Jean-Christophe Farges

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.

Dental Pulp Defence and Repair Mechanisms in Dental Caries.

Dental caries is a chronic infectious disease resulting from the penetration of oral bacteria into the enamel and dentin. Microorganisms subsequently trigger inflammatory responses in the dental pulp. These events can lead to pulp healing if the infection is not too severe following the removal of diseased enamel and dentin tissues and clinical restoration of the tooth. However, chronic inflammation often persists in the pulp despite treatment, inducing permanent loss of normal tissue and reducing innate repair capacities. For complete tooth healing the formation of a reactionary/reparative dentin barrier to distance and protect the pulp from infectious agents and restorative materials is required. Clinical and in vitro experimental data clearly indicate that dentin barrier formation only occurs when pulp inflammation and infection are minimised, thus enabling reestablishment of tissue homeostasis and health. Therefore, promoting the resolution of pulp inflammation may provide a valuable therapeutic opportunity to ensure the sustainability of dental treatments. This paper focusses on key cellular and molecular mechanisms involved in pulp responses to bacteria and in the pulpal transition between caries-induced inflammation and dentinogenic-based repair. We report, using selected examples, different strategies potentially used by odontoblasts and specialized immune cells to combat dentin-invading bacteria in vivo.

Human dental pulp stem cells cultured in serum-free supplemented medium

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.

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.

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.

Expression and localisation of αv integrins in human odontoblasts

Integrin αβ heterodimers mediate adhesion to the extracellular matrix and at cell-cell contacts and initiate intracellular signalling cascades in response to a variety of inductive factors. Apart from the expression of αvβ3 that we have previously reported, little is known about the expression of integrins in odontoblasts. Here, we investigated the expression of αv-binding β integrin subunits in healthy human dental pulp in vivo and in odontoblasts differentiated in vitro. Reverse transcription/polymerase chain reaction analysis revealed the expression of αv, β1, β5 and β8 integrin mRNA, but not β6, in whole pulp cells. Flow cytometry showed that the αv and β1 subunits were the most intensely expressed. Immunohistochemistry demonstrated that the β1 subunit was localised in newly differentiated odontoblasts in the root and in mature odontoblasts in the crown, including their intradentinal cell processes. The αv chain was predominantly expressed by mature odontoblasts and αvβ5 was only observed in mature odontoblasts. In vitro differentiated odontoblasts expressed genes for αv, β1 and β5, but not for β6 and β8. A comparison of integrin profiles between cultured pulp cells and in vitro differentiated odontoblasts revealed that odontoblast maturation was characterised by a significant increase in the expression of αv and β1 subunits and αvβ5 integrin. The β8 subunit was detected in nerve cells only. Histological analysis of teeth from αv knockout mice showed no obvious structural modification in the odontoblast layer. Thus, human mature odontoblasts express αvβ3, αvβ5 and perhaps αvβ1 integrins, with the possible presence of α-β1 pairs. The roles that these molecules play in the exchange of information throughout the odontoblast layer remain to be determined.

Manufacturing of dental pulp cell-based products from human third molars: current strategies and future investigations

In recent years, mesenchymal cell-based products have been developed to improve surgical therapies aimed at repairing human tissues. In this context, the tooth has recently emerged as a valuable source of stem/progenitor cells for regenerating orofacial tissues, with easy access to pulp tissue and high differentiation potential of dental pulp mesenchymal cells. International guidelines now recommend the use of standardized procedures for cell isolation, storage and expansion in culture to ensure optimal reproducibility, efficacy and safety when cells are used for clinical application. However, most dental pulp cell-based medicinal products manufacturing procedures may not be fully satisfactory since they could alter the cells biological properties and the quality of derived products. Cell isolation, enrichment and cryopreservation procedures combined to long-term expansion in culture media containing xeno- and allogeneic components are known to affect cell phenotype, viability, proliferation and differentiation capacities. This article focuses on current manufacturing strategies of dental pulp cell-based medicinal products and proposes a new protocol to improve efficiency, reproducibility and safety of these strategies.

Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies

High doses of zoledronic acid (ZOL), one of the most potent inhibitors of bone resorption, are currently evaluated in phase III clinical trials in Europe for the treatment of malignant pediatric primary bone tumors. The impact of such an intensive treatment on the craniofacial skeleton growth is a critical question in the context of patients with actively growing skeleton; in particular, in light of our previous studies evidencing that endochondral bone formation was transiently disturbed by high doses of ZOL. Two protocols adapted from pediatric treatments were developed for newborn mice (a total of 5 or 10 injections of ZOL 50μg/kg every two days). Their impact on skull bones and teeth growth was analyzed by X-rays, microCT and histology up to 3months after the last injection. ZOL administrations induced a transient delay of skull bone growth and an irreversible delay in incisor, first molar eruption and root elongation. Other teeth were affected, but most were erupted by 3months. Root histogenesis was severely impacted for all molars and massive odontogenic tumor-like structures were observed in all mandibular incisors. High doses of ZOL irreversibly disturbed teeth eruption and elongation, and delayed skull bone formation. These preclinical observations are essential for the follow-up of onco-pediatric patients treated with ZOL.

Phenotypic analysis of immunocompetent cells in healthy human dental pulp.

INTRODUCTION Like other tissues in the body, the human dental pulp is equipped with a network of immune cells that can be mobilized against pathogens when they invade the tooth. Very little data, mostly obtained with classic histologic methods, have reported their quantities and relative percentages. The objective of this study was to characterize and precisely quantify immunocompetent cells in healthy human dental pulp by using fluorescence-activated cell sorting, together with identifying specific cell subsets in the leukocyte (CD45(+)) cells. METHODS Healthy human third molars were collected from 42 young patients. Dental pulps were separated from the hard tissues and prepared for flow cytometry or immunostaining analyses. RESULTS CD45(+) cells represented 0.94% ± 0.65% of cells obtained from the enzymatic digestion of whole dental pulps (n = 34). CD16(+)CD14(+) granulocytes/neutrophils (50.01% ± 9.08%, n = 7) were found to represent the major subpopulation in CD45(+) cells followed by CD3(+) T lymphocytes (32.58% ± 11%, n = 17), CD14(+) monocytes (8.93% ± 5.8%, n = 7), and HLA-DR(high) Lin1(-) dendritic cells (4.51% ± 1.12%, n = 7). Minor subpopulations included CD3(-)CD56(+) natural killer cells (2.63% ± 1.15%, n = 7) and CD19(+) B lymphocytes (1.65% ± 0.89%, n = 17). We further identified cells harboring a phenotype compatible with Foxp3/CD25-expressing regulatory T lymphocytes (CD45(+)CD3(+)CD4(+)CD127(low)). Fluorescence-activated cell sorting analysis and confocal microscopy also revealed expression of HO-1 in HLA-DR(+) cells. CONCLUSIONS For the first time, this study identifies and precisely quantifies the relative proportion of immunocompetent cells potentially involved in tissue homeostasis of healthy human dental pulp.

Immunocytochemical localization of fibronectin and a 165-kDa membrane protein in the odontoblast layer under initial carious lesions in man

The possible role of fibronectin in dental tissue repair was investigated by comparing its distribution and that of the 165-kDa fibronectin-binding membrane protein (165 kDa-FnBP) in odontoblasts underlying carious and sound dentine. By immunoperoxidase and light microscopy, fibronectin was localized in the dentine underlying the carious lesion, mainly on the surface of the tubule walls, whereas it could not be detected in neighbouring sound zones. The antibody to the 165 kDa-FnBP strongly reacted with the membrane of odontoblasts underlying the lesion, although those facing sound dentine did not express this antigen. Ultrastructurally the 165 kDa-FnBP was localized in the cell membrane at the apical portion of odontoblasts, including the process membrane, beneath the initial lesion; fibronectin was detected in the dentinal area close to the process, and also in contact with its external surface. By a high-resolution immunogold procedure, the proteins were colocalized at the external surface of odontoblast processes. These data suggest that fibronectin present in human carious dentine could modulate the behaviour of underlying odontoblasts by means of newly expressed 165 kDa-FnBP.