The goal of our research is to develop a dental material that is capable of inducing reactionary dentin by surviving odontoblasts at the floor of cavity preparations. The development of such materials has been hampered by cumbersome in vivo animal studies. Objective: to develop a new rapid in vitro dentin induction assay that would determine the potential of various test compounds for reparative dentin formation. Methods: A 2.6 kp segment of the mouse dentin sialophosphoprotein (mDSPP) promoter was cloned into the pGL3 luciferase (Luc) basic expression vector and co-transfected with a second vector containing the hydromycin resistance gene (Hyr) (selection marker) into human dental pulp-derived (HDP-J) or mouse odontoblast (MO6-G3) cell lines. Stably transfected cells were established by selecting in hydromycin supplemented media and screening for basal Luc activity. A known inducer, BMP-7 (OP-1), and an inhibitor, TGF-b, were used as controls with BMP2 serving as the test compound. Cells were plated in 12-well culture plates and grown in a-MEM supplemented with 10% FCS, antibiotics, and 50 mg/mL ascorbic acid in 95% air and 5% CO2. The test compounds were added (0.0 ng/mL-200 ng/mL) and cells harvested at 24 or 48 hours. Cells were lysis, centrifuged and Luc activity of the supernatants measured with a luminometer normalized by protein concentrations. The data was analyzed using a regression curve and the accuracy determined by the variance of regression. Results: BMP-4 was found to up-regulate DSPP expression at 24 hours, but the inductive effect was lost by 48 hours of treatment. BMP-7 up-regulated DSPP expression at all time points while TGFb-1 down-regulated expression within 24 hours. Conclusions: This study demonstrates the proof-of-concept of a novel rapid, sensitive dentin inductive assay that will be useful for screening novel molecules for reparative dentin potential. Support: NIDCR COSTAR DE14318 and DE11688.

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