Methods: Forty male adult Holtzman rats were subjected to orchiectomy to suppress testosterone production (Ocx). Twenty of the rats received 250mg/kg testosterone esters (Tes). An additional twenty rats served as sham-operated controls. Experimental periodontitis was induced in ten animals in each group by ligature placement around the lower first molar teeth 30 days post-surgery and maintained for 15 days, while the remaining 10 animals in each group served as controls. Changes in alveolar bone height were radiographically quantified. Serum levels of Ca+2, alkaline phosphatase (ALP), cytokines and chemokines were measured. For evaluation of the impact of testosterone on osteoclastogenesis in vitro, RAW 264.7 cells were used as osteoclast (OC) precursors and OC RANKL-mediated differentiation and activity were evaluated in the presence or absence of 1nM, 10nM, 100nM or 1µM testosterone. Flutamide (100nM) was used as a specific blocker to assess the role of androgen receptors. One-way ANOVA or Student’s t- test was used for statistical analyzes as indicated.
Results: Mean serum testosterone levels were significantly reduced in the Ocx group (0.2±0.01 ng/ml) and increased in the Tes group (29.96±3.3) when compared to sham controls (1.2±0.23; p<0.05). In the absence of periodontitis, there was significant bone loss in the Tes group (0.74±0.04 mm) when compared to Ocx (0.49±0.07) or Sham (0.55±0.05; p<0.05). Ocx alone increased serum IL-2 levels (+90%) and Tes increased IL-1β (+174%), IL-2 (+200%) and IFN-γ (+73%). Ligature-induced periodontal disease resulted in significantly higher bone loss in the Ocx group (1.69±0.14) compared to Sham controls (1.32±0.09; p<0.05). Periodontitis also led to a significant reduction in ALP levels in Ocx compared to Sham controls (p<0.05), and diminished IL-10 (-44 and -47%) in Ocx and Tes groups, respectively. A decrease in IL-4 (-52%) and an increase in IL-1β (+173%) accompanied these changes in the Tes group. In vitro, testosterone dose-dependently inhibited OC numbers and area (p<0.05), although the highest dose resulted in an increase in OC formation. The addition of flutamide rescued the 100nM testosterone inhibition restoring the number of OC.
Conclusions: Sub- and supraphysiological testosterone levels predispose to inflammatory changes with a significantly higher periodontal destruction in rats. These clinical observations are accompanied by cytokine/chemokine and ALP modulation. Testosterone presents an optimal range (10-100nM) to reduce OC formation and activity in vitro, which is mediated, at least in part, via the androgen receptor.