Identification of a novel frameshift mutation (383insT) in the RUNX2 (PEBP2 alpha/CBFA1/AML3) gene in a Japanese patient with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.

Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement.

The mechanical force generated during tooth movement creates compressed and cell-free areas in the periodontal membrane. The way in which periodontal ligament cells disappear at the compressed area during tooth movement remains unclear. In the present study we examined whether periodontal ligament cells undergo apoptosis by mechanical stress during tooth movement using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end-labelling method (TUNEL). TUNEL-positive stainings of periodontal ligament cells began to appear at the compressed areas 12 h after tooth movement, and the number of those cells reached maximum at 24 h after tooth movement. Thereafter TUNEL-positive cells disappeared at 48 h, and direct and undermining bone resorption began at the same area 72 h after tooth movement. These results showed that compressed periodontal ligament cells were eliminated by apoptosis in the early phase of tooth movement.

Histological reaction of auditory bulla bone to synthetic auditory ossicle (Apaceram) in rats.

To investigate the biocompatibility of a synthetic auditory ossicle to host bone, small thin Apaceram disks composed of dense hydroxyapatite were implanted under the periosteum of the left auditory bulla in 32 rats for periods ranging from 1 day to 270 days. A sham operation performed on 10 rats served as a control. Decalcified histological sections stained with hematoxylin and eosin were observed using light microscopy. The experiment showed: 1) a time-dependent mature fibrous connective tissue surrounding the Apaceram disk, 2) no evidence of inflammatory reaction caused by the implant from 90 days after implantation until the end of the experiment, 3) no evidence of osteolysis by osteoclasts caused by the implant, and 4) direct contact of bone to the implant on the bone-disk interface at 180 and 270 days after implantation. The findings suggest that Apaceram has a high degree of implant biocompatibility, making it a satisfactory substitute biomaterial for otological reconstructive surgeries.

Preservation of rat palatal scar tissue myofibroblasts in organ culture.

In order to modulate palatal scar tissue, especially its myofibroblastic component, there is a pressing need for an in vitro model of this tissue. In the present, study we established an organ culture model of the rat palatal scar tissue. After excision of palatal mucoperiosteum, explants from the developing immature scar tissue and from the normal palatal mucosa were used to observe myofibroblasts in vivo and their maintenance in organ culture. Explants were cultured at the gas-liquid interface in serum-free Waymouth's MB 752/1 medium and in a humid atmosphere containing 55% O2/5% CO2 in air at 37 degrees C for 3 days. Viability of the cultured explants was evaluated with morphological and histological criteria and BrdU incorporation. After organ culture, the scar tissue showed good preservation of the in vivo histology. The myofibroblasts and smooth muscle cells of the cultured scar tissues showed continuous expression of alpha-smooth muscle actin (alpha-SMA), mimicking the in vivo situation. In the normal tissues, only smooth muscle cells of the blood vessels expressed alpha-SMA. These results demonstrate that the established model provides a useful in vitro experimental tool for investigating the palatal scar tissue in general and its myofibroblasts in particular.

A case of Japanese cleidocranial dysplasia with a CBFA1 frameshift mutation.

Cleidocranial dysplasia (CCD), which is caused by mutations of the core binding factor alpha 1 (CBFA1)/runt-related gene 2 (Runx2), is an autosomal, dominantly inherited disorder of high penetrance affecting skeletal ossification and tooth development. Recently, we found a novel frameshift mutation 383-T-insertion (S128F) in exon 3 in the CBFA1 gene of a Japanese classic CCD patient. We describe our detailed investigation of the patient with CCD associated with the CBFA1 mutation. The patient showed the characteristic expression of CCD, such as dysplasia of the clavicles, patent fontanelles, short stature, impacted supernumerary teeth, and delayed eruption of the permanent teeth. In addition to these characteristics, orthopantomography delayed ossification of the mandibular symphysis and a three-dimensional computed tomograph (3D-CT) analysis showed hypoplasia of the zygomatic arch. Furthermore, the acellular cementum of an impacted supernumerary tooth was absent in this patient. Thus, the CBFA1 mutation was critical for the pathogenesis of CCD in this patient.

Interferon-gamma inhibits the myofibroblastic phenotype of rat palatal fibroblasts induced by transforming growth factor-beta1 in vitro.

Interferon-gamma (IFN-gamma), 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-beta1 (TGF-beta1) 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-gamma on TGF-beta1-pretreated palatal fibroblasts for the purpose of clarifying the suppressive potency against myofibroblastic phenotype expression in vitro. IFN-gamma significantly altered the spindle morphology of TGF-beta1-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 alpha-smooth muscle actin protein, a marker for myofibroblast, as determined by immunoblot analysis. Northern blot analysis showed that IFN-gamma inhibited proalpha2(I) collagen mRNA expression that was stimulated by TGF-beta1 pretreatment for 24 h. Furthermore, IFN-gamma decreased the cell contractility enhanced by TGF-beta1 pretreatment for 24 h in a three-dimensional collagen gel culture system. These results suggest that IFN-gamma may have negative effects with regard to controlling the myofibroblastic phenotype induced by TGF-beta1 in palatal fibroblasts.

Transforming growth factor-beta 1 modulates myofibroblastic phenotype of rat palatal fibroblasts in vitro.

The effects of transforming growth factor-beta 1 (TGF-beta 1) on normal rat palatal fibroblasts in vitro were investigated in the present study in order to unravel the precise mechanisms by which the phenotypic modulation of fibroblasts occurs during the scar formation process. TGF-beta 1 dramatically changed the morphology of normal palatal fibroblasts from polygonal into an elongated shape, which was very similar to that of fibroblasts derived from experimental immature scar tissue in rat palatal mucosa. This morphological transition was concomitant with an increase in the expression of alpha-smooth muscle (alpha-SM) actin protein, a marker for myofibroblasts, when determined by immunocytochemistry. An immunoblot study also revealed that alpha-SM actin expression in palatal fibroblasts became evident after 24 h of TGF-beta 1 treatment and increased time-dependently up to 72 h. Northern blot analysis showed that TGF-beta 1 stimulated endogenous TGF-beta 1 mRNA expression in palatal fibroblasts within 24 h. Neither epidermal growth factor nor basic fibroblast growth factor had any effect on either alpha-SM actin expression or TGF-beta 1 mRNA expression. Pretreatment of palatal fibroblasts with TGF-beta 1 significantly increased the contractile capacity in a three-dimensional collagen gel culture, even when the culture medium was deprived of TGF-beta 1 for 72 h of the experimental period. Moreover, the contractility of scar fibroblasts, which highly expressed alpha-SM actin protein and TGF-beta 1 mRNA, was significantly lowered by a neutralizing antibody to TGF-beta 1. These data strongly suggest that TGF-beta 1 is a potential inducer of phenotypic expression of myofibroblasts in palatal fibroblasts and that auto-induction of TGF-beta 1 mRNA expression may play an important role in the scar formation process in palatal mucosa.