Proanthocyanidin Treatment Enhances Structural-Integrity and Osteogenic Differentiation of Microtissue Spheroids

Abstract

<p>Objectives: To surface precondition the microtissue spheroids (MS) of dental pulp stem cells (DPSCs) with a natural collagen cross-linker, proanthocyanidin (PA), in order to maintain structural integrity and promote osteogenesis.<br>Methods: MS of DPSCs were fabricated using 12-series micro-molds (MicroTissues, Inc.). Day 2 MS were treated with selected 1-4 mg/ml concentrations of PA solution for 1-60 minutes and assessed for viability using MTT assay. MS treated with 1mg/mL PA for 1-60 minutes were stained with live/dead viability staining and observed under confocal microscopy on Day1, Day3, and Day7. The diameters of PA-treated MS were measured every 48-hours for 18-days and the diameter change and percentage change were analyzed to measure structural stability. PA-treated MS were induced in osteogenic differentiation medium for 14-days and assessed for Alkaline Phosphatase (ALP) activity and mineralization by Alizarin red staining. The surface topology and ultrastructure of PA-treated MS were observed using scanning electron microscopy and H&E staining.<br>Results: No immediate cytotoxic effects on MS were observed up to 4mg/mL PA (<em>p</em>>0.05). 1mg/mL was identified as the safest concentration as the dead cell numbers were minimal over 7 days indicating the stability of the 1mg/mL PA-treated MS (<em>p</em>>0.05). The MS which did not receive any surface preconditioning reduced in diameter consistently over time and it was 20% less than the original size over 18 days (<em>p</em><0.05). However, 30-minute and 60-minute treatment with 1mg/mL PA showed only a 5% and 10% reduction from the original diameter (<em>p</em><0.05), respectively. After 7-day osteogenic induction, significant increase (<em>p</em><0.05) in mineralization was observed in 30-minute PA-treated group as shown by Alizarin red staining, however, the increase in ALP levels was not significant compared to control groups (<em>p</em>>0.05).<br>Conclusions: Surface preconditioning of MS with 1mg/mL PA for 30-minutes could promote maintenance of the structural integrity and osteogenic differentiation potential of MS of DPSCs.<br></p>