EDTA Irrigation Protocols Influence the Liberation of Dentine Matrix Components: a Proteomics Insight

Abstract

Background: Vital pulp treatment and regenerative procedures aim to preserve dental pulp vitality and support tissue healing using biomaterials and endogenous stem/progenitor cells. However, clinical success is limited by an incomplete understanding of pulp-dentine biology, particularly the effects of irrigation solutions on dentine matrix proteins and dental pulp stem cells.

Aim: This thesis aimed to determine optimal ethylenediamine tetra acetic acid (EDTA) irrigation conditions for releasing bioactive dentine proteins, using proteomic and protein detection techniques to assess their potential effects on dental pulp healing and regeneration.

Methods: Fresh bovine incisors were sectioned at the cemento-enamel junction, the pulp tissue was removed, and the inverted crowns were mounted in tissue culture plates. The pulp chambers served as reservoirs for a series of irrigation-based experiments designed to investigate the extraction and identification of dentine extracellular matrix (ECM) components under varying conditions. Different irrigation protocols were applied using EDTA. The first experiment assessed the effect of irrigation duration, with EDTA applied for 20 minutes, 2 hours, and 24 hours to compare the quantity and profile of released dentine matrix proteins. The second experiment compared protein extraction using EDTA and sodium dodecyl sulphate gel (SDS), a laboratory detergent known for its strong protein-solubilising properties. The third experiment evaluated the release of water-soluble proteins through sequential water washing of dentine surfaces followed by EDTA irrigation on the same samples, allowing assessment of cumulative protein extraction efficiency. The extraction lysates from these three experiments were analysed using sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and in the first experiment, semi- quantification was performed using ImageJ. The final experiments aimed to identify and characterise the extracted proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to analyse proteins. Samples were treated with EDTA and water under different temperature conditions (4 °C, 37 °C, and 60 °C). Each condition was prepared in triplicate (n = 18), and the total irrigation time was 30 minutes for all samples. Analysis using MaxQuant identified 2,557 proteins, which were refined using Perseus filters to exclude entries identified only by site, reverse hits, or contaminants. Only proteins detected in at least two replicates were retained, yielding a total of 1,878 proteins for inclusion in this study. Western blot analysis was subsequently conducted to validate the LC–MS/MS findings and to confirm the presence of transforming growth factor beta 1 (TGF-β1) in water- and EDTA-treated dentine samples at 4 °C.

Results: Increasing the duration of EDTA irrigation resulted in greater protein extraction from the dentine matrix. An irrigation period of 20 minutes was sufficient to extract detectable dentine matrix proteins; however, a 2 hours application released more proteins, and the 24 hours irrigation period produced the highest yield among all time intervals assessed. A potent agent such as SDS extracted higher amounts of protein within a shorter application time compared with EDTA irrigation. Water-soluble proteins were readily released when dentine tissue was exposed to a liquid, likely comprising a mixture of dentine matrix and pulp proteins, as the dentine-pulp complex functions as a unified biological system. Temperature also influenced protein release, with EDTA at 4 °C yielding the greatest quantity and diversity of proteins. Forty-one proteins were commonly upregulated in EDTA-treated samples compared with water at all temperatures, including TGF-β1, IGFBP5, DSPP, BGN, COL11A2, and CTHRC1. Seventeen proteins were consistently downregulated in EDTA-treated samples relative to water across all temperature conditions. Notably, at least thirteen growth factors were exclusively identified in EDTA extracts but were absent in water-treated groups. Finally, western blot analysis confirmed the presence of TGF-β1 at multiple molecular weights across all EDTA conditions and in water extracts at 4 °C.

Conclusion of the thesis: EDTA irrigation effectively releases bioactive dentine matrix components, with protein yield increasing proportionally to exposure duration. While a 20 minute application extracted detectable protein levels, prolonged irrigation (up to 24 hours) notably enhanced protein recovery. Temperature also influenced extraction efficiency, with EDTA at 4 °C producing the greatest protein quantity and diversity. Proteomic analysis identified forty-one proteins consistently upregulated in EDTA-treated samples compared with water, including several growth factors such as TGF-β1, IGFBP5, DSPP, and BGN. Western blot validation confirmed the presence of TGF-β1 across all EDTA conditions. Another notable finding was that EDTA released at least thirteen growth factors that were not detected in the water-treated samples, highlighting its superior ability to extract matrix-bound bioactive molecules. These findings indicate that EDTA efficiently liberates bioactive dentine components associated with tissue repair and regeneration. Maintaining EDTA at 4 °C and applying it as a final rinse in vital pulp and regenerative endodontic procedures may provide a biologically favourable environment that supports healing and enhances treatment outcomes.