Ryo Kunimatsu

Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow

Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.

Effects of mechanical stimuli on the synthesis of superficial zone protein in chondrocytes.

Superficial zone protein (SZP) has been demonstrated to contribute to the boundary lubrication in synovial joints. This study was designed to clarify the modulation of SZP expression by mechanical stress in articular chondrocytes. Cyclic tensile strains of 7 and 21% cell elongation were applied to cultured chondrocytes obtained from porcine mandibular condylar cartilage. The mRNA levels of SZP, IL-1 beta, and TGF-beta1 were examined by a quantitative real-time PCR analysis. Protein level of SZP was examined by Western blotting. The SZP mRNA level was significantly upregulated after 12, 24, and 48 h by 7% elongation. Although SZP mRNA level was upregulated by 21% elongation after 12 h, it decreased to a lower level than the control after 48 h. The TGF-beta1 mRNA level exhibited an almost similar change to SZP. The IL-1 beta mRNA level was not changed markedly by 7% elongation. However, the IL-1 beta mRNA level was significantly increased by a 12-h application of 21% elongation. Western blot analysis revealed that the SZP expression was increased by 7% elongation, but decreased remarkably by 21% elongation. It is suggested from these findings that the SZP expression level in the chondrocytes is enhanced by optimal mechanical stimuli, but inhibited by excessive loading partly affected by TGF-beta1 and IL-1 beta, leading to the deterioration of joint lubrication.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system.

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.

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.

Amelogenin Enhances the Proliferation of Cementoblast Lineage Cells

BACKGROUND It is well known that enamel matrix proteins play a crucial role in tooth root formation and amelogenesis. Because amelogenin is a major enamel matrix protein, it is assumed that amelogenin also affects the metabolism of cementum. However, the biologic functions of amelogenin in cementoblasts remain unclear. The purpose of this study is to examine the effect of recombinant human full-length amelogenin (rh174) on the proliferation of cultured human cementoblast-like (HCEM) and human periodontal ligament (HPDL) cells. METHODS HCEM and HPDL cells were cultured and treated with 100 ng/mL rh174 in the presence or absence of an anti-cluster of differentiation (CD) 63 blocking antibody. Cell proliferation was evaluated using a cell proliferation enzyme-linked immunosorbent assay 5-bromo-2-deoxyuridine kit and quantification of the cell number by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium-inner salt assay. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 was measured by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS The proliferation of HCEM and HPDL cells was enhanced significantly (P <0.05) by treatment with rh174, and inhibited significantly (P <0.05) by the addition of anti-CD63 blocking antibody. In addition, the ratio of phosphorylated ERK1/2 to total ERK1/2 became significantly larger (P <0.05) by treatment with rh174, and was reduced significantly by the addition of anti-CD63 blocking antibody in both HCEM and HPDL cells. CONCLUSION The results show that rh174 interacts with CD63, and rh174/CD63 interaction activates the ERK1/2 signaling pathway, enhancing the proliferation activities of HCEM and HPDL cells.

Modulation of hyaluronan catabolism in chondrocytes by mechanical stimuli

Hyaluronan (HA) is a component of the extracellular matrices of cartilage contributing to the structural and functional integrity. HA metabolism is regulated by both anabolic and catabolic processes; however, a great deal more of the detail has been unknown yet. The purpose of this study was to clarify the effect of excessive mechanical load on the expression and activity of hyaluronidase (HYAL) in chondrocytes with a special reference to the expressions of IL-1beta and tumor necrosis factor (TNF)-alpha. A cyclic tensile load of 22.8% cell elongation, regarded as an excessive mechanical stimulus, was applied to cultured rabbit knee articular chondrocytes. HYAL1, HYAL2, IL-1beta, and TNF-alpha mRNA levels were examined by quantitative real-time PCR analysis. The HYAL activity in culture medium was examined by HA zymography. Both HYAL1 and HYAL2 mRNA levels were upregulated significantly by the loading in cultured chondrocytes. HYAL activity was also enhanced as compared with unloaded controls. The IL-1beta mRNA level was upregulated significantly by the loading, and TNF-alpha mRNA level was slightly upregulated. HYAL1 and HYAL2 mRNA levels were upregulated significantly by IL-1beta treatment, resulting in a slight increase in HYAL activity. These results show that the expression of HYAL1 and HYAL2 in articular chondrocytes is enhanced by excessive mechanical stimuli and affected in part by induction of IL-1beta, leading to HA catabolism in articular cartilage.

Differential Effects of Amelogenin on Mineralization of Cementoblasts and Periodontal Ligament Cells

BACKGROUND Amelogenin is a major component of developing extracellular enamel matrix proteins and plays a crucial role during the formation of tooth enamel. In addition, amelogenins are suggested to exert biologic functions as signaling molecules through cell-surface receptors. The purpose of this study is to examine the effect of recombinant human full-length amelogenin (rh174) on the mineralization of human cementoblasts (HCEMs) and human periodontal ligament cells (HPDLs). METHODS HCEMs, namely, a cell line immortalized by transfection of human telomerase reverse transcription gene, and HPDLs isolated from human first premolars were cultured and treated with 0 to 1,000 ng/mL rh174. The messenger ribonucleic acid (mRNA) levels of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were examined by real-time polymerase chain reaction analysis. The protein levels of OCN and BSP were examined by Western blot analysis. ALP activity and calcium deposition of cell cultures were also determined. Mineralization of cells was evaluated by red dye staining. RESULTS The treatment of HCEMs with rh174 upregulated the ALP, OCN, and BSP mRNA levels. In addition, the protein levels of OCN and BSP, ALP activity, and calcium deposition were enhanced, resulting in enhanced mineralization. Conversely, there were no significant effects of rh174 on the mineralization of HPDLs. CONCLUSION The present study shows that rh174 enhances mineralization accompanied by upregulation of mineralization markers in HCEMs, whereas it has no effect on that in HPDLs, suggesting different effects of amelogenin on PDL and cementum.

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.

Effects of hyaluronan oligosaccharide on the expression of MMP-1 in periodontal ligament cells

It is well known that low-molecular weight hyaluronan (HA) is detected in human periodontal tissue under inflammatory conditions. HA oligosaccharide (HAoligo) has been demonstrated to induce matrix metalloproteinase (MMP)-1 expression in dendritic cells and chondrocytes, however, the bioactivities of HAoligo in periodontal ligament (PDL) cells remain unclear. In this study, we investigated the effect of HAoligo on MMP-1 expression in human PDL (HPDL) cells and the mechanisms in terms of the signal transmission. HAoligo was generated and purified from commercial human umbilical cord HA. HPDL cells were isolated from healthy ligaments, and cultured with HAoligo for 0-24h. The expression of MMP-1, tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 was analyzed by real-time PCR and Western blot analyses. Effects of specific inhibitors of p38 mitogen-activated protein kinase (MAPK) activity, MAPK kinase activity and NFkB activity on HAoligo-induced MMP-1 expression in HPDL cells were also investigated by real-time PCR and Western blot analyses. HAoligo remarkably enhanced MMP-1 expression in both mRNA and protein levels, but no effect was shown on the expression of TIMP-1 and TIMP-2 mRNAs. Inhibition of p38MAPK activity decreased the MMP-1 expression. Neither inhibition of NFkB nor MAPKK activity affected the MMP-1 expression. It was suggested that HAoligo induces MMP-1 expression in HPDL cells, and p38MAPK plays a crucial role in signal transduction for MMP-1 inducted by HAoligo.

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1β) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1β on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0-10 ng/ml IL-1β or 0-1000 ng/ml prostaglandin (PGE(2)) for 0-24h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE(2) and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE(2) were enhanced by exogenous IL-1β in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE(2) treatment dose-dependently. It is shown that the expression of COX-2/PGE(2) was enhanced by IL-1β in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE(2), suggesting that IL-1β-induced COX-2/PGE(2) play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.