Effect of Hypoxia on Dental Pulp Mesenchymal Stem Cells in a Purpose of Tissue Engineering

International audienceDuring life, teeth are exposed to severe injuries (decay, traumatisms…), which can result in dental pulp necrosis. Creating a “pulp tissue equivalent” constitutes a promising therapeutic approach to replace the current invasive treatments. Dental pulp of deciduous teeth contains mesenchymal stem cells (SHEDs: Stem cells from Human Exfoliated Deciduous teeth), shown to have a high proliferation and differentiation potential. Our approach aims to assess the effect of severe hypoxia on these cells, mimicking the clinical conditions of the matrix implantation in the pulp space. 3D collagen matrices seeded with SHEDs (1.5 million of cells/ml) were cultivated under severe hypoxia (1% O2) during 3 days. Then, to mimic the kinetics of revascularization, the matrices were replaced in normoxic conditions (21% O2). Induced mRNA and protein modifications were studied by qPCR, ELISA, Western Blot and immunocytochemistry, at several time points. A transcriptomic analysis (DNA affymetrix chips “gene” type) of the samples was then performed at the time point with the highest VEGF mRNA expression. The capacity of SHEDs exposed to hypoxia to induce angiogenenis was then tested in a tubulogenesis model. Finally, SHEDs pretreated by hypoxia were induced toward osteogenic differentiation in 3D plastic compressed collagen matrix. Our data show that hypoxic conditions induce: 1) an increase of the transcription factor HIF‐1 alpha observed in the cell nucleus, 2) a x4 increase of VEGF mRNA expression at 24 h (qPCR), confirmed by ELISA analysis, 3) the up‐regulation of numerous genes activated by HIF‐1 alpha and involved in angiogenesis, apoptosis and glycolysis regulation. Furthermore, SHEDs pretreated by hypoxia enhanced capillary formation by endothelial cells. In parallel, osteogenic differentiation assay showed that pretreatment by hypoxia did not impair matrix mineralization by SHEDs, which was slightly enhanced.These cells are good candidate for tissue engineering approaches, in particular for treating damaged dental tissues

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.status: publishe

Common SNPs of AmelogeninX (AMELX) and Dental Caries Susceptibility

International audienceGenetic approaches have shown that several genes could modify caries susceptibility; AmelogeninX (AMELX) has been repeatedly designated. Here, we hypothesized that AMELX mutations resulting in discrete changes of enamel microstructure may be found in children with a severe caries phenotype. In parallel, possible AMELX mutations that could explain resistance to caries may be found in caries-free patients. In this study, coding exons of AMELX and exon-intron boundaries were sequenced in 399 individuals with extensive caries (250) or caries-free (149) individuals from nine French hospital groups. No mutation responsible for a direct change of amelogenin function was identified. Seven single-nucleotide polymorphisms (SNPs) were found, 3 presenting a high allele frequency, and 1 being detected for the first time. Three SNPs were located in coding regions, 2 of them being non-synonymous. Both evolutionary and statistical analyses showed that none of these SNPs was associated with caries susceptibility, suggesting that AMELX is not a gene candidate in our studied population

Dental Caries and Enamelin Haplotype

International audienceIn the literature, the enamelin gene ENAM has been repeatedly designated as a possible candidate for caries susceptibility. Here, we checked whether ENAM variants could increase caries susceptibility. To this aim, we sequenced coding exons and exon-intron boundaries of ENAM in 250 children with a severe caries phenotype and in 149 caries-free patients from 9 French hospital groups. In total, 23 single-nucleotide polymorphisms (SNPs) were found, but none appeared to be responsible for a direct change of ENAM function. Six SNPs had a high minor allele frequency (MAF) and 6 others were identified for the first time. Statistical and evolutionary analyses showed that none of these SNPs was associated with caries susceptibility or caries protection when studied separately and challenged with environmental factors. However, haplotype interaction analysis showed that the presence, in a same variant, of 2 exonic SNPs (rs7671281 and rs3796704; MAF 0.12 and 0.10, respectively), both changing an amino acid in the protein region encoded by exon 10 (p.I648T and p.R763Q, respectively), increased caries susceptibility 2.66-fold independent of the environmental risk factors. These findings support ENAM as a gene candidate for caries susceptibility in the studied population

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

<div><p>Mutations in <i>PHEX</i> (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides <i>in vitro</i> and <i>in vivo</i> on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation <i>in vitro</i>, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.</p> </div

Summary of the role of the MEPE-derived ASARM peptide in the etiology of tooth dentin abnormalities in XLH patients.

<p>A: SIBLING proteins containing the ASARM peptide are processed by a multitude of proteolytic enzymes, some of which may release the ASARM peptide or larger protein fragments containing the ASARM peptide into the extracellular matrix (ECM). ASARM and ASARM-containing peptides are inhibitory for mineralization, binding directly to hydroxyapatite (HAP) mineral crystals in bones and teeth. In normal conditions, neutralizing PHEX cleavage of ASARM releases extracellular matrices from this inhibition and mineralization proceeds appropriately. B: In XLH tooth dentin, inactivating mutations in the <i>PHEX</i> gene result in nonfunctional PHEX enzyme that allows HAP crystal-binding, ASARM-containing peptides to accumulate in the dentin thus inhibiting tooth mineralization (pathway 1). Normal PHEX also protects full-length MEPE from cleavage by proteases (cathepsin B), thereby preventing release of mineralization-inhibiting ASARM. In XLH, excessive cleavage of MEPE by proteases (in the absence of functional PHEX) to release the inhibitory ASARM peptide might also contribute to the impaired mineralization of dentin. Finally, ASARM accumulation in XLH may impair dentinogenesis by decreasing odontoblast differentiation and downregulating genes encoding for secreted ECM proteins (pathways 2 and 3), while increasing MEPE expression (pathway 4) which in turn would further exacerbate the XLH hypomineralization tooth phenotype.</p

Light and electron microscopy of the cells, matrix and mineral in 3D SHED cell cultures.

<p>SHED cell cultures maintained in nonmineralizing (NM) or mineralizing (M) conditions in the absence or presence of 20 µM of either phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) ASARM peptide for 21 days were visualized by light microscopy and by scanning (SEM) and transmission (TEM) electron microscopy. <b>A,B:</b> SEM reveals SHEDs (arrows) well integrated into the collagen scaffold. Mineralization of the cultures appears as nodules within the collagen scaffold (arrowheads) only in the M and the M+np-ASARM conditions. Energy-dispersive X-ray spectroscopy (EDX) for compositional microanalysis of the nodules (performed at the white square) shows major spectral peaks for calcium (Ca) and phosphorus (P) with an acquired Ca/P ratio of 1.67+/−0.05 in both mineralizing conditions where nodules appeared. <b>C:</b> Light microscopy (left panel) and TEM (center and right panel) of the mineralized cultures (M and M+np-ASARM). Mineralized nodules (black box, left panel) are often in close proximity to the SHED cells, and consist of aggregates of multiple, smaller mineralization nodules (arrowheads) and occasional mineralized collagen fibrils (white box center panel, and right panel).</p

Inhibition of mineralization by phosphorylated MEPE-ASARM peptide in a 3D collagen/tooth slice culture model. A:

<p>Schematic of the 3D culture model. Stem cells from human deciduous exfoliated teeth (SHEDs) were obtained for cell cultures studies from the pulp of caries-free human third molars. Passaged cells (10⁶ SHEDs) seeded into a type I collagen gel scaffold were applied to a human tooth slice with a pulp chamber cavity to mimick the tooth/dentin environment, all of which were supported peripherally by a steel wire mesh to minimize collagen gel contraction. <b>B:</b> Calcium content in the cell/matrix layer determined by flame atomic absorption spectrometry of cultures maintained in NM or M conditions in the absence or presence of p-ASARM or np-ASARM at a concentration of 5, 10 or 20 µM for 21 days. Calcium content significantly decreased in the presence of 20 µM of p-ASARM while the nonphosphorylated form of the peptide had no effect on calcium content of the cultures. <b>C-D:</b> SHED cultures were maintained in nonmineralizing (NM) or mineralizing (M) conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056749#s2" target="_blank">Materials and Methods</a>) in the absence or presence of 20 µM phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) peptide for 21 days. Mineralized nodules in the extracellular matrix were clearly visible by light microscopy after von Kossa staining in the M and M+np-ASARM. Quantification of mineralized nodules (<b>D</b>) shows that they were essentially undetectable in presence of the p-ASARM peptide, whereas about 40 nodules were counted per slice in the M and M+np-ASARM samples. <i>n</i> = 3, error bars +/− SD, ** indicates significant difference (p<0.01) relative to mineralizing condition without peptide.</p