Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-β signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix.

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In response to microbial invasion of dentin odontoblasts secrete an altered calcified matrix termed reactionary dentin (Rd). 3D reconstruction of focused-ion-beam scanning electron microscopy (FIB-SEM) image slices revealed helical tubular structures in Rd that contrasted with regular cylindrical tubules characteristic of dentin from healthy teeth and affected so-called physiological dentin (Pd) lying exterior to Rd. This helical structure in Rd provided effective constriction of tubule lumen diameter that formed a barrier to bacterial advance towards the dental pulp. SEM of resin cast preparations revealed altered extension of odontoblast processes through Rd. The distribution of key mineral elements was studied by combination of 3D reconstruction of focused-ion-beam based X-ray microanalysis (FIB-EDS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). There was a marked redistribution of calcium and phosphorous in Rd together with an increase of diffusely deposited magnesium compatible with the mineral deposition phase of synthesis of this altered matrix. Changes in tubule structure and mineral content characteristic of Rd are consistent with reduced hardness and lower elastic modulus reported for this matrix. Findings provide insight into the unique structure of Rd synthesised as a primary response to infection.