Search

The differences between oral mucosa and skin wound healing involving hypoxic responses of fibroblasts are poorly elucidated. In this study, we aimed to study the different hypoxic responses between oral and skin fibroblasts embedded in a three-dimensional (3D) collagen matrix to address the early stage of wound healing. Primary oral mucosa fibroblasts (OMFs) obtained from the retromolar area and skin fibroblasts (SFs) obtained from the abdomen were cultured in the 3D ‘floating model’ under either 21%, 5% or 1% O2 for 2 days. Cell viability under hypoxia was higher in the OMFs than in the SFs. Collagen gel contraction was suppressed under hypoxic conditions in both fibroblasts, consistent with the reduction of alpha smooth muscle actin expression, except for SFs under 1% O2. Subsequently, their gene expression profiles between 21 and 1% O2 concentrations were compared via microarray technology, and the expression profiles of the extracellular matrix (ECM)-associated proteins, including matrix metalloproteinases and collagens, were evaluated. The OMFs were more susceptible to 1% O2, and more of their genes were downregulated than the SFs’. Although the production and expression levels of ECM-associated proteins in both fibroblasts diminished under hypoxia, those levels in OMFs were significantly higher than those in SFs. In the case of single origin OMFs and SFs, our findings suggest that OMFs possess a higher baseline production capacity of several ECM-associated proteins than SFs, except type III collagen. The intrinsic hypoxic responses of OMFs may be attributed to a more favourable wound healing in oral mucosa.

The present study aimed to develop a more biomimetic tissue-engineered oral mucosa equivalent comprising 1% type I tilapia scale collagen scaffold having microstructures mimicking the dermal-epidermal junction of oral mucosa and oral keratinocytes as graft materials for human use. We designed four micropattern prototypes mimicking the dermal-epidermal junction. Using a semiconductor process and soft lithography, negative molds were fabricated to develop microstructures using both polydimethylsiloxane and silicon substrates. Micropattern configurations of dermal-epidermal junctions manufactured from fish collagen consisting of a fibril network using our micropatterning system were well preserved, although the internal fibril network of the pillar pattern was sparse. Mixing 1% chondroitin sulfate with the collagen matrix minimized tissue-engineered oral mucosa equivalent contraction. Histologic examinations showed a flattening of the vertical dimensions of all microstructures and expansion of their pitches, indicating changes in the originally designed configurations. Nonetheless, histologic examinations revealed that a fully differentiated and stratified epithelial layer was developed on all scaffolds, suggesting that the microstructured fish scale collagen scaffolds have potential in the manufacturing of tissue-engineered oral mucosa equivalents for clinical use; however, enhancement of the mechanical properties of micropatterns is required. Our micropatterning technology can also apply to the development of oral mucosa

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, β-D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2 mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin α6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.

Summary 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 β1 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.