[François Gros, my boss]. / François Gros, mon patron.

This article shares the author's memories of his collaborations with François Gros when the latter was Professor at the University of Paris 7, then Director of the Institut Pasteur, then President of the Pasteur-Weizmann Council. He underlines the major contributions of "his boss" in the exercise of these functions.

Cet article fait part de souvenirs de l'auteur relatifs à ses collaborations avec François Gros lorsque ce dernier était professeur à l'université Paris 7, puis directeur de l'Institut Pasteur, puis président du Conseil Pasteur-Weizmann. Il souligne les contributions majeures de «  son patron  ¼ dans l'exercice de ces fonctions.

Irma Thesleff-Orthodontist who became a developmental biologist.

Irma Thesleff is one of the leading scholars in developmental biology. She and her research group have clarified the mysteries of tooth development. For several decades, her research of very high quality has focused on morphogenesis and resulted in an understanding of the highly complex signaling networks. Irma Thesleff has been duly recognized both in the domestic and international context. Her research continues despite her retirement.

Stem Cell and Oxidative Stress-Inflammation Cycle.

Under a variety of physical and experimental settings, stem cells are able to self-renew and differentiate into specialized adult cells. MSCs (mesenchymal stromal/stem cells) are multipotent stem cells present in a wide range of fetal, embryonic, and adult tissues. They are the progenitors of a variety of specialized cells and are considered crucial tools in tissue engineering. MSCs, derived from various tissues, including cord blood, placenta, bone marrow, and dental tissues, have been extensively examined in tissue repair, immune modulation, etc. Increasing the vitality of MSCs and restoring cellular mechanisms are important factors in treatment success. Oxidative stress harms cellular molecules such as DNA, proteins, and lipids due to the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cells and tissues or insufficiency of antioxidant systems that can inactivate them. Oxidative stress has a close link with inflammation as a pathophysiological process. ROS can mediate the expression of proinflammatory genes via intracellular signaling pathways and initiate the chronic inflammatory state. At the same time, inflammatory cells secrete a large number of reactive species that cause increased oxidative stress at sites of inflammation. In inflammatory diseases, the differentiation of stem cells and the regenerative and wound healing process can be affected differently by the increase of oxidative stress. Recent studies have indicated that dental pulp stem cells (DPSCs), as a resource of adult stem cells, are an attractive option for cell therapy in diseases such as neurological diseases, diabetes, cardiological diseases, etc., as well as its treatment potential in pulp inflammation. The future of oxidative stressinflammation cycle and/or ageing therapies involves the selective elimination of senescent cells, also known as senolysis, which prevents various age-related diseases. Most pathologies are implicated on the effects of ageing without exerting undesirable side effects.

Adaptive amelogenesis during unimpeded eruption of rat mandibular incisor.

The aim of the study was to see whether the length of the enamel secretion zone in unimpeded rat incisors, measured precisely, is in agreement with the observed decrease in enamel thickness. Unimpeded eruption of mandibular incisors of five experimental and two control rats was induced by cutting off the erupted part of the incisors three times per week for 5 weeks. The length of the zone of enamel secretion in unimpeded and impeded control incisors was measured on longitudinal and serial transverse histological sections of fixed, demineralised and embedded hemimandibles. Impeded contralateral incisors were also included in the study. The length of the zone of enamel secretion in unimpeded incisors showed an increase to 8,398 ± 558 µm, that is 161% of the length in control incisors (5,213 ± 95 µm). The contralateral incisor showed a reduction in eruption rate, in length of the secretion zone, and the whole tooth was shifted somewhat apically. The measured length of the secretion zone is in agreement with the observed thickness of enamel (98 µm) in unimpeded incisors. The reduced eruption rate and the apical shift of the contralateral incisor are probably due to an increased occlusal load.

Adipose Tissue as a Strategic Source of Mesenchymal Stem Cells in Bone Regeneration: A Topical Review on the Most Promising Craniomaxillofacial Applications.

Bone regeneration in craniomaxillofacial surgery represents an issue that involves both surgical and aesthetic aspects. The most recent studies on bone tissue engineering involving adipose-derived stromal/stem cells (ASCs) have clearly demonstrated that such cells can play a crucial role in the treatment of craniomaxillofacial defects, given their strong commitment towards the osteogenic phenotype. A deeper knowledge of the molecular mechanisms underlying ASCs is crucial for a correct understanding of the potentialities of ASCs-based therapies in the most complex maxillofacial applications. In this topical review, we analyzed the molecular mechanisms of ASCs related to their support toward angiogenesis and osteogenesis, during bone regeneration. Moreover, we analyzed both case reports and clinical trials reporting the most promising clinical applications of ASCs in the treatment of craniomaxillofacial defects. Our study aimed to report the main molecular and clinical features shown by ASCs, used as a therapeutic support in bone engineering, as compared to the use of conventional autologous and allogeneic bone grafts.

Thank you letters from patients in an intensive care unit: From the expression of gratitude to an applied ethic of care.

INTRODUCTION: Patients' perception of an intensive care unit stay can lead to a better understanding of the expectations and needs of patients hospitalised in intensive care so that care for critically ill patients can be adapted and improved. Thank you letters are sources of original information which come directly and spontaneously from patients. OBJECTIVE: The objective of this study was to analyse the thank you letters from patients who required intensive care treatment and to identify messages that could be intended for the intensive care unit team. DESIGN: We conducted a qualitative study according to a thematic analysis. The body of research consisted of 17 letters from patients hospitalised in intensive care unit. SETTING: The study took place in the medical-surgical intensive care unit of a French general hospital. The intensive care unit is made up of 16 resuscitation beds and four continuous monitoring beds. FINDINGS: Two main themes emerged: (i) expression of gratitude through a description of the caregivers' behaviour (humanity and professionalism) and recognition for surviving (ii) the narrative of the intensive care unit experience. CONCLUSION: Thank you letters give a rich insight into how the patients perceived their stay in intensive care unit. Letters from patients give direct feedback on the quality of care provided, contribute to give meaning to work and raise the question of what the core values of care should be for all concerned in the healthcare providers-patients relationship.

Is Pulp Inflammation a Prerequisite for Pulp Healing and Regeneration?

The importance of inflammation has been underestimated in pulpal healing, and in the past, it has been considered only as an undesirable effect. Associated with moderate inflammation, necrosis includes pyroptosis, apoptosis, and nemosis. There are now evidences that inflammation is a prerequisite for pulp healing, with series of events ahead of regeneration. Immunocompetent cells are recruited in the apical part. They slide along the root and migrate toward the crown. Due to the high alkalinity of the capping agent, pulp cells display mild inflammation, proliferate, and increase in number and size and initiate mineralization. Pulp fibroblasts become odontoblast-like cells producing type I collagen, alkaline phosphatase, and SPARC/osteonectin. Molecules of the SIBLING family, matrix metalloproteinases, and vascular and nerve mediators are also implicated in the formation of a reparative dentinal bridge, osteo/orthodentin closing the pulp exposure. Beneath a calciotraumatic line, a thin layer identified as reactionary dentin underlines the periphery of the pulp chamber. Inflammatory and/or noninflammatory processes contribute to produce a reparative dentinal bridge closing the pulp exposure, with minute canaliculi and large tunnel defects. Depending on the form and severity of the inflammatory and noninflammatory processes, and according to the capping agent, pulp reactions are induced specifically.

In vitro bioactivity of Bioroot™ RCS, via A4 mouse pulpal stem cells.

OBJECTIVE: To evaluate the biocompatibility and osteoinductive properties of Bioroot™ RCS (BR, Septodont, France) compared to Kerr's Pulp Canal Sealer™ (PCS, Kerr, Italy) using the mouse pulp-derived stem cell line A4, which have an osteo/odontogenic potential in vitro and contribute to efficient bone repair in vivo. METHODS: A4 cells were cultured at the stem cell stage in the presence of solid disks of BR or PCS, whereas untreated A4 cells were used as control. After 3, 7, 10 days of direct contact with the sealers, cell viability was quantified using Trypan Blue exclusion assay. Immunolabelings were performed to assess the expression of odontoblast markers i.e. type 1 collagen, DMP1 or BSP. Finally, sealer-treated cells were induced toward osteo/odontogenic differentiation to assess the impact of the sealers on mineralization by Von Kossa staining. Statistical significance was evaluated by one-way analysis of variance and t-test (p<0.05). RESULTS: BR did not alter the viability and morphology of A4 pulpal cells compared to control group (p>0.05); however, living cell percentage of PCS was significantly lower compared to control and BR groups (p<0.05). BR preserved the intrinsic ability of A4 cells to express type 1 collagen, DMP1 or BSP at the stem cell stage. It did not alter the integrity of collagen fibers surrounding the cells and promoted overexpression of BSP and DMP1 at the cell surface. In contrast to PCS, BR did not compromise the mineralization potential of pulpal A4 stem cells. SIGNIFICANCE: Bioroot™ RCS was not as cytotoxic as PCS. It did not recruit the pulpal stem cells toward differentiation but preserve their osteo-odontogenic intrinsic properties. Bioroot™ RCS might provide more suitable environment to induce stem cells for hard tissue deposition.

Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets.

Characterizing stem cell intrinsic functions is an ongoing challenge for cell therapies. Here, we report that two independent A4 and H8 stem cell lines isolated from mouse molar pulp display the overall functions of bioaminergic cells. Both clones produce neurotrophins and synthesize, catabolize, store, and transport serotonin (5-hydroxytryptamine [5-HT]) and dopamine (DA). They express 5-HT1D,2B,7 and D1,3 autoreceptors, which render pulpal stem cells competent to respond to circulating 5-HT and DA. We show that injury-activated platelets are the source of systemic 5-HT and DA necessary for dental repair since natural dentin reparation is impaired in two rat models with monoamine storage-deficient blood platelets. Moreover, selective inhibition of either D1, D3, 5-HT2B, or 5-HT7 receptor within the pulp of wild-type rat molars after lesion alters the reparative process. Altogether our data argue that 5-HT and DA coreleased by pulp injury-activated platelets are critical for stem cell-mediated dental repair through 5-HT and DA receptor signalings.

Distribution of the amelogenin protein in developing, injured and carious human teeth.

Amelogenin is the major enamel matrix protein with key roles in amelogenesis. Although for many decades amelogenin was considered to be exclusively expressed by ameloblasts, more recent studies have shown that amelogenin is also expressed in other dental and no-dental cells. However, amelogenin expression in human tissues remains unclear. Here, we show that amelogenin protein is not only expressed during human embryonic development but also in pathological conditions such as carious lesions and injuries after dental cavity preparation. In developing embryonic teeth, amelogenin stage-specific expression is found in all dental epithelia cell populations but with different intensities. In the different layers of enamel matrix, waves of positive vs. negative immunostaining for amelogenin are detected suggesting that the secretion of amelogenin protein is orchestrated by a biological clock. Amelogenin is also expressed transiently in differentiating odontoblasts during predentin formation, but was absent in mature functional odontoblasts. In intact adult teeth, amelogenin was not present in dental pulp, odontoblasts, and dentin. However, in injured and carious adult human teeth amelogenin is strongly re-expressed in newly differentiated odontoblasts and is distributed in the dentinal tubuli under the lesion site. In an in vitro culture system, amelogenin is expressed preferentially in human dental pulp cells that start differentiating into odontoblast-like cells and form mineralization nodules. These data suggest that amelogenin plays important roles not only during cytodifferentiation, but also during tooth repair processes in humans.

Comparative studies between mice molars and incisors are required to draw an overview of enamel structural complexity.

In the field of dentistry, the murine incisor has long been considered as an outstanding model to study amelogenesis. However, it clearly appears that enamel from wild type mouse incisors and molars presents several structural differences. In incisor, exclusively radial enamel is observed. In molars, enamel displays a high level of complexity since the inner part is lamellar whereas the outer enamel shows radial and tangential structures. Recently, the serotonin 2B receptor (5-HT2BR) was shown to be involved in ameloblast function and enamel mineralization. The incisors from 5HT2BR knockout (KO) mice exhibit mineralization defects mostly in the outer maturation zone and porous matrix network in the inner zone. In the molars, the mutation affects both secretory and maturation stages of amelogenesis since pronounced alterations concern overall enamel structures. Molars from 5HT2BR KO mice display reduction in enamel thickness, alterations of inner enamel architecture including defects in Hunter-Schreger Bands arrangements, and altered maturation of the outer radial enamel. Differences of enamel structure were also observed between incisor and molar from other KO mice depleted for genes encoding enamel extracellular matrix proteins. Thus, upon mutation, enamel analysis based exclusively on incisor defects would be biased. In view of the functional relationship between enamel structure and tooth morphogenesis, identification of molecular actors involved in amelogenesis requires comparative studies between mice molars and incisors.

Plithotaxis, a collective cell migration, regulates the sliding of proliferating pulp cells located in the apical niche.

Using the proliferating cell nuclear antigen (PCNA) immunostaining, we previously identified, after pulp exposure, three zones of proliferating cells in the rat molar pulp. Zones I and II were in the crown near the pulp. Zone III was near the apex revealing a recruitment of mitotic cells at distance from the lesion. To gain further insight into the spatio-temporal evolution of proliferating pulp cells of zone III, we performed a longitudinal study of PCNA staining in rat molar mesial root at 3, 8, and 15 d after pulp exposure associated to implantation of unloaded or amelogenin loaded agarose beads. At day 3 after implantation, PCNA-positive cells were located in the central part of the radicular pulp. At day 8, PCNA-labeled cells were aligned in the lateral part of the pulp beneath the odontoblast/sub-odontoblast layer. At day 15, PCNA labeling became undetectable in the root and was located in the coronal pulp. These results suggest that after pulp exposure, PCNA-positive cells may migrate from the central part of the radicular pulp to the sub-odontoblast cell layer and then from the apical root to the crown. Electron microscopy and immunostaining analysis showed that pulpal cells were linked by desmosome-like and gap-junctions. Extracellular matrix was composed of thin collagen fibrils associated with glycosaminoglycans favoring cell mobility. These data suggest that the syncytium-like structure formed by pulp radicular cells may be a pre-request for plithotaxis, a collective cell migration process. This emergent mechanism may govern pulp healing and regeneration after dental lesion.

Pulp stem cells: implication in reparative dentin formation.

Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

Deletion of serotonin 2B receptor provokes structural alterations of mouse dental tissues.

Rampant caries and periodontal diseases occur in patients treated with antidepressants such as serotonin reuptake inhibitors (SRIs; e.g., Prozac) which target the serotonin transporter (SERT). As the serotonin 2B receptor (5HT2BR) regulates SERT functionality and capacity to recognize SRIs, we investigated the potential role of 5HT2BR on dental tissues by exploiting 5HT2BR knockout (KO) mice. Compared to wild-type (WT) mice, several structural differences were identified in the teeth of KO mice. In the molar of KO mice, rod curvatures and twisting were altered compared to WT mice, suggesting involvement of 5HT2BR at early stages of enamel formation. The volume of the KO enamel layer was also reduced, and larger porosities were observed in the prismatic enamel, with smaller crystallite thickness. Crystallite pattern disorganization and occlusal abrasion were enhanced in female KO mice, indicating a sexual dimorphism. In the incisor, no difference was detected in the width of the enamel layer between KO and WT mice; however, enamel maturation differed in absence of 5HT2BR. Specifically, the outer aprismatic enamel border was 1.5- to 2-fold larger in KO compared to WT mice, together with a decreased etching pattern. Finally, although no noticeable difference was observed in dentin, the micro-CT three-dimensional pulp reconstruction evidenced a decrease in both length and width of dentin formation in the root canals of the KO versus WT mice. These data provide evidence that 5HT2BR-mediated signaling pathways are involved in enamel formation and dentinogenesis.

TGF-ß regulates enamel mineralization and maturation through KLK4 expression.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.

Amelogenin exons 8 and 9 encoded peptide enhances leucine rich amelogenin peptide mediated dental pulp repair.

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair.

Mechanism of heparin acceleration of tissue inhibitor of metalloproteases-1 (TIMP-1) degradation by the human neutrophil elastase.

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s⁻¹) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s⁻¹). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k₋1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.