Keiji Moriyama

The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement

A novel 90-kDa protein named periostin, which is preferentially expressed in the periosteum and the periodontal ligament (PDL), may play a role in bone metabolism and remodeling. However, the precise role of periostin in the PDL remains unclear. Therefore, we examined the expression of periostin mRNA during experimental tooth movement. Experimental tooth movement was achieved in 7-week-old male Sprague-Dawley rats. In control specimens without tooth movement, the expression of periostin mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The periostin mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The divergent expression of periostin mRNA in the PDL began to be observed at 3xa0h and continued up to 96xa0h after tooth movement. The maximum changes, which showed stronger staining in the pressure sites than in the tension sites, were observed at 24xa0h. The expression of periostin mRNA in the PDL 168xa0h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggest that periostin is one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.

Severe Destructive Autoimmune Lesions with Aging in Murine Sjögren’s Syndrome through Fas-Mediated Apoptosis

When we evaluated the age-associated changes in autoimmune exocrinopathy in a NFS/sld murine model for primary Sjögrens syndrome (SS), severe destructive autoimmune lesions developed in the salivary and lacrimal glands in the aged mice, compared with those observed in the younger model. We detected a decreased secretion of saliva and tear flow in the aged group. A significant increase of TUNEL(+)-apoptotic epithelial duct cells in the salivary glands was detected in the aged SS animal model. A higher proportion of mouse salivary gland cells bearing Fas was found in the aged group, whereas no significant changes were seen on tissue-infiltrating CD4(+) T cells bearing FasL in the salivary glands from young and aged mice. We detected an increased cleavage product of organ-specific autoantigen, 120-kd alpha-fodrin, in the aged salivary gland tissues on immunoblotting, and an increase in serum autoantibody production against 120-kd alpha-fodrin by enzyme-linked immunosorbent assay. An increase in the proliferative response of splenic T cells against organ-specific autoantigen was observed, whereas nonspecific concanavalin A responsiveness was decreased in the aged mice. In addition, a decrease in Fas expression was found on splenic CD4(+) T cells in the aged mice, and anti-Fas mAb-stimulated apoptosis was down-regulated on CD4(+) T cells. These results indicate that age-associated dysregulation of CD4(+) T cells may play a crucial role on acceleration of organ-specific autoimmune lesions in a murine model for primary SS through Fas-mediated apoptosis.

Interferon-γ inhibits the myofibroblastic phenotype of rat palatal fibroblasts induced by transforming growth factor-β1 in vitro

Interferon‐γ (IFN‐γ), a multifunctional cytokine, has been noted as a potential therapeutic agent for various fibrotic disorders, including excessive scar tissue formation. We previously reported that transforming growth factor‐β1 (TGF‐β1) induced the myofibroblastic phenotype in palatal fibroblasts derived from palatal mucosa, and that such effects might have a close link to palatal scar formation. In the present study, we examined the effects of IFN‐γ on TGF‐β1‐pretreated palatal fibroblasts for the purpose of clarifying the suppressive potency against myofibroblastic phenotype expression in vitro. IFN‐γ significantly altered the spindle morphology of TGF‐β1‐pretreated palatal fibroblasts into the polygonal one that was similar to the non‐treated palatal fibroblasts. This change was parallel with a decrease in the expression of α‐smooth muscle actin protein, a marker for myofibroblast, as determined by immunoblot analysis. Northern blot analysis showed that IFN‐γ inhibited proα2(I) collagen mRNA expression that was stimulated by TGF‐β1 pretreatment for 24 h. Furthermore, IFN‐γ decreased the cell contractility enhanced by TGF‐β1 pretreatment for 24 h in a three‐dimensional collagen gel culture system. These results suggest that IFN‐γ may have negative effects with regard to controlling the myofibroblastic phenotype induced by TGF‐β1 in palatal fibroblasts.

Mechanism for bone invasion of oral cancer cells mediated by interleukin-6 in vitro and in vivo

Osteoclastic bone resorption is an important step in bone invasion in several malignancies. Although interleukin (IL)‐6 accelerates osteoclastic bone resorption, it remains unclear whether IL‐6 may be involved in bone invasion of oral cancer.

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Expression of cathepsin K mRNA during experimental tooth movement in rat as revealed by in situ hybridization

The expression of cathepsin K. a novel collagenolytic enzyme specifically expressed in osteoclasts, was investigated in the rat maxillary dentoalveolar unit during experimental tooth movement by in situ hybridization histochemistry with a non-radioisotopic cRNA probe for rat cathepsin K. Orthodontic elastics were inserted into the interproximal space between the maxillary first and second molars of 7-week-old male SD rats according to Waldos method and sections prepared from tissues obtained at 12 hr, 1, 2, 3, 4, 7, and 12 days after orthodontic force application. Cathepsin K mRNA expression was detected in the mono- and multinuclear osteoclasts on the pressure side of the alveolar bone at 12 hr after force application, and the distribution and number of cathepsin K mRNA-positive osteoclasts increased time-dependently on the pressure side. At 3-4 days, a marked increase in cathepsin K mRNA-positive osteoclasts was found not only on the pressure side but also on the tension side of the alveolar bone in response to tooth movement. At 7-12 days, the cathepsin K mRNA-positive osteoclasts on both sides had disappeared. These findings suggest that the recruitment of osteoclasts on the pressure side begins during the initial stage of orthodontic tooth movement and the site-specific early induction of cathepsin K mRNA may cause an imbalance in the relative resorption activities on the pressure and tension side incident to such movement.

Evidence for Apoptosis Induction in Myofibroblasts during Palatal Mucoperiosteal Repair

Apoptosis is thought to be a requisite event for maintaining kinetic homeostasis within continually renewing tissues such as the oral mucosa and skin. However, no systematic study of the apoptotic process in fibroblasts in the oral mucosa following injury has been performed. In this study, we have assessed the expression of transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF), which are among the most important modulators of wound repair, during wound healing following mucoperiosteal injury in the rat plate. In addition, we have investigated fibroblast differentiation and apoptosis by immunohistochemical analysis for a-smooth-muscle (a-SM) actin or DNA strand breaks, respectively, to clarify the mechanisms of the wound healing process. TGF-β1-positive cells were noted in the subepithelium from Day 2 to Day 14 after injury, by which time the wounds were completely re-epithelialized. Strong expression of bFGF was observed, mainly in macrophages and monocytes at the injured site, from Day 10 to Day 14 after injury. TGF-β1 and bFGFimmunostaining was significantly lower during the later phase of wound healing. In addition, the number of myofibroblasts expressing a-SM actin increased (peak at Day 14), and thereafter gradually decreased. In parallel, the apoptosis in myofibroblasts was prominent on Day 14. These results suggest that TGF-β1 and bFGF may be potential stimulators of apoptosis in myofibroblasts after re-epithelialization in the palatal wound healing process. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring.

Cloning and expression of the chick p63 gene

We have isolated a chick cDNA for p63, a member of the p53 transcription factor family. This cDNA encodes a protein of 582 amino acids for an alpha isoform in the C-terminal region, while lacking the N-terminal transactivation domain. The chick p63 gene is first expressed in the prospective cutaneous ectoderm at stage 6 and later in the developing epithelia. The p63 expression is intense in specialized epithelial structures, such as apical ectodermal ridge of the limb bud, epithelia of branchial arches and feather buds. Furthermore, we have found that the transcripts are detected in the interdigital epithelium, intersomite epithelium, and epaxial dermamyotome.

Prognostic implications of nasal cavity and cleft morphology in secondary bone grafting

OBJECTIVEnTo examine the prognostic significance of the skeletal morphology around the nasal cavity and the alveolar cleft in secondary bone grafting (SBG).nnnDESIGN AND SETTINGnFifty-one alveolar clefts in 41 patients (10 bilateral and 31 unilateral cleft lips and palates) registered in the Tokushima University Dental Hospital were examined in this study.nnnMETHODnEvaluation of the bony bridge after SBG using dental radiographs at 1 year after surgery. The clefts were divided into two groups: group I (54.9%) in which the upper border of the bony bridge was preferably maintained on or above the horizontal reference line (RL) constructed at the level of the root apex of the upper central incisor adjacent to the cleft, and group II (45.1%) in which the bone level was lower than the RL. Presurgical cleft width was determined by the dental radiographs. The cleft/nasal cavity ratio; the value of the cleft width divided by the nasal cavity width on the cleft side, which was analyzed by frontal cephalograms before the SBG; and the cleft/apertura piriformis ratio, the value analyzed by computed tomography, were used.nnnRESULTS AND CONCLUSIONnThe age, sex, and eruptive stage of the canine teeth at the time of the SBG showed no significant difference between groups. The presurgical cleft width also showed no significant difference between group I (6.6 +/- 3.1 mm) and group II (7.9 +/- 3.3 mm). The cleft/nasal cavity ratio showed a significant difference between groups I and II (0.42 +/- 0.14, 0.75 +/- 0.25; p < .05). Furthermore, the cleft/apertura piriformis ratio also showed a significant difference between groups I and II (0.32 +/- 0.12, 0.65 +/- 0.26; p < .05). These results suggested that measurements of the skeletal morphology around the nasal cavity and alveolar cleft might aid in predicting the stability of the bony bridge after SBG.