Cyclic mechanical pressure-loading alters epithelial homeostasis in a three-dimensional in vitro oral mucosa model: clinical implications for denture-wearers.

Denture-wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure-loading mimicking denture-wearing using an organotypic culture system to develop a three-dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50 kPa) for 7 days (cycles of 60 min on, 20 s off to degas and inject air). Upon initiation of an air-liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7 days. Paraffin-embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure-loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki-67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure-loaded 3DOMMs. Pressure-loading attenuated integrin ß1 expression and increased matrix metalloproteinase-9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure-loaded 3DOMM. Cyclic pressure-loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture-wearing could compromise oral epithelial homeostasis.

Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvß6 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 αvß6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-ß1) production into the supernatant, but down-regulated TGF-ß 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 αvß6 expression, cause the down-regulation of TGF-ß/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.

Migration behaviour and pathogenesis of five ascarid nematode species in the Mongolian gerbil Meriones unguiculatus.

To understand the characteristic features of the Mongolian gerbil, Meriones unguiculatus, as an animal model of ascarid infections, the migration behaviour and pathogenesis of larvae were investigated in experimentally infected gerbils. Embryonated eggs from each of Toxocara canis, Baylisascaris procyonis, B. transfuga, Ascaris suum, and A. lumbricoides were orally inoculated into gerbils and larvae were recovered from various organs at designated periods. In T. canis-infected gerbils, larvae were present in the liver 3 days after infection and in the skeletal muscle and brain via the heart and lungs at a similar rate. In B. procyonis- and B. transfuga-infected gerbils, larvae were present in the lungs within 24 h after infection, with some having reached the brain by that time. After 24 h, larvae of B. procyonis tended to accumulate in the brain, while those of B. transfuga accumulated in skeletal muscles. In A. suum- and A. lumbricoides-infected gerbils, larvae remained in the liver on day 5 post-infection and elicited pulmonary haemorrhagic lesions, which disappeared 7 days after initial infection. Thereafter, no larvae of any type were recovered. Ocular manifestations were frequently observed in T. canis- and B. procyonis infected gerbils, but were rare in B. transfuga-infected gerbils. In the cases of A. suum and A. lumbricoides, migration to the central nervous system and eyes was extremely rare, and larvae had disappeared by 2 weeks post-infection. Fatal neurological disturbances were observed in B. procyonis-infected gerbils, whereas irreversible non-fatal neurological symptoms were observed in the case of B. transfuga.

On the formation of the temporomandibular joint cavity in the human fetus.

The purpose of this study was to clarify the mechanism of articular cavity formation by means of observing the temporomandibular joint (TMJ) in human fetuses (9th-30th gestational weeks) using light microscope. In the 9th-11th week, although neither cavity had been formed, several small blood vessels running postero-anteriorly on the lower surface of the articular disk that would be the future lower cavity were recognized. However, such blood vessels were not seen in the future upper cavity beneath the glenoid fossa. In the 12th week, when the lower cavity formation commenced, the cavity was filled with blood corpuscles, and a series of apertures was observed throughout the condyle, from the posterior portion to a part of the anterior portion. Tracing posteriorly, the lower cavity diminished gradually until it had at last united to one blood vessel. On the other hand, in upper cavity formation, which was recognized only at the posterior portion of the TMJ, no blood corpuscles were seen in the upper cavity but several small clusters of collagen fibers. Blood corpuscles in the lower cavity disappeared after the formation of synovial membrane (20th week) and vascular canals in the condyle (21st week). From these findings, the mechanism of lower cavity formation appears to differ from that of upper cavity formation. Lower cavity formation involved small blood vessels running postero-anteriorly on the lower surface of the articular disk. As for upper cavitation, there was no evidence of blood vessels, which would suggest that upper cavitation depends on another mechanism of formation.

Plus/minus screening of rabbit corneal endothelial cDNA library.

Plus/minus screening of the rabbit corneal cDNA library was performed using corneal and iris RNA as probes. Thirteen clones were isolated: three ferritin H-chains, a NADH-ubiquinone oxidoreductase B22 subunit, an alpha 1 type VIII collagen, a 25 KDa FKBP-506 binding protein (FKBP25), a thrombospondin 2, and six unknown clones. Although proteins translocated from these isolated mRNA are not corneal specific, they play an important role in the cornea. None of the isolated known mRNAs maps to chromosome 1, 16, or 20. These clones, thrombospondin excepted, were not observed in the high frequency clones in the profile of the aortic endothelial cDNA library.