The Effects of Fibroblasts and Keratinocyte Growth Factor on Morphogenesis of Reconstituted Human Oral Epithelium

Objective: To examine the effects of fibroblasts and KGF on human oral epithelial morphogenesis.

Methods: Two types of primary human oral mucosal equivalents were prepared: organotypic mono-cultures, with keratinocytes grown on collagen matrix in the absence of fibroblasts, and organotypic co-cultures, with keratinocytes grown on fibroblast-containing collagen biomatrix. Exogenous KGF was added in culture media at day 1 of culture. The cultures were grown in serum-free medium for 10 days. Tissue architecture (histomorphometry), cell proliferation (Ki67), cell differentiation (CK13, CK14, CK19), and cell death (TUNEL) were assessed.

Results: In fibroblast deprived cultures (OTK), epithelium (28.03 ± 5.03) was thinner (p=0.037) than the one grown in presence of fibroblasts (OTKF)(66.06 ± 8.68). Basal cell proliferation was significantly lower in OTK than in OTKF (p=0,025). Addition of KGF induced a dose-dependent increase of total epithelial thickness and basal cell proliferation in both types of culture. The distribution of the various cell layers in the total epithelial thickness and cytokeratin expression showed major differences dependent on the presence/absence of fibroblasts. Additional KGF did not modify the pattern of epithelial stratification or the cytokeratin distribution. Cell death was higher in basal layer of epithelium in OTK compared with OTKF (p=0.012). KGF induced a decrease in cell death of the basal cell layer and an increase of cell death in the superficial cell layer, although not statistically significant (p=0,378).

Conclusion: Fibroblasts have a crucial effect on proliferation and differentiation of human oral epithelium, especially for the development of stratum spinosum. KGF influences epithelial proliferation, but has little or no effect on epithelial differentiation, suggesting that other factors synthesised by fibroblasts are responsible for the differentiation of human oral epithelium.