Naoto Hirose

Effects of low-intensity pulsed ultrasound on the expression of cyclooxygenase-2 in mandibular condylar chondrocytes.

AIMS To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1β). METHODS Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1β. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1β, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1β-treated chondrocytes were examined in terms of the expression of p-integrin β1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS COX-2 mRNA level was upregulated by the treatment with IL-1β and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin β, and it inhibited the expression of p-ERK 1/2. CONCLUSION LIPUS exposure inhibited IL-1β-induced COX-2 expression through the integrin β1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.

P2X7 receptor in the trigeminal sensory nuclear complex contributes to tactile allodynia/hyperalgesia following trigeminal nerve injury

The present study directly addresses the roles of the P2X7 receptor (P2X7R), an ionotropic adenosine triphosphate (ATP) receptor, and cytokines in the induction of orofacial pain following chronic constriction injury (CCI) of the infraorbital nerve (IoN).

An adolescent patient with multiple impacted teeth.

Multiple impacted permanent teeth is uncommon and rarely reported in the literature. This article reports the treatment of an adolescent patient with multiple impacted teeth without systemic disease. A 9-year 2-month-old boy complained of a delay of eruption of the first molars. All first molars were unerupted, and the left deciduous second molar was a submerged tooth. The panoramic radiograph showed all permanent teeth except the incisors were unerupted and, especially for the first molars, spontaneous eruption was not expected. His medical history was uneventful. A lingual arch appliance and a segmental arch were placed on the mandibular and maxillary dentitions, respectively, to guide eruption of the impacted first molars. After traction of the first molars, eruption of the impacted lower premolars was induced. Furthermore, at 15 years the impacted mandibular second molars were also positioned properly by use of the lingual arch with auxiliary wires. After achieving traction of the impacted teeth, tooth alignment was initiated using multibracket appliances after the bilateral extraction of the second premolars. After 22 months of treatment with multibracket appliances, an acceptable occlusion was achieved with a Class I molar relationship. After 2 years of retention an acceptable occlusion was maintained without any relapse in the occlusion. Since a delay in the treatment of impacted teeth may induce secondary problems such as root dilacerations and ankylosis, it is highly recommended to perform early treatment of multiple impacted teeth during adolescence.

Effects of Enzymatic Degradation after Loading in Temporomandibular Joint

Synovial fluid of the joint decreases friction between the cartilage surfaces and reduces cartilage wear during articulation. Characteristic changes of synovial fluid have been shown in patients with osteoarthritis (OA) in the temporomandibular joint (TMJ). OA is generally considered to be induced by excessive mechanical stress. However, whether the changes in synovial fluid precede the mechanical overloading or vice versa remains unclear. In the present study, our purpose was to examine if the breakdown of joint lubrication affects the frictional properties of mandibular condylar cartilage and leads to subsequent degenerative changes in TMJ. We measured the frictional coefficient in porcine TMJ by a pendulum device after digestion with hyaluronidase (HAase) or trypsin. Gene expressions of interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), matrix metalloproteinases (MMPs), type II collagen, and histology were examined after prolonged cyclic loading by an active pendulum system. The results showed that the frictional coefficient increased significantly after HAase (35%) or trypsin (74%) treatment. Gene expression of IL-1β, COX-2, and MMPs-1, -3, and -9 increased significantly in enzyme-treated TMJs after cyclic loading. The increase in the trypsin-treated group was greater than that in the HAase-treated group. Type II collagen expression was reduced in both enzyme-treated groups. Histology revealed surface fibrillation and increased MMP-1 in the trypsin-treated group, as well as increased IL-1β in both enzyme-treated groups after cyclic loading. The findings demonstrated that the compromised lubrication in TMJ is associated with altered frictional properties and surface wear of condylar cartilage, accompanied by release of pro-inflammatory and matrix degradation mediators under mechanical loading.

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21Cip1 and p27Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.

P2X7 Receptor and Cytokines Contribute to Extra-territorial Facial Pain

The whisker pad area (WP) is innervated by the second branch of the trigeminal nerve and experiences allodynia and hyperalgesia following transection of the mental nerve (MN; the third branch of the trigeminal nerve). However, the mechanisms of this extra-territorial pain remain unclear. The ionotropic P2X7 ATP receptor (P2X7) in microglia is known to potentiate, via cytokines, the perception of noxious stimuli, raising the possibility that P2X7 and cytokines are involved in this extra-territorial pain. One day after MN transection (MNT), WP allodynia/hyperalgesia developed, which lasted for > 8 wks. Activation of microglia and up-regulation of P2X7, membrane-bound tumor necrosis factor (TNF)-α (mTNF-α), and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC) were evident for up to 6 wks after MNT. Allodynia/hyperalgesia after MNT was blocked by intracisternal administration of etanercept, a recombinant TNF-α receptor (p75)-Fc fusion protein. Intracisternal A438079, a P2X7 antagonist, also attenuated allodynia/hyperalgesia and blocked up-regulation of mTNF-α and sTNF-α in the TNC. We conclude that sTNF-α released by microglia following P2X7 activation may be important in both the initiation and maintenance of extra-territorial pain after MNT.

Synthesis of hyaluronan and superficial zone protein in synovial membrane cells modulated by fluid flow

Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied.

Effects of C-Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells.

BACKGROUND Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. METHODS Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full-length amelogenin (rh174); 2) amelogenin cleavage product lacking the C-terminal (rh163); 3) amelogenin cleavage product lacking the N-terminal (rh128); and 4) the C-terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast-like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium assay and cell proliferation enzyme-linked immunosorbent assay. Mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) (MAPK-ERK) pathway was examined by Western blot analysis. RESULTS Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). CONCLUSION C-terminal amelogenin cleavage product increased proliferation of HCEM through MAPK-ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.

The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells

BACKGROUND AND OBJECTIVES Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

Semaphorin 3A Inhibits Inflammation in Chondrocytes under Excessive Mechanical Stress

Background Excessive mechanical stress causes inflammation and destruction of cartilage and is considered one of the cause of osteoarthritis (OA). Expression of semaphorin 3A (Sema3A), which is an axon guidance molecule, has been confirmed in chondrocytes. However, there are few reports about Sema3A in chondrocytes, and the effects of Sema3A on inflammation in the cartilage are poorly understood. The aim of this study was to examine the role of Sema3A in inflammation caused by high magnitude cyclic tensile strain (CTS). Methods Expression of Sema3A and its receptors neuropilin-1 (NRP-1) and plexin-A1 (PLXA1) in ATDC5 cells was examined by Western blot analysis. ATDC5 cells were subjected to CTS of 0.5 Hz, 10% elongation with added Sema3A for 3 h. Gene expression of IL-1β, TNF-ɑ, COX-2, MMP-3, and MMP-13 was examined by qPCR analysis. Furthermore, the phosphorylation of AKT, ERK, and NF-κB was detected by Western blot analysis. Results Added Sema3A inhibited the gene expression of inflammatory cytokines upregulated by CTS in a dose-dependent manner. Addition of Sema3A suppressed the activation of AKT, ERK, and NF-κB in a dose-dependent manner. Conclusions Sema3A reduces the gene expression of inflammatory cytokines by downregulating the activation of AKT, ERK, and NF-κB pathways in ATDC5 cells under CTS.