Extracted Dentine Matrix Proteins and Dental Pulp Stem Cell Differentiation

Studies have demonstrated the ability of calcium hydroxide to stimulate release of signalling molecules from dentine which mediate dentine-pulp complex regeneration. Clinical practice has moved towards use of adhesive restorative materials even in deep cavities, despite a lack of biological evidence to support this trend. Objectives: To investigate the effects of dentine matrix components extracted by etching/conditioning agents on the differentiation of dental pulp stem cells (DPSCs). Methods: Dentine matrix proteins (DMPs) were extracted from human powdered dentine with 10% EDTA, 10% citric acid and 37% phosphoric acid over 14 days at 4^oC. Extracts from each material were pooled, exhaustively dialysed for 10 days against distilled water prior to lyophilisation. A clonal population of DPSCs were cultured for 24hrs, serum starved for a further 24hrs and cultured in the presence of DMPs extracted by the above agents (10µg/ml in culture media) for up to 20 days. Effects of the extracts on DPSC differentiation and cell behaviour was assessed by gene and protein expression (CD105, ostepontin, alkaline phosphatase, PCNA, Collagen1a, DSP) and Alizarin red staining. Results: Down regulation of CD105 in cultures exposed to phosphoric and citric acid extracted DMPs was observed after 20 days of culture. Alkaline phosphatase expression was detectable in phosphoric acid DMP cultures at day 14 and in EDTA and citric acid DMP cultures on day 20 with no ALP expression observed in unstimulated controls. A significant increase in osteopontin, with a decrease in PCNA expression was observed in cells exposed to phosphoric and citric acid DMPs between days 2 and 14. Alizarin red staining confirmed appearance of mineralised nodules in all test groups from day 5. Immunocytochemistry revealed expression of Collagen1 and DSP in all stimulated cultures after 2 days. Conclusions: DMPs extracted by phosphoric and citric acid demonstrate a potent stimulatory response in DPSCs.