Immunolocalization of Smad-4 in developing molar roots of alendronate-treated rats

Abstract Introduction During root formation, Smad-4 plays a key role during the epithelial–mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate. Methods Newborn Wistar rats were daily injected 2.5 mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope. Results In ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation. Conclusion The immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation.