Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia.

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via ß2-adrenoceptor (ß2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that ß2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (µCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.

Effects of fluoride in bone repair: an evaluation of RANKL, OPG and TRAP expression.

The objective of this study was to evaluate comparatively the effect of fluoride in the expression of the receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG) and tartrate-resistant acid phosphatase (TRAP) in alveolar bone repair in rats. We used 3 groups of male Wistar rats (n = 5/group), which received drinking water containing different doses of F (NaF): 0, 5 and 50 ppm, for 60 days before the incisors extraction. The upper incisors were extracted and the animals were killed 7, 14, 21 and 30 days after extraction. The hemi-maxillae were collected for microscopic examination (histomorphometric and immunostaining for RANKL, OPG and TRAP). Histomorphometric analysis confirmed an increase in the volume density of neoformed bone between 7 and 30 days for groups control, 5 and 50 ppm of F, with a concomitant decrease in the volume density of connective tissue and blood clot. Higher blood clot for groups 5 and 50 ppm of F at 30 days was observed. The RANKL and OPG expressions were not changed by chronic exposure to fluoride in the drinking water during the studied periods; on the other hand, TRAP expression was changed (at 7 days) by chronic exposure to fluoride (p < 0.05). It was concluded that F in high concentrations can slow the blood clot remission and bone repair, and alter the TRAP expression in the beginning of the bone tissue repair. However, a better understanding about this blood clot remission phenomenon is required.

Fluoride effect on the process of alveolar bone repair in rats: evaluation of activity of MMP-2 and 9

Objective: The aim of this study was to evaluate comparatively the effect of fluoride (F) on the activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) involved in process of alveolar bone repair. Material and methods: This study used 4 groups of Wistar rats with 80 days of life (n = 160) which received drinking water containing different doses of fluoride (NaF): 5, 15, 50 ppm and deionized water (control) throughout the experiment. These animals had their right upper incisors extracted. After extraction, the animals were euthanized at 7, 14, 21 and 30 days and the hemi-maxillae were collected for microscopic analysis (Hematoxylin and Eosin and immunohistochemistry for MMP-9) and zymography (MMP-2 and 9).Results: Microscopically the process of bone repair was similar in all groups, being noted only a delay of the blood clot resorption and bone formation in the group of 50 ppm F. The expression for MMP-9 showed differences betweengroups only during the initial repair (7 days). However, the zymography showed no significant differences between treated and control groups. Conclusion: Ours results suggest an effect of fluoride on the activity of MMPs 2 and 9 at the initial period of alveolar repair which could be associated to the process of blood clot remission and delay in bone repair. Further studies are needed to establish the relationship between the initial process of resorption of the blood clot, and the involvement of MMPs 2 and 9 and its regulators/tissue inhibitors.