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The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α–induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

The complement system is an efficient plasma immune surveillance system that controls tissue injury and infection. Although the liver constitutes the primary circulating complement protein synthesis site, extrahepatic synthesis is known to optimize local tissue inflammatory reaction. Because dentin–pulp regeneration is known to be regulated locally, we investigated activation of the local complement system within the dental pulp and its role in initiating the regeneration process. Membrane attack complex (C5b-9) formation and Gram's staining revealed that complement activation is correlated with the presence of Gram-positive bacteria in carious human teeth. RT-PCR analysis demonstrated that cultured human pulp fibroblasts stimulated with lipoteichoic acid produce all the proteins required for efficient complement activation. This was demonstrated in vitroby C5b-9 formation and C5a active fragment production in the absence of plasma proteins. Finally, the dynamic migration assays performed in μ-Slide chemotaxis chambers and use of a C5aR-specific antagonist (W54011) demonstrated that the activation of complement proteins synthesized by pulp fibroblasts and the subsequent release of C5a specifically induced pulp progenitor cell recruitment. Our study reveals human pulp fibroblasts as the first nonimmune cell type capable of synthesizing all complement proteins. These fibroblasts cells contribute significantly to tissue regeneration by recruiting pulp progenitors via complement activation, which suggests to a potential therapeutic strategy of targeting pulp fibroblasts in dentin–pulp regeneration.

It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study, using bactericidal assays, we demonstrated that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureusand Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, using a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking, we showed that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. We have therefore elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration.

Cadherins are calcium-dependent cell adhesion molecules involved in the regulation of various biological processes such as cell recognition, intercellular communication, cell fate, cell polarity, boundary formation, and morphogenesis. Although previous studies have shown E-cadherin expression during rodent or human odontogenesis, there is no equivalent study available on N-cadherin expression in dental tissues. Here we examined and compared the expression patterns of E- and N-cadherins in both embryonic and adult (healthy, injured, carious) human teeth. Both proteins were expressed in the developing teeth during the cap and bell stages. E-cadherin expression in dental epithelium followed an apical-coronal gradient that was opposite to that observed for N-cadherin. E-cadherin was distributed in proliferating cells of the inner and outer enamel epithelia but not in differentiated cells such as ameloblasts, whereas N-cadherin expression was up-regulated in differentiated epithelial cells. By contrast to E-cadherin, N-cadherin was also expressed in mesenchymal cells that differentiate into odontoblasts and produce the hard tissue matrix of dentin. Although N-cadherin was not detected in permanent intact teeth, it was re-expressed during dentin repair processes in odontoblasts surrounding carious or traumatic sites. Similarly, N-cadherin re-expression was seen in vitro, in cultured primary pulp cells that differentiate into odontoblast-like cells. Taken together these results suggest that E- and N-cadherins may play a role during human tooth development and, moreover, indicate that N-cadherin is important for odontoblast function in normal development and under pathological conditions.

Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivodistribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitroassay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.