Hisashi Ichimiya

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-κB ligand) in a mouse monocyte cell line (RAW 264.7). HA at Mr < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at Mr > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-κB) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Heparin inhibits osteoclastic differentiation and function

We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF‐κB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate‐resistant acid phosphatase (TRAP) and detected using a phenyl‐phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGICTM kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c‐Src protein was determined by immunoblot analysis. Heparin suppressed TRAP‐positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c‐Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz‐crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL. J. Cell. Biochem. 103: 1707–1717, 2008.

Advanced Clinical Usefulness of Ultrasonography for Diseases in Oral and Maxillofacial Regions

Various kinds of diseases may be found in the oral and maxillofacial regions and various modalities may be applied for their diagnosis, including intra-oral radiography, panoramic radiography, ultrasonography, computed tomography, magnetic resonance imaging, and nuclear medicine methods such as positron emission tomography. Of these modalities, ultrasound imaging is easy to use for the detection of noninvasive and soft tissue-related diseases. Doppler ultrasound images taken in the B-mode can provide vascular information associated with the morphology of soft tissues. Thus, ultrasound imaging plays an important role in confirming the diagnosis of many kinds of diseases in such oral and maxillofacial regions as the tongue, lymph nodes, salivary glands, and masticatory muscles. In the present article, we introduce three new applications of ultrasonography: guided fine-needle aspiration, measurement of tongue cancer thickness, and diagnosis of metastasis to cervical lymph nodes.

Clinical significance of ultrasonographic examination including detection of thyroid gland diseases when surveying cervical lymph nodes in subjects with oral squamous cell carcinoma.

OBJECTIVE The objective of this study was to evaluate the clinical significance of ultrasonography (US) for screening for thyroid gland diseases when surveying the cervical lymph nodes to detect metastasis in subjects with oral squamous cell carcinoma (SCC). METHODS The detection rates and characteristics of abnormal thyroid findings detected by US in 301 subjects with oral SCC were analyzed. Subjects with abnormal findings were referred to thyroid specialists and the diagnoses and treatments from these specialists were noted. The ratio of subjects who consulted a thyroid specialist after indications of thyroid gland abnormalities to analyze subject compliance was also examined. Follow-up examinations were regularly conducted to assess any changes in the thyroid gland. RESULTS Of the 301 subjects with oral SCC, 91 had abnormal thyroid gland findings on US. As subject age increased, the rate of detection of abnormal thyroid gland findings on US significantly increased. The rate of detection of abnormal findings in women by US was significantly higher than that in men, but the male-to-female ratio was lower compared with that in previous reports. It was demonstrated that oral SCC in the floor of the mouth was associated with the highest prevalence of abnormal findings in the thyroid gland (40%), followed by oral SCC of the maxillary gingiva (39%). Of the 91 subjects with abnormal findings, 10 showed enlargement in the size of the lesion on follow-up examination with US. Eleven subjects with no abnormal findings on initial examination showed abnormal findings on follow-up examination. CONCLUSION The results suggest that subjects with oral SCC have a relatively high rate of abnormal findings in the thyroid gland that can be detected by US. Scans that include the thyroid gland should be performed when surveying cervical lymph nodes for metastasis during US examination. Particular attention should be paid to thyroid gland-related diseases in older men who have oral cancer and to thyroid gland abnormalities if patients had oral SCC in the floor of the mouth or of the maxillary gingiva. It is recommended that follow-up US examinations be regularly performed in cases of oral SCC.