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Aim: The aim of the study was to investigate the effect of dentine conditioning agents on growth factor liberation and settlement of dental pulp progenitor cells (DPSCs) on dentine surfaces. Methodology: The agents used included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH < 1), citric acid (10%, pH 1.5) and polyacrylic acid (25%, pH 3.9). Human dentine slices were conditioned for exaggerated conditioning times of 5 and 10 min, so that the growth factor liberation reached quantifiable levels above the limit of detection of the laboratory methods employed. Transforming growth factor beta‐1 (TGF‐β1) release and surface exposure were quantified by enzyme‐linked immunosorbent assay (ELISA) and immunogold labelling. Scanning electron microscopy (SEM) was used to assess the morphology of cells and coverage by DPSCs cultured on dentine surfaces for 8 days. Results: After 5‐min conditioning of dentine slices, citric acid was the most effective agent for growth factor release into the aqueous environment as measured by ELISA (Mann–Whitney U with Bonferroni correction, p <.01 compared with phosphoric and polyacrylic acid). As well as this, dentine slices treated with phosphoric acid for the same period, displayed significantly less TGF‐β1 on the surface compared with the other agents used, as measured by immunogold labelling (MWU with Bonferroni correction, p <.05). After 8 days, widespread coverage by DPSCs on dentine surfaces conditioned with citric acid and EDTA were evident under SEM. On dentine surfaces conditioned with phosphoric and polyacrylic acid, respectively, less spread cells and inconsistent cell coverage were observed. Conclusions: Based on the findings of this in vitro study, a desirable biological growth factor‐mediated effect may be gained when conditioning dentine by milder acidic or chelating agents such as citric acid and EDTA. The results must be interpreted in the context that the potential of the applied materials inducing a desirable biological response in DPSCs is only one consideration amongst other important ones in a clinical setting. However, it is crucial to look beyond the mere physical effects of materials and move towards biologically based treatment approaches as far as the restorative management of teeth with viable dental pulps are concerned. [ABSTRACT FROM AUTHOR]