Adaptive calcified matrix response of dental pulp to bacterial invasion is associated with establishment of a network of glial fibrillary acidic protein+/glutamine synthetase+ cells

Abstract

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein(-) cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP(+)/GS(+) cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP(+)/GS(+) interodontoblastic cells. A regulatory role for the networks of GFAP(+)/GS(+) cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response.

Figure 1

A: Photograph of a split tooth demonstrating the carious lesion and the regions of pulp adjacent to carious lesion (A-site) and remote from that (R-site). B: A-site showing physiological dentin (top) with a regular tubular structure (arrowhead), and reactionary dentin (bottom) marked with irregularity and scarcity of dentinal tubules (Toluidine blue staining). C:A-site showing Acridine orange staining of bacteria (orange-red fluorescence) within the dentinal tubules (green staining), demonstrating accumulation at the interface of physiological and reactionary dentin with few penetrating the reactionary dentin. Specificity for bacteria was confirmed by fluorescence ISH staining using a universal bacterial amplicon on an adjacent section. In addition this pattern of staining was not observed in healthy teeth. D: Toluidine blue staining of dental pulp (from A) with the odontoblastic layer, cell-free zone, and central pulp. Note the absence of inflammatory infiltrate. E: Toluidine blue staining of odontoblasts in the A-site of the carious tooth (A) demonstrating plump nuclei and interodontoblastic cells (arrowhead) with small nuclei and granular cytoplasm (inset). F: Toluidine blue staining of interodontoblastic granular cell extending fine processes (arrowhead). A, A-site; R, R-site; Od, odontoblast; Cf, cell-free zone; Cr, cell-rich zone; Cp, central pulp; Pd, physiological dentin; Rd, reactionary dentin; and Gc, interodontoblastic granular cell.

Figure 2

A: Immunohistochemical localization of DSP (A) and DMP-1 (B) in odontoblasts of a healthy tooth. (4′,6′-diamidino-2-phenylindole, blue fluorescence counterstaining). DSP (C) and DMP-1 (D) immunostaining in the A-site of a carious tooth. E: FISH identification of dspp expression in odontoblasts (green fluorescence) also demonstrating nonreactive interodontoblastic cells. Specificity of staining for mRNAs was confirmed by controls (see Materials and Methods). F: Expansion of inset in E indicating odontoblasts (arrow) and interodontoblastic cells (arrowhead). G: The relative expression of dspp and dmp-1 in healthy teeth and A- and R-sites of carious teeth. Showing pooled data (n = 4). Error bar represents SEM; **P < 0.0001. Od, odontoblast; Cf, cell-free zone; and Rd, reactionary dentin.

Figure 3

A: Immunohistochemical localization of TGF-β1 in the pulp of a carious tooth. TGF-β1 is expressed mainly by odontoblasts in a gradient mode decreasing from the R-site to the A-site. Inset shows double staining of dotted region for TGF-β1 and nuclear 4′,6′-diamidino-2-phenylindole (blue fluorescence). B: Schematic representation of a gradient mode of expression of TGF-β1 in ROI. C: Statistical analysis of TGF-β1 comparing immunolabeling intensity of healthy versus carious teeth from A-sites to R-sites (n = 4). *P < 0.05. D: The relative expression of tgf-β1 in healthy teeth and A- and R-sites of carious teeth showing pooled data (n = 4). Error bar represents SEM; **P < 0.0001. A, A-site; R, R-site; and cp, central pulp.

Figure 4

A: Expression of vimentin in odontoblasts and interodontoblastic cells in the A-site of a carious tooth. Note extension of vimentin filaments into the odontoblastic process within dentin (arrowhead). B: GFAP staining of interodontoblastic cells, extending between the odontoblasts in the A-site of a carious tooth. Note the lack of staining in odontoblast process (arrowhead). C: Coincidence of staining (arrowheads) for GFAP (C) and glutamine synthetase (D) in a healthy tooth. od, odontoblast; and rd, reactionary dentin.

Figure 5

A: The expression of occludin (arrowhead) in the R-site. B: Lack of expression of occludin in A-site. C: Peripheral expression of N-cadherin in odontoblasts in the R-site of a carious tooth (arrowhead). D: Expression of connexin-43 in the odontoblastic layer of a healthy tooth. Lack of expression of connexin-32 (E) and −26 (F) in the odontoblastic layer of a healthy tooth. Expression of connexin-43 (G), −32 (H), and −26 (I) in the odontoblastic layer of the A-site of a carious tooth. J: After ISH identification of dspp expression in odontoblasts and nonexpressing interodontoblastic cells in the A-site of a carious tooth, a region was chosen at random and the outlines of these two cell types overlaid with connexin-43 (black dots), −32 (yellow dots), and −26 (red dots) from sequential 1-μm sections stained for these markers (K). Note the presence of connexin-43 in a network of interodontoblastic cells that do not express dspp (black arrowheads), odontoblastic-odontoblastic connections (blue arrow) and odontoblastic-interodontoblastic communications (black arrow). L: Schematic drawing representing connexin-43 (black dots), −32 (yellow dots), and −26 (pink dots) expression in K. Note the networks of GFAP⁺ (pink) cells (black arrowheads). Od, odontoblast; and Cz, cell-free zone.

Figure 6

The relative expression of occludin (ocln), N-cadherin (cdh2), connexin-26 (gjb2), connexin-32 (gjb1), and connexin-43 (gja1) in healthy teeth and in A- and R-sites of carious teeth showing pooled data (n = 4). Error bar represents SEM; **P < 0.01.

Figure 7

A: Electron micrograph of the A-site of a carious tooth, demonstrating intimate association of an interodontoblastic process with an odontoblast. B: Specialized junction (arrow) between an interodontoblastic process rich in intermediate filaments and an odontoblast. C: Interodontoblastic cell with several slender processes rich in intermediate filaments. Inset refers to higher magnification of region marked by arrow. D: Immunogold labeling of GFAP in interodontoblastic cell processes equivalent to those in Figure 7C. Od, odontoblast; and P, interodontoblastic cell process.

Figure 8

Schematic representation showing response of pulpal cells to invading bacteria. Healthy tooth: processes of the odontoblasts occupy dentinal tubules. Odontoblasts express both dmp-1 (red granules) and dspp (blue granules). There is also strong expression of tgf-β1 in these cells (black granules). Intercellular connections between odontoblasts include occludin (blue) and connexin-43 (green). Odontoblasts also connect to interodontoblastic cells through connexin-43. No intercellular connexins detected in interodontoblastic granular cells. These cells show moderate reactivity for GFAP (yellow filaments). A-site, carious tooth: zone of invasion by bacteria and extensive deposition of reactionary dentin (orange). Tubules show retracted odontoblastic processes. Marked upregulation of dspp; no detectable expression of dmp-1. No detectable staining for occludin. Reactivity in odontoblasts for N-cadherin (red) and connexins 43 (green), 32 (yellow), and 26 (orange). Evidence for intercellular communication in interodontoblastic granular cells mediated by expression of connexin-43. Prominent GFAP⁺ staining (yellow filaments) in interodontoblastic cells. R-site, carious tooth: no invasion by bacteria; reduced deposition of reactionary dentin. Expression of dspp; no detectable expression of dmp-1. Preservation of intercellular adhesion. Evidence for intercellular communication in interodontoblastic granular cells mediated by expression of connexin 43 (green).