The Inhibition of Sulfated Glycosaminoglycans on Cultured Palatal Fibroblast Behavior

Sulfated glycosaminoglycans (GAGs) have been implicated in wound healing, but little is known about their roles at the cellular level. Objective: To investigate the cellular behavior of rabbit palatal fibroblasts in in vitro wound healing when sulfated GAGs are inhibited. Methods: Fibroblasts were isolated from the palatal mucosa of an adult female New Zealand White rabbit. Passage 2 cells were cultured with 30mM chlorate, a competitive inhibitor of GAG sulphation (experimental group) and without (control group). Fibroblast morphology was first observed under phase contrast microscopy at 4, 6, 12, 18 and 24 hours after cell seeding to determine if the chlorate concentration used was toxic to the cells. Subsequently, adherent fibroblast number was quantified by MTS assay at 4, 6, 8 and 10 hours after cell seeding. Fibroblast proliferation was assessed at days 3, 5, and 7 after cell seeding. Cell migration was observed under phase contrast microscopy at 4, 6, 12, 18 and 24 hours after creation of an in vitro wound-healing model on a six-well plate. Statistical analyses were carried out with one-way ANOVA for cell adhesion and proliferation. Results: There was no apparent difference in fibroblast morphology between the control and chlorate-treated group during the entire experiment. The experimental group of palatal fibroblasts exhibited fewer adherent cells at 4, 6, 8 and 10 hours (p<0.05). Fibroblast proliferation was consistently slower in the chlorate-treated group at days 3, 5 and 7 (p<0.05). In the in vitro wound healing model, the wounded edges in the control fibroblast culture closed completely at 18 hours, whereas the wounded edges of the chlorate-treated group were closed only at 24 hours. Conclusion: The addition of 30mM chlorate suppressed rabbit palatal fibroblast adhesion, proliferation and migration. This study highlighted the roles sulfated GAGs play in fibroblast behavior that would likely influence clinical wound healing.