks.
HA hydrogels were synthesized by the crosslinking reaction between carboxyl groups of HA and amine groups of gelatin (GEL). Then, artificial bone grafts were prepared by mixing MGSB with HA-GEL hydrogels. In vitro proliferation of preosteogenic cells was enhanced with increasing molecular weight of HA. In addition, histological analysis of dissected tissues with hematoxylin and eosin staining confirmed the effective in vivo bone regeneration by the MGSB/HA-GEL hydrogel complex. The MGSB/HA-GEL hydrogels were well resorbed and partially substituted to the lamellar bone after implantation for 8 weeks.
The novel artificial bone graft of MGSB/HA-GEL hydrogel complex for effective bone regeneration might be clinically feasible for further development.
[Show abstract][Hide abstract] ABSTRACT: Human amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated.
In order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced human AFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxilin and eosin, immunocytochemistry, and formation of neuro-muscular junction.
MYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD, and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1, and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more neuro-muscular junction, indicating functional restoration of muscle injury by hAFS cells expressing by MYOD.
Collectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.
[Show abstract][Hide abstract] ABSTRACT: An ideal scaffold for bone tissue regeneration should be dissolved at the same rate of host bone growth into the defect. Therefore, to produce such a scaffold, it is necessary to obtain a standard healing rate of bone defects. In this study, we compared healing rate of bone defects in calvarial and long bones, which have differential developmental and regenerative mechanisms. In the calvaria and tibia, 3 mm defects were made, and healing was analyzed using microcomputed tomography (microCT) and histology up to six weeks. MicroCT analysis showed that in calvarial defects, an unhealed gap remained until six weeks, whereas tibial defects had healed after three weeks. H&E and Trichrome staining consistently showed that calvarial defects were not completely healed by six weeks, however, a tibial defect started to heal from three weeks. Results of histomorphometric analysis showed that 60% of calvarial defects had healed at six weeks after surgery, whereas 80% of tibial defects showed regeneration at three weeks. Cartilage formation was detected only in tibial defects, suggesting endochondral regeneration in long bone, but not in flat bone. Collectively, these results demonstrate that healing of a long bone defect is faster than that of flat bone by approximately two folds. Therefore, our data suggest that dissolution of scaffold should be optimized based on the type of bone defect.
Tissue Engineering and Regenerative Medicine 12/2013; 10(6). DOI:10.1007/s13770-013-1094-9 · 1.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 10-year-old boy presented with a swelling on the right side of the jaw. He had undergone excision of the lesion about 10 months ago at a private dental clinic and the swelling began to regrow 4 months after surgery. A panoramic radiograph revealed 4 sclerotic round masses with radiolucent rims surrounded by sclerosis of the right posterior mandible. Computed tomography scan showed 4 round bony masses centered on the buccal cortex and bone marrow space, sclerosis of the adjacent bone and periosteal reaction. He underwent a marginal resection under general anesthesia and the final histopathological report confirmed the diagnosis of osteoid osteoma. Postoperative course was uneventful, and there was no evidence of recurrence at the 5.5-year follow-up. In the review of the literature, 20 osteoid ostemas were found in the jaw and to the best of our knowledge, the present case is the only one showing multifocal nidi.
[Show abstract][Hide abstract] ABSTRACT: Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.
[Show abstract][Hide abstract] ABSTRACT: Ectopic mineralization of soft tissues is known to be a typical response to systemic imbalance of various metabolic factors as well as tissue injury, leading to severe clinical consequences. In this study, coxsackievirus B3 (CVB3) infection in mice resulted in significant tissue injury, especially in the heart and pancreas. Inflammatory damage and apoptotic cell death were observed in CVB3-infected heart and pancreas tissues. Along with tissue damage, substantial ectopic calcification was detected in CVB3-infected heart, pancreas, and lung tissues, as determined by von Kossa staining and calcium content quantification. In addition, CVB3 infection induced upregulation of osteogenic signals, including six genes (BMP2, SPARC, Runx2, osteopontin, collagen type I, and osterix) in the heart, three genes (SPARC, osteopontin, and collagen type I) in the pancreas, and two genes (BMP2 and alkaline phosphatase) in the lung, as determined by quantitative real-time PCR analysis. Intriguingly, we showed that α-lipoic acid diminished CVB3-mediated inflammatory and apoptotic tissue damage, subsequently ameliorating ectopic calcification via the suppression of osteogenic signals. Collectively, our data provide evidence that ectopic calcification induced by CVB3 infection is implicated in the induction of osteogenic propensity, and α-lipoic acid may be a potential therapeutic agent to treat pathologic calcification.
Biochemical and Biophysical Research Communications 01/2013; 432(2). DOI:10.1016/j.bbrc.2013.01.061 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was aimed to evaluate the effects of low-level laser therapy (LLLT) in the treatment of trabecular bone loss induced by skeletal unloading. Twelve mice have taken denervation operation. At 2 weeks after denervation, LLLT (wavelength, 660 nm; energy density, 3 J/cm(2)) was applied to the right tibiae of six mice (LASER) for 5 days/week over 2 weeks by using a minimally invasive laser needle system (MILNS) which consists of a 100 μm optical fiber in a fine needle (diameter, 130 μm). Structural parameters and histograms of bone mineralization density distribution (BMDD) were obtained before LLLT and at 2 weeks after LLLT. In addition, osteocyte, osteoblast, and osteoclast populations were counted. Two weeks after LLLT, bone volume fraction, trabeculae number, and trabeculae thickness were significantly increased and trabecular separations, trabecular bone pattern factor, and structure model index were significantly decreased in LASER than SHAM (p < 0.05). BMDD in LASER was maintained while that in SHAM was shifted to lower mineralization. Osteocyte and osteoblast populations were significantly increased but osteoclast population was significantly decreased in LASER when compared with those in SHAM (p < 0.05). The results indicate that LLLT with the MILNS may enhance bone quality and bone homeostasis associated with enhancement of bone formation and suppression of bone resorption.
Lasers in Medical Science 01/2013; 28(6). DOI:10.1007/s10103-013-1265-x · 2.49 Impact Factor