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.
Tooth development has attracted the attention of researchers since the 19th century. It became obvious even then that morphogenesis could not fully be appreciated from two‐dimensional histological ...sections. Therefore, methods of three‐dimensional (3D) reconstructions were employed to visualize the surface morphology of developing structures and to help appreciate the complexity of early tooth morphogenesis. The present review surveys the data provided by computer‐aided 3D analyses to update classical knowledge of early odontogenesis in the laboratory mouse and in humans. 3D reconstructions have demonstrated that odontogenesis in the early stages is a complex process which also includes the development of rudimentary odontogenic structures with different fates. Their developmental, evolutionary, and pathological aspects are discussed. The combination of in situ hybridization and 3D reconstruction have demonstrated the temporo‐spatial dynamics of the signalling centres that reflect transient existence of rudimentary tooth primordia at loci where teeth were present in ancestors. The rudiments can rescue their suppressed development and revitalize, and then their subsequent autonomous development can give rise to oral pathologies. This shows that tooth‐forming potential in mammals can be greater than that observed from their functional dentitions. From this perspective, the mouse rudimentary tooth primordia represent a natural model to test possibilities of tooth regeneration.
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.
Vascularization of Engineered Teeth Nait Lechguer, A.; Kuchler-Bopp, S.; Hu, B. ...
Journal of dental research,
12/2008, Volume:
87, Issue:
12
Journal Article
Peer reviewed
The implantation of cultured dental cell-cell re-associations allows for the reproduction of fully formed teeth, crown morphogenesis, epithelial histogenesis, mineralized dentin and enamel ...deposition, and root-periodontium development. Since vascularization is critical for organogenesis and tissue engineering, this work aimed to study: (a) blood vessel formation during tooth development, (b) the fate of blood vessels in cultured teeth and re-associations, and (c) vascularization after in vivo implantation. Ex vivo, blood vessels developed in the dental mesenchyme from the cap to bell stages and in the enamel organ, shortly before ameloblast differentiation. In cultured teeth and re-associations, blood-vessel-like structures remained in the peridental mesenchyme, but never developed into dental tissues. After implantation, both teeth and re-associations became revascularized, although later in the case of the re-associations. In implanted re-associations, newly formed blood vessels originated from the host, allowing for their survival, and affording conditions organ growth, mineralization, and enamel secretion.
Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells ...need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.
During four days of prenatal development in the mouse, the morphology of the first lower molar moves from the early cap to the bell stage. Five phenomena characterize this period: growth of the tooth ...germ; development of the cervical loop; histogenesis of the enamel organ; folding of the epithelial‐mesenchymal junction associated with cusp formation; and change in cellular heterogeneity in the mesenchyme. All these processes are controlled by epithelial‐mesenchymal interactions.
These complex histo‐morphogenetic events have been documented using histological sections and 3D reconstructions. When combined with functional tests in vitro, this approach allowed searching for possible relationships between simultaneous changes occurring in both the epithelial and ecto‐mesenchymal compartments.
Parallel changes that occur in the two tissues could result from different mechanisms, as illustrated by the increasing number of pre‐odontoblasts and pre‐ameloblasts during crown growth. Cell division was involved mainly in the ecto‐mesenchyme, while proliferation and cell re‐organization occurred in the inner dental epithelium. 3D reconstructions also raised still unsolved questions, such as the possible relationship between cusp size and spatial specification of cell kinetic parameters, changes in cell position within the inner dental epithelium, and tracing cell migration in the mesenchyme during development.
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.
The hypohidrotic ectodermal dysplasias (HED) belong to a large and heterogenous nosological group of polymalfomative syndromes characterized by dystrophy or agenesis of ectodermal derivatives. ...Molecular etiologies of HED consist of mutations of the genes involved in the Ectodysplasin (EDA)-NF-κB pathway. Besides the classic ectodermal signs, craniofacial and bone manifestations are associated with the phenotypic spectrum of HED. The dental phenotype of HED consists of various degrees of oligodontia with other dental abnormalities, and these are important in the early diagnosis and identification of persons with HED. Phenotypic dental markers of heterozygous females for EDA gene mutation—moderate oligodontia, conical incisors, and delayed dental eruption—are important for individuals giving reliable genetic counseling. Some dental ageneses observed in HED are also encountered in non-syndromic oligodontia. These clinical similarities may reflect possible interactions between homeobox genes implicated in early steps of odontogenesis and the Ectodysplasin (EDA)-NF-κB pathway. Craniofacial dysmorphologies and bone structural anomalies are also associated with the phenotypic spectrum of persons with HED patients. The corresponding molecular mechanisms involve altered interactions between the EDA-NF-κB pathway and signaling molecules essential in skeletogenic neural crest cell differentiation, migration, and osteoclastic differentiation. Regarding oral treatment of persons with HED, implant-supported prostheses are used with a relatively high implant survival rate. Recently, groundbreaking experimental approaches with recombinant EDA or transgenesis of EDA-A1 were developed from the perspective of systemic treatment and appear very promising. All these clinical observations and molecular data allow for the specification of the craniofacial phenotypic spectrum in HED and provide a better understanding of the mechanisms involved in the pathogenesis of this syndrome.
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.
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.
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