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The ultimate goal of periodontal treatment is the regeneration of periodontal tissues which have been lost due to periodontal disease. Recently, enamel matrix derivatives (EMD) have been introduced as an agent with the potential to enhance the regenerative process. However, there is limited knowledge of the biological mechanisms underlying the activity of EMD. The purpose of the present study was to evaluate the EMD effects on regulation of alternative splicing of fibronectin (FN) mRNA in two regions (EDA, EDB) using human gingival fibroblasts (GFs) GFs were grown to confluence and treated with EMD (0, 5, 25, 50, or 100 ,ug/ml) for 24 h prior to mRNA or protein assessments. Semi-quantitative RT-PCR with "P-end labeled sense primers was used to determine the effects of EMD on total and splice variant mRNA levels. "P-labeled products were excised from polyacrylamide gels following electrophoresis, and radioactivity measured using scintillation spectroscopy. Immunocytochemistry with fluorescent labeling was used to evaluate the expression of FN and EDA+at the protein level under confocal laser scanning microscopy. Our results showed that EMD treatment significantly increased the relativelevels of EDA-FmRNA over baseline levels (without EMD) in a dose-dependent manner (0, 5, 25, 50, 100 ,ug/ml; p<0.01, ANOVA) . In contrast, the relative levels of EDB+mRNA and total FN mRNA level were unchanged with EMD treatment. On the protein level, immunolocalization of FN and EDA+did not show any significant changes following EMD treatment relative to baseline levels. In conclusion, EMD specifically increased EDA+mRNA levels in a concentration-dependent manner, with no apparent effects on the protein level. These results suggest that EMD has functional activities similar to those previously reported for TGF-ƒÀ in the regulation of FN splicing. J. Jpn. Soc. Periodontol., 43: 107-117, 2001.

Regulation of Alternative Splicing of Fibronectin mRNA by Enamel Matrix Derivative in Human Gingival Fibroblasts.