In-vitro Model of Gingival Fibroblast Differentiation in Spheroid Culture

Objective: This study investigates the morphology and histology of human cell line gingival fibroblasts in spheroid culture to ascertain if such a three-dimensional system promotes differentiation that resembles the in vivo situation.

Method: Cell line of gingival fibroblasts was seeded in tissue culture flasks until confluent. Then they were trypsinized and cultured in multi well plates by liquid overlay technique. Each plate was divided into three sections, each section containing a different number of cells; 25 000, 50 000, 100 000 cells, and allowed to form into cohesive spheroids. The spheroids were fixed and processed for histological examination after 2, 14 and 30 days in vitro.

Result: Gingival fibroblasts formed spheroids in a period between 12 and 48 hours. After 2 weeks an inner dead cell zone, an outer live cell zone and a mixed cell zone could be observed in spheroids of different ages and different cell number. Alcian Blue staining showed the presence of glycosaminoglycans at the centre of the spheroids at 14 and 30 days.

Conclusion: This study concluded that cell line gingival fibroblasts can be grown consistently in a spheroid form. However the cells appeared to differentiate along a chondrogenic rather than fibrous connective tissue lineage. This result may be used for further investigation of gingival fibroblast cells in this 3-dimensional model.