In Vitro Assessment of Biomimetic Dentin Conditioning Using Citric Acid

Objectives: Current approaches in regenerative endodontics involve cleaning of the root canal system followed by controlled bleeding into the pulp space. EDTA is applied to liberate fossilized growth factors from the canal wall dentin. However, under natural conditions, i.e. caries, dentin dissolution and subsequent recruitment of regenerative cells occur in an acidic environment generated by the release of organic acids from cariogenic biofilms. We thus hypothesized that an organic acid such as citric acid (CA) could be better than EDTA with its neutral to slightly alkaline pH to condition the root dentin for the recruitment and differentiation of pluripotent cells towards a functional pulp.
Methods: Human dentin disks were immersed in 17% Na-EDTA (pH 7), 10% CA (pH 2), or PBS, and afterwards washed twice with PBS (n=5). Migration of human bone marrow-derived stem cells towards these disks was assessed using a modified Boyden chamber assay. In a second experiment expression of mineralization markers (COL1, DSPP, RUNX2) in cells that were in contact with pre-treated dentin disks was determined by RT-qPCR. Statistical analysis was performed using one-way analysis of variance, α= 0.05.
Results: Citric acid conditioning of dentin disks strongly promoted migration of stem cells towards the dentin, with a three-fold difference in mean cell numbers compared to EDTA (p=0.0012). Expression of mineralization associated genes in adhered cells was similar between disks treated with CA or EDTA: COL1 (p=0.536), DSPP (p=0.2771) and RUNX2 (p=0.1888).
Conclusions: Under the current conditions, biomimetic demineralization of human root dentin using citric acid was superior to EDTA treatment in terms of the attraction of stem cells, but not their differentiation towards mineralizing cells.