Anne Baudry
email: anne.baudry@parisdescartes.fr

Benoit Schneider

Jean-Marie Launay

Odile Kellermann

Serotonin in stem cell based-dental repair and bone formation: a review

Keywords: Serotonin, Stem cells, Bone mineralization, Tooth repair

Genetic and pharmacological studies provided evidence that serotonin (5-HT) is an important signaling molecule for the development and the maintenance of mineralized tissues. However, how 5-HT takes part to the homeostasis of teeth and bone remains elusive. In the dental field, a major breakthrough comes from the identification of 5-HT but also dopamine (DA) as "damage" signals necessary for stem cell-based tooth repair. Pulpal stem cells express the overall functions of 5-HT and DA neurons including a definite set of functional 5-HT/DA receptors that render cells responsive for circulating bioamines. Upon tooth injury, activated platelets release bulks of 5-HT/DA that mobilize pulpal stem cells for natural dental repair.

The contribution of 5-HT to bone metabolism is more documented with description of both anabolic and resorptive effects. By controlling the tissue-non specific alkaline phosphatase (TNAP), 5-HT2B receptors exert an anabolic function and a pivotal role in mineralization processes. Increasing our understanding of the role of 5-HT receptors in bone metabolism may pave the road for the development of therapeutic strategies towards skeletal-associated pathologies and ectopic calcification.

Introduction

Serotonin (5-hydroxytryptamine ; 5-HT) is a tryptophan-derived bioamine synthesized by neurons of the raphe nuclei in the central nervous system and gut enterochromaffin cells in the periphery, this last site accounting for 95% of 5-HT produced in the human body. In the brain, 5-HT acts as a neurotransmitter and mediates a wide variety of behavioral and physiological processes such as sleep, memory, cognition, appetite or mood [START_REF] Jonnakuty | What do we know about serotonin?[END_REF]. Peripheral 5-HT stored in platelet-dense granules is involved in the regulation of cardiovascular, gastrointestinal, smooth muscle and endocrine functions [START_REF] Jonnakuty | What do we know about serotonin?[END_REF]. This monoamine influences such broad physiological processes thanks to the presence at the surface of recipient cells of a serotonin transporter (SERT) and/or serotonergic receptors (5-HTRs). To date, 15 serotonergic receptor subtypes were identified, divided in seven classes (5-HT1-7R) [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF]. They all belong to the Gprotein coupled receptor (GPCR) superfamily except for the 5-HT3R, a ligand gated ion channel [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

At early stages of embryonic mammalian development, 5-HT of maternal origin [START_REF] Cote | Maternal serotonin is crucial for murine embryonic development[END_REF] was shown to exert a trophic role [START_REF] Gaspar | The developmental role of serotonin: news from mouse molecular genetics[END_REF][START_REF] Moiseiwitsch | The role of serotonin and neurotransmitters during craniofacial development[END_REF][START_REF] Lauder | Serotonin and morphogenesis. I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo[END_REF], notably in craniofacial development [START_REF] Choi | 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells[END_REF][START_REF] Shuey | Serotonin and morphogenesis. Transient expression of serotonin uptake and binding protein during craniofacial morphogenesis in the mouse[END_REF]. Shuey et al. reported craniofacial malformations, including mandibular and maxillary hypoplasia, in mouse embryos exposed to antidepressants such as sertraline or fluoxetine (Prozac ® ) that target and block the SERT [START_REF] Shuey | Serotonin as a regulator of craniofacial morphogenesis: site specific malformations following exposure to serotonin uptake inhibitors[END_REF]. Similar morphological defects were observed when mouse embryos were treated with the 5-HT2R inverse agonist ritanserin [START_REF] Choi | 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells[END_REF]. Supporting the role of 5-HT in skeleton development and tooth formation, 5-HT would drive the migration and differentiation of neural crest cells which are at the origin of oral facial mineralized tissues.

Following migration from the rostral hindbrain (rhombomeres 1 and 2) and caudal midbrain, neural crest cells populate the first branchial arch to give rise to the ectomesenchyme required for the formation of (i) mandibular and maxillary bones and (ii) the central core dentin and root lining cementum in teeth [START_REF] Thesleff | Epithelial-mesenchymal signalling regulating tooth morphogenesis[END_REF][START_REF] Santagati | Cranial neural crest and the building of the vertebrate head[END_REF] (Fig. 1). The ectomesenchyme functionaly interacts with the dental epithelium to promote formation of the tooth bud preceding cell differentiation into dentin-secreting odontoblasts and enamel-secreting ameloblasts. Interestingly, the presence of 5-HT2BR at day 9 of mouse embryogenesis in the first branchial arch argues for an involvement of this receptor in the migration of neural crest cells and their commitment towards osteo-odontogenic lineages. Consistent with this, by using the alternative vertebrate model Xenopus laevis, Reisoli et al. showed that 5-HT2BR contributes to morphogenesis of the first branchial arch and formation of the jaw joint [START_REF] Reisoli | Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus[END_REF], reinforcing the key role of the 5-HT2BR in craniofacial development.

Expression of the SERT and diverse serotonergic receptors has been evidenced at the mRNA or protein levels in osteoblast primary cultures, osteoblastic cell lines or RANKL-induced RAW264.7 osteoclasts as well as in tooth tissues [START_REF] Bliziotes | Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake[END_REF][START_REF] Westbroek | Expression of serotonin receptors in bone[END_REF][START_REF] Battaglino | Serotonin regulates osteoclast differentiation through its transporter[END_REF][START_REF] Collet | The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation[END_REF][START_REF] Harichane | Enamel alterations in serotonin 2B receptor knockout mice[END_REF][START_REF] Riksen | Serotonin and fluoxetine receptors are expressed in enamel organs and LS8 cells and modulate gene expression in LS8 cells[END_REF][START_REF] Warden | The emerging role of serotonin (5-hydroxytryptamine) in the skeleton and its mediation of the skeletal effects of low-density lipoprotein receptor-related protein 5 (LRP5)[END_REF][START_REF] Yun | Peripheral serotonin-mediated system suppresses bone development and regeneration via serotonin 6 G-protein-coupled receptor[END_REF]. This suggests that bone-forming osteoblasts, bone-resorbing osteoclasts, ameloblasts and odontoblasts are directly influenced by circulating 5-HT. Accordingly, Gustafsson et al. showed that 5-HT stimulates proliferation of human osteoclasts and primary osteoblasts as well as murine preosteoblasts in vitro [START_REF] Gustafsson | Serotonin and fluoxetine modulate bone cell function in vitro[END_REF]. Two-months old rats treated by daily 5-HT administration for 3 months display higher bone mineral density, altered bone architecture and higher femoral stiffness [START_REF] Gustafsson | Long-term serotonin administration leads to higher bone mineral density, affects bone architecture, and leads to higher femoral bone stiffness in rats[END_REF]. Further, clinical studies have reported that the use of selective serotonin reuptake inhibitors (SSRIs) antidepressants such as fluoxetine, for a long time period, is associated with altered bone mineral density, osteoporosis or increased risks of bone fracture [START_REF] Haney | Skeletal effects of serotonin (5-hydroxytryptamine) transporter inhibition: evidence from clinical studies[END_REF][START_REF] Rizzoli | Antidepressant medications and osteoporosis[END_REF][START_REF] Tsapakis | The adverse skeletal effects of selective serotonin reuptake inhibitors[END_REF]. Four-weeks old wild-type mice treated with fluoxetine exhibit reduced bone mass accrual during growth [START_REF] Warden | Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth[END_REF].

SERT knockout mice also display reduced bone mass, altered skeletal architecture and decreased femur mechanical properties [START_REF] Warden | Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth[END_REF]. Yun et al. recently reported that activation of 5-HT6R by the agonist ST1936 inhibits osteoblast differentiation in vitro and calvarial bone regeneration in vivo [START_REF] Yun | Peripheral serotonin-mediated system suppresses bone development and regeneration via serotonin 6 G-protein-coupled receptor[END_REF]. The 5-HT1BR was also involved in the negative control of bone mass. 5-HT1BR knockout mice display increased osteoblast proliferation and bone formation resulting in high bone mass [START_REF] Yadav | Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum[END_REF]. In contrast, pharmacological blockade of the 5-HT2AR with MDL11939 leads to loss of bone mass in mice, due to an impairment of bone formation [START_REF] Tanaka | Modulation of osteoblast differentiation and bone mass by 5-HT2A receptor signaling in mice[END_REF].

The deletion of 5-HT2BR provokes an age-dependent reduction of bone mineral density, but only in females [START_REF] Collet | The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation[END_REF]. The osteopenia observed in 5-HT2BR -/-mice can be accounted for by a defect of osteoprogenitor recruitment and a decrease of osteoblast proliferation [START_REF] Collet | The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation[END_REF]. The bony phenotype of 5-HT2BR -/-mice is reminiscent to that of SERT -/-mice [START_REF] Warden | Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth[END_REF], but the damages on skeleton caused by the absence of 5-HT2BR are less pronounced and occur later compared to SERT -/-mice. Of note, 5-HT2AR is up-regulated in 5-HT2BR -/-calvaria-derived osteoblasts [START_REF] Collet | The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation[END_REF]. 5-HT2AR may, at least partly, compensate for the absence of the 5-HT2BR. Besides, we monitored structural alterations of dental tissues in 5-HT2BR knockout mice [START_REF] Harichane | Enamel alterations in serotonin 2B receptor knockout mice[END_REF][START_REF] Dimitrova-Nakov | Deletion of serotonin 2B receptor provokes structural alterations of mouse dental tissues[END_REF]. Electron microscopy analysis revealed that teeth of both male and female 5-HT2BR -/-mice exhibit numerous enamel porosities and reduced thickness of the crystallite, which are worsened in female (Fig. 2).

In this review, we will highlight how the use of stem cell lines, which combine the capacities to maintain a stable immature phenotype and to recapitulate in vitro upon induction the spatio-temporal features of odontogenic or osteogenic differentiation, has contributed to our understanding of the role of 5-HT and serotonergic receptors in bone homeostasis and tooth repair.

Serotonin in tooth repair: a dialogue between odontogenic stem cells and platelets

To rational the development of stem cell-based tooth regenerative strategies, a prerequisite is to define the intrinsic properties of odontogenic stem cells and to identify the physiological signals leading to the recruitment of those pulpal progenitors for reparative dentin formation upon tooth injury. Since the 2000's, odontogenic stem cells were isolated from the pulp of human permanent third molars that referred to as dental pulp stem cells (DPSCs) [START_REF] Gronthos | Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo[END_REF] and from exfoliated deciduous teeth called SHED cells [START_REF] Miura | SHED: stem cells from human exfoliated deciduous teeth[END_REF]. Upon induction, DPSCs and SHEDs can differentiate in vitro into odontoblasts, osteoblasts, adipocytes and neuron-like cells. In vivo, they give rise to dentin-like structures when transplanted subcutaneously with hydroxyapatite/tricalcium phosphate (HA/TCP) powder into the dorsal surface of immunocompromised mice [START_REF] Gronthos | Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo[END_REF][START_REF] Huang | Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine[END_REF]. Other stem cells have been derived from the periodontal ligament of permanent tooth (PDLCs) [START_REF] Seo | Investigation of multipotent postnatal stem cells from human periodontal ligament[END_REF]. PDLCs showed the capacity to differentiate into cementoblasts, adipocytes and osteoblasts in vitro and to form cementum and periodontal ligament-like structures after transplantation in vivo [START_REF] Seo | Investigation of multipotent postnatal stem cells from human periodontal ligament[END_REF]. SCAP cells, derived from the root apical papilla of human tooth, are able to differentiate into osteoblasts and adipocytes in vitro [START_REF] Sonoyama | Mesenchymal stem cell-mediated functional tooth regeneration in swine[END_REF]. Upon transplantation with HA/TCP in immunocompromised mice, SCAP cells formed dentin [START_REF] Sonoyama | Mesenchymal stem cell-mediated functional tooth regeneration in swine[END_REF]. These dental stem cells are heterogeneous cell populations and the lack of specific markers render their isolation difficult. This limits their use for defining the proper identity of odontogenic stem cells. Establishing homogenous dental stem cell lines will help to challenge this issue.

Isolation of clonal pulpal stem cell lines

In 2005, our laboratory isolated clonal cell lines from the first molar tooth germs of day 18 mouse embryos [START_REF] Priam | New cellular models for tracking the odontoblast phenotype[END_REF], which display stem cell properties [START_REF] Lacerda-Pinheiro | Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation[END_REF]. They maintain a stable phenotype under long-term standard culture conditions, even in 10% fetal calf serum and exhibit the hallmarks of the odontogenic lineage. They express the transcription factors Lhx6 and Lhx7, two LIM-domain homeobox genes that specify the first branchial arch where the tooth bud is formed, as well as low levels of the odontoblast markers, type 1 collagen, dentin matrix protein (DMP1) and dentin sialoprotein (DSP). After implantation in a mouse incisor or rat molar, two pulpal stem cell lines (A4 and H8) induce spatio-temporally controlledosteodentin formation and promote efficient tooth repair [START_REF] Lacerda-Pinheiro | Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation[END_REF][START_REF] Harichane | Pulpal progenitors and dentin repair[END_REF]. A4 and H8 cells are however precursors at different stages of commitment along the odontogenic lineage. The A4 cell line behaves as a multipotent mesoblastic stem cell, which undergo in vitro odonto/osteogenic, chondrogenic or adipogenic differentiation, depending on the inducers. H8 cells are monopotent progenitors whose potential is restricted towards the odontogenic fate [START_REF] Lacerda-Pinheiro | Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation[END_REF].

Odontogenic pulpal stem cells display a mixed serotonergic/dopaminergic phenotype

As human DPSCs that secrete diverse neurotrophic factors [START_REF] Martens | Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissueengineered collagen construct in vitro[END_REF][START_REF] Ratajczak | The Neurovascular Properties of Dental Stem Cells and Their Importance in Dental Tissue Engineering[END_REF], A4 and H8 pulpal cell lines display high contents of Brain-Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) as well as insulin-like growth factor-1 (IGF1), which are all stored in large dense-core vesicles [START_REF] Baudry | Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets[END_REF]. Of note, the dental pulp is a tissue highly innervated. BDNF, NGF and IGF-1 are all well known to promote axon guidance and elongation as well as neuronal survival [START_REF] Lykissas | The role of neurotrophins in axonal growth, guidance, and regeneration[END_REF][START_REF] Decourtye | IGF-1 Induces GHRH Neuronal Axon Elongation during Early Postnatal Life in Mice[END_REF]. These neurotrophins released by pulpal stem cells might contribute to tooth innervation along root formation and tooth repair. Surprisingly, we discovered that odontogenic stem cells have a dual serotonergic and dopaminergic identity [START_REF] Baudry | Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets[END_REF]. Both A4 and H8 pulpal cell lines are able to synthesize 5-HT and dopamine (DA) and express all functional enzymes ensuring complete 5-HT/DA metabolisms. This includes the tryptophane hydroxylase and the tyrosine hydroxylase, the rate-limiting enzymes in 5-HT and DA synthesis, respectively, as well as monoamine oxidases involved in the catabolism of these bioamines. Further, both cell lines express functional SERT and vesicular transporters (VMATs), which confer the ability to co-store 5-HT and DA. Until now, such a mixed 5-HT/DA phenotype is an intriguing finding that has never been described. Finally, the properties of pulpal stem cells to produce neurotrophins as well as overall bioaminergic functions led foundation for their use for therapeutic purposes, including the treatment of neural tissue injury [START_REF] Sakai | Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms[END_REF] or neurodegenerative diseases [START_REF] Volponi | Stem cell-based biological tooth repair and regeneration[END_REF][START_REF] Wang | Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells[END_REF].

Functional serotonergic 5-HT1D,2B,7 and dopaminergic D1,3 autoreceptors are expressed by odontogenic stem cells

A striking feature of A4 and H8 pulpal stem cells is the expression at the plasma membrane of the same specific subset of serotonergic and dopaminergic receptors. This includes the 5-HT2B, 5HT1D and 5-HT7 receptor subtypes and dopaminergic D1 and D3 receptor subtypes, which are all G-protein coupled receptors (GPCRs). Of note, the three 5-HT receptors selectively expressed by odontogenic stem cells have been shown to mediate trophic effects of 5-HT during early embryogenesis [START_REF] Gaspar | The developmental role of serotonin: news from mouse molecular genetics[END_REF][START_REF] Lauder | Expression of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors in the mouse embryo[END_REF]. 5-HT1DR and 5-HT7R are detected in preimplantation mouse embryos [START_REF] Vesela | Expression of serotonin receptors in mouse oocytes and preimplantation embryos[END_REF]. The 5-HT2BR is expressed at embryonic day 9 by neural crest-derived tooth buds [START_REF] Lauder | Expression of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors in the mouse embryo[END_REF]. All these bioaminergic receptors are functional in pulpal cell lines since they are coupled to downstream signaling effectors (Fig. 3). For instance, 5-HT7R and D1R positively control adenylate cyclase activity while 5-HT1DR and D3R are negatively coupled to cAMP production. The pleiotropic 5-HT2BR couplings to phospholipase Cdependent inositol 1, 4, 5-triphosphate (IP3), NO and phospholipase A2 (PLA2)-mediated eicosanoid productions put forward some preferential role of this receptor in dental repair since all these regulatory molecules are known to contribute to mechanical stress, inflammation and vasodilatation mechanisms in other tissues [START_REF] Aoki | Prostaglandins and chronic inflammation[END_REF][START_REF] Li | Mechanical stress-initiated signal transduction in vascular smooth muscle cells in vitro and in vivo[END_REF][START_REF] Nizar | In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase[END_REF]. Such specific expression of 5-HT/DA receptors at the cell surface of pulpal stem cells suggests these receptors take part to the fine-tuning of odontogenic stem cell functions in an autocrine and/or paracrine manner. An overexpression of 5-HT1D,2B,7R and D1,3R has been reported in various types of cancer stem cells [START_REF] Pai | Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival[END_REF][START_REF] Sachlos | Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells[END_REF]. In a physiological context, whether 5-HT and DA receptordependent signalings act together to maintain pulpal stem cell quiescence and/or influence the balance between self-renewal and differentiation remains to be delineated.

Stem cell behavior is regulated by a local microenvironment referred as to "the stem cell niche". This anatomic space includes (i) stem cells and (ii) stromal-supporting cells that interact with the stem cells via secreted factors and/or extracellular matrix (ECM), which allows the diffusion of mechanical and chemical signals [START_REF] Jones | No place like home: anatomy and function of the stem cell niche[END_REF][START_REF] Mitsiadis | Dental pulp stem cells, niches, and notch signaling in tooth injury[END_REF][START_REF] Pagella | Investigation of orofacial stem cell niches and their innervation through microfluidic devices[END_REF][START_REF] Oh | The Perivascular Niche and Self-Renewal of Stem Cells[END_REF]. The distribution of tissue stem cells is not random, and, several distinct stem cell niches (apex, isthmus between the mesial and the central pulp chambers, mesial part below the pulp exposure site,...) potentially exist within the dental pulp [START_REF] Harichane | Pulpal progenitors and dentin repair[END_REF][START_REF] Baudry | Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets[END_REF][START_REF] Mitsiadis | Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp[END_REF]. In these specialized niches, whether the maintenance of the odontogenic stem cell state and/or differentiation of odontogenic progenitors would relate to 5-HT/DA receptors-dependent control of developmental signals such as BMP, FGF, Notch, Hedgehog and Wnt remains to be explored. Finally, the role of 5-HT and DA signaling in the recruitment of pulpal stem cells, their self-renewal, migration and differentiation for tooth repair deserves further in vitro and in vivo investigations.

Serotonergic and dopaminergic receptors relay the action of platelets-derived 5-HT and DA to mobilize pulpal stem cells for tooth repair

The presence of a specific repertoire of serotonergic and dopaminergic receptors at the plasma membrane of odontogenic stem cells renders these cells competent to respond to both 5-HT and DA, and in particular to those circulating bioamines. In the periphery, 5-HT and DA are mainly stored in blood platelets. In platelets, 5-HT and DA contents are about 25,000 and 100-300 times higher than the concentration of free circulating 5-HT and DA in plasma, respectively [START_REF] Prada | Content and subcellular localization of catecholamines and 5-hydroxytryptamine in human and animal blood platelets: monoamine distribution between platelets and plasma[END_REF]. Of note, the dental pulp is highly vascularized [START_REF] Matthews | Microvascular architecture and exchange in teeth[END_REF]. This raised the question of whether 5-HT and DA released by injury-activated platelets would represent "warning" signals necessary for mobilizing pulpal stem cells for tooth repair. Such issue was tackled using Fawn-hooded and reserpine-treated rats, two rat models whose blood platelets are deficient in bioamine storage. In wild-type rats, after pulp exposure of the mesial part of the first maxillary molar, the formation of a reparative dentin was observed at the lesion site after 30 days and the thickness of the dental bridge increased until 3 months. By contrast, with Fawn-hooded and reserpine-treated rats, natural dentin formation was not detected [START_REF] Baudry | Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets[END_REF]. Thus, natural dental repair depends on platelet-stored bioamines that recruit genuine stem cells expressing 5-HT/DA receptors (Fig. 4). The pharmacological inhibition of each of the 5-HT2B,7 and D1,3 receptors upon introduction of selective antagonists within the pulp also altered the reparative process [START_REF] Baudry | Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets[END_REF]. Upon tooth injury, these receptors are involved in pulpal stem cell-based dental repair by transducing 5-HT and DA co-released by activated platelets.

The identification of 5-HT/DA as crucial "damage" signals for stem cell-mediated tooth repair paves the road for novel therapeutic approaches in the dental field.

Bone mineralization: a major role of 5-HT2BR signaling

Bone development, growth, remodeling and repair are highly regulated processes ensuring skeleton homeostasis. Despite considerable efforts to dissect the molecular pathways sustaining osteogenesis, the regulatory mechanisms governing collagen matrix mineralization still remain enigmatic. It is now clear that the serotonergic system contributes to bone metabolism [START_REF] Lavoie | Regulation of Bone Metabolism by Serotonin[END_REF], but our understanding of the role of serotonergic receptors in bone mineralization is still scarce. Notably, what are the signaling pathways and targets governed by 5-HT receptors to orchestrate bone homeostasis? Tackling this issue in vivo or with primary cultures is rendered difficult by the heterogeneity of cell types and the multiplicity of signals at play. Clonal osteogenic cell lines with homogenous phenotypic properties permit to overcome these limitations.

The C1 mesoblastic cell line selectively expresses 5-HT2BRs during osteogenic differentiation

The C1 tripotential mesoblastic cell line maintains a stable undifferentiated phenotype even in 10% serum. Upon addition of appropriate inducers, C1 cells engage at high frequency (>90%), in a mutually exclusive and synchronous manner, into the osteogenic, chondrogenic or adipogenic programs [START_REF] Poliard | Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways[END_REF]. For osteogenic differentiation, C1 cells must first form threedimensional nodules mimicking the in vivo mesodermal stem cell condensation required for skeleton development and bone repair. After addition of ß-glycerophosphate (ßGP) and ascorbate, C1 cells reach terminal differentiation within 12 days (Fig. 5). Till day 4 of the osteogenic program, close to 100% C1 aggregates direct the deposition of a profuse type 1 collagen extracellular matrix, consistent with what observed during intramembranous bone formation in vivo. At day 7, hydroxyapatite crystals start to be formed and deposited on collagen fibrils surrounding C1 osteoblasts leading to progressive mineralization of the extracellular matrix. At day 12, C1 cells convert into mature osteocyte-like cells embedded in a calcified matrix and cease to divide [START_REF] Poliard | Lineage-dependent collagen expression and assembly during osteogenic or chondrogenic differentiation of a mesoblastic cell line[END_REF].

Pharmacological studies led to show that a functional 5-HT2BR is implemented at day 5 of the osteogenic program, just before matrix mineralization [START_REF] Locker | Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation[END_REF][START_REF] Baudry | Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C[END_REF]. Of note, the mRNAs encoding 5-HT2BR are already present at the stem cell state in condensing C1 mesoblastic cells and their level do not vary during the 12 day differentiation time course. The selective expression of 5-HT2BR is an intrinsic event of the C1 osteogenic program and requires posttranscriptional regulation mechanisms that have yet-to-be identified. 5-HT2BR signaling optimizes matrix mineralization since blockade of 5-HT2BR by ritanserin from day 5 reduces by 40% Ca 2+ incorporation within the bone matrix [START_REF] Locker | Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation[END_REF], in agreement with the osteopenic phenotype of 5-HT2BR -/-mice [START_REF] Collet | The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation[END_REF].

5-HT2BRs are coupled to NO and eicosanoid productions along C1 osteogenic program

From day 5 to terminal differentiation, 5-HT2BRs exhibit an agonist dependent-and independent-coupling to the nitric oxide (NO) signaling pathway (Fig. 5). The critical role of NO radical in the dynamic process of bone remodeling has been described for more than a decade [START_REF] Klein-Nulend | Nitric oxide signaling in mechanical adaptation of bone[END_REF]. In bone, the endothelial isoform of nitric oxide synthase (eNOS) is constitutively expressed while the inductible NOS (iNOS) is only activated in inflammatory conditions. eNOS knockout mice display osteoporosis, which sustains an anabolic role of NO in bone metabolism [START_REF] Armour | Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase[END_REF][START_REF] Aguirre | Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity[END_REF]. Resorptive effects of NO on the skeleton have also been reported [START_REF] Van't Hof | Nitric oxide and bone[END_REF].

The importance of a tight regulation of NO production for bone homeostasis is further supported by bone defects in patients suffering of inflammatory diseases like rheumatoid arthritis, associated with strong NO production [START_REF] Van't Hof | Nitric oxide and bone[END_REF].

In addition to its coupling to NOS, the 5-HT2BRs are coupled to the phospholipase A2 (PLA2)/acid arachidonic (AA) signaling pathway. 5-HT2BR/PLA2 coupling contributes to osteogenic differentiation by controlling the cyclooxygenase (COX) and lipoxygenase (LOX) pathways in a stepwise manner. During the initial phase of mineralization process, i.e. from day 5 to day 10, 5-HT2BRs control a COX-dependent prostaglandin PGE2 synthesis (Fig. 5).

At day 10, when osteoblasts are converted into osteocytes, COX activity switches off. 5-HT2BR/PLA2-derived AA becomes substrates for LOX enzymes and osteocytes produce the leukotriene LTB4. As for NO, both PGE2 and LTB4 eicosanoids are known to be involved in the regulation of bone metabolism by exercising either bone-forming and/or bone-resorbing actions [START_REF] Hikiji | The roles of prostanoids, leukotrienes, and platelet-activating factor in bone metabolism and disease[END_REF]. Of note, COX knockout mice display reduced bone formation [START_REF] Zhang | Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair[END_REF].

NO/PGE2/LTB4 can either act as intra-and/or inter-cellular second messengers. This raises the question of the nature of the osteoblast specific-functions governed by these effectors in bone homeostasis.

5-HT2BRs govern bone mineralization through control of TNAP activity via eicosanoids and phosphatidylinositol-specific phospholipase C

Although not an osteoblast specific enzyme, the tissue-non specific alkaline phosphatase (TNAP) is a key actor in bone mineralization that governs the inorganic phosphate (Pi)/pyrophosphate (PPi) ions ratio. The essential role of this glycosylphosphatidylinositol (GPI)-anchored enzyme in mineral crystal formation has been evidenced in human patients with TNAP loss-of-function mutation [START_REF] Whyte | Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization[END_REF] or TNAP knockout mice [START_REF] Narisawa | Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia[END_REF], which are defective for bone and teeth mineralization. Furthermore, transgenic mice, which ectopically express TNAP specifically in the dermis enriched in type 1 collagen, develop pathological skin calcification [START_REF] Murshed | Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone[END_REF]. The dynamics of the C1 cell differentiation allowed to show that TNAP activity is fully controlled by 5-HT2BR-dependent PLA2 signaling pathway (Fig. 5). As for 5-HT2BR transcripts, TNAP mRNAs are dormant at the stem cell stage and their levels remain constant along the osteogenic program. To date, the signals that turn on 5-HT2BR and TNAP translation at day 5 are still missing. While expressed, TNAP enzyme remains inactive until day 7. Its activation depends on post-translational modifications elicited by 5-HT2BR signaling. 5-HT2BR coupling to PLA2 stimulates phosphatidylinositol-specific phospholipase C (PIPLC) activity, which cleaves the GPI anchor of TNAP and thereby unlocks TNAP activity. Such a functional link between 5-HT2BR, PIPLC and TNAP has also been evidenced in primary calvarial cells [START_REF] Baudry | Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C[END_REF]. 5-HT2BR -/-osteoblasts also revealed a hitherto-unkown interaction between 5-HT2BRs and the nuclear PPARβ/δ receptors via prostacyclins [START_REF] Chabbi-Achengli | Serotonin 2B receptor (5-HT2B R) signals through prostacyclin and PPAR-ss/delta in osteoblasts[END_REF].

While the functional impact of 5-HT in the skeleton is still debated, the 5-HT2BR plays a key role in mineralization processes.

Conclusions and future directions

This review reports some recent aspects of the implication of 5-HT and serotonergic receptors in mineralized tissues homeostasis. As concern dental tissues, tooth decay is one of the widest spread infections around the world (almost 100%). Root canal sealers used for endodontic treatments aim to eradicate bacterial infection and protect tooth against re-contaminations. These filling materials are not designed to preserve and/or mobilize resident pulpal stem cells for tooth repair. The identification of a dialogue mediated by 5-HT and DA between blood platelets and odontogenic stem cells provides a rational for the development of novel therapeutic strategies targeting pulpal stem cells and therefore enhancing natural tooth repair.

Regarding the skeleton, the integrity of this dynamic tissue is ensured by a constant remodeling through the coordinated action of bone-forming osteoblasts and bone-resorbing osteoclasts. Although, it is becoming clear that the 5-HT system contributes to bone metabolism, the action of 5-HT on skeleton homeostasis is still controversed. A genetic approach developed by Yadav et al. revealed a low bone mass phenotype in mice depleted for TPH2, the brain TPH isoform. These data suggest that brain-derived 5-HT contributes to skeleton homeostasis through the hypothalamus by suppression of sympathetic tone [START_REF] Yadav | A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure[END_REF].

Yadav et al. have also shown that gut-specific TPH1 conditional knockout mice display increased bone formation and high bone mass suggesting an inhibiting effect of circulating 5-HT on bone formation through 5-HT1BRs [START_REF] Yadav | Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum[END_REF]. However, others studies reported that adult TPH1 -/-mice had no change in bone mass density [76,[START_REF] Chabbi-Achengli | Decreased osteoclastogenesis in serotonin-deficient mice[END_REF]. Further analyses indicate altered bone resorption in TPH1 -/-mice due to osteoclastic differentiation defect [START_REF] Chabbi-Achengli | Decreased osteoclastogenesis in serotonin-deficient mice[END_REF]. Additional works are thus required to better characterize the role of 5-HT in bone metabolism taking into account the relative affinity of serotonergic receptors for 5-HT and the diversity of their couplings. Among the 5-HT receptors, the 5-HT2BR exerts a pivotal role in mineralization processes by controlling TNAP activity through its PLA2/eicosanoids/PIPLC coupling.

Several lines of evidence support the notion that physiological and pathological mineralizations mobilize similar pathways [START_REF] Murshed | Molecular determinants of extracellular matrix mineralization in bone and blood vessels[END_REF]. Currently, molecular mechanisms involved in ectopic mineralization of soft tissues are unsolved. Of note, deregulations of PGE2 and LTB4 levels have been associated with pathological situations such as bone fracture, rheumatoid arthritis, osteoarthritis, osteoporosis [START_REF] Hikiji | The roles of prostanoids, leukotrienes, and platelet-activating factor in bone metabolism and disease[END_REF]. Whether alteration of 5-HT2BR signaling would drive ectopic pathological calcifications deserves further investigations.

Contents 1 . Introduction 2 .

 12 Serotonin in tooth repair: a dialogue between odontogenic stem cells and platelets 2.1. Isolation of clonal pulpal stem cell lines 2.2. Odontogenic pulpal stem cells display a mixed serotonergic/dopaminergic phenotype 2.3. Functional serotonergic 5-HT1D,2B,7 and dopaminergic D1,3 autoreceptors are expressed by odontogenic stem cells 2.4. Serotonergic and dopaminergic receptors relay the action of platelets-derived 5-HT and DA to mobilize pulpal stem cells for tooth repair 3. Bone mineralization: a major role of 5-HT2BR signaling 3.1. The C1 mesoblastic cell line selectively expresses 5-HT2BRs during osteogenic differentiation 3.2. 5-HT2BRs are coupled to NO and eicosanoid productions along C1 osteogenic program 3.3. 5-HT2BRs govern bone mineralization through control of TNAP activity via eicosanoids and phosphatidylinositol-specific phospholipase C 4. Conclusions and future directions

Fig. 1 .

 1 Fig. 1. Schematic representation of the migration of neural crest cells from the rostral hindbrain and caudal midbrain to reach the first branchial arch at the origin of teeth and mandibular (mb) and maxillary (mx) bones.

Fig. 2 .

 2 Fig. 2. Enamel structural defects in molar of 5-HT2BR knockout mice observed by microcomputed tomography and electron microscopy analysis.

Fig. 3 .

 3 Fig. 3. 5-HT and DA receptors present at the surface of odontogenic stem cells and their couplings.

Fig. 4 .

 4 Fig. 4. 5-HT and DA co-released by activated platelets upon tooth injury are "warning" signals that mobilize pulpal stem cells for tooth repair through 5-HT/DA receptors.

Fig. 5 .

 5 Fig. 5. Diagram summarizing the regulation of TNAP activity by 5-HT2BR through its PLA2/eicosanoids/PIPLC coupling during osteogenic differentiation.

Acknowledgments

This work was supported by Inserm.

Conflicts of interest

The authors declare no conflict of interest.