Development and characterization of an in vitro gingival epithelial model

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Abstract
A 3-dimensional gingival epithelial model has been developed and characterized. Oral epithelial cells and connective tissue fibroblasts were isolated from human gingival tissue and used to create an in vitro oral mucosa co-culture model. Fibroblasts were seeded on a scaffold of nylon mesh, allowed to proliferate and secrete collagen and extracellular matrix proteins to form a stroma capable of supporting the growth of epithelial cells. Epithelial cells were seeded on top of a confluent stromal layer, proliferated and differentiated to form a stratified squamous epithelium. Resident epithelial cells were stimulated, by manipulation of growth medium and culture conditions, to form a multi-layered oral mucosa-like tissue. Histologic analyses revealed cellular architecture exhibiting stromal-epithelial interaction which supports the growth and differentiation of an epithelial layer. lmmunohistochemical analyses confirmed production of types I and III collagen. Immunofluorescence of the stromal layer identified type IV collagen and fibronectin. Fibronectin was also detected on surface epithelium. Differentiation of basal, spinous and granular cells was observed, and the presence of differentiation markers, acidic (K10, 14–16, 19) and basic (Kl–8) cytokeratins were confirmed using broad spectrum cytokeratin antibodies, AE1 and AE3. Development of a discontinuous basal lamina zone, with hemidesmosomes, was observed by electron microscopy. The co-culture was metabolically active, as measured by the thiazoyl blue (MTT) assay for mitchondrial function and [3H] thymidine incorporation into DNA. The human gingival epithelial co-culture model was viable up to 35 days post-epithelial seed. This model may offer opportunities for limited study of periodontal tissue responsiveness.