Manabu Kanyama

Involvement of CTGF, a Hypertrophic Chondrocyte-Specific Gene Product, in Tumor Angiogenesis

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-β. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis.

Quality of life assessment of bone-anchored fixed partial denture patients with unilateral mandibular distal-extension edentulism

STATEMENT OF PROBLEM Dental implants are expanding their use among partially edentulous patients. However, whether implants can promote the quality of life (QOL) of these patients has not been sufficiently examined. PURPOSE This study compared the QOL level among implant denture, removable partial denture, and no restoration patients with distal extension type unilateral mandibular edentulism. MATERIAL AND METHODS Three groups (n = 12 each) of subjects with unilateral mandibular distal-extension edentulism who were matched for age, sex, and missing teeth were studied. The groups were (1) implant denture, (2) removable partial denture, and (3) no restoration. QOL levels of these 3 groups were compared using a self-administered questionnaire with 3 major subscales: oral condition, general condition, and dental treatment. RESULTS The implant denture group showed higher oral condition related QOL score than the other groups. There was no significant difference in oral condition-related QOL scores between the removable partial denture and no restoration groups. There was no significant difference in the general condition related QOL score and dental treatment-related score among the 3 groups. CONCLUSION In unilateral mandibular distal extension edentulous patients, oral-condition-related QOL levels for dental implant patients were higher than those of removable partial denture or no restoration patients. The QOL levels of the removable partial denture patients were almost identical to those of no restoration patients.

Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction

OBJECTIVE To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. DESIGN Five weeks old wild type male rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. RESULTS CTGF was expressed strongly in the endothelial cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important role in angiogenesis and granulation tissue formation specifically at early healing stage after tooth extraction to initiate alveolar bone repair. CONCLUSION CTGF was expressed at early healing stage of the rat tooth extraction wound.

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

Matrix metalloproteinase-8 is the major potential collagenase in active peri-implantitis

PURPOSE Current clinical procedures to control or regenerate bone loss due to peri-implantitis are not predictable neither accomplish complete resolution. Therefore, early detection of the onset and the active periods of bone loss are crucial for prevention of extensive peri-implant bone resorption. This study aimed to determine a possible association between the presence of collagenases (MMP-1, MMP-8 and MMP-13) in peri-implant sulcular fluid (PISF) and active periods of bone loss by annually adjusted vertical bone loss (AVBL) measurements. METHODS Intended sample consisted of 76 consecutive patients who received oral implant treatment at the Fixed Prosthodontic Clinic of Okayama University Hospital from 1990 to 2000. Twelve subjects were lost to follow-up or refused to participate. Consequently, the actual sample consisted of 64 patients who were followed-up for at least one year. Those patients with AVBL>0.6mm were included in the severe peri-implantitis group, and randomly selected, age-, gender- and implantation site-matched healthy patients (AVBL<0.3mm) comprised the control group. PISF samples were collected from both groups and further analyzed by western blot for detection of collagenases. RESULTS Four patients presented severe peri-implantitis. MMP-8 was the only collagenase detected in peri-implant sites with ongoing bone loss. PISF samples from control group showed no positive reactions to any collagenase. CONCLUSION This study showed MMP-8 as a possible marker for progressive bone loss in peri-implantitis.

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.

Comparison of inter-twin concordance in symptoms of temporomandibular disorders: A preliminary investigation in an adolescent twin population

Abstract There is controversy as to the genetic contribution to the pathogenesis of temporomandibular disorders (TMD). Several reports reveal a marked familial aggregation in the signs and symptoms of TMD, while others do not. Therefore, our goal was to investigate the hypothesis using a sophisticated research design, which was a well-known genetic survey inter-twin concordance assessment in the symptoms of TMD. This study is the first step to survey TMD symptoms of a twin group in junior and high schools as a preliminary trial. The 63 twins were asked to participate in this study in junior and senior high schools affiliated with the University of Tokyo, Japan, schools which kept ten twins in each grade for the purpose of several genetic survey programs. After excluding incomplete filling out of questionnaire sheets and data from male-female pairs, 43 monozygotic (MZ) (15.3±1.7 yrs, male/female = 17/26 pairs) and nine dyzygotic (DZ) (15.2±1.8 yrs, male/female = 6/3 pairs) twins were studied. Outcomes consisted of a prevalidated 14-item self-administered questionnaire, which assessed proband- and pair-wise concordance levels in the MZ and DZ twins. These results demonstrated that the MZ twins had a higher tendency of inter-twin concordance than DZ twins in terms of jaw pain in wide mouth opening (proband-wise concordance = 66.7% in MZ, 0% in DZ), difficulty in mouth opening (20% in MZ, 0% in DZ) and difficulty in mouth closing (50.0% in MZ, 33.3% in DZ), while there was no significant difference between the MZ and DZ concordance levels in other general health-related and behavior- related items, except toothache. However, the pair-wise concordance rates of jaw pain in wide mouth opening and difficulty in mouth opening in the MZ twins were not significantly higher compared to the DZ rate. Possibly, a genetic factor contributed to the pathogenesis of TMD in an adolescent population. The sample size needs to be increased, and there are plans to survey the next sample in the same schools.

Detection of specific antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint

To find specific humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Band-recognition frequency and the peak optical density of the band were compared between groups by chi2 and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was specific for the OA sera. In two OA patients whose treatment outcome was less favorable, the reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these findings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology.

Regulation of CCN2 gene expression and possible roles in developing tooth germs

CCN proteins are extracellular and cell-associated molecules involved in several developmental processes, but their expression patterns and regulation in tooth development remain unclear. Here we first determined the expression patterns of CCN genes in mouse tooth germs. We found that at early stages CCN2 was detected in dental lamina, dental mesenchyme, and primary enamel knot, while other CCN family members were expressed broadly. By the bell stage, all members were expressed in differentiating odontoblasts and ameloblasts, but CCN1 and CCN2 transcripts were conspicuous in differentiating osteoblasts in dental follicle. Next, we asked what signalling molecules regulate CCN2 expression and what roles CCN2 may have. We found that upon surgical removal of dental epithelium CCN2 was not longer expressed in dental mesenchyme in cultured bud stage germs. Implantation of beads pre-coated with BMPs and FGFs onto E12-13 mandibular explants induced CCN2 expression in dental mesenchyme. There was a dose-dependent effect of BMP-4 on CCN2 induction; a concentration of 100 ng/μl was able to induce strong CCN2 expression while a minimum concentration of 25 ng/μl was needed to elicit appreciable expression. Importantly, Noggin treatment inhibited endogenous and BMP-induced CCN2 expression, verifying that CCN2 expression in developing tooth germs requires BMP signalling. Lastly, we found that rCCN2 stimulated proliferation in dental mesenchyme in a dose-dependent manner. Together, the data indicate that expression of CCN genes is spatio-temporally regulated in developing tooth germs. CCN2 expression appears to depend on epithelial and mesenchymal-derived signalling factors, and CCN2 can elicit strong proliferation in dental mesenchyme.