Effect of transgenic <i>Rank</i> on lower molar growth in <i>Msx2</i>⁻^/− mice.

<p>Microradiographs were taken at 2 (a–c), 3 (d–f), and 10 (g–i) weeks for <i>Msx2</i>^−/− mice overexpressing (b, e, and h) or not expressing (a, d, and g) transgenic <i>Rank</i>, and for <i>WT</i> mice (c, f, and i). At 2 and 3 weeks, eruption of the first and second molars was more advanced in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice than in <i>Msx2</i>^−/− mice, as shown by their positions relative to the vestibular bone crest (arrows in a, b, d, and e). At 3 weeks, the most significant feature of the progression in second molar growth was the more advanced root elongation in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice compared to <i>Msx2</i>^−/− mice (squares in e versus d). However, the root lengths did not match those of <i>WT</i> mice (square in f). At 10 weeks, while the third molars of <i>Msx2</i>^−/− mice were completely surrounded by and indistinguishable from bone on the microradiograph (square in g), the third molars of <i>Msx2</i>^−/−<i>Rank^Tg</i> mice were fully erupted and functional (square in h). Measures of the first molar mesial root length and width at the median position (in µm) were performed on histological sections and presented in a table form (j). A higher length and lower width were observed at 2 weeks for <i>Msx2</i>^−/−<i>Rank^Tg</i> molar root comparatively to <i>Msx2</i>^−/− molar root. However, <i>Msx2</i>^−/−<i>Rank^Tg</i> molar root length and width remained respectively lower and superior to those observed for WT molar root. At 3 weeks, no significant difference of length and width was observed between <i>Msx2</i>^−/−<i>Rank^Tg</i> and <i>Msx2</i>^−/− molar roots.</p

Effect of transgenic <i>Rank</i> on lower first molar and incisor root formation in <i>Msx2</i>⁻^/− mice.

<p>Van Gieson histology staining (a–f) and keratin immunohistochemistry (g–r) were respectively performed on mandibular frontal sections of 3- and 2-week-old <i>Msx2</i>^−/− mice either overexpressing or not expressing transgenic <i>Rank</i>. At 3 weeks, <i>Rank</i> overexpression had induced a normalization in the size of most epithelial cell rests of Malassez (Ma) (a and c versus d and f), and at 2 weeks it had induced a better commitment of Hertwig epithelial root sheath (HERS) cells, specifically in the labial area (j and k versus g and h). Occasionally and independently of <i>Rank</i> overexpression, epithelial cyst–like structures were observed in the lingual area of <i>Msx2</i>^−/− mandibular first molars (j, i). Cytokeratin-14 immunolabelling revealed that these cyst-like structures were associated with abnormal continuity between dental and oral epithelia (j) and the presence of a periodontal pocket (square in j enlarged in l). Another defect observed at 3 weeks in the lingual root of <i>Msx2</i>^−/− mice, also independent of <i>Rank</i> overexpression, was a lacuna-like structure in the dentine facing the site of transition between crown and root epithelia (squares in a and d enlarged in b and e, respectively). In the incisor root equivalent, normalization of the size of the epithelial cell rests of Malassez (Ma) was observed in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice (squares in m versus p enlarged in n and o and q and r, respectively).</p

<i>Rank</i> overexpression stimulates alveolar bone osteoclastogenesis.

<p>TRAP activity assays were performed on frontal sections of the mandibles of 2 (a, b) and 3 (c–f) week-old mice to determine the effect of <i>Rank</i> overexpression on osteoclast numbers. At 2 weeks, the number of TRAP-positive cells was significantly increased around the first molar root in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice (b, e). The root appeared longer but thinner in <i>Msx2</i>^−/−<i>Rank^Tg</i> than in <i>Msx2</i>^−/− mice, and advanced eruption was also clearly visible. At 3 weeks, no significant difference in the number of TRAP-expressing cells was observed (c, d, e). While the length of the first molar roots of <i>Msx2</i>^−/− mutants expressing or not expressing <i>Rank</i> was similar, it remained thinner in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice (c, d). Asterisk in (d): Epithelial cyst on the lingual part of the root of a <i>Msx2</i>^−/−<i>Rank^Tg</i> mouse. M1, first molar; I, incisor. (e) Numbering of the TRAP positive cells in the alveolar bone surface performed on 7 µm thick sections (n>8) and presented as a table with statistical analyses.</p

Gene signature induced by <i>Rank</i> overexpression in the dental epithelium and alveolar bone of <i>Msx2</i>⁻^/− mice.

<p>RT-PCR of total RNA extracted from dental epithelium (a) and alveolar bone (c, e) revealed <i>Rank</i> expression in the epithelium and alveolar bone of <i>Msx2</i>^−/−<i>Rank^Tg</i> mice. Increased <i>Rankl</i> expression was associated with <i>Rank</i> overexpression in <i>Msx2</i>^−/− mice (a, c). In <i>Msx2</i>^−/−<i>mice</i>, <i>Rank</i> overexpression induced a decrease in <i>Opg</i> transcriptional activity in the epithelium, while in alveolar bone, expression of <i>Opg</i> increased slightly (a, c). Expression levels of <i>Runx2</i> and <i>Ocn</i> in alveolar bone were unaffected by <i>Rank</i> overexpression (c), but expression of the T lymphocyte marker <i>Cd3e</i> and the monocyte and macrophage markers <i>Csf1r</i>, <i>F4/80</i> and <i>Cd11b</i> were increased (e). Also shown are 4-fold or higher increases in gene expression in <i>Msx2</i>^−/−<i>Rank^Tg</i> mice compared to <i>Msx2</i>^−/− mice not expressing <i>Rank^Tg</i>, as quantified by RT-qPCR TaqMan arrays in dental epithelium (b) and alveolar bone (d).</p

Combined effects of loss of <i>Msx2</i> and <i>Rank</i> overexpression on mouse mandibular bone phenotype.

<p>Microradiographs and scans of 16-week-old <i>WT</i> (a), <i>Msx2</i>^−/− (b), and <i>Msx2</i>^−/−<i>Rank^Tg</i> (c) mouse skulls were performed to compare characteristics of the bone of the mandible. While the <i>Msx2</i>^−/− mouse mandibular features (b) presented no major alterations compared to WT animals (a), <i>Msx2</i>^−/−<i>Rank^Tg</i> mice had marked disruptions in the architecture of the mandibular bone (c). These disruptions were either mono- or bilateral and were associated with conversion of the incisor epithelium toward massive osteolytic tumors (asterisks in c). Basal bone around these tumors was thinner (arrows in c), porous (arrow in c), and displaced, as seen in the upper view of the mandibular scan (c).</p

Additional file 1: of Translation and validation of the French version of the Child Perceptions Questionnaire for children aged from 8 to 10 years old (CPQ 8-10)

Final french version of the (CPQ 8-10) questionnaire. (DOC 73 kb

Cytoskeleton and major extracellular matrix proteins were analyzed by immunofluorescence.

<p>Actin was observed in DPSC (A), DPSC with BD (B) and in DPSC with BR (C); vimentin and tubulin (D, E and F); and type 1 collagen and fibronectin (G, H and I). The observed decrease in type 1 collagen, when DPSC were in contact with biomaterials, was confirmed using RT-qPCR (5J), whereas protein secretion was not modified by the experimental conditions (Fig 5K). Scale Bar: 100μm.</p

Media used in the study.

<p>Media used in the study.</p

Composition and properties of the biomaterials.

<p>Composition and properties of the biomaterials.</p

Primer sequences used for the study.

<p>Primer sequences used for the study.</p