Miho Inoue

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin α5β1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast‐two‐hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma‐derived chondrocytic cell line HCS‐2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS‐2/8 cells to FN in a dose‐dependent manner. The CCN2/CTGF‐enhancing effect on cell adhesion to FN was abolished by a blocking antibody against α5β1 integrin (α5β1), but not by one against anti‐αvβ3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C‐terminal CT domain with FN, and that α5β1 is involved in this adhesion.

Secreted frizzled related protein (sFRP)-2 inhibits bone formation and promotes cell proliferation in ameloblastoma

Secreted frizzled related protein (sFRP)-2, a Wnt antagonist, was strongly expressed by both stromal and tumor cells of ameloblastoma. The aim of this study is to evaluate whether sFRP-2 secreted from tumor cells have any direct role in suppressed bone formation or not. A pre-osteoblastic cell line, KUSA/A1 cells, cultured in conditioned medium of an ameloblastoma-derived cell line (AM-1CM) was used in the study. Alkaline phosphatase (ALP) activity, alizarin red staining, mineral quantification and MTS assay was performed. Wnt-canonical pathway is a major pathway for osteoblasts. Antagonists of this pathway, sFRP-1, 2 and 3, were detected by immunohistochemistry and western blot analysis. KUSA/A1 cells cultured in AM-1CM showed high cell proliferation, low ALP activity without mineralized matrix deposition. sFRP-2 was strongly expressed in ameloblastoma tissue and AM-1 cells. After sFRP-2 depletion, the cells showed diffuse mineralization. In this study, it was confirmed that ameloblastoma cells have a major role in decreased bone formation by secreting sFRP-2 in cell culture model. Though, sFRP-2 has great effect on tumor progression, inhibition of sFRP-2s anti-bone formation activity and cell proliferative activity may reduce the invasive property of ameloblastoma and possibility of recurrence rate.

Effect of a New Titanium Coating Material (CaTiO3-aC) Prepared by Thermal Decomposition Method on Osteoblastic Cell Response

Titanium and hydroxyapatite (HA) are widely used as biomaterials for dental and medical applications. HA-coated titanium implants have excellent biocompatibility and mechanical properties. However, the adherence of HA film formed on titanium substrate is weak because of the lack of chemical interaction between HA and titanium. A solution to this problem is to form an intermediate film on titanium substrate, which provide excellent adherence to both titanium substrate and HA. We developed a novel biomaterial called calcium titanate-amorphous carbon (CaTiO3-aC) coating prepared by modified thermal decomposition method. The purpose of this study was to evaluate the effect of CaTiO 3-aC and HA coating (positive control), and Ti (negative control) on osteoblastic (MT3T3-E1) cell responses. An increased cellular proliferation was observed in CaTiO3-aC coating compared to HA coating. The maximum expressions of ALP activity, Col I and ALP mRNA were higher and achieved in shorter period of time in CaTiO3-aC coating compared to others. These results demonstrated that CaTiO3-aC promoted better cell attachment, cellular proliferation, and osteoblastic differentiation compared with HA. In conclusion, we suggested that CaTiO3-aC could be considered as an important candidate as a coating material.

Stromal cells promote bone invasion by suppressing bone formation in ameloblastoma

Aims:  To study the stromal variation and role of stromal–tumour cell interaction in impaired bone formation as well as enhanced bone resorption in ameloblastoma.

Tyrosine kinase type-receptor ErbB4 in chondrocytes : interaction with connective tissue growth factor and distribution in cartilage

In order to identify receptor molecules that participate in the growth and differentiation of chondrocytes, we cloned a number of cDNA fragments from HCS‐2/8 chondrocytic cells, by using tyrosine kinase‐specific primers for amplification. The mRNA expression of one such receptor, ErbB4, was increased by connective tissue growth factor/hypertrophic chondrocyte‐specific gene product (CTGF/Hcs24), which promotes all stages of the endochondral ossification in vitro. ErbB4 expression was observed through all stages of chondrocytic differentiation in vitro, corresponding to the wide distribution of CTGF/Hcs24 target cells. Furthermore, positive signals for erbB4 mRNA were detectable throughout most populations of chondrocytes, in growth and articular cartilage in vivo. These results demonstrate for the first time that ErbB4 is expressed in chondrocytes and may play some roles in chondrocytic growth and differentiation along with CTGF/Hcs24.

Locally Injected Dexmedetomidine Inhibits Carrageenin-induced Inflammatory Responses in the Injected Region

BACKGROUND:Dexmedetomidine, a highly selective agonist of &agr;2-adrenoceptors, is a commonly used sedative; however, a potent anti-inflammatory effect has also been found. In the present study we evaluated the inhibitory effect of locally injected dexmedetomidine on inflammatory responses in the injected region. METHODS:Local inflammation was induced in the hindpaws of male mice (aged 6–8 weeks) by intraplantar injection of lambda-carrageenin. To offset the central effect of tested agents, different agents were blindly injected into the left and right paws in the pairs of comparison. The effect of dexmedetomidine on edema (increase in paw volume), the accumulation of leukocytes, and production of tumor necrosis factor-&agr; (TNF-&agr;) and cyclooxygenase-2 (COX-2) were evaluated after carrageenin injection, using water displacement plethysmometry, histological imaging, immunohistochemistry, and Western blotting analysis. Furthermore, we also evaluated the effect of yohimbine, a full antagonist of &agr;2-adrenoceptors, and phenylephrine, an agonist of the &agr;1-adrenoceptor, on dexmedetomidine’s action on inflammatory responses. RESULTS:Paw volume and amount of leukocytes in the injected region significantly increased after the injection of carrageenin. Similarly, TNF-&agr; and COX-2 production was found in the subcutaneous region injected with carrageenin, 4 hours after injection. Dexmedetomidine significantly inhibited all increases in paw volume, leukocytes, and production of TNF-&agr; and COX-2. Furthermore, yohimbine significantly antagonized the anti-inflammatory effects of dexmedetomidine, whereas phenylephrine did not significantly alter them. CONCLUSIONS:The findings suggest that locally injected dexmedetomidine exhibits an anti-inflammatory effect against local acute inflammatory responses, mediated by &agr;2-adrenoceptors.

Effect of fluoride-substituted apatite on in vivo bone formation.

Biological apatites are characterized by the presence of minor constituents such as magnesium (Mg), chloride (Cl), or fluoride (F) ions. These ions affect cell proliferation and osteoblastic differentiation during bone tissue formation. F-substituted apatites are being explored as potential bonegraft materials. The aim of the present study is to investigate the mechanism of bone formation induced by fluoride-substituted apatite (FAp) by analyzing the effect of FAp on the process of in vivo bone formation. FAps containing different F concentrations (l-FAp: 0.48wt%, m-FAp: 0.91wt%, h-FAp: 2.23wt%) and calcium-deficient apatite (CDA), as positive control, were implanted in rat tibia and bone formation was evaluated by histological examination, immuhistochemistry, in situ hybridization and tartrate-resistant acid phosphatase examinations. The results showed that l-FAp, m-FAp, h-FAp, and CDA biomaterials allowed migration of macrophages, attachment, proliferation, and phenotypic expression of bone cells leading to new bone formation in direct apposition to the particles. However, the l-FAp preparation allowed faster bone conduction compared to the other experimental materials. These results suggest that FAp with low F concentration may be an efficient bonegraft material for dental and medical application.

Distribution, gene expression, and functional role of EphA4 during ossification

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles.

Effects of Periodontal Cell Grafts and Enamel Matrix Proteins on the Implant–Connective Tissue Interface: A Pilot Study in the Minipig

We have developed an experimental model to help identify and characterize factors necessary for periodontal connective tissue attachment formation on dental implants. In this pilot study, we report the effect of autogenous periodontal cell grafts, with and without the a pplication of enamel matrix derivative (EMD), on the implant-connective tissue interface. Periodontal ligament (PDL) and gingival connective tissue (GCT) cultures were established from an adult minipig. Implants were placed in osteotomies prepared with exaggerated countersinks that served as recipient sites for autogenous cell grafts in bilateral edentulated posterior mandibular sextants. In addition, 1 side received an application of EMD before placement of the autogenous cell grafts. A bioabsorbable membrane covering the coronal portion of the implants was placed before closure. After 8 weeks, quantitative histomorphometric and qualitative light microscopic analyses revealed that the implants that received gelatin vehicle alone were surrounded by bone, whereas the implants that received GCT cell grafts were mostly surrounded by fibrous connective tissue. In contrast, implants that received PDL cells without the application of EMD demonstrated good bone contact, but strands of epithelium were observed in the implant-connective tissue interface. Implants that received PDL cells and EMD also had good bone contact but without evidence of epithelium. A cementum-like interface was not observed in any of the groups. Results of this pilot study suggest that EMD and the type of cell populations present in the implant wound-healing environment may alter the implant-connective tissue interface.

Localization of CD44 (Hyaluronan Receptor) and Hyaluronan in Rat Mandibular Condyle

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.