Caspase-9 inhibition decreases expression of Mmp9 during chondrogenesis.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Expression dynamics of metalloproteinases during mandibular bone formation: association with Myb transcription factor.

As the dentition forms and becomes functional, the alveolar bone is remodelled. Metalloproteinases are known to contribute to this process, but new regulators are emerging and their contextualization is challenging. This applies to Myb, a transcription factor recently reported to be involved in bone development and regeneration. The regulatory effect of Myb on Mmps expression has mostly been investigated in tumorigenesis, where Myb impacted the expression of Mmp1, Mmp2, Mmp7, and Mmp9. The aim of this investigation was to evaluate the regulatory influence of the Myb on Mmps gene expression, impacting osteogenesis and mandibular bone formation. For that purpose, knock-out mouse model was used. Gene expression of bone-related Mmps and the key osteoblastic transcription factors Runx2 and Sp7 was analysed in Myb knock-out mice mandibles at the survival limit. Out of the metalloproteinases under study, Mmp13 was significantly downregulated. The impact of Myb on the expression of Mmp13 was confirmed by the overexpression of Myb in calvarial-derived cells causing upregulation of Mmp13. Expression of Mmp13 in the context of other Mmps during mandibular/alveolar bone development was followed in vivo along with Myb, Sp7 and Runx2. The most significant changes were observed in the expression of Mmp9 and Mmp13. These MMPs and MYB were further localized in situ by immunohistochemistry and were identified in pre/osteoblastic cells as well as in pre/osteocytes. In conclusion, these results provide a comprehensive insight into the expression dynamics of bone related Mmps during mandibular/alveolar bone formation and point to Myb as another potential regulator of Mmp13.

X-linked and autosomal recessive Hypohidrotic Ectodermal Dysplasia: genotypic-dental phenotypic findings.

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of ectodermal structures and its molecular etiology corresponds to mutations of EDA-EDAR genes. The aim of this study was first to investigate the genotype and dental phenotype associated with HED and second, to explore possible correlations between dental features and molecular defects. A total of 27 patients from 24 unrelated families exhibiting clinical signs of HED (22 XLHED males, 5 autosomal recessive forms) were retrospectively included. In the sample, 25 different mutations on EDA and EDAR genes were detected; 10 were not previously described. EDA and EDAR mutations corresponded respectively to 80.0% and 20.0% of the mutations. The dental phenotype analysis revealed a mean number of primary and permanent missing teeth ranging respectively from 14.5 (4-20) to 22.5 (10-28); the majority of the patients exhibited dysmorphic teeth. Overall, no differential expression in the degree of oligodontia according to either the mutated gene, the mutated functional sub-domains, or the mutation type, could be observed. Nevertheless, the furin group exhibited severe phenotypes unobserved in the TNF group. Significant differences in the number of some primary missing teeth (incisor and canine) related to EDA-EDAR genes defects were detected for the first time between XLHED and autosomal recessive HED, suggesting differential local effects of EDA-EDAR genes during odontogenesis. The present genotypic-phenotypic findings may add to the knowledge of the consequences of the molecular dysfunction of EDA-NF-kB in odontogenesis, and could be helpful in genetic counseling to distinguish autosomal forms from other HED syndromes.

Bone Marrow Stromal Cells Promote Innervation of Bioengineered Teeth.

Transplantation of bone marrow mesenchymal stem cells (BMDCs) into a denervated side of the spinal cord was reported to be a useful option for axonal regeneration. The innervation of teeth is essential for their function and protection but does not occur spontaneously after injury. Cultured reassociations between dissociated embryonic dental mesenchymal and epithelial cells and implantation lead to a vascularized tooth organ regeneration. However, when reassociations were coimplanted with a trigeminal ganglion (TG), innervation did not occur. On the other hand, reassociations between mixed embryonic dental mesenchymal cells and bone marrow-derived cells isolated from green fluorescent protein (GFP) transgenic mice (BMDCs-GFP) (50/50) with an intact and competent dental epithelium (ED14) were innervated. In the present study, we verified the stemness of isolated BMDCs, confirmed their potential role in the innervation of bioengineered teeth, and analyzed the mechanisms by which this innervation can occur. For that purpose, reassociations between mixed embryonic dental mesenchymal cells and BMDCs-GFP with an intact and competent dental epithelium were cultured and coimplanted subcutaneously with a TG for 2 wk in ICR mice. Axons entered the dental pulp and reached the odontoblast layer. BMDCs-GFP were detected at the base of the tooth, with some being present in the pulp associated with the axons. Thus, while having a very limited contribution in tooth formation, they promoted the innervation of the bioengineered teeth. Using quantitative reverse transcription polymerase chain reaction and immunostainings, BMDCs were shown to promote innervation by 2 mechanisms: 1) via immunomodulation by reducing the number of T lymphocytes (CD3+, CD25+) in the implants and 2) by expressing neurotrophic factors such as NGF, BDNF, and NT3 for axonal growth. This strategy using autologous mesenchymal cells coming from bone marrow could be used to innervate bioengineered teeth without treatment with an immunosuppressor such as cyclosporine A (CsA), thus avoiding multiple side effects.

Origin of the deciduous upper lateral incisor and its clinical aspects.

The upper lateral incisor in humans is often affected by dental anomalies that might be explained developmentally. To address this question, we investigated the origin of the deciduous upper lateral incisor (i2) in normal human embryos at prenatal weeks 6-8. We used serial frontal histological sections and computer-aided 3D reconstructions. At embryonic days 40-42, two thickenings of the dental epithelia in an "end-to-end" orientation were separated by a groove at the former fusion site of the medial nasal and maxillary processes. Later, these dental epithelia fused, forming a continuous dental lamina. At the fusion site, i2 started to develop. The fusion line was detectable on the i2 germ until the 8th prenatal week. The composite origin of the i2 may be associated with its developmental vulnerability. From a clinical aspect, a supernumerary i2 might be a form of cleft caused by a non-fusion of the dental epithelia.

Bone marrow cells can give rise to ameloblast-like cells.

Post-eruptive loss of ameloblasts requires identification of alternative sources for these cells to realize tooth-tissue-engineering strategies. Recent reports showed that bone-marrow-derived cells can give rise to different types of epithelial cells, suggesting their potential to serve as a source for ameloblasts. To investigate this potential, we mixed c-Kit(+)-enriched bone marrow cells with embryonic dental epithelial cells and cultured them in re-association with dental mesenchyme. Non-dividing, polarized, and secretory ameloblast-like cells were achieved without cell fusion. Before basement membrane reconstitution, some bone marrow cells migrated to the mesenchyme, where they exhibited morphological, molecular, and functional characteristics of odontoblasts. These results show, for the first time, that bone-marrow-derived cells can be reprogrammed to give rise to ameloblast-like cells, offering novel possibilities for tooth-tissue engineering and the study of the simultaneous differentiation of one bone marrow cell subpopulation into cells of two different embryonic lineages.

Three-dimensional Micro-culture System for Tooth Tissue Engineering.

The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development. Progress is being made to regenerate teeth by reassociating dissociated embryonic dental cells and implanting them in vivo. In the present study, we tested the hanging drop method to study mixed epithelial-mesenchymal cell reorganization in a liquid instead of semisolid medium to see whether it could lead to tooth histogenesis and organogenesis. This method allowed the control of the proportion and number of cells to be used, and the forming microtissues showed homogeneous size. The liquid environment favored cell migrations as compared with collagen gels. Three protocols were compared. The one that sequentially combined the hanging drop and semisolid medium cultures prior to in vivo implantation gave the best results. Indeed, after implantation, teeth developed, showing a well-formed crown, mineralization of dentin and enamel, and the initiation of root formation. Vascularization and the cellular heterogeneity in the mesenchyme were similar to what was observed in developing molars. Finally, after coimplantation with a trigeminal ganglion, the dental mesenchyme, including the odontoblast layer, became innervated. The real advantage of this technique is the small number of cells required to make a tooth. This experimental model can be employed to study the development, physiology, metabolism, or toxicology in forming teeth and test other cell sources.

Synthesis of collagen type I, type I trimer and type III by embryonic mouse dental epithelial and mesenchymal cells in vitro.

Epithelial and mesenchymal dental cells were grown in primary monolayer culture and the ability of both cell types to synthesize interstitial collagens was investigated. Pepsin-solubilized collagens were analyzed by CM-cellulose chromatography and both cell types were found to synthesize collagen type I, type III and type I trimer. The collagen phenotype of mesenchymal cells (type I: 82.4%, type III: 8.5%, type I trimer: 9.1%) was different from that of epithelial cells (type I: 71.8%, type III: 9.5%, type I trimer: 18.7%). The radioactivity incorporated into collagen molecules by mesenchymal cells was 34-times greater than the radioactivity incorporated by epithelial cells. This result agreed with previous observations obtained from tissue culture experiments (Lesot, H. and Ruch, J.V. (1979) Biol. Cell. 34, 23--37) which indicated a low synthesis of interstitial collagens by isolated dental epithelia when compared to isolated dental mesenchymes.

Increased apoptosis during morphogenesis of the lower cheek teeth in tabby/EDA mice.

In wild-type (WT) mice, epithelial apoptosis is involved in reducing the embryonic tooth number and the mesial delimitation of the first molar. We investigated whether apoptosis could also be involved in the reduction of tooth number and the determination of anomalous tooth boundaries in tabby (Ta)/EDA mice. Using serial histological sections and computer-aided 3D reconstructions, we investigated epithelial apoptosis in the lower cheek dentition at embryonic days 14.5-17.5. In comparison with WT mice, apoptosis was increased mainly mesially in Ta dental epithelium from day 15.5. This apoptosis showed a similar mesio-distal extent in all 5 morphotypes (Ia,b,c and IIa,b) of Ta dentition and eliminated the first cheek tooth in morphotypes IIa,b. Apoptosis did not appear to play any causal role in positioning inter-dental gaps. Analysis of the present data suggests that the increased apoptosis in Ta mice is a consequence of impaired tooth development caused by a defect in segmentation of dental epithelium.

Dental Epithelial Histomorphogenesis in vitro.

Recent developments in tooth-tissue engineering require that we understand the regulatory processes to be preserved to achieve histomorphogenesis and cell differentiation, especially for enamel tissue engineering. Using mouse first lower molars, our objectives were: (1) to determine whether the cap-stage dental mesenchyme can control dental epithelial histogenesis, (2) to test the role of the primary enamel knot (PEK) in specifying the potentialities of the dental mesenchyme, and (3) to evaluate the importance of positional information in epithelial cells. After tissue dissociation, the dental epithelium was further dissociated into individual cells, re-associated with dental mesenchyme, and cultured. Epithelial cells showed a high plasticity: Despite a complete loss of positional information, they rapidly underwent typical dental epithelial histogenesis. This was stimulated by the mesenchyme. Experiments performed at E13 demonstrated that the initial potentialities of the mesenchyme are not specified by the PEK. Positional information of dental epithelial cells does not require the memorization of their history.

The supernumerary cheek tooth in tabby/EDA mice-a reminiscence of the premolar in mouse ancestors.

OBJECTIVE: A supernumerary cheek tooth occurs mesially to the first molar in tabby/EDA (Ta) mice affected by hypohidrotic ectodermal dysplasia. The supernumerary tooth (S) has been hypothetically homologized to the premolar, which has disappeared during mouse evolution. DESIGN: This hypothesis was tested using available morphological data on the lower cheek teeth in wild type (WT) and Ta mice. RESULTS: The presence of S is accompanied by a reduction in the mesial portion of the M(1) in mutant mice. 3D reconstructions suggest that the S in Ta homo/hemizygous embryos originates from a split off the mesial portion of the first molar (M(1)) cap. In WT embryos, two vestigial tooth primordia are transiently distinct in front of the M(1). The distal vestige has the form of a wide bud and participates during the development of the mesial portion of the M(1). This bud has been homologized with the vestigial primordium of the fourth premolar of mouse ancestors. The premolar disappearance coincided with a mesial lengthening of the M(1) during mouse evolution. The incorporation of the distal premolar vestige into the mesial part of the M(1) in WT embryos can be regarded as a repetition of the premolar disappearance during evolution. CONCLUSION: : Ontogenetic and phylogenetic data support that the S in Ta mice arises due to the segregation of the distal premolar vestige from the molar dentition and thus represents an evolutionary throwback (atavism).

Cell-matrix interactions and cell-cell junctions during epithelial histo-morphogenesis in the developing mouse incisor.

The continuously growing rodent incisor develops mainly along its antero-posterior axis. The labio-lingual asymmetry which characterizes this tooth is initiated at the cap stage and increases further during the cap to bell transition (ED14 to ED16) when histogenesis of the enamel organ proceeds. Histology, transmission electron microscopy (TEM), and immunostaining were used to document the changes in the basement membrane (BM) as well as the modifications of epithelial cell-matrix and cell-cell interactions during this period. The expression of plakoglobin, desmoglein and E-cadherin at ED14 suggested that the main cell-cell junctional complexes were adherens junctions. The expression of desmoglein and TEM observations suggested a progressive antero-posterior stabilization of the enamel organ by means of desmosomes from ED14 to ED18. alpha6 integrin, BP 230 and laminin gamma2 chain were all expressed in the developing incisor but were not always co-distributed. Immunostaining and TEM suggested that only primitive type II hemidesmosomes were present. At ED14, cells of the enamel knot (EK) did not show any specific expression for antigens involved in cell-cell interaction. However, strong staining for the laminin gamma2 chain characterized the BM in contact with EK cells. The BM in the labial part of the cervical loop demonstrated ultrastructural changes: the presence of loops of the lamina densa in this region preceeded the differential expression of the integrin alpha6 subunit and that of the laminin gamma2 chain in the labial/lingual parts of the cervical loop. Apoptosis was transiently observed in the contiguous mesenchyme. This affected osteoblasts and also nerve cells close to the labial part of the cervical loop.

Odontoblast differentiation.

Odontoblasts are post-mitotic, neural crest-derived, cells which overtly differentiate according to tooth specific temporo-spatial patterns and secrete predentin-dentin components. Neither the timing nor the molecular mechanisms of their specification are known and the problem of their patterning in the developing jaws is far from being solved. On the other hand, some significative strides were made concerning the control of their terminal differentiation. Fibronectin interacting with a 165 kDa, non integrin, membrane protein intervenes in the cytoskeletal reorganization involved in odontoblast polarization and their terminal differentiation can be triggered in vitro by immobilized members of the TGF beta family. Histological aspects and the transcriptional phenotypes (transcripts of TGF beta s, BMPs, msxs, IGF1, fibronectin, osteonectin, bone sialoprotein genes) are very similar in vivo and in vitro. In vivo members of the TGF beta super family secreted by preameloblasts, trapped and activated by basement membrane associated components, might initiate odontoblast terminal differentiation.

Effects of hepatocyte growth factor anti-sense oligodeoxynucleotides or met D/D genotype on mouse molar crown morphogenesis.

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelio-mesenchymal interactions during early organogenesis and to be also involved in the development of murine molars. In the developing tooth, HGF is expressed in the cells of the dental papillae, and c-Met, its receptor, in the cells of dental epithelia. In order to study the functional role played by HGF in tooth development, we tested the effects of HGF translation arrest by anti-sense phosphorothioate oligodeoxynucleotides on E-14 molars cultured in vitro. We also analyzed the histo-morphogenesis and crown cytodifferentiation of transgenic met E-14 molars cultured in vitro. 3D reconstructions revealed perturbations of the cusp pattern. However, histo-morphogenesis and crown cytodifferentiation were normal at the histological level.

Mouse odontogenesis in vitro: the cap-stage mesenchyme controls individual molar crown morphogenesis.

Day 14 ICR mouse first lower (M1) and upper molars (M1) as well as heterotopic recombinations of M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme were cultured for 6, 8 and 10 days on semi-solid medium. Computer-assisted 3D reconstructions were performed to follow the in vitro development of these explants. In vitro culture of cap-stage molars allowed for the emergence of unequivocal morphological features distinctive for M1 versus M1 including the cusp pattern, cusp inclination and tooth specific chronology for odontoblast and ameloblast terminal differentiations. Both M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme recombinations developed according to the known developmental fate of the mesenchyme. Our data demonstrate that the cap-stage dental ecto-mesenchyme not only directs tooth class specific morphogenesis, but also individual molar crown features. Furthermore, the mesenchyme apparently also controls the typical mirror symmetry of right and left handed teeth.

Mouse molar morphogenesis revisited by three dimensional reconstruction. I. Analysis of initial stages of the first upper molar development revealed two transient buds.

Early stages of tooth development in the maxillary cheek region in the mouse were investigated by combined analysis of histological sections, computer assisted 3D reconstructions and morphometry. In ED 12.5 embryos, 3D reconstructions revealed an accessory epithelial bud (R1) and a large bud (R2), which appeared as a single bud-shaped epithelium in frontal sections. This developmentally most advanced dental epithelium in the mouse embryonic maxilla until ED 13.5, generally considered as the bud of the first molar, regressed during later development. Meanwhile the bud and cap of the first upper molar originated more posteriorly, from ED 13.5. The regression of R1 and R2 was associated with epithelial apoptosis. Apoptotic cells and bodies were apparent on sections in the R1 epithelium from ED 12.5. The R2 epithelium maintained the large bud-shaped appearance on sections, representing the largest part of the dental epithelium in the maxillary cheek region until ED 14.0; apoptoses were detected there as late as from ED 13.5. During regression, the R2 rudiment was transformed into the medial and lateral epithelial ridges, posteriorly in continuity with the arising cap of the first molar. The reduced R1 epithelium seemed to contribute to the medial ridge. These results should be taken into consideration in the interpretation of early odontogenesis in the upper jaw in the mouse. The interesting problem of the identification of tooth homology of the rudiments should be elucidated by further comparative morphological and paleontological investigations.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell staged first and second upper molar teeth.

Tooth morphogenesis is a complex multifactorial process in which differential mitotic activities and cell death play important roles. Upper first (m1) and second (m2) molars from mouse embryos were investigated from early cap to bell stage. m2 differed from m1 by delayed origin of the enamel grooves delimiting the protrusion of the cap bottom towards the dental papilla, and retardation of the enamel knot formation. The width of the m2 enamel organ was conspicuously smaller during cap formation and length remained smaller throughout the period of observation. Formation of the cap depression was comparable in m1 and m2, however margins delimiting the enamel organ cavity arose in m1 and m2 as mirror images. Attempts were made to correlate changes in the distribution of apoptotic cells and bodies and/or mitoses with morphogenesis. These cellular activities were recorded from histological sections and represented in space using computer-assisted three-dimensional reconstructions. Mitoses in the epithelial compartment were associated with the development of the cervical loop. In the mesenchyme of m1 at early bell stage, a postero-anterior increasing gradient of mitoses was observed which might be correlated with the anterior growth of the molar. Cells in the enamel knot demonstrated a high level of apoptosis, retarded in m2, but absolutely no division. Apoptotic processes were also involved in the anterior delimitation of the m1 epithelium. Apoptosis might correspond to the programmed destruction of cells whose function had to be suppressed or whose potential activity had to be avoided.

Reactionary dentinogenesis.

Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by surviving odontoblast cells in response to an appropriate stimulus. Whilst this stimulus may be exogenous in nature, it may also be from endogenous tissue components released from the matrix during pathological processes. Implantation of isolated dentine extracellular matrix components in unexposed cavities of ferret teeth led to stimulation of underlying odontoblasts and a response of reactionary dentinogenesis. Affinity chromatography of the active components prior to implantation and assay for growth factors indicated that this material contained significant amounts of TGF-beta 1, a growth factor previously shown to influence odontoblast differentiation and secretory behavior. Reactionary dentinogenesis during dental caries probably results from solubilization of growth factors, TGF-beta in particular, from the dentine matrix which then are responsible for initiating the stimulatory effect on the odontoblasts. Compositional differences in tertiary dentine matrices beneath carious lesions in human teeth have also been shown indicating modulation of odontoblast secretion during reactionary and reparative dentinogenesis.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. III. Spatial distribution of mitoses and apoptoses up to bell-staged first lower molar teeth.

Computer-assisted 3D reconstructions were used to follow the development of the embryonic mouse first lower molar (M1). At ED 12.5, the thickening of the oral epithelium, which was thought to correspond to the molar dental lamina, regressed in its anterior part as a result of apoptosis. Only the posterior part later gave rise to molars. The transition to the cap stage entailed medial and lateral extensions of the dental epithelium. The growth and histo-morphogenesis of the enamel organ as well as cervical loop formation proceeded more rapidly in the anterior part of the M1 during the cap and early bell stages producing significant morphological differences along the antero-posterior axis. Apoptosis was temporarily intensive in the anterior part of the bud- and cap-shaped epithelium and thus pointed domains which do not participate in the formation of the final M1 enamel organ. In the well-formed cap, apoptoses displayed maximum concentration in the enamel knot (EK). No increase in the number of metaphases could be detected in the vicinity of the EK. Mitoses were distributed throughout the epithelial compartment until cap stage and then mainly concentrated in the inner dental epithelium at the early bell stage. At this later stage, either lateral views or thick virtual sections performed in the reconstruction demonstrated a clear cut distribution of mitoses and apoptoses in the enamel organ. At the early bell stage, mitoses in the mesenchyme demonstrated an increasing postero-anterior gradient.