Interactions of Emdogain, Nicotine and Glutathione in Fibroblasts and Osteoblasts

Objectives: This investigation aims to assess the effect of enamel matrix protein (Emdogain) on human periosteal fibroblasts (HPF) and MG63 osteoblast like cells in culture and its modulation by nicotine and the anti-oxidant glutathione (G); by assaying androgen metabolites as biomarkers of oxidative stress relevant to healing responses. Methods: Monolayer cultures of MG63 osteoblasts were incubated in Eagle's MEM with radiolabelled 14C-testosterone to establish optimal concentrations of Emdogain (E), nicotine and glutathione. Subsequent incubations were performed in replicates with either 14C-testosterone or 14C-4-androstenedione as substrate, and optimal concentrations of E (30mg/ml), nicotine (N200mg/ml) and G (3mg/ml) alone and in combination for 24h. Similar experiments were done with HPF. The medium was solvent extracted with ethyl acetate for radioactive steroid metabolites and separated by thin layer chromatography (TLC) in a benzene: acetone solvent system (4:1 v/v), and quantified using a radioisotope scanner. Results: The substrates were metabolised mainly to diols, 5alpha-dihydrotestosterone (DHT) and 4-androstenedione or testosterone, with the two substrates used for both cell types which showed similar trends. With HPF and 14C-Testosterone as substrate there was a 34% increase in yields of DHT in response to E and 2.6-fold inhibition in response to N compared with controls. This inhibition was overcome by 70% and 63% in combination with E and G respectively increasing to 97% when combined, being significantly greater than with either E or G combined with N (n=8; p<0.0001). There were similar trends with 14C-4-A as substrate and for both substrates in osteoblast cultures. The baseline yields and response to agents were significantly greater for HPF in comparison with osteoblast cultures. Conclusion: The ‘in vitro' findings of this study can be extrapolated to ‘in vivo' applications of Emdogain as an adjunctive regenerative therapeutic agent for management of periodontal diseases associated with oxidative stress and complicated by environmental factors.