Growth Factors Enhance Minocycline Accumulation by Gingival Fibroblasts

Objectives: Gingival fibroblasts actively accumulate minocycline, a process that could enhance redistribution of systemic minocycline to the gingiva and increase its concentration in gingival fluid. To determine whether fibroblast minocycline accumulation can be up-regulated, we examined the effect of growth factors on transport activity in cultured human gingival fibroblasts. Methods: Fibroblasts were grown to confluence in 24-well plates containing MEM supplemented with 10% fetal bovine serum. Cells were starved overnight in medium containing 0.5% serum, then treated for 3, 6 or 24 hours with human transforming growth factor b1 (TGF, 1-30 ng/ml), fibroblast growth factor-basic (FGF, 0.3-10 ng/ml) or platelet derived growth factor-BB (PDGF, 0.3-10 ng/ml). Minocycline transport was assayed by measuring the increase in cell-associated fluorescence at 37° C. Lineweaver-Burk analysis was used to assess effects on transport affinity and velocity. Results: All three factors significantly enhanced minocycline transport in a dose-dependent manner (P < 0.02, ANOVA), primarily by inducing significant increases in affinity of transport (P < 0.02). Treatment with 10 ng/ml TGF, FGF or PDGF enhanced intracellular minocycline content by 11%, 15% and 21%, respectively, after 3 hours, by 21%, 25% and 23%, respectively, after 6 hours and by 25%, 44% and 39%, respectively, after 24 hours. To determine whether protein kinase C (PKC) could play a role in signaling for increased minocycline transport, we examined the effects of the PKC activator phorbol myristate acetate (PMA). Treatment with 100nM PMA for 15 minutes induced a 28% increase in fibroblast minocycline content (P < 0.05) by significantly enhancing the affinity of transport (P = 0.006). Conclusions: Through a process that may involve PKC, TGF, FGF and PDGF significantly enhance minocycline accumulation by gingival fibroblasts. Thus, redistribution of systemic minocycline to the gingiva could be enhanced by growth factors associated with injury and healing. Supported by NIDCR Grant DE12601