Table 1.
The primer pairs for RT-PCR.
Figure 1.
Light micrographs illustrating root development and Hertwig’s epithelial root sheath (HERS) in postnatal day 15 mice.
(A) Hematoxylin and eosin staining of the mandible. (B) Immuno-staining with ameloblastin (AMBN). AMBN is localized in ameloblasts of the outer enamel epithelium in the first and the second molars (arrow) and incisor (arrow-head). (C) Hematoxylin and eosin staining of the distal root of the lower first molar. (D) Immuno-staining with cytokeratin 5. Ameloblasts, epithelial cell rests of Malassez, and HERS positively reacted against cytokeratin 5 immunohistochemistry. (E) Immuno-staining with AMBN. Ameloblasts around enamel (asterisk) and in the basal portion of HERS (arrow) reacted with AMBN antibody, but not in the tip (arrowhead). am, ameloblasts; od, odontoblasts; en, enamel; de, dentin; pl, periodontal ligament; erm, epithelial call rests of Malassez; HERS, Hertwig’s root sheath. Red circle marks the location of HERS. Scale bars: 500 µm in (A) and (B), and 100 µm in (C) – (E).
Figure 2.
Expression of AMBN in HERS derived cells and suppression of AMBN expression by treatment with AMBN siRNA.
HERS derived cells were isolated from postnatal day 10 mice and maintained in MCDB153 medium. (A) Immuno-staining with AMBN. (B) Immuno-staining without AMBN. (C) Immuno-staining with cytokeratin 5. (D) Immuno-staining without cytokeratin 5. Scale bars: 10 µm. (E) Expression of AMBN was analyzed with total RNA from cells treated with either AMBN siRNA or control siRNA. MW; 100 bp molecular weight marker; C, treated with control siRNA; Si, treated with AMBN siRNA. Expression of AMBN was inhibited by 60% when treated with AMBN siRNA. (F) The BrdU incorporation rate of control siRNA group was higher than that of the siRNA treated group two days after siRNA treatment. Statistical analysis was performed using Student’s t-test, **; p<0.01.
Figure 3.
Histological observation of the lower first molar treated with siRNAs.
Distal root length of the lower first molar in AMBN siRNA treated was shorter than that of controls. (A) Stereomicroscopic appearance of the lower first molar treated with AMBN siRNA. (B) Hematoxylin and eosin staining in control and AMBN siRNA. Treatment with AMBN siRNA caused irregularities in dentin form at the tip of the root. (C) Average root length in control and AMBN siRNA treated groups. Root length in treatment with AMBN siRNA (n = 10) was shorter than that of control siRNA (n = 10) (Two headed arrow indicates root length in Figure B.). Statistical analysis was performed using Student’s t-test, *; p<0.05.
Figure 4.
Light micrographs illustrating HERS treated with siRNAs.
AMBN siRNA injected mice revealed a multilayered appearance in the basal portion of HERS and increased BrdU positive cells in that of the outer layer. Treatment with control siRNA (A–C) and AMBN siRNA (D–F). (A) and (D) Hematoxylin-eosin staining. (B) and (E) Immuno-staining with cytokeratin 5. Treatment with AMBN siRNA revealed a multilayered appearance in the basal portion of HERS (arrow). (C) and (F) Immuno-staining with AMBN. Treatment with AMBN siRNA increased BrdU positive cells in the outer layer of HERS (arrows). de, dentin; od, odontoblasts; pl, periodontal ligament; HERS, Hertwig’s root sheath. Red dotted lines mark the location of HERS. Scale bars: 100 µm. (G) The percentage of BrdU positive cells per total HERS cells was counted and compared between treatments with control and AMBN siRNA. The percentage of BrdU positive cells was higher with treatment with AMBN siRNA (n = 10) than that with control siRNA (n = 10). Statistical analysis was performed using Student’s t-test, *; p<0.05.
Figure 5.
Comparison between AMBN expressing and non expressing cells.
Full length AMBN cDNA transfected cells expressed AMBN at a high intensity whereas control cells had no AMBN mRNA. (A) and (B) Positive cell cycle regulatory factors, CDK1, CDK4 and CDK6, and negative cell cycle regulatory factors, p21Cip1 and p27Kip1, were examined by RT-PCR analysis. AMBN non expressing cells had lower expression levels of negative cell cycle regulators than those of AMBN expressing cells. (C) BrdU positive cells were counted and compared between AMBN expressing and non-expressing cells. Cell numbers in the AMBN non-expressing group was lower than those of the AMBN expressing group. Statistical analysis was performed using Student’s t-test, **; p<0.01.