Cellular and molecular mechanisms during the early development of rat alveolar bone

Abstract

Bone tissue, a specialized connective tissue, is constituted by different cell types (lining cells, osteoblasts, osteocytes and osteoclasts) and a mineralized extracellular matrix. The periodontium comprises the structures responsible for maintaining the tooth in the maxillary and mandibular bones and consists of: cementum, periodontal ligament, alveolar bone and gingiva. The alveolar process is formed concomitantly to the development and eruption of teeth; its formation begins in the fetal life, with mineral deposition on mesenchymal matrix surrounding the tooth germs. The alveolar bone, which covers internally the alveolar process, is formed from the ectomesenchymal cells from dental follicle. Furthermore, alveolar bone formation occurs only after the initial root formation. Runx2 is a specific regulator of osteoblast differentiation and Osterix, a important transcription factor in the bone tissue formation. Although alkaline phosphatase is considered as a osteoblast marker, it is also expressed in pre-osteoblasts (osteoblast in differentiation). On the other hand, it has been suggested that osteocalcin is expressed in mature osteoblasts. Considering that alveolar process and alveolar bone are formed at different period, although they are closely associated, we intend to investigate the immunoexpression of osteoblast markers at different phases of these tissues formation. Furthermore, we intend to evaluate the initial phase of bone resorption of the process/alveolar bone and if there is a correlation between the phenomenon of apoptosis of osteoblasts and osteocytes and bone resorption. Thus will be used fetuses of pregnant rats that will be killed at 16, 18 and 20 days of pregnancy, and rats with 5, 10 and 15 days of age (newborn) also will be used. After sacrifice, the heads of embryos and newborns will be collected, trimmed and immediately fixed in formaldehyde. After descalcification, the fragments will be dehydrated and embedded in paraffin. The 6¼m sections, containing the process/alveolar region of the 1st molar, will be stained with hematoxylin & eosin (HE) or Masson's trichrome for morphological analysis. Some sections will be submitted to immunohistochemistry reactions for alkaline phosphatase, Runx2, Osterix, osteocalcin, VEGF and Ki-67 detection; the TUNEL method for detection and quantitation of osteoblasts and osteocytes apoptosis will be performed. Other sections will be submitted to histochemical reaction for tartrate resistant acid phosphatase (TRAP), used as an osteoclast marker, and the number of TRAP-positive osteoclasts in the linear alveolar bone surface will be estimated. Maxillar bone fragments containing the process/alveolar bone of the first molars will be immersed in glutaraldehyde/formaldehyde solution and embedded in araldite for ultrastructural analysis. Fragments will be fixed in glutaraldehyde/formaldehyde, dehydrated and embedded in LR White, and the ultrathin sections will be sumitted to immunocytochemistry for osteocalcin detection. (AU)