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Adhesive Resin Induces Apoptosis and Cell-cycle Arrest of Pulp Cells

¹ Department of Cariology, Restorative Sciences, and Endodontics, and
² Department of Oral Medicine, Oral Pathology, and Oral Oncology, University of Michigan School of Dentistry, 1011 N. University, Rm. 5211, Ann Arbor, MI 48109-1078, USA;

View larger version (26K): [in a new window]   Figure 1. Adhesive resin induces apoptosis of pulp cells and macrophages. Percentage of apoptotic mouse odontoblast-like cells (MDPC-23) (a), undifferentiated pulp cells (OD-21) (b), or macrophages (RAW 264.7) (c) after a 12- or 24-hour exposure to unpolymerized, partially polymerized (light-cured for 10 sec), or polymerized (light-cured for 40 sec) SingleBond, respectively. Apoptotic cells were identified as a sub-G1 population after being stained with propidium iodide and sorted by flow cytometry. Asterisk indicates statistical significance at p 0.05, as compared with untreated controls (white bars). Data represent mean values (± SD) of triplicate samples per condition and cell type, and each sample consisted of approximately 10,000 cells.

View larger version (49K): [in a new window]   Figure 2. Micromorphology and flow cytometry profile of pulp cells and macrophages exposed to an adhesive resin. MDPC-23, OD-21, or macrophages were exposed for 24 hrs to SingleBond light-cured for 0 sec (unpolymerized), 10 sec (partially polymerized), or 40 sec (polymerized), or untreated controls. (a–l) Phase-contrast photomicrographs of representative fields at 200x. (m–z) Flow cytometry profile of approximately 10,000 cells/condition stained with propidium iodide. The proportion of apoptotic cells per condition ("A") is depicted in the top left-hand corner of each graph.

View larger version (31K): [in a new window]   Figure 3. Translocation of phosphatidylserine and activation of Caspase-3 constitute early stages of adhesive resin-induced apoptosis of pulp cells and macrophages. Percentage of annexin-V-positive MDPC-23 (a), OD-21 (b), or macrophages (c) after a zero- to six-hour exposure to partially polymerized (light-cured for 10 sec) SingleBond. Asterisk indicates statistical significance at p 0.05, as compared with untreated controls. Caspase-3 activity was determined in lysates of MDPC-23 (d), OD-21 (e), or macrophages (f) after a four-hour exposure to partially polymerized SingleBond (), or untreated controls (•). Positive control was 1 ng of purified recombinant Caspase-3 (). The specificity of Caspase-3 activation was determined by the addition of the inhibitor Ac-DEVD-CHO to the reaction (). Data represent mean values (± SD) of triplicate samples per condition and cell type.

View larger version (24K): [in a new window]   Figure 4. Cell-cycle analysis of viable pulp cells and macrophages. Flow cytometry profile of MDPC-23, OD-21, or macrophages exposed to partially polymerized SingleBond (light-cured for 10 sec) and stained with propidium iodide. (a) Percentage of viable cells at each phase of the cell cycle (G1, S, G2). Oblique line bars depict untreated control cells, and solid black bars depict cells exposed for 12 hrs to the adhesive resin (b–g). Cell-cycle profiles of cells exposed for 12 hrs to partially polymerized SingleBond and untreated controls. Data represent mean values (± SD) of triplicate samples per condition and cell type, and each sample consisted of approximately 10,000 cells.