## 4 Discussion
Before starting the statistical analysis, we verified that all the concentrations obtained were higher than the LoD of the analytical method, a concentration characterized by a high variability. The data reported on the analytical variability were verified with the EP15A2 protocol; however, the awareness remains that in the calculation of biological variability, unlike the pre-analytical variability, this cannot be completely removed. Components related to biological variability were typically specific for each analyte of bone metabolism and showed a tendency to vary between subjects. The variations of the values obtained in the single subject (CVI), which are physiological in the saliva of the biomarker concentrations, are the result of a dynamic control and attributable to individual biological factors, reflect the random fluctuation of the analyte concentration around the homeostatic set point and were specific for individual biomarkers: P1 PN (5.6%), TRAcP 5b (20%) and PTHrP (23%), respectively. Biological variation between subjects (CVG) i.e., the difference between homeostatic set points for the same analyte in different individuals gave different values for P1 PN (18%), TRAcP 5b (35%) and PTHrP (19%9) respectively. The differences found in the expression of the components of the biological variability of P1 NP, TRAcP 5b and PTHrP may be an expression of the different mechanisms of action in the bone regeneration process that occurs in the growth phase. Bone is a dynamic and metabolically active tissue that is continuously remodeling by the coordinated actions of osteoclasts and osteoblasts in cellular packages. PINP is an indicator of bone formation and is produced during the formation of the bone matrix by cleavage of type I collagen; TRAcP 5b reflects the number of osteoclasts and is correlated to bone resorption; PTHrP instead intervenes in the physiological regulation of bone remodeling (endocrine mechanism) but it is also locally produced (paracrine function) by osteoblast progenitors; it promotes the differentiation of mature osteoblasts by inhibiting the apoptosis process that allows bone formation; moreover it stimulates the differentiation of the osteoclasts that is responsible of the bone resorption [35]. PTHrP is present in the enamel organ and is responsible for the formation of the eruptive pathway [36]. This observation has inspired various studies that have focused on the role of PTHrP in mediating osteoclastogenesis [37]. In subjects in the development age, the values on the biological variability of PTHrP in saliva, the low individuality, the higher critical difference with respect to the other evaluated biomarkers and the concentrations with secretion dynamics similar to those of the serum could be explained according to the constant local paracrine production related to the need for a regulatory mechanism on bone formation and tooth growth [35; 36; 37; 38]. P1 NP and TRAcP 5b [38] show high individuality (individuality index (II) \(<\) 0.6) equal to 0.12 and 0.56 respectively; the variation of values for each individual assessed occupies only a small part of the distribution of values for the whole group. In these cases, there is the risk of considering the value of an analyte in an individual to be improperly physiological and significantly far from its homeostatic set point but still included within the range of values obtained. On the contrary, the PTHrP shows low individuality and the dispersion of value in each individual covers most of the dispersion between individuals (ranging from 0.33 ng/mL to 0.90 ng/mL) making it useful for the interpretation of determinations a greater variation than 68.44% [36; 37; 38; 39]  
The evaluated calculation of the biological variability of the bone biomarkers was of particular interest as it provides parameters for the calculation of the critical difference (RCV). RCV is of particular interest in evaluating variations in the concentrations of bone biomarkers on salivary matrix during pathology and/or dental therapy.