Effects of C-Xylopyranoside Derivative on Expression of the Basement Membrane-related Molecules of Oral Keratinocytes and Fibroblasts

[Problem statement] Our research team successfully developed a tissue-engineered, human oral mucosa equivalent that has been transplanted as a graft to reconstruct mucosa defects. Phase I clinical trial to increase keratinized gingiva in Japan have been implemented under current good manufacturing practices standard. Because the specific and rigorous release testing for quality assurance/control is required on the day of grafting prior to the product release, we currently measure the glucose consumption level in the culture medium to detect the defectives in this protocol. However, a few substandard products such as a thin epithelium formation is unavoidably formed, which is due to “variations” within the grafts. In parallel with the development of different non-invasive assessments, pharmacological manipulation would be one way to increase the quality of the grafts and minimize the “variations” during manufacturing. Glycosaminoglycans (GAGs) and proteoglycans are one of the major components of extracellular matrix including basement membrane in oral mucosa and have many biological functions. Thus, they play an important role in homeostasis of the epithelium. Recently, C- Xylopyranoside derivatives is reported to increase GAGs and PGs biosynthesis and improve tissue morphogenesis in a skin organotypic culture model.

[Purpose] We hypothesized that one of the C-Xylopyranoside derivatives β-D-xylopyranoside-n-propane-2-one (XPP) stimulates the ability to produce basement membrane related molecules such as basement membrane components and integrins in oral mucosa keratinocytes and fibroblasts, resulting in improvement of epithelial structure. The present study aimed to assess the effects of XPP on the expression level of GAGs and basement membrane related molecules produced by oral keratinocytes and fibroblasts, and the differences of histological appearance in a reconstructed oral mucosa model.

[Methods] Primary oral keratinocytes and fibroblasts were isolated and serially cultured in EpiLife culture medium containing 0.06 mM Ca⁺⁺ (growth medium) and Dulbecco’s Modified Eagle Medium containing 10% serum (DMEM), respectively. First, we tested the cell viability of oral keratinocytes cultured in growth medium and EpiLife containing 1.2 mM Ca⁺⁺(hereafter differentiation medium)  as well as oral fibroblasts cultured in differentiation medium and DMEM supplemented with 0, 2 and 10mM XPP for up to 48 hours using a Cell-Counting Kit. Next, total amount of GAGs produced by oral keratinocytes and fibroblasts into the culture media was quantitated using Blyscan GAG assay. Concurrently, oral keratinocytes and fibroblasts were lysed and immunoblotting was performed to detect the expression levels of basement membrane related molecules. Using an organotypic culture system, three-dimensional oral mucosa models (3DOMM) in which oral keratinocytes and fibroblasts were incorporated were fixed, embedded in paraffin, and then histologically and immunohistochemically examined.

[Results] The concentration of 2 and 10mM XPP did not affect cell viability of oral keratinocytes and fibroblasts grown in those culture media. Blyscan assay showed the amount of GAGs secreted from both cells into those culture media significantly increased when XPP was added, particularly from keratinocytes cultured in differentiation medium. The expression level of type ‡W collagen, Nidogen, Integrin α6 and β1 in oral keratinocytes increased when cultured in growth medium. While the histological examinations did not showed significant differences between the XPP-treated and untreated 3DOMMs, more intense expression of laminin and type ‡W collagen was seen in the XPP-treated one. Moreover, basal layer cells of the XPP treated 3DOMM strongly expressed integrin α6 and β1, ligands of laminin and type ‡W collagen, compared with untreated 3DOMM.

[Conclusion] These results suggested XPP stimulate GAG synthesis in oral keratinocytes and fibroblasts, leading to increase in the expression of basement membrane related molecules in the XPP-treated 3DOMM.

[References]

·Izumi K et al; Int J Oral Maxillofac Implants. 2013 28:e295-e303.

·Sok J et al;Eur J Dermatol 2008 18:297-302