Losartan improves alveolar bone dynamics in normotensive rats but not in hypertensive rats

Abstract Hypertension is one of the main causes of premature death in the world; also, it is associated with several bone alterations. Preclinical studies have demonstrated delayed alveolar bone healing in hypertensive rats. However, losartan has been favorable for consolidation of bone grafts and reduction in active periodontitis. Therefore, losartan is suggested to be effective in bone formation stages, as well as in the synthesis of matrix proteins and mineralization. Objectives: To evaluate the alveolar bone dynamics in hypertensive rats treated with losartan by laser confocal microscopy and histological analysis. Methodology: Thirty-two rats, 16 spontaneously hypertensive rats (SHR) and 16 Wistar albinus rats, treated or not with losartan (30 mg/kg/day) were used. Calcein fluorochrome at 21 days and alizarin red fluorochrome at 49 days were injected in rats (both 20 mg/kg). The animals were submitted to euthanasia 67 days after treatment, and then the right maxilla was removed for laser confocal microscopy analysis and the left maxilla for histological analysis. Results: This study showed a greater calcium marking in normotensive animals treated with losartan in relation to the other groups. Laser confocal microscopy parameters showed higher values of bone volume formed, mineralized surface, active surface of mineralization and bone formation rate in normotensive animals treated with losartan. However, a smaller mineralized surface was observed in all hypertensive animals. Conclusion: Losartan can improve bone mineralization parameters under normal physiological conditions, but the same anabolic effect does not occur under hypertension.


Introduction
Hypertension is a chronic disease with the highest number of premature deaths and care assistance in the world. 1,2 Its high prevalence reaches about one in three young adults in the world, and this number is estimated to increase. 3 In addition to cardiovascular diseases, hypertension is associated with chronic renal failure, stroke, and bone abnormalities, such as: abnormal calcium metabolism, 4 altered alveolar bone quality, 5 delayed alveolar bone healing, 6 bone density loss, 7 increased risk of fractures 8 and, consequently, osteoporosis. 9 The question arises whether bone changes may be associated with the vasoconstriction present in hypertension. 10 Thus, plasma calcium supply would be reduced, impairing alveolar bone mineralization. Finally, alveolar bone dynamics can interfere direct in the success of clinical procedures, such as: tooth extraction, consolidation of bone grafts and implant osseointegration. Renin-angiotensin system inhibition is a target for hypertension control. 11 Then, angiotensin II receptor blockers can prevent vasoconstriction and high blood pressure. 12 In addition, angiotensin II enhances bone resorption by increasing RANKL and osteoclastic activation. 6,13,14 Also, it has effects of osteoblastic lineage changing, 15,16 by decreasing the expression of osteogenesis-related transcription factors via the AT1 receptor, such as Runx2,Msx2,and osteocalcin. 15 Losartan, as an angiotensin II receptor blocker, 17 has attracted interest not only for being a vasodilator, but also for its positive effects in bone metabolism. 18,19 Preclinical studies have reported benefits of losartan on bone fracture healing and reduction in bone fracture risk. 20 They also reported improved bone graft healing. 10 In in vitro 18 and in vivo studies, 21 losartan significantly enhanced bone density by decreasing osteoclastogenesis and increasing osteoblastic activity. Therefore, a topic to be evaluated referred to how antihypertensive drugs interfere in alveolar bone dynamics.
The action of losartan in the renin-angiotensin system, and its consequent influence on bone metabolism, is widely studied. 19,22,23  Fluorochrome application 21 days after beginning drug treatment, the fluorochrome calcein was administered intramuscularly (20 mg/kg). Alizarin red fluorochrome was also used intramuscularly at a dose of 20 mg/kg for each animal after more 28 days, according to previous studies. [25][26][27] The first injected calcein fluorochrome indicates calcium deposition in old bone and the subsequent alizarin fluorochrome in the newly-formed bone.

Euthanasia
All animals were submitted to euthanasia 67 days after drug treatment with a intravenously lethal excessive anesthetic dose (60 mg/kg, Tiopental Cristália Ltda; Itapira, SP, Brazil). Therefore, this experimental period is adequate to characterize the modeling or remodeling process of old bone into new bone indicated by fluorochormes and histology.

Laboratory processing
The right side of the maxilla was removed and Overlapping of the two layers of fluorochromes was performed, representing calcium deposition in both periods ( Figure 1C). Therefore, this overlap shows the conversion of old bone into new bone.
Images were saved in TIFF format and moved to the Image J program (Processing Software and Image Analysis, Ontario, Canada). Using the "color selection" tool, each image was standardized according to hue, saturation, and brightness. Both bone types were observed in the same setting on a single slide for measure the parameters: Bone volume formed,

Statistical analysis
The GraphPad Prism 7.0 profiler (GraphPad Software, La Jolla, USA) was used for statistical test.
Shapiro-Wilk test (p<0.05) was performed to verify homoscedasticity and whether the results were parametric. After confirming the normal distribution, Anova test was performed followed by Tukey post-test for multiple comparisons when necessary. For all tests, the p-value considered as significant was p<0.05.

Results
Systolic blood pressure and body weight