Delayed posterior frontal suture closure in <i>Ambn</i> overexpressing mice.

<p>(A and B) Whole mount alcian blue/alizarin red staining of wildtype (WT) (A) and <i>Ambn</i> transgenic (TG) (B) animals at embryonic stage day 18. The interfrontal gap was wider in the transgenic skulls compared to controls. (C and D) Higher magnification of the skulls shown in A and B. Note that the margins of the frontal bone osteogenic front were not well defined, and the interfrontal and parietal spaces were wider in transgenic mice compared to controls. (E and F) Dried skulls of WT and TG animals at 35 days postnatal. The PF suture was closed in WT mice (E), but remained patent in transgenic mice (F). (G and H) Higher magnification of the skulls shown in A and B. In TG mice, sutures remained open and adjacent bones overlapped (H). Cross-sections of the PF suture revealed the ossified areas of WT mice (I) and TG mice (J) at age 35 day postnatal. The PF suture was sealed by ossification in WT mice, while the left and right parietal bones were linked by soft tissue in TG mice.</p

Weight and size comparison between wildtype and <i>Ambn</i> transgenic mice dried skulls.

<p>Dried skulls were prepared from 60 days postnatal wildtype (WT) and <i>Ambn</i> transgenic (TG) mice. Weight of dried skulls (A), width of frontal bone and suture (B), length of interfrontal suture (C) and thickness of frontal bone (D) were measured, and values were graphed as mean+/− standard deviation (SD). * represents significant difference, p<0.05 (Kruskall-Wallis one-way analysis). (E) Anatomical reference points for skull morphological measurements, including width of frontal bone and suture (a), length of interfrontal suture (b), and overall skull length (c).</p

Presence of miRNA in healthy human crevicular fluid.

<p>“gel-like” images of R6K ScreenTape (A) after subjected to Tapestation bioanalyzer showed RNA ladder (L) on left lane and only small-sized RNA present in the sample lanes (middle and right lane). The positive bands approximately 50 nucleotides (nt) were present in representative GCF samples (A1 and B1). Electropherogram (B) corresponding to the gel-like images on the (A) figure. The <i>x</i>-axis on the electropherogram represents RNA size (nt), while the <i>y</i>-axis represents the measurement response of fluorescence units (FUs).</p

Secretory microRNA-29 expression in gingival crevicular fluid during orthodontic tooth movement

<div><p>Secretory microRNAs (miRNAs) have been used increasingly as biomarkers for cancers, autoimmune diseases and inflammatory diseases. They are reported as being freely circulated or encapsulated in microvesicles such as exosomes. This study was performed to elucidate the presence of miRNAs with exosomes in human gingival crevicular fluid (GCF), and the expression profile of <i>miRNA-29</i> during orthodontic tooth movement. Four healthy volunteer and fifteen orthodontic patients were enrolled in the study. Secretory miRNA in GCF was collected and analyzed using a bioanalyzer, realtime PCR and Western blot analysis. The expression profile of secretory <i>miR-29</i> family in GCF was analyzed during the course of canine retraction for 6 weeks. The results demonstrated the presence of miRNAs in the GCF. After series of ultracentrifugation and RT-PCR array, exosome-depleted fractions and pellets were isolated and we found that secretory miRNAs were detected in both the exosome-associated fraction and the exosome-depleted supernatant fraction; however, the concentration of miRNAs was higher in the exosome-associated fraction than in the exosome-depleted fraction suggesting a close association between the secretory miRNAs and exosomes in GCF. We also demonstrated the increased expression profiles of <i>miR-29</i> family during six weeks of orthodontic tooth movement in humans. Secretory miRNAs are present in GCF and secretory <i>miRNA-29</i> family expression profiles increase during the tooth movement in humans. Secretory <i>miRNA-29</i> in GCF could serve as potential biomarkers for periodontal remodeling.</p></div

Distribution of miRNAs in exosome pellet and exosome-depleted supernatant fractions from crevicular crevicular fluid.

<p>Crevicular fluid miRNAs in all subjects are predominantly in exosomes; however, some miRNAs are present in exosome-depleted supernatant (A). The higher CT cycles are detected in exosome-depleted supernatant than in exosome pellet after normalized with amount of RNA (ng) (B) and the ΔCT is the difference between CT of supernatant-CT of exosome pellet after normalization with let-7d and g. Positive numbers show higher concentrations in the exosome pellet whereas negative numbers indicate higher concentrations in the exosome-depleted supernatant (C).</p

Ambn expression in cranial bone and dura mater.

<p>(A) RT-PCR analysis of Ambn mRNA expression in the posterior frontal suture region from 1, 3, 10, and 20 days postnatal WT mice. The β-Actin gene was used as internal control. (B) Western blot analysis of AMBN protein expression in postnatal day 3 posterior-frontal (PF) suture tissues and teeth. Two bands at 55 and 50 kDa were recognized. β-Actin was used as loading reference. (C) Immunostained sections of cranial bone and dura mater from 3 day postnatal wildtype (WT) mice demonstrated Ambn protein localization in calvarial bone (cb), dura mater (dm), and condensed mesenchymal cells (me). Note the high levels of Ambn protein in the dura mater and in calvarial osteoblasts. Bar = 20 µm.</p

Electron microscope images and Western blot of isolated exosome from healthy human gingival crevicular fluid.

<p>Electron microscopy (A and B) of the ultracentrifugation pellet from GCF shows the characteristic spherical shape and size (50-100nm) of exosomes, Western blot (C) shows strong staining of the ultracentrifugation pellet with the exosomal membrane markers anti-CD63 and anti-CD9.</p

Relative fold change (ΔT) of each studied microRNA at different timepoints.

<p>Relative fold change (ΔT) of each studied microRNA at different timepoints.</p

The expression profile of miRNA-29 family in GCF during tooth movement in human.

<p>The expression of miRNA-29a, -29b, and -29c were shown as gradually increase profile toward the last timepoint (6-wk). The significant differences are detected between T0 (pretreatment) and T4 (6-wk) in all studied miRNAs. Note that the significant differences of miRNA-29b expression are detected between T0 and T1 (1-hr), T0 and T3 (7-day) (P<0.05).</p

Role of Ambn in suture development.

<p>(A and B) Cross-sections of the PF suture revealed the ossified areas of wildtype (WT) and Ambn transgenic (TG) mice at age of 35 days postnatal. The PF suture was fused by ossification in WT mice (A), while the left and right parietal bones were interrupted by soft tissue in TG mice (B). (C and D) BrdU labeling of proliferative cells in developing sutures. Note the accumulation of BrdU-labeled cells in the suture center of WT mice (C) compared to TG mice (D). (E and F) TUNEL staining in developing sutures. Note the red labeling for TUNEL-positive cells in WT and TG mice. (G and H) DAPI counterstaining of (E and F).</p