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.

A problem-based learning tutorial for dental students regarding elderly residents in a nursing home in Japan.

This educational trial was an eight-day problem-based learning (PBL) course for fourth-year predoctoral students at Okayama University's dental school who interviewed elderly residents living in a nursing home. The purpose of this PBL course was to introduce geriatric dentistry to the students by allowing them, independently, to discover the clinical problems of elderly patients as well as the solutions. The sixty-five students were divided into nine small groups and received patient information (age, gender, degree of care needed, medical history, food type, medications, and oral condition) in datasheets before visiting the nursing home. Each group of students directly interviewed one patient and the caregivers and identified the patient's medical, psychological, and social problems. After the interview, the students participated in a PBL tutorial to delineate a management approach for the patient's problems. To measure the efficacy of this program, the students completed a questionnaire before and after the course regarding their level of understanding of and attitudes toward geriatric dentistry, clinical research, and self-study. The results showed that student's perceptions of their knowledge about and attitudes toward oral health care for the elderly significantly increased after the PBL course, which suggests that such tutorials should be an option for dental curricula.

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.

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

There is controversy as to the genetic contribution to the pathogenesis of temporo-mandibular 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.

[A case report of a speech disturbance patient treated by a magnetic attachment overdenture].

PATIENT: A 50-year-old female who wore a cast-clasp-supported removable partial denture (RPD) complained of speech disturbance, because the clasps disturbed her tongue movement during speech. She wished to receive dental implant treatment, but it was difficult to solve the esthetic problem by the planned implant-supported fixed partial denture. Therefore, the RPD (overdenture) supported by magnetic attachments was inserted to recover the speech disturbance. DISCUSSION: The treatment outcome of this patient was evaluated with a self-administered questionnaire. The results suggested that the RPD with magnetic attachments improved the patient's pronunciation, esthetics and quality of life. CONCLUSION: It was suggested that the magnetic attachment overdenture effectively solved the speech disturbance.

Expression and roles of connective tissue growth factor in Meckel's cartilage development.

Meckel's cartilage is a prominent feature of the developing mandible, but its formation and roles remain unclear. Because connective tissue growth factor (CTGF, CCN2) regulates formation of other cartilages, we asked whether it is expressed and what roles it may have in developing mouse Meckel's cartilage. Indeed, CTGF was strongly expressed in anterior, central, and posterior regions of embryonic day (E) 12 condensing Meckel's mesenchyme. Expression decreased in E15 newly differentiated chondrocytes but surged again in E18 hypertrophic chondrocytes located in anterior region and most-rostral half of central region. These cells were part of growth plate-like structures with zones of maturation resembling those in a developing long bone and expressed such characteristic genes as Indian hedgehog (Ihh), collagen X, MMP-9, and vascular endothelial growth factor. At each stage examined perichondrial tissues also expressed CTGF. To analyze CTGF roles, mesenchymal cells isolated from E10 first branchial arches were tested for interaction and responses to recombinant CTGF (rCTGF). The cells readily formed aggregates in suspension culture and interacted with substrate-bound rCTGF, but neither event occurred in the presence of CTGF neutralizing antibodies. In good agreement, rCTGF treatment of micromass cultures stimulated both expression of condensation-associated macromolecules (fibronectin and tenascin-C) and chondrocyte differentiation. Expression of these molecules and CTGF itself was markedly up-regulated by treatment with transforming growth factor-beta1, a chondrogenic factor. In conclusion, CTGF is expressed in highly dynamic manners in developing Meckel's cartilage where it may influence multiple events, including chondrogenic cell differentiation and chondrocyte maturation. CTGF may aid chondrogenesis by acting down-stream of transforming growth factor-beta and stimulating cell-cell interactions and expression of condensation-associated genes.

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.

Cbfa1/Runx2 gene expression in articular chondrocytes of the mice temporomandibular and knee joints in vivo.

Healthy articular cartilage is thought to be maintained by the modulation of Cbfa1 expression, although little is currently known about Cbfa1 expression in such tissues. Therefore, we examined in vivo Cbfa1 transcript levels in the temporomandibular (TM) and knee joints of 3- and 10-week-old male ICR mice (weighing 50-70 g). A digoxigenin-11-UTP-labeled single-stranded RNA probe (0.6 kbp PstI-HindIII fragment of the 3' of untranslated region in exon 8 of mouse Cbfa1 cDNA) was prepared and in situ hybridization was performed on paraffin-embedded TM and knee joint sections. The antisense probe detected Cbfa1 transcripts in prehypertrophic chondrocytes, but not in the articular surface layer chondrocytes of 3- and 10-week-old mice TMJs. Despite the intense Cbfa1 expression in prehypertrophic chondrocytes, articular surface layer chondrocytes of the knee joints expressed low and undetectable level of Cbfa1 in the 3- and 10-week-old mice, respectively. These results indicate that Cbfa1 are highly expressed in the prehypertrophic chondrocytes presumably for articular tissue remodeling during the entire lifespan of the mouse, whereas Cbfa1 expression is suppressed in the articular surface chondrocytes in the adult mouse TM and knee joints to obtain the permanent cartilage phenotype.

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.

Quality of life assessment in patients with implant-supported and resin-bonded fixed prosthesis for bounded edentulous spaces.

OBJECTIVES: Dental implants have become increasingly popular in the prosthetic rehabilitation of patients with bounded edentulous spaces. Oral condition-related quality of life (QOL) levels have rarely been assessed in these patients. MATERIAL AND METHODS: Two groups of subjects with bounded edentulous spaces were studied: an implant-supported fixed prosthesis group (11 patients) and a resin-bonded fixed prosthesis group (33 patients). The two groups were well matched in terms of sex, age, missing units and location of missing units. The patients were requested to answer a self-administered QOL questionnaire with two major subscales - oral condition- and general condition-related QOL scores. The test-retest reliability of each question was pre-examined and found acceptable (mean Spearman rank correlation coefficient was 0.55 +/- 0.16). Mean QOL score differences between the two groups were analyzed by the Mann-Whitney U-test. RESULTS: Mean oral condition-related QOL scores of the implant-supported and resin-bonded fixed prosthesis groups were 87.8 +/- 9.5 and 87.1 +/- 12.3% (P = 0.85), and mean general condition-related QOL scores were 73.8 +/- 14.8 and 71.6 +/- 15.2% (P = 0.95), respectively. No significant QOL differences between the two groups were observed in the two subscales. CONCLUSION: In patients with bounded edentulous spaces, multidimensional QOL levels of patients with an implant-supported fixed prosthesis do not exceed those of patients with a resin-bonded fixed prosthesis in a short follow-up period.