Three Dimensional Reconstitution of Oral Mucosal Keratinocytes and Its Biologic Characteristics

ABSTRACT

The purposes of this study were to develop an in vitro co-culture model of epithelial tissue with dermal equivalent, cultured at an air-liquid interface, and to evaluate the effects of extracellular matrix and concentration of calcium and fetal bovine serum in medium to find optimized culture condition. Oral keratinizing epithelial cells in monolayer culture were grown in Mitomycin-treated 3T3 feeder. Primary cultured oral epithelial cells were reconstituted onto the dermal equivalents consisting of 3T3 fibroblast and type I collagen, and co-culture was grown at the air-liquid interface. The histomorphological development of reconstituted oral epithelium in vitro for 21 days revealed 10~12 layered statified epithelium, closely similar to the parakeratinized gingival epithelium. Neither laminin nor type IV collagen was able to induce keratinocyte differentiation. But a mixture of laminin and type IV collagen induced well-polarized keratinizing tissue with anchoring structure of basal cells. When the reconstituted oral epithelium was incubated in 1.0% and 0.5% serum-containing medium, the granular cell layers with orthokeratinization developed. The reconstituted epidermis generated in serum-free keratinocyte growth medium (KGM)-containing pituitary extract showed features of incomplete differentiation. The present study shows that the dermal equivalents containing fibroblasts will support epidermal morphogenesis and differentiation. And these results suggest that extracellular matrix and calcium concentration are important factors during the reconstitution of keratinizing epithelium in vitro.