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.

Muscle activities in the hand and arm during tooth brushing and the regulation of brushing movements by oral sensory perception.

Investigations were made of the forces during brushing of the teeth, the patterns of brushing movements, and muscle activities, with and without blockage of sensory perception in the oral cavity, while each subject brushed by the rolling method, scrubbing method, and by his own individual habitual brushing method. We learned from electromyograms that the scrubbing method primarily used the muscles of the palm of the hand and the forearm, while the rolling method cleaned principally by action of the muscles of the upper arm and shoulder. With the rolling method, the rhythmic pattern of the brushing movement and muscle activity were lost when sensory perception in the oral cavity was blocked and, in those subjects who had a low periodontal membrane tactile threshold, the brushing force was greater than when sensory perception was not blocked. However, no effect was observed with blockage of the sensory perception in the oral cavity when the scrubbing method was used. Since brushing movements with the scrubbing method resemble the movements of writing, we also carried out investigations on the relationship between the forces during writing and brushing. As a result we found that with the scrubbing method, the brushing forces were strong for the subjects who had strong writing forces, while they were weak for those with weak writing forces. It was clear from the above results that the force of brushing with the rolling method was affected more by factors relating to oral sensory perception than muscle activity, while with the scrubbing method it was affected more by factors relating to muscle activity of the palm of the hand and forearm than by oral sensory perception. There was a clear difference in the brushing forces, pattern of brushing movements, and muscle activity between experienced and inexperienced subjects with both the scrubbing and rolling methods. Thus we learned that the effect of brushing instructions could be evaluated not only by the efficiency of plaque removal, brushing force or skill of brushing movements, but also by muscle activity. The phenomena observed with the scrubbing and rolling methods were not seen with the subjects' individual habitual brushing method.