Bioengineered tooth emulation systems for regenerative and pharmacological purposes.

Genetic conditions, traumatic injuries, carious lesions and periodontal diseases are all responsible for dental pathologies. The current clinical approaches are based on the substitution of damaged dental tissues with inert materials, which, however, do not ensure full physiological recovery of the teeth. Different populations of dental mesenchymal stem cells have been isolated from dental tissues and several attempts have already been made at using these stem cells for the regeneration of human dental tissues. Despite encouraging progresses, dental regenerative therapies are very far from any clinical applications. This is tightly connected with the absence of proper platforms that would model and faithfully mimic human dental tissues in their complexity. Therefore, in the last decades, many efforts have been dedicated for the development of innovative systems capable of emulating human tooth physiology in vitro. This review focuses on the use of in vitro culture systems, such as bioreactors and "organ-on-a-chip" microfluidic devices, for the modelling of human dental tissues and their potential use for dental regeneration and drug testing.

The effect of extracellular acidosis on the behaviour of mesenchymal stem cells in vitro.

The stem cell fraction of a cell population is finely tuned by stimuli from the external microenvironment. Among these stimuli, a decrease of extracellular pH (pHe) may occur in a variety of physiological and pathological conditions, including hypoxia and inflammation. In this study, by using bone marrow stem cells and dental pulp stem cells, we provided evidence that extracellular acidosis endows the maintenance of stemness in mesenchymal cells. Indeed, continuous exposure for 21 d to low pHe (6.5-6.8) conditions impaired the osteogenic differentiation of both cell types. Moreover, the exposure to low pHe, for 1 and up to 7 d, induced the expression of stemness-related genes and proteins, drove cells to reside in the quiescent G0 alert state and enhanced their ability to form floating spheres. The pre-conditioning with extracellular acidosis for 7 d did not affect the differentiation potential of dental pulp stem cells since, when the cells were cultured again at physiological pHe, their multilineage potential was almost unmodified. Our data provided evidence of the role of extracellular acidosis as a modulator of the stemness of mesenchymal cells. This condition is commonly found both in systemic and local bone conditions, hence underlining the relevance of this phenomenon for a better comprehension of bone healing and regeneration.

Investigation of orofacial stem cell niches and their innervation through microfluidic devices.

Stem cell-based mediated therapies represent very promising approaches for tissue regeneration and are already applied with success in clinics. These therapeutic approaches consist of the in vitro manipulation of stem cells and their consequent administration to patients as living and dynamic biological agents. Nevertheless, the deregulation of stem cells function might result in the generation of pathologies such as tumours or accelerated senescence. Moreover, different stem cells sources are needed for regeneration of specific tissues. It is thus fundamental to understand the mechanisms regulating the physiology of stem cells. Microfluidic technology can be used to mimic in vivo scenarios and allow the study of stem cell physiology at both single cell and whole stem cell niche levels.This review focuses on the potential sources of stem and progenitor cells for orofacial regeneration and the use of microfluidic technologies for the study of stem cells behaviour and stem cell niches, in the light of regenerative medicine.

Fighting for territories: time-lapse analysis of dental pulp and dental follicle stem cells in co-culture reveals specific migratory capabilities.

Stem cell migration is a critical step during the repair of damaged tissues. In order to achieve appropriate cell-based therapies for tooth and periodontal ligament repair it is necessary first to understand the dynamics of tissue-specific stem cell populations such as dental pulp stem cells (DPSC) and dental follicle stem cells (DFSC). Using time-lapse imaging, we analysed migratory and proliferative capabilities of these two human stem cell lines in vitro. When cultured alone, both DPSC and DFSC exhibited low and irregular migration profiles. In co-cultures, DFSC, but not DPSC, spectacularly increased their migration activity and velocity. DFSC rapidly surrounded the DPSC, thus resembling the in vivo developmental process, where follicle cells encircle both dental epithelium and pulp. Cell morphology was dependent on the culture conditions (mono-culture or co-culture) and changed over time. Regulatory genes involved in dental cell migration and differentiation such as TWIST1, MSX1, RUNX2, SFRP1 and ADAM28, were also evaluated in co-cultures. MSX1 up-regulation indicates that DPSC and DFSC retain their odontogenic potential. However, DPSC lose their capacity to differentiate into odontoblasts in the presence of DFSC, as suggested by RUNX2 up-regulation and TWIST1 down-regulation. In contrast, the unchanged levels of SFRP1 expression suggest that DFSC retain their potential to form periodontal tissues even in the presence of DPSC. These findings demonstrate that stem cells behave differently according to their environment, retain their genetic memory, and compete with each other to acquire the appropriate territory. Understanding the mechanisms involved in stem cell migration may lead to new therapeutic approaches for tooth repair.

NANOG priming before full reprogramming may generate germ cell tumours.

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Genetic basis for tooth malformations: from mice to men and back again.

Teeth arise from sequential and reciprocal interactions between the oral epithelium and the cranial neural crest-derived mesenchyme. Their formation involves a precisely orchestrated series of molecular and morphogenetic events. Numerous regulatory genes that have been primarily found in organisms such as Drosophila, zebrafish, xenopus and mouse are associated with all stages of tooth formation (patterning, morphogenesis, cytodifferentiation and mineralization). Most of these genes belong to evolutionary conserved signaling pathways that regulate communication between epithelium and mesenchyme during embryonic development. These signaling molecules together with specific transcription factors constitute a unique molecular imprint for odontogenesis and contribute to the generation of teeth with various and function-specific shapes. Mutations in several genes involved in tooth formation cause developmental absence and/or defects of teeth in mice. In humans, the odontogenic molecular program is not as well known as that of mice. However, some insight can be obtained from the study of mutations in regulatory genes, which lead to tooth agenesis and/or the formation of defective dental tissues.

Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials.

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.

Stem cells for tooth engineering.

Tooth development results from sequential and reciprocal interactions between the oral epithelium and the underlying neural crest-derived mesenchyme. The generation of dental structures and/or entire teeth in the laboratory depends upon the manipulation of stem cells and requires a synergy of all cellular and molecular events that finally lead to the formation of tooth-specific hard tissues, dentin and enamel. Although mesenchymal stem cells from different origins have been extensively studied in their capacity to form dentin in vitro, information is not yet available concerning the use of epithelial stem cells. The odontogenic potential resides in the oral epithelium and thus epithelial stem cells are necessary for both the initiation of tooth formation and enamel matrix production. This review focuses on the different sources of stem cells that have been used for making teeth in vitro and their relative efficiency. Embryonic, post-natal or even adult stem cells were assessed and proved to possess an enormous regenerative potential, but their application in dental practice is still problematic and limited due to various parameters that are not yet under control such as the high risk of rejection, cell behaviour, long tooth eruption period, appropriate crown morphology and suitable colour. Nevertheless, the development of biological approaches for dental reconstruction using stem cells is promising and remains one of the greatest challenges in the dental field for the years to come.

Two- versus three-dimensional in vitro differentiation of human pulp cells into odontoblastic cells.

The spatial organization of the pulp cells may modify the cytodifferentiation process. The purpose of this study was to compare the two- versus three-dimensional cell culture systems for differentiation of human odontoblastic cells in vitro. Pulpal cores from freshly extracted human third molars were cultured in vitro in a perfusion device on two types of membranes: polyester membrane (two-dimensional [2D] cell culture) and nylon mesh (three-dimensional [3D] cell culture). The cells were incubated with minimum essential medium containing (a) substitute serum, (b) 10% fetal calf serum (FCS), (c) 10% fetal calf serum + 2 mM beta-glycerophosphate (beta GP), and (d) 10% fetal calf serum + transforming growth factor (TGF) beta 1. Immunohistochemistry was used to evaluate the expression of collagen I, osteonectin, and nestin. Small differences were observed between 2D and 3D cell culture systems. This was particularly evident in the 10% FCS group. beta-Glycerophosphate in the 3D system seems to stimulate the osteogenic cell phenotype, as a considerable induction of osteonectin is observed.

Expression of Deltex1 during mouse embryogenesis: comparison with Notch1, 2 and 3 expression.

The Notch signalling pathway defines a phylogenetically conserved cell-cell communication process that enables cell-fate specification in multicellular organisms. Deltex is a component of the Notch signalling network that physically interacts with the ankyrin repeats of Notch. Here, we report on the expression pattern of the Deltex1 gene during mouse embryonic development and, furthermore, we compare its expression with that of the Notch1, 2 and 3 genes. Complementary and combinatorial expression patterns between Deltex1 and the three Notch genes were observed throughout embryogenesis since Deltex1 expression was related either to cytodifferentiation (i.e. neuronal tissues) or to cell proliferation events (i.e. eye, vascular structures, hematopoiesis).

Molecular aspects of tooth pathogenesis and repair: in vivo and in vitro models.

Several growth factors and extracellular matrix molecules, which are expressed during embryonic tooth development, are re-expressed in dental tissues under pathological conditions. Pathological conditions such as caries lesions and dental injuries are often lethal to the odontoblasts, which are then replaced by other pulp cells. These cells are able to differentiate into odontoblast-like cells and produce a reparative dentin. Here we demonstrate the in vivo distribution of several molecules in human permanent teeth under normal and pathological conditions. The intermediate filament protein nestin, which is a marker of young odontoblasts, is absent from old permanent teeth. Similarly, the Notch protein, which is involved in cell fate specification and is localized in the sub-odontoblastic cell layer during odontogenesis, is not detected in adult dental tissues. In carious and injured teeth, nestin is expressed in a selective manner in odontoblasts surrounding the injury site, while Notch is expressed in the sub-odontoblastic layer of cells. We reproduced this physiological event in an in vitro culture system. Pulp cells cultured in the presence of beta-glycerophosphate formed mineralization nodules. As odontoblasts, pulp cells contributing to the nodule formation express type I collagen, osteonectin, dentin sialophosphoprotein, and nestin. In this in vitro assay system, nestin is up-regulated after local application of Bone Morphogenetic Protein 2 and 4. Fourier transform infrared microspectroscopy showed that both the organic and the mineral compositions of the nodules have the characteristics of human dentin and differ from those of enamel and bone. These findings show that both the molecular and the mineral characteristics of the human dentin matrix are respected in the in vitro culture conditions.

Expression pattern of notch1, 2 and 3 and Jagged1 and 2 in lymphoid and stromal thymus components: distinct ligand-receptor interactions in intrathymic T cell development.

The suggested role of Notch1 or its mutants in thymocyte differentiation and T cell tumorigenesis raises the question of how the different members of the Notch family influence distinct steps in T cell development and the role played by Notch ligands in the thymus. We report here that different Notch receptor-ligand partnerships may occur inside the thymus, as we observed differential expression of Notch1, 2 and 3 receptors, their ligands Jagged1 and 2, and downstream intracellular effectors hairy and Enhancer of Split homolog 1 (HES-1) and hairy and Enhancer of Split homolog 5 (HES-5), depending on ontogenetic stage and thymic cell populations. Indeed, while Jagged2 is expressed in both stromal cells and thymocytes, Jagged1 expression is restricted to stromal cells. Moreover, a differential distribution of Notch3, with respect to Notch1, was observed in distinct age-related thymocyte subsets. Finally, Notch3 was preferentially up-regulated in thymocytes, following the induction of their differentiation by interaction with thymic epithelial cells expressing the cognate Jagged1 and 2 ligands, suggesting that, besides Notch1, Notch3 may also be involved in distinct steps of thymocyte development. Our results suggest that the Notch signaling pathway is involved in a complex interplay of T cell developmental stages, as a consequence of the heterogeneity and specific expression of members of the Notch receptor family and their cognate ligands, in distinct thymic cell compartments.

Reactivation of Delta-Notch signaling after injury: complementary expression patterns of ligand and receptor in dental pulp.

The evolutionarily conserved Notch-mediated intercellular signaling pathway is essential for proper embryonic development of many tissues and organs. Recent data suggest that Notch receptors and their membrane-bound ligands Delta and Serrate are involved in both patterning and cell fate determination during odontogenesis. It remains, however, uncertain if Notch signaling is important for tooth homeostasis and regeneration. Here we report on the expression of Notch receptors and the Delta1 ligand in dental pulp of normal and injured adult rat teeth. Notch receptors were absent from normal adult dental tissues, whereas expression was upregulated after injury. In injured teeth, Notch2 was expressed in mesenchymal cells of the pulp both close to the site of injury (i.e., in the dental crown) and at a distance from it (i.e., in the dental roots), Notch3 expression was mainly associated with vascular structures, while Notch1 expression was restricted to few pulpal cells close to the lesion. None of them was expressed in odontoblasts. Expression of Delta1 was upregulated in odontoblasts of the injured teeth, as well as in vascular structures. These results demonstrate the reactivation of the Notch signaling pathway during wound healing and, furthermore, highlight the similarity between developmental and regenerative processes.

Expression of the transcription factors Otlx2, Barx1 and Sox9 during mouse odontogenesis.

The molecular mechanisms governing the decision between molariform and incisiform patterns of rodent dentition are not yet known. Transcription factors are regulators of regionally specific morphogenesis and key co-ordinators of gene activity during developmental processes. Here, we analysed the expression of several transcription factors during mouse tooth development. Otlx2/Rieg is a homeobox gene involved in Rieger syndrome, a human disorder characterized by dental hypoplasia. Otlx2/Rieg expression distinguishes stomatodeal epithelium well before tooth initiation, and thereafter its expression becomes restricted to the epithelia of both molar and incisor primordia. The recently identified homeodomain transcription factor Barx1 is first expressed in mesenchyme of the first branchial arch, but during advanced developmental stages the gene is exclusively expressed in the mesenchyme of molar primordia. Finally, the Sry-related transcription factor Sox9 is expressed in epithelial components and to a lesser degree in condensed mesenchyme of the developing teeth. These results suggest that Otlx2/Rieg, Barx1, and Sox9 participate in the hierarchical cascade of factors involved in the regulation of tooth morphogenesis.

Mouse Otlx2/RIEG expression in the odontogenic epithelium precedes tooth initiation and requires mesenchyme-derived signals for its maintenance.

The mouse Otlx2 gene is a new member of the paired-like family of homeobox genes whose human homologue, RIEG, is involved in Rieger syndrome, an autosomal-dominant disorder. One of the cardinal features of Rieger syndrome is dental hypoplasia, indicating that Otlx2/RIEG activity is essential for normal tooth development. Here, we analyzed the expression of Otlx2 during mouse tooth development and studied its regulation in dental explants. Otlx2 expression distinguishes stomatodeal from other ectoderm as early as Embryonic Day 8.5, well before tooth initiation. Thereafter, its craniofacial expression becomes restricted to the tooth-forming areas and to the epithelial components of molar and incisor primordia. Although Otlx2 induction precedes the specification of odontogenic mesenchyme, tissue recombination experiments show that the maintenance of its expression requires signals from the mesenchyme and that dental mesenchyme has the capacity to induce ectopic expression of Otlx2 in nondental epithelium. Finally, we compare Otlx2 expression with that of the recently identified homeodomain transcription factor Barx1 expressed in molar mesenchyme. Their strictly complementary expression patterns in the epithelial and mesenchymal components suggest that both genes participate in the reciprocal tissue interactions which are a hallmark of odontogenesis.

Mouse Serrate-1 (Jagged-1): expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4.

Serrate-like genes encode transmembrane ligands to Notch receptors and control cell fate decisions during development. In this report, we analyse the regulation of the mouse Serrate-1 gene during embryogenesis. The Serrate-1 gene is expressed from embryonic day 7.5 (E7.5) and expression is often observed at sites of epithelial-mesenchymal interactions, including the developing tooth, where Serrate-1 is first (E11.5) expressed in all cells of the dental epithelium, but not in mesenchyme. A transient upregulation in dental mesenchyme (E12.5-15.5) is correlated with down-regulation of Serrate-1 expression in epithelial cells contacting the mesenchyme, i.e. in the cells destined to become ameloblasts. This expression pattern is reproduced in explants of dental epithelium and mesenchyme in vitro: epithelium induces Serrate-1 expression in mesenchyme, while epithelium in close proximity to this mesenchyme does not express detectable levels of Serrate-1 mRNA, suggesting that down-regulation of Serrate-1 expression in preameloblasts is caused by mesenchyme-derived signals. Finally, regulation of Serrate-1 expression differs from that of Notch genes. The Serrate-1 gene is induced in dental mesenchyme by fibroblast growth factor-4, but not by bone morphogenetic proteins, while the converse is true for Notch genes. This indicates that, at least during tooth development, the expression patterns observed for receptors and ligands in the Notch signaling pathway are generated by different induction mechanisms.

Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate.

Notch 1, Notch 2, and Notch 3 are three highly conserved mammalian homologues of the Drosophila Notch gene, which encodes a transmembrane protein important for various cell fate decisions during development. Little is yet known about regulation of mammalian Notch gene expression, and this issue has been addressed in the developing rodent tooth during normal morphogenesis and after experimental manipulation. Notch 1, 2, and 3 genes show distinct cell-type specific expression patterns. Most notably, Notch expression is absent in epithelial cells in close contact with mesenchyme, which may be important for acquisition of the ameloblast fate. This reveals a previously unknown prepatterning of dental epithelium at early stages, and suggests that mesenchyme negatively regulates Notch expression in epithelium. This hypothesis has been tested in homo- and heterotypic explant experiments in vitro. The data show that Notch expression is downregulated in dental epithelial cells juxtaposed to mesenchyme, indicating that dental epithelium needs a mesenchyme-derived signal in order to maintain the downregulation of Notch. Finally, Notch expression in dental mesenchyme is upregulated in a region surrounding beads soaked in retinoic acid (50-100 micrograms/ml) but not in fibroblast growth factor-2 (100-250 micrograms/ml). The response to retinoic acid was seen in explants of 11-12-d old mouse embryos but not in older embryos. These data suggest that Notch genes may be involved in mediating some of the biological effects of retinoic acid during normal development and after teratogenic exposure.

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.

Midkine (MK) is the first cloned gene in a new family of heparin-binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar.

Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.