Françoise Bleicher

Characterization and Gene Expression of High Conductance Calcium-activated Potassium Channels Displaying Mechanosensitivity in Human Odontoblasts

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the KCa channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the α-subunit of these channels on odontoblasts in vivo. In inside-out patches, KCa channels were identified on the basis of their K+ selectivity, conductance, voltage, and Ca2+ dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to KCa α-subunit mRNA was constructed and used forin situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the KCa channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

Voltage-gated Sodium Channels Confer Excitability to Human Odontoblasts POSSIBLE ROLE IN TOOTH PAIN TRANSMISSION

Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na+ currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of α2 and β2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of α2 and β2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts β2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. AnkyrinG and β2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.

Odontoblast: a mechano-sensory cell.

Odontoblasts are organized as a single layer of specialized cells responsible for dentine formation and presumably for playing a role in tooth pain transmission. Each cell has an extension running into a dentinal tubule and bathing in the dentinal fluid. A dense network of sensory unmyelinated nerve fibers surrounds the cell bodies and processes. Thus, dentinal tubules subjected to external stimuli causing dentinal fluid movements and odontoblasts/nerve complex response may represent a unique mechano-sensory system giving to dentine-forming cells a pivotal role in signal transduction. Mediators of mechano-transduction identified in odontoblast include mechano-sensitive ion channels (high conductance calcium-activated potassium channel--K(Ca)--and a 2P domain potassium channel--TREK-1) and primary cilium. In many tissues, the latter is essential for microenvironment sensing but its role in the control of odontoblast behavior remains to be elucidated. Recent evidence for excitable properties and the concentration of key channels to the terminal web suggest that odontoblasts may operate as sensor cells.

Odontoblast primary cilia: facts and hypotheses

Odontoblasts, the cells responsible for the dentine formation, are organized as a single layer of highly polarized and differentiated post‐mitotic cells along the interface between the dental pulp and the mineralized tubules. They lay down the physiological secondary dentine throughout the life of the teeth. Odontoblasts play a central role in the transportation of calcium to the dentine and they possibly mediate early stages of sensory processing in teeth. A primary cilium, 9+0 configuration, have been regularly identified in a supra nuclear location. Calbindin D28k has been detected at the base of the cilium membrane. The cilium structure was positive with detyrosinated α tubulin antibodies in vivo and in cultured human odontoblasts. Transcripts of tektin, a protein involved in ciliogenesis, were expressed in vitro. The putative role of the primary cilium constituting a critical link between external teeth stimuli and odontoblast responses is extensively discussed.

TGFβ1 Signaling and Stimulation of Osteoadherin in Human Odontoblasts In Vitro

Transforming growth factor beta 1 (TGF g 1) is generally considered to be a potent inducer of dentin formation. In order to further assess this role, we studied the influence of this factor in human dental pulp cells on the expression of osteoadherin (OSAD), a newly described proteoglycan found in bone and dentin and suspected to play a role in mineralization events. We performed TGF g 1 stimulation both in cultures of human tooth thick slices including mature odontoblasts and in pulp explant cultures giving rise to early secretory odontoblasts or pulpal fibroblasts. We first showed by immunohistochemistry that molecules involved in TGF g 1 signal transduction, that is, membrane receptors T g RI and T g RII and intracellular proteins SMAD-2, SMAD-3, and SMAD-4, were present in human dental cells in vivo and were all maintained after culture of thick-sliced teeth in cells undergoing TGF g 1 stimulation. In this culture system, OSAD synthesis was increased in mature odontoblasts close to the TGF g 1 delivery system. In explant cultures, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the growth factor stimulated OSAD gene expression in early secretory odontoblasts and in pulpal fibroblasts. Taken together, these results indicate that OSAD expression is stimulated by TGF g 1 in pulpal fibroblasts and in early secretory and mature odontoblasts. We suggest that TGF g 1 in this way could control the organization and the mineralization of the extracellular matrix deposited by these cells during dentin formation.

General expression profiles of human native odontoblasts and pulp-derived cultured odontoblast-like cells are similar but reveal differential neuropeptide expression levels

OBJECTIVES Odontoblasts play a central role during the dentin formation by organic matrix production and mineralisation. Recently, suitable in vitro techniques for studying mature primary odontoblasts and the newly differentiated odontoblasts have been developed. Firstly, the gene expression profiles of native and cultured odontoblasts were compared at large-scale to investigate the similarities and differences between the samples. Secondly, differential expression levels of the genes encoding neuronal proteins were analyzed to study odontoblasts sensory function. DESIGN Microarray analysis was performed to mature native and cultured pulp-derived odontoblast-like cells to compare their transcriptome. Then, the probes positive only in one sample were divided into gene ontology categories. Expression levels of selected neuronal proteins were further studied with quantitative PCR, and at the protein level by immunofluorescence of mature and newly differentiated odontoblasts in developing tooth. RESULTS Remarkable similarities between the general and neuronal protein gene expression profiles were observed. Higher cortistatin, galanin, somatostatin receptor 1 (SSTR1) and tyrosine phosphatase receptor type Z1 (PTPRZ1) expression was detected in native than in cultured odontoblast at the mRNA level. Pronociceptin was more abundantly expressed in cultured than in native odontoblasts. Immunofluorescence of mature and newly differentiated odontoblasts on human tooth germs confirmed the results. CONCLUSIONS Cultured odontoblasts used in this study have similar general gene expression pattern to native odontoblasts, and therefore offer a valuable tool for the in vitro odontoblast studies. The expression of PTPRZ1 and galanin, which participate in sensory signal transduction, supports the previously suggested role of odontoblasts as sensory cells.

Odontoblast control of dental pulp inflammation triggered by cariogenic bacteria.

Inflammation is part of the normal protective immune response of the host to tissue infection. It promotes the recruitment of circulating immunocompetent blood cells and their migration through the endothelial barrier to gain access to the damaged site and eliminate injurious pathogens. If kept uncontrolled, inflammation can result in a wide range of acute, chronic, and systemic inflammatory disorders (Serhan and Petasis, 2011). Therefore, higher organisms have evolved protective mechanisms to ensure the inflammatory response is resolved in a specific time-limited manner (Serhan et al., 2008). Resolution of inflammation requires the elimination of injurious agents and the removal of pro-inflammatory mediators that initiate host defense against microbial invasion. In addition, anti-inflammatory agents including steroids, IL-1 receptor antagonist, soluble TNF receptor, interleukin-10 (IL-10), nitric oxide (NO), heme oxygenase-1, as well as regulatory T lymphocytes (Tregs), are produced to limit tissue damage and promote return to homeostasis (Gilroy et al., 2004; Eming et al., 2007; Blancou and Anegon, 2010; Buckley et al., 2013). Recent studies have revealed that endogenous lipid mediators, such as lipoxins and resolvins, synthesized locally during the inflammatory phase, stimulate cellular and molecular events that define the resolution of inflammation and repair (Serhan and Petasis, 2011). Complete cessation of inflammation is thus an active, multifactorial and highly orchestrated process (Uddin and Levy, 2011; Rius et al., 2012). A major cause of inflammation in human dental pulp is the presence, in the affected dentine, of the oral bacteria responsible for carious lesion development (Love and Jenkinson, 2002). Pulp inflammation accompanies the hosts innate and adaptive immune responses to these bacteria and/or to their components released during bacterial growth that diffuse to the pulp through dentine tubules. It generally dampens after pathogen removal by the dental practitioner and neutralization of diffusing components by the pulp immune system, which both decrease the production of pro-inflammatory mediators (Hahn and Liewehr, 2007). However, in cases of important dentine damage, pulp inflammation does not resolve completely but becomes chronic with moderate inflammatory infiltrate, collagenous fibrosis and premature tissue aging, and sometimes leads to pulp necrosis and dental abscess development. These evolutions induce permanent loss of normal tissue function and reduction of pulp defense capacities to future injuries. Conversely, cessation of pulp inflammation generally induces the re-establishment of homeostasis and accurate tissue healing characterized by maintenance of pulp vitality, absence of inflammatory infiltrate and fibrosis, and formation of a barrier of reactionary dentine by surviving original odontoblasts and/or reparative dentine by newly differentiated odontoblast-like cells (Lesot et al., 1994). Dentine neoformation moves the pulp tissue away from the dentine injury and the crown filling biomaterial, thus reducing the risk of permanent irritation by external chemical or bacterial agents. In the light of what happens in other healing tissues, it is reasonable to speculate that the more rapidly dentine neoformation is initiated, the quicker pulp homeostasis and health are re-established. Pulp inflammation resulting from carious lesions is characterized by a strong increase in the production of pro-inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, CXCL8, and IL-18. Interestingly, IL-10, a cytokine that plays a central role in limiting host immune response to pathogens by promoting the development of Tregs is also upregulated (Farges et al., 2011). An increase in the production of NO, a free radical anti-inflammatory at high concentration (Connelly et al., 2001), is also observed in bacteria-challenged, inflamed dental pulps (Di Nardo Di Maio et al., 2004; Korkmaz et al., 2011). The role of NO in this context remains unclear but experiments have suggested that, besides its well-known roles in vascular tone and nociceptive input modulation, it may be implicated in dental pulp healing by promoting odontoblast-like cell differentiation and subsequent formation of reparative dentine (Mei et al., 2007; Yasuhara et al., 2007). Recently, special attention was paid to lipopolysaccharide-binding protein (LBP), an acute-phase protein known to attenuate pro-inflammatory cytokine production by macrophages activated with bacterial components. LBP was shown to prevent binding of several bacterial cell wall components including lipopolysaccharides, lipoteichoic acids, lipopeptides, and peptidoglycan to host cells (Schumann, 2011; Lee et al., 2012). Interestingly, it was found to transfer lipopolysaccharides to high-density lipoproteins in the plasma for neutralization (Wurfel et al., 1995). We recently detected LBP synthesis and accumulation in bacteria-challenged inflamed pulp, whereas this protein was not found in healthy pulp (Carrouel et al., 2013). We proposed this molecule is involved in the neutralization of bacterial components before they gain access to pulp cells, thus limiting activation of the pulp immune system and the associated inflammatory response. Despite these important findings, the effects of IL-10, NO, and LBP in the control of dental pulp inflammation and promotion of pulp healing remain largely unknown. Studies are thus warranted to evaluate their importance in these processes and elucidate their putative therapeutic potential. Bacterial components that trigger innate immune responses are mostly represented by a limited number of evolutionary-conserved, structural motifs found in a wide range of microbes and called Pathogen-Associated Molecular Patterns (PAMPs) (Beutler, 2009). PAMP recognition (or sensing) is mediated by a set of specific germline-encoded host receptors referred to as Pattern Recognition Receptors (PRRs). PRRs are mainly localized at the cell surface or are present in the cytosol or in endosomes (Takeuchi and Akira, 2010). Owing to their specific localization at the pulp-dentin interface and the entrapment of their long cell processes in dentine tubules, odontoblasts are the first cells challenged by intradentinal PAMPs and its been proposed they are involved in the PAMP recognition process (Figure ​(Figure1).1). Immunolocalization of PRRs of the Toll-like receptor family in the odontoblast cell membrane (TLR2 and TLR4), as well as their activation in odontoblasts and odontoblast-like cells in vitro by specific PAMPs, argue in favor of this hypothesis (Durand et al., 2006; Veerayutthwilai et al., 2007). Odontoblasts thus constitute, in the tooth, the first line of defense for the host and are suspected to be involved in the initiation, development and maintenance of the pulp immune and inflammatory responses to dentine-invading pathogens. Studies have shown that upon TLR2 stimulation odontoblasts activate specific intracellular signaling pathways involving NF-κB and p38 MAP kinase (Carrouel et al., 2013). This activation leads to odontoblast down-regulation of dentine formation, production of pro-inflammatory molecules including interleukin-6 (IL-6) and CCL2, CXCL1, CXCL2, and CXCL8 chemokines, as well as immature dendritic cell accumulation into the odontoblast layer close to the carious dentine (Farges et al., 2009). The immunosuppressive cytokine IL-10 is up-regulated, suggesting odontoblasts may participate in limiting the inflammatory process in bacteria-challenged pulps (Farges et al., 2011). Up-regulation of LBP in vitro in PAMP-stimulated odontoblast-like cells (unpublished results) and in vivo in odontoblasts challenged by intradentinal cariogenic bacteria (Carrouel et al., 2013) could also contribute to shorten pulp inflammation duration. It is currently unknown whether these inflammation-dampening effects, by modulating specific intracellular signaling pathways, allow odontoblasts to recover their dentinogenic functions, an important event for dentine neoformation at the pulp-lesion interface and pulp healing. Figure 1 Possible role of odontoblasts in the initiation of the dental pulp immune response to cariogenic bacteria. Oral bacteria that degrade dentine during carious lesions release pathogen-associated molecular patterns (PAMPs; brown dots) that may diffuse through ... Resolution of inflammation is essential to maintain host health and several families of specialized “pro-resolving” local mediators (SPMs) including lipoxins, resolvins, protectins, and maresins have been involved in the clearance and regulation of inflammatory exudates to restore tissue homeostasis (Serhan et al., 2008). SPMs are biosynthesized from ω-3 poly-unsaturated fatty acids and provide local control over the execution of an inflammatory response toward resolution. In particular, they inhibit NF-κB and MAP kinase signaling pathways and pro-inflammatory cytokine production (Serhan and Petasis, 2011; Uddin and Levy, 2011). In spite of the importance of these lipid mediators, no studies have been published on their production in the inflamed dental pulp. In conclusion, we propose that identifying odontoblast molecules and mechanisms involved in the cessation of dental pulp inflammation is a crucial step for developing natural, host-derived agents able that promote rapid return to dental pulp homeostasis and health after pathogens are removed from caries-affected dental tissues.

Spatial and temporal expression of KLF4 and KLF5 during murine tooth development.

OBJECTIVE KLF4 and KLF5, members of the Krüppel-like factor (KLF) family, play key roles in proliferation, differentiation and apoptosis during development. In order to determine if these transcription factors are associated with tooth development, we examined the expression pattern of KLF4 and KLF5 during murine tooth development. DESIGN In situ hybridization and immunohistochemistry were performed to detect the expression pattern of KLF4 and KLF5 from E12.5 to PN3 during murine tooth development. RESULTS In situ hybridization analysis revealed that Klf4 was specifically expressed in polarizing odontoblasts from E16.5 (incisor) or E18.5 (first molar) to PN3. Immunohistochemistry staining showed that KLF4 was specifically expressed in both polarizing odontoblasts and ameloblasts at the same stages. KLF5 was mainly expressed from E18.5 to PN3 in secretory ameloblasts when enamel mineralization occurs and in secretory odontoblasts. However, an expression of KLF5 was also observed at earlier stages (E14.5 and E16.5) mainly in proliferating epithelial cells. CONCLUSIONS These results suggest that the expression of KLF4 is closely correlated to the growth-arrest and the first step of odontoblast and ameloblast differentiation. Furthermore, KLF5 maybe involved in proliferation at the early stages of tooth development and related to mineralization of both enamel and dentin matrices at later stages.

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.

Microtubule-associated protein 1b, a neuronal marker involved in odontoblast differentiation.

INTRODUCTION Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubule-associated protein highly expressed during the development of the nervous system. Its expression, regulated by the fragile X mental retardation protein (FMRP), is essential to stabilize microtubules during the elongation of dendrites and neurites. Other microtubules-associated molecules such as tau or MAP2 seem to act similarly. The aim of this work was to identify the MAP-1B expression in in vitro and in vivo human odontoblasts during development and carious processes. The expression of MAP2 and tau was also studied. MATERIALS AND METHODS In cultured cells, MAP-1B expression was analyzed by real-time polymerase chain reaction, flow cytometry, and Western blot. Its distribution was visualized by in situ hybridization and immunochemistry both in vitro and in vivo. The expression of FMRP, MAP2, and tau was identified by real-time polymerase chain reaction and immunochemistry. RESULTS MAP-1B is specifically expressed in odontoblasts from adult third molars as well as incisor germs from human embryos. In adult carious teeth, it is also expressed in newly differentiated dentin-forming cells. In vitro, MAP-1B expression is related to the differentiation state of odontoblasts. MAP-1B clearly underlines the cellular architecture of cell bodies and processes of differentiated cells. FMRP, MAP2, and tau are also detected in vivo. CONCLUSION On the basis of these data, MAP-1B could be considered as a new protein involved in the terminal differentiation of odontoblasts.