_Applied_  
_sciences_  
_Article_  
Quantification of Salivary Nitric Oxide in Patients with Fixed Orthodontic Treatment  
**[PERSON] 1, [PERSON] 2, [PERSON] 3,4,5,6,***,  
and microfractures, periodontal problems, and intra- and extra-oral lesions [1]. Lately, aligners have been developed as a less visible, less irritating, easier to clean and more comfortable treatment than conventional fixed therapy. The characteristics of the forces transmitted by the aligners depend largely on the properties of the materials used, their thickness and profile complexity, the degree of activation, and the use of accessory elements. Thermoplastic devices made of thicker materials produce a higher force, and thinner ones a lower force [2].  
Nitric oxide (NO) is a pleiotropic biological mediator involved in intestinal motility, platelet aggregation and adhesion, bone tissue formation and destruction, apoptosis, neurotransmission, the regulation of vascular tone, and immunological functions [3, 4]. It has a short in vivo half-life (a few seconds or less). Thus, levels of more stable metabolites such as nitrite (NO2\({}^{-}\)) and nitrate (NO3\({}^{-}\)) anions have been used for the indirect quantification of NO in biological fluids. Altered NO levels have been associated with septic status, sexual intercourse, infections, hypertension, exercise, type II diabetes mellitus, hypoxia, and cancer [4, 5].  
Since it is fat-soluble, NO is not stored but is synthesised de novo and diffuses freely across lipid membranes. NO has the potential to exert its effects on target cells through various mechanisms. For example, the NO-mediated activation of the enzyme guanylyl cyclase (GC) catalyses the formation of the secondary messenger 3\({}^{\prime}\),5\({}^{\prime}\)-cyclic guanosine monophosphate (cGMP). cGMP is involved in several biological functions such as the regulation of smooth muscle contractility, cell survival, cell proliferation, axonal guidance, synapse plasticity, inflammation, and angiogenesis [5].  
Nitric oxide is also considered a regulator of bone response to mechanical stress as it mediates adaptive bone formation (increased levels reduce osteocl activity and vice versa) and the pathological effects of lipopolysaccharidesaccharides (LPS), tumour necrosis factor (TNF), interleukin 1 (IL-1) and other cytokines; regulates leukocytes and epithelial cell adhesion; inhibits T cell proliferation; and enhances natural killer (NK) cell activity, among other immune-related processes [6, 7].  
Given that any local oral change involves oxidative stress, we hypothesised that salivary nitric oxide may be a specific and sensitive marker for changes produced by orthodontic appliances.  
Saliva is easy to collect and store and contains high quality DNA. However, research on its usage for diagnostic purposes is still in its early stages, and progress is limited by the lack of strict study protocols that allow a direct comparison of results from several independent laboratories [1].  
The aim of the study is to quantify salivary NO levels by both rapid and specific methods and to demonstrate that it is a valid, specific, and sensitive biomarker for bone changes determined by the application of different types of braces. Moreover, the study aims to highlight the impact of fixed orthodontic equipment on the oxidative status of the oral cavity in comparison to thermoplastic aligners, as fixed bracers exert stronger forces and more friction on the oral soft tissue, which can cause inflammation, lesions, and a shift in the oxidative status of the oral cavity.