The contact of the bone that invaded the dental follicle space in the ALN specimens may have occasioned the earlier immunoexpression of Smad-4 by dental follicle cells, since the bony crypt is the source of signalling molecules: the cervical portion CHIR99021 of the dental follicle expresses BMP-2,24 while the basal alveolar crypt bone expresses TGF β-1 during the molar root formation of young rats;25 these molecules exert signalling functions over the dental follicle cells to induce their differentiation into cementum secreting cells, which was confirmed by the immunodetection of Smad-4 in the present study. These events are coordinated by the dental follicle,1 and 11 which

has been severely disrupted due to the effects of alendronate treatment on bone remodelling. However, despite the evidences of TGF-β/BMP signalling in ALN specimens, confirmed by the positive immunolabelling for Smad-4 BTK inhibition at all time points, it apparently did not stimulate HERS

cells and the downstream cascade of root odontoblast differentiation, as well as root elongation. Indeed, it was detected several TUNEL-positive odontoblasts and ectomesenchymal cells in the dental papilla of ALN unerupted molars at 30 days. Additionally, sodium alendronate increases the amelogenin deposition within the forming enamel and even promotes its accumulation into mantle dentine during crown formation of rat molars.18 As small amounts of amelogenin are secreted by epithelial diaphragm cells during the differentiation of ectomesenchymal cells into root odontoblasts,26, 27 and 28 it is possible that alendronate may interfere in the root formation, besides the above commented effects on dental follicle. The present findings indicate that resorption of the basal portion of the bony crypt is necessary to root formation. Smad-4

is a marker of the differentiation of ectomesenchymal cells from the dental follicle into the cementum secreting cells Flavopiridol (Alvocidib) cementoblasts and fibroblasts, which occurs in alendronate-treated tooth germs despite the disruption of dental follicle and HERS. These results contribute to the current understanding of periodontal development, as well as to the understanding of bisphosphonate therapy of young patients suffering bone diseases such as osteogenesis imperfect, Paget’s diseases, and others, for the risk of disturbing the tooth development, eruption and root formation. The authors declare no conflict of interest in this study. This study was authorized by the Ethical Committee for Animal Research of the University of São Paulo, Brazil (Protocol # 16/2007). This research was supported by the São Paulo Research Foundation (FAPESP – grants 06/60094-5 and 09/54853-9) and the National Council for Scientific and Technological Development (CNPq) – Brazil.