Yoshiro Kawano

Immunolocalization of aquaporin-1 in the mechanoreceptive Ruffini endings in the periodontal ligament.

Previous ultrastructural studies have suggested an axon-Schwann cell interaction in the periodontal Ruffini ending, a primary mechanoreceptor. However, no information is available on the transport mechanism between them. The present study examined the immunolocalization of aquaporin-1 (AQP1) and -4 (AQP4), a member of the water-selective channel, in the periodontal Ruffini endings of the rat incisors and trigeminal ganglion. In addition, the expression of mRNA for AQP1 and 4 was detected in the trigeminal ganglion by a RT-PCR technique. A single PCR product of the sizes anticipated for AQP1 and 4 was detectable in a reverse transcripted cDNA sample from the trigeminal ganglion, whose neurons innervate the periodontal Ruffini endings. An AQP1 immunoreaction was recognizable in the axon terminals of the periodontal Ruffini endings as well as their associated terminal Schwann cells, as confirmed with a double staining with AQP1 and either PGP9.5 or S-100 protein. However, no immunoreaction for AQP4 was found in periodontal Ruffini endings. Although the AQP4 immunoreaction was localized in some satellite cells - but never in neurons - of the trigeminal ganglion, 16.1% trigeminal neurons showed the AQP1 immunoreaction. Furthermore, the AQP1 immunoreaction was found in certain satellite cells which surrounded AQP1-positive or -negative neurons. An analysis of a cross-sectional area of these positive neurons demonstrated that approximately 66.9% of the positive neurons were 400-1000 microm2 (671.4+/-172.4 microm2), indicating that they could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that AQP1 controls water transport in the periodontal Ruffini endings.

Zoledronic acid induces S-phase arrest via a DNA damage response in normal human oral keratinocytes

OBJECTIVE This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct. DESIGN Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 μM ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (γ)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2. RESULTS ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of γ-H2A.X was also seen. CONCLUSIONS These results indicate that a 10-μM ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiquitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover.

Detection of acid-sensing ion channel 3 (ASIC3) in periodontal Ruffini endings of mouse incisors.

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion - which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite†

This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the β1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.

Expression of 25 kDa heat shock protein by synovial type B cells of the mouse temporomandibular joint

Earlier studies have demonstrated immunoreactivity for heat shock protein 25 (Hsp25) in type B synovial lining cells of the rat temporomandibular joint, and also the presence of characteristic cytoplasmic processes in these cells, but it is unclear whether or not the type B cells in other animals possess such elaborate cytoplasmic projections and as there is as yet no evidence for the synthesis of this protein by these cells. For these reasons, the expression of Hsp25 was investigated in the synovial membrane of the mouse temporomandibular joint by immunocytochemistry and by in situ hybridization using a specific cRNA probe. Intense immunoreaction for Hsp25 was found in the cytoplasm of certain synovial lining cells that were identified as type B by immunoelectron-microscopy. These Hsp25-positive cells had slender cytoplasmic processes, either projecting towards or covering the synovial surface. Morphological differences between cytoplasmic processes seemed to depend on the location of the type B cell bodies. In situ hybridization showed intense signals for Hsp25 mRNA in the synovial lining cells, suggesting that the type B cells produce, rather than resorb, Hsp25. These findings indicate that Hsp25 is a useful marker for the identification of the synovial type B cells in the temporomandibular joint. It is further hypothesized that Hsp25 in type B cells is involved in maintaining their specific profile and epithelial-like arrangement, and in protecting against mechanical stress.

Three-dimensional direction and interrelationship of prisms in cuspal and cervical enamel of dog tooth.

The three‐dimensional architecture of enamel prisms was examined in cuspal enamel and compared with that in cervical enamel by light and electron microscopy as well as computer‐assisted reconstruction using the developing enamel of several dog teeth.

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvβ6 and transforming growth factor beta signalling in a three-dimensional in vitro wound healing model

This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 × 10(5)cm(2) each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2mM Ca(2+) EpiLife for 5 days, and then in an air-liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 μM zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin αvβ6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-β1) production into the supernatant, but down-regulated TGF-β receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin αvβ6 expression, cause the down-regulation of TGF-β/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.

Involvement of neurotrophin-4/5 in regeneration of the periodontal Ruffini endings at the early stage

Little is known about the role of neurotrophin‐4/5 (NT‐4/5) in the regeneration of mechanoreceptors. Therefore, the present study examined the regeneration process of Ruffini endings in the periodontal ligament in nt‐4/5‐deficient and wildtype mice following transection of the inferior alveolar nerve by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by computer‐assisted quantitative image analysis. Furthermore, rescue experiments by a continuous administration of recombinant NT‐4/5 were performed and analyzed quantitatively. At postoperative day 3 (PO 3d), almost all PGP 9.5‐positive neural elements had disappeared; they began to appear in both types of animals at PO 7d. At PO 10d, almost all nerve fibers showed a beaded appearance, with fewer ramifications in both types of mice. Although the regeneration proceeded in the wildtype, a major population of the periodontal Ruffini endings continued to display smooth outlines at PO 28d in the nt‐4/5 homozygous mice. The reduction ratio of neural density reached a maximum at PO 3d, decreased at PO 10d, and later showed a plateau. In a rescue experiment, an administration of NT‐4/5 showed an acceleration of nerve regeneration in the homozygous mice. These findings indicate that the nt‐4/5‐depletion causes a delay in the regeneration of the periodontal Ruffini endings, but the delay is shortened by an exogenous administration of NT‐4/5. Combined with our previous findings of bdnf‐deficient mice (Harada et al. [ 2003 ] Arch Histol Cytol 66:183–194), these morphological and numerical data suggest that multiple neurotrophins such as NT‐4/5 and brain‐derived neurotrophic factor (BDNF) play roles in their regeneration in a stage‐specific manner. J. Comp. Neurol. 501:400–412, 2007.

Phenotypes of articular disc cells in the rat temporomandibular joint as demonstrated by immunohistochemistry for nestin and GFAP

The articular disc is a dense collagenous tissue containing disc cells that are phenotypically described as chondrocyte‐like cells or fibrochondrocytes. Despite the possible existence of these phenotypes in systemic joints, little is known about the detailed classification of the articular disc cells in the temporomandibular joint. In this immunocytochemical study we examined the localization and distribution patterns of nestin and glial fibrillary acidic protein (GFAP) in the articular disc of the rat temporomandibular joint at postnatal day 1, and weeks 1, 2, 4 and 8, based on the status of tooth eruption and occlusion. Nestin and GFAP are intermediate filament proteins whose expression patterns are closely related to cell differentiation and cell migration. Both types of immunopositive cell greatly increased postnatally to a stable level after postnatal week 4, but they showed different distribution patterns and cell morphologies. Nestin‐reactive disc cells, which were characterized by a meagre cytoplasm and thin cytoplasmic processes, were scattered in the articular disc, whereas GFAP‐positive cells, characterized by broader processes, existed exclusively in the deeper area. In mature discs, the major proportion of articular disc cells exhibited GFAP immunoreactivity. Furthermore, a double‐immunostaining demonstrated that the nestin‐negative cells, consisting of GFAP‐positive and ‐negative cells, exhibited immunoreactions for heat shock protein 25. These findings indicate that the articular disc cells comprise at least three types in the rat temporomandibular joint and suggest that their expressions closely relate to mechanical loading forces within the joint, including occlusal force, as observed through postnatal development.