The expression of TRAF6 protein and TRAF6 mRNA during different stages of deciduous tooth root physiological absorption was observed. Deciduous teeth with physiological absorption period were divided into 3 groups according to the length of deciduous tooth root absorption: early stage of deciduous tooth root physiological absorption (I group), intermediate stage of deciduous teeth roots’ physiological absorption (II group), final stage of deciduous tooth root physiological absorption (III group). TRAF6 antibody and TRAF6 hybridization in situ agent were used to detect the expression of TRAF6 protein and TRAF6 mRNA of odontoclasts of deciduous teeth with different physiological absorption stages. Im-pro-plas 6.0 analytical system software was used to measure the optical density of odontoclasts stained in three groups. SPSS13.0 software was used to analyze the regularity of expression. Optical density value of TRAF6 immunohistochemistry stain and TRAF6 mRNA in situ hybridization stain of odontoclasts was larger in I and II group compared with the value in III group. It indicated that odontoclasts in early and intermediate stages showed more activity than in the final stage. Expression of TRAF6 protein and TRAF6 mRNA during different stages of deciduous tooth root physiological absorption was consistent with the activity of odontoclast.

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We examined the change of HSP70 expression in the mouse dental pulp which was exposed to experimental teeth separation by immunohistochemistry as a model for conservative dental treatment. Eight week-old 18 male ddY mice were used and a wedge was inserted between upper 1^st and 2^nd molars for 3 hours. In the experimental group, HSP70 expression increased in the dental pulp tissues. The expression was the greatest at 24 hours after which it gradually decreased to become similar to the level of the control group at 1 week. These results suggest that when immunohistochemical expression of HSP70 is used as an index, almost no severe damage occurs upon clinical application of a wedge insertion.

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Mineral trioxide aggregate (MTA) is a commonly used endodontic repair material reported to exhibit calcified-tissue conductive activity. The effect of MTA on osteoblast differentiation has been well-defined; however, its effect on mesenchymal cells is not fully understood. Recently, we reported that MTA converts the differentiation pathway of pluripotent mesenchymal cells (C2C12 cells) into osteoblast lineages, possibly through the influx of elution components, such as calcium ions released from MTA. The purpose of this study was to investigate whether calcium ions released from MTA flow directly into C2C12 cells and whether this influx increases intracellular calcium levels. C2C12 cells were loaded with fluo-3 acetoxymethyl ester, which acts as a calcium-sensitive fluorescent probe. The intracellular calcium levels of C2C12 cells were assessed by confocal laser scanning microscopy. MTA induced a rapid increase in fluorescence intensity (FI) in C2C12 cells; however, application of Dulbecco’s modified Eagle’s medium (DMEM) as the growth medium had no such effect. To clarify the specific effects of MTA-derived calcium ions, we used calcium chloride as an exogenous calcium ion source and ethylene glycol tetraacetic acid (EGTA) as a calcium chelator. DMEM containing calcium chloride elicited a rapid rise in FI, whereas MTA plus EGTA did not affect FI. Moreover, MTA did not affect FI in C2C12 cells pre-incubated with verapamil, an L-type voltage-dependent calcium channel (VDCC_L) blocker. Our data suggest that calcium ions released from MTA flow directly into C2C12 cells via VDCC_L.

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The reciprocal and highly regulated processes of cellular proliferation, cellular differentiation, and progression to a postmitotic state during embryogenesis generate the cellular diversity in the developing nervous system. Growth factors, in part, can regulate these proliferative or differentiation processes by analogous mechanisms. Basic fibroblast growth factor (bFGF), a member of FGF family, has broad biological functions involving the regulation of cell growth, differentiation, and proliferation. As extension and remodeling of neurites play essential roles in development and neuronal plasticity, we investigated a role for nerve growth factor receptor (NGFR) on bFGF-induced neurite outgrowth in rat pheochromocytoma cell line, PC12. Our goal in the present study was to determine if there is a causal link between bFGF and NGFR. Results of these studies indicate that bFGF is required for NGFR-induced changes in morphology and transcriptional induction of the gene. We have provided convincing evidence that inhibitor of bFGF, PD173074, completely inhibited NGFR protein expression, whereas it partially blocked the NGFR protein expression in response to bFGF in PC12 cells. Another important finding of our study provides the data on the involvement of bFGF in MAPK-dependent signaling pathways and neurite outgrowth in PC12 cells, which suggests a central role of MAPK in the neuronal induction by bFGF. Taken together, these results raise the possibility that bFGF activates a MAPK-mediated pathway related to NGFR expression.

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The gingival epithelium is a physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues which requires protection from exposure to bacteria and their products. Gingival inflammation results from the coordinated activation of cascades of signaling pathways initiated by growth factors and their receptors. Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, belong to the neurotrophin family of growth factors and mediate vital physiological functions in the brain, and they are expressed in a wide variety of non-neuronal tissues. Aberrant neurotrophin signaling underlies the pathogenesis of several inflammatory conditions, yet little is known about the role of neurotrophins in the gingival inflammation. To fill this gap in knowledge, we analyzed the patterns of BDNF and TrkB expression in the inflamed tissues of human and in experimental rat periodontitis tissues. Gingival tissues from patients with periodontitis demonstrated greater levels of BDNF and TrkB expression than the tissues from healthy individuals, while an in vivo rat study showed increased expression of BDNF and TrkB in experimental rat periodontitis tissues. Interleukin-1β(IL-1β), a pro-inflammatory cytokine, elevated the expression of BDNF and TrkB mRNAs in human gingival fibroblast (HGF) and human gingival epithelial (HGE) cells. Inducible NOS (iNOS) mRNA expression was elevated in HGF cells that were stimulated by IL-1β. Also, IL-1β stimulated protein expression of BDNF and iNOS in HGF cells. ELISA confirmed that the expression of BDNF by HGF was upregulated in the presence of IL-1β. Addition of S-Methylisothiourea Sulphate (SMT), a specific inhibitor of iNOS, profoundly reduced the production of BDNF in HGF. Together, these results support a role for BDNF as a new modulator of gingival inflammation. Gingival fibroblast cells within inflammatory environment can significantly increase the production of neurotrophic factors, which may be involved with the anti-inflammatory mechanisms.

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Increasing evidence suggests an association between periodontal disease and low birthweight (LBW). We previously studied the effect of lipopolysaccharide (LPS) from periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) on human trophoblasts, and found that Aa-LPS induces apoptosis via the mitochondria-dependent pathway. This effect may contribute to the pathogenesis of periodontitis-associated LBW. In the present study, we observed the human placental trophoblast-like BeWo cells’ response to Aa-LPS. Human placental trophoblast-like BeWo cells were cultured and treated with Aa-LPS. The time-dependant effects of Aa-LPS on cultured BeWo cells were studied using the Affymetrix GeneChipTM system (human; ca. 39500 genes). Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR analysis of gene expression were also carried out to confirm the DNA microarray results. In BeWo cells, Aa-LPS altered many gene expressions including the alpha subunit of the heterotrimeric G protein that stimulates adenylyl cyclase (GNAS) mRNA level. The reduction of GNAS gene expression by Aa-LPS was successfully confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In conclusion, since GNAS was found to be particularly important to placental and fetal growth, the reduction of GNAS gene expression by Aa-LPS may be involved in growth retardation of the fetus, and may be the molecular basis of the mechanism of periodontitis-associated LBW.

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The effect of compression force on periodontal ligament (PDL) plays a critical role in orthodontic tooth movement. However, little is known about this effect on apoptosis and the underlying reactive mechanisms in PDL cells. This study focuses on the application of compression force associated with reactive oxygen species (ROS)-induced apoptosis in PDL cells. Following the application of orthodontic force to maxillary first molars, the molars were investigated using immunohistochemical analysis. The cell cycle and apoptosis were assessed in compression force-treated PDL cells using flow cytometry. Furthermore, we assayed the effect on ROS generation by DCFDA fluorescence. In vivo study revealed an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 8-positive cells in the orthodontic force group. In our in vitro study model, the compression force increased the G1 phase and apoptotic cells. A large increase in the intracellular ROS levels in human (h) PDL cells was observed in the compression force group. Taken together, these results provide new information on compression force-induced G1 arrest and apoptosis in hPDL cells.

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Objective: To investigate the expression and correlation analysis of β-adrenoceptors (AR), VEGF, MMP-9 and caspase-3 in the different phases of infantile hemangioma (IH) and to find out the possible mechanism of β-AR antagonist propranolol treatment for IH. Methods: 52 fresh operative IH specimens were collected and were divided into 2 groups: 32 patients of proliferating phase and 20 patients of involuting phase. Ten specimens of normal skin were also collected as the control group. The expression of β-AR, VEGF, MMP-9 and caspase-3 were detected by immunohistochemistry. The integral optical density and positive area rate of β-AR, VEGF, MMP-9 and caspase-3 were examined using a microscopic image analysis system. The correlations among the above markers were also analyzed using Pearson correlation coefficients. Results: β1-AR and β2-AR were both strongly expressed in the proliferating and involuting hemangioma tissue. There was a significant difference (P<0.01) for the expression of β1-AR and β2-AR between the hemangioma tissue and the normal skin. VEGF and MMP-9 were strongly expressed in the proliferating hemangioma and weakly expressed in the involuting hemangioma, and there was a significant difference (P<0.01) for the expression of VEGF and MMP-9 between the proliferating hemangioma and involuting hemangioma or between the proliferating hemangioma and the normal skin. There was a significantly positive correlation between the expression of β-AR and the expression of VEGF and MMP-9 in the proliferating phase of IH. There was a significant difference (P<0.01) for the expression of caspase-3 between the proliferating hemangioma and involuting hemangioma or between the involuting hemangioma and the normal skin. There was a significantly negative correlation between the expression of β-AR, VEGF and MMP-9 and the expression of caspase-3 in the proliferating phase of IH. Conclusions: β-AR, VEGF, MMP-9 and caspase-3 have an effect on the pathogenesis of hemangioma. β-AR antagonist propranolol might treat IH by the mechanism of regulating the β-AR/VEGF/MMP-9/caspase-3 pathway, inhibit the proliferation of hemangioma, promote the apoptosis of hemangioma, and eventually bring about the regression of IH.

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Oral mucosa consists of the superficial epithelium and the underlying lamina propria, and it functions as a barrier against exogenous substances. In development, interactions of stem/progenitor cells of the epithelium and mesenchyme are crucial to the morphogenesis of oral mucosa. Our previous work in low level fluoride-induced cell motility of epithelial cells has yielded important clues for periodontal physiology. This study focuses on a working concept of low level fluoride to provide a conducive oral environment for pivotal epithelial-mesenchymal interactions. Cultured human primary gingival epithelial cells treated with 50 μM NaF were investigated by DNA microarray. Quantitative real-time PCR and an in vivo experimental rat skin wound model were employed to confirm the findings obtained with the microarray analysis. Microarray data revealed that low level fluoride-treated human gingival epithelial cells elevated various biological processes. The key proliferation markers, FGF2 and FGF7, and mesenchymal marker, Twist1, expression was up-regulated and quantitative real-time PCR confirmed this observation. Our in vivo study revealed that low concentration of NaF increased FGF2, FGF7, and Twist1 protein expression in fluoride-treated skin wound tissues compared with controls. These results provide new information on low level fluoride-induced epithelial-mesenchymal interactions and may thus aid in the understanding of oral mucosa development.

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The aim of this study was to develop experimental titanium implants sputter-coated from a hydroxyapatite target and to evaluate peri-implant tissue responses in an animal experiment. The experimental implants were prepared from plastic rods, each of which was 1.6 mm in diameter and 7 mm in length. A thin titanium film was deposited onto the rods by DC magnetron sputtering. The surface of each rod was subsequently sputter-coated from a hydroxyapatite target by RF magnetron sputtering. The experimental implants were placed in the tibiae of 8-week-old male SD rats. Titanium-coated implants were placed as controls using the same method. Tissue samples were obtained 3, 5, 7, 10, 14, and 28 days after implant placement. Histological evaluation was performed using a light microscope, and the bone-implant contact ratios (BICs) were measured. Microstructural observations of implant-bone interfaces were made using a transmission electron microscope (TEM). Peri-implant osteoblastic activity was evaluated using samples obtained 5, 7, and 10 days after implant placement. Immunohistochemical evaluation of type I collagen, osteopontin, and osteocalcin was performed. Uniform smoothness of experimental implant surfaces was confirmed by scanning electron microscope(SEM) images. In the calcium phosphate layer, the compositional ratio of the experimental implant surface was Ca:P:O=1.0:0.79:2.8 based on the results of X-ray photoelectron spectroscopy(XPS). In the trabecular bone region, the BIC ratio was significantly higher in the experimental group than in the control group at 5, 7, 10, and 28 days after implant placement. Type I collagen, osteopontin, and osteocalcin immunostaining revealed that the experimental group tended to show positive results earlier than the control group. The results of this study suggest that surface treatment using a hydroxyapatite target and RF magnetron sputtering can enhance new bone formation during implant osseointegration.

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The aim of this study was to examine histomorphologically how bisphosphonate (BP) injected before and after implant placement surgery affects the peri-implant bone in vitamin D-deficient animal model. This study used 60 six-week-old male Sprague-Dawley (SD) rats which were given a vitamin D-deficient diet. These experimental animals were divided into Group 1 (BP administration starting before implant placement), Group 2 (control group) and Group 3 (BP administration starting after implant placement). Threaded titanium implant was placed 0.50 mm mesial to the first molar. The samples were tissues from Groups 1 and 2 obtained at 1, 2, 4 and 8 weeks after implant placement and tissues from Group 3 obtained after the start of BP administration. Evaluation by light microscope and micro-CT imaging were performed. In Group 1, bone density around implants significantly increased as time progressed from after implant placement. In Group 3, bone density significantly decreased as the duration of bisphosphonate use increased. Group 1 had a significantly higher proportion of lacunae without osteocytes compared with Group 2 after implant placement. In Group 3, the proportion of these lacunae increased significantly from one to two weeks after the start of bisphosphonate administration. There were significantly higher proportions at 4 weeks and 8 weeks after the start of administration compared with 1 week after the start. This histological result suggests the need to exercise caution in using implant treatment on patients taking bisphosphonates and in administering bisphosphonates in patients after implant placement.

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This study aimed to investigate whether adenovirus-mediated VEGF gene therapy could improve bone healing, and it compared the effects of an in vivo versus ex vivo approach using rats. A replication-deficient adenovirus containing human VEGF165 and green fluorescence protein (GFP) genes AdGFP/hVEGF165 was used in this study. The rats were divided into 5 groups, and a bone fracture model was used in all of the rats. In the in vivo group, the adenovirus particles were directly injected infraperiosteally. In the ex vivo group, the rat bone marrow stromal cells (BMSC) that had been infected by AdGFP/hVEGF165 in vitro were seeded onto a resorbable matrix and used to treat the bone fracture by injection infraperiosteally at specific anatomic sites. Three groups served as control subjects. Two groups were treated either with adenovirus containing green fluorescence protein gene (AdGFP) or with BMSCs that had been transfected by AdGFP. Another group was left untreated. Fracture healing was evaluated radiographically and histologically. The results showed that VEGF gene treated rats had more ossification at the fracture site than did the control groups 2 weeks after surgery. Both ex vivo and in vivo group showed extensively ossification, and the newly formed bone exhibited normal bone histology at 28 days after surgery. There was no difference in the union of the bone between the in vivo and the ex vivo group. In summary, we have demonstrated that VEGF gene therapy can enhance bone fracture healing, and both the in vivo and ex vivo approaches may be effective and suitable for introducing this transgene to the fracture site.

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The objective of this study was to investigate the osteogenic potential of human dental follicle cells (hDFC) in vivo. hDFC were isolated from dental follicle tissue by enzymatic digestion and cultured in growth medium (GM). hDFC were grown in a three-dimensional (3D) culture using gelatin sponges in osteogenic induction medium (OIM) or GM. The cells were transplanted onto calvaria bone of immunodeficient rats (n = 3). Hematoxylin and eosin (H-E) staining and immunohistochemistry were performed, and bone formation was analyzed with micro-computed tomography (micro-CT). H-E staining showed newly formed bone after transplantation of hDFC grown in a 3D culture in OIM. Immunohistochemistry showed BMP-2, Runx2, and Osterix expression in the hDFC transplantation site during the early stage of bone formation. Moreover, micro-CT showed that the transplanted hDFC that were 3D-cultured in OIM promoted good bone quality and bone volume compared to the other two groups. Thus, human dental follicles may be a potentially useful cell source for regenerative therapy.

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Films were prepared from the powders of DNA/poly-L-arginine, DNA/poly-L-lysine, and DNA/chitosan complexes via hydrothermal hot-pressing in an effort to make more useful dental materials. The contact angles of water were between 37.6° and 49.9°. Zeta potentials at pH 7.4 were between 10.96 mV and 30.44 mV. The DNA/chitosan complex film showed the lowest contact angle and highest zeta potential. All films showed poor attachment of dermal fibroblast cells, with cells forming spheroids on all films. However, the cells survived on the films after a 5-day cultivation period. The DNA/poly-L-arginine and DNA/poly-L-lysine complex films were almost completely biodegraded within 14 days and most of the DNA/chitosan complex films were biodegraded 90 days after implantation in soft tissues of rats. These results suggest that films formed from DNA/polycation complexes will be useful for clinical treatments requiring thin membranes or films, such as protective membranes for stomatitis and incised oral wounds.

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Identification of the specific subset of periodontal ligament-derived mesenchymal stem cells (PDLSCs) will permit the development and improvement of current periodontal regeneration therapy. A popular approach for the isolation of a subset of PDLSCs would be to use cell surface markers to identify these cells, which are effectively enriched in order to isolate stem cells. The CD146 marker is most commonly used to isolate PDLSCs from human periodontal ligament tissues (hPDL). Previous studies have shown that CD146-positive (CD146+) cells possess potencies of high self-renewal and multi-lineage differentiation. However, the capability and potency of mesenchymal stem cells (MSCs) in hPDL-derived CD146-negative (CD146-) cells have not been well established.
In this study, CD146+ and CD146- cells were isolated from hPDL, and their properties, including their MSCs potential, were characterized. hPDL was obtained from healthy premolars (n = 10) extracted for orthodontic reasons. Flow cytometry analysis revealed that the average proportion of CD146+ cells was about 50%. An approximately 20% fraction of cells with the highest CD146 expression was sorted as CD146+ cells and an approximately 20% fraction with the lowest CD146 expression was sorted as CD146- cells using fluorescence-activated cell sorting. Cell cultures were assessed for their colony-forming efficiency, proliferation and differentiation into osteoblasts, adipocytes and chondrocytes. Approximately 20% of CD146+ cells had replicative potential and formed single-cell colonies. The colony-forming efficiency of CD146+ cells was approximately twofold higher than for CD146- cells. Cell proliferation measured by cell-cycle analysis and cell counting showed that the proliferative potential of CD146+ cells was higher than that of CD146- cells. Cell differentiation potential in vitro was determined by real-time PCR and cell staining with alkaline phosphatase and Alizarin Red S for osteogenic differentiation, Oil Red O for adipogenic differentiation, and Alcian Blue for chondrogenic differentiation. The levels of osteogenic and adipogenic differentiation were significantly higher in CD146+ cells than in CD146- cells under appropriate conditions. The level of glycosaminoglycan and gene expression of cartilage oligomeric matrix protein in CD146- cell pellets were higher than in CD146+ cell pellets. These results suggest that CD146+ cells possess bi-potent differentiation potential whereas CD146- cells possess unipotent differentiation potential.

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The purpose of this study was to compare the stability of porous TiNi alloy miniscrew implants with different force levels (0 g, 200 g, 400 g and 600 g) loaded immediately after placement. Using a randomized split-mouth design, we placed 16 miniscrews into 3 skeletally mature male beagle dogs. All the miniscrews were equally divided into 4 groups. The result showed that there was more fibrous tissue repair between the miniscrew-bone interfaces with the increase of the force levels. The 600 g load caused serious bone trabecular damage on the neck of miniscrew, whereas no significant effect on tissue around miniscrews has been apical. Not only was the miniscrew-bone interface completely mechanically integrated, but it also formed osteoid-like tissue. There was more fibrous healing, with the increasing of the force levels. Bone trabecular around the neck of the miniscrew destroyed, when the case of 600 g loaded.

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To gain insight into the molecular mechanisms associated with development of tongue, gene expression profiles of the tongue at embryonic day 13.25 (E13.25) and 15.5 was investigated using Affymetrix Mouse GeneChip. Using the twice significance of difference as the standard, the molecular mechanisms of tongue development were studied and several pathways and networks of essential secreted signaling molecules and downstream transcription factors were identified. The significant pathways of differentially expressed genes were involved in Wnt signaling pathway, Hedgehog signaling pathway, Cadherin signaling pathway, etc. Gene functional categories, particularly involved in muscle contraction, muscle organ development, cytoskeleton as well as calcium ion binding were identified. Furthermore, some of transcription factors such as Myf6, Tead4, Cebpd were activated. These data proved that E15.5 was the early differentiation and maturation stage of tongue, meanwhile, cytoskeleton, structural constituent of cytoskeleton, transcription factor activity, calcium ion related activity were involved in cell motion, muscle organ development and muscle contraction, through transcription factors and the interaction of Wnt pathway with Hedgehog pathway. It had highlighted potential cascades and important candidates for further investigation on the genetic mechanism and clinical therapy of tongue related diseases.

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We report a case of bisphosphonate-related osteonecrosis of the jaw (BRONJ) that caused cellulitis extending from the submandibular to temporal region. An 81 year-old man was referred to our department because of trismus and painful swelling of the left mandible, for which extraction of the wisdom tooth had been performed 3 months prior. He suffered from bone metastasis of prostate cancer, and was being administered anticancer drugs and zoledronate. A physical examination revealed an exposed jaw bone, and our clinical diagnosis was BRONJ with secondary infection. Five days after his first visit, the patient suffered from rapidly increased painful swelling extending from the submandibular to temporal region. Following admission, incision of the submandibular abscess was performed and acute phase inflammation was tentatively relieved. However, painful swelling in the temporal region recurred and an incision of the temporal skin for drainage was performed, which caused persistent purulent discharge. We performed a computed tomography examination, which excluded temporal bone necrosis. Thereafter, irrigation of the abscess cavity and systemic antibiotic chemotherapy were maintained. Although the exposed jaw bone persisted, there was no further pus discharge from the incision wound and the patient was discharged 45 days after admission.

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The object of this study was to estimate the influence of radiation therapy (RT) and hyperbaric oxygen therapy (HBO) on osteoradionecrosis of the jaw (ORNJ). This retrospective study included patients who were diagnosed with ORNJ from 1998 to 2012 (15 years) and who recieved a total dose of RT (Gy) for the duration of the study. The duration of ORNJ development and the healing period of ORNJ were examined. The mean dose of RT was 64.0 Gy. The mean healing period of ORNJ was 30.3 months in the continuous RT group, and 20.8 months in the pre- and post-operative irradiation group. The healing term for ORNJ with HBO was approximately the same as that without HBO. We concluded continuous RT may induce more severe ORNJ than pre- and post-operative RT, and that the effectiveness of HBO on ORNJ is suspicious.

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