**1. Introduction**

Recent studies show that changes in the oxidative state and the regulation of redox homeostasis affect bone turnover and remodeling [1–3]. Excessive production of reactive oxygen species (ROS), not counterbalanced by endogenous antioxidant defense systems, induces oxidative stress with consequent abnormal osteocyte apoptosis, which activates the osteoclasts and inhibits osteoblast osteogenic activity [1,4–6]. This is related to estrogen deficiency, aging, or bone inflammatory processes in which oxidative stress induces low bone mineral density and loss of bone mass [3,7–9]. A few recent clinical studies showed that an imbalance at a cellular level between ROS and antioxidants seems to be involved in the pathophysiology of bone-related diseases [1,10–12]. Oxidative stress and a decreased reduced glutathione/oxidized glutathione (GSH/GSSG) ratio are associated with the inhibition of osteoblast differentiation and the mineralization process and alter the levels of specific osteogenic markers [13]; furthermore, the ROS increase activates osteoclast differentiation [10,14]. These events are often associated with bone metabolic diseases such as osteoporosis, the most common bone disease, in which oxidative stress is considered a significant risk factor for its development [7–9]. Increased ROS production is responsible for diverting bone precursor cell differentiation toward the formation of other cell types rather than bone tissue cells [15,16]. On the contrary, antioxidants counteract these negative effects and favor the activity of osteoblasts, the viability of bone stem cells, and the maintenance of a normal bone remodeling process [1,3,6,11,12,16].

Literature data obtained in animals or cell lines showed the antioxidant activity of various natural substances [1]. In particular, diets rich in vegetables and/or fruits with high content of active antioxidants, such as polyphenols including flavonols, isoflavones, and phytosterols/phytoestrogens, can have an important role in prevention and/or managemen<sup>t</sup> of osteoporosis and bone inflammatory diseases related to oxidative stress [17–21]. Indeed, plasma antioxidant content was found diminished in osteoporotic women [11,22]. These antioxidant compounds scavenge ROS [1,6,16,23,24] and reduce oxidative stress in many diseases including bone diseases and, in particular, osteoporosis [17,22,24–27]. Many literature data sugges<sup>t</sup> a positive relationship between antioxidant intake and prevention of bone loss often associated with increased bone fractures [18,20,24,27]. For this, osteoporosis is considered a highly debilitating and socially relevant pathology; in fact, among the elderly, the pathological consequences of osteoporosis are among the main causes of mortality [28,29]. Recently, it was shown that diets containing blueberry (BB) prevent osteoporosis in ovariectomized rats [19,26,30]; indeed, BB and, in particular, *Vaccinuim myrtillus* (VM) have a wide variety and high concentrations of well-characterized polyphenols such as anthocyanins, coumarins, flavonols, flavanols, and their phenolic derivatives [31,32], with beneficial properties in bone anabolism [17–21]. Moreover, recent studies sugges<sup>t</sup> VM as a "functional food" and, as such, of benefit for dietary supplementation [31,32]; today, VM, together with *Vaccinium corymbosum*, is among the main species of BB used in the food industry. Recently, we demonstrated that blueberry juice (BJ), mainly obtained from VM, exhibits osteogenic action, through its antioxidant and antiosteoclastogenic effect, in murine osteocytes, MLO-Y4. Moreover, it shows a protective effect in bone marrow mesenchymal stromal cells (MSCs), fundamental for cell therapy in bone diseases, by preventing oxidative stress-induced toxicity [33].

The aim of this study was to evaluate the effect of BJ containing certain quantities of soluble polyphenols on the factors related to differentiation and the mineralization process of osteoblasts in the presence or absence of oxidative stress. In fact, the role of these dietary polyphenols on osteogenic activity of osteoblasts and on redox-regulated molecular processes involved in bone formation and regeneration is still little known. In particular, no data are reported on the molecular mechanisms involved in the protective action of BJ phytochemicals against oxidative stress-induced damage on osteogenic activity of osteoblasts. Moreover, it is interesting to assess effects of complex mixture of phytochemicals on osteoblast activity, considering that individuals consume fruit and vegetables rich in a variety of polyphenols. Indeed, some studies show that various polyphenols and their derivatives are bioavailable from BB and they are also absorbed in humans in intact form [34–36]. Finally, this study was performed to elucidate, at a cellular and molecular level, the beneficial effects of BJ polyphenols on bone regeneration, before suggesting their use as a dietary and pharmacological supplement for the prevention and/or managemen<sup>t</sup> of osteoporosis and other bone diseases related to oxidative stress.

This study was performed in human osteoblast-like cell line SaOS-2 in which oxidative stress was induced by an intracellular depletion of GSH by butionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase that regulates GSH synthesis [13], before starting osteogenic differentiation and during the early phases of the mineralization process. We used SaOS-2 cells given that, in these cells, BSO-induced oxidative stress inhibits osteogenic factors involved in the final

stage of osteoblast activity and related to di fferentiation and the mineralization process [13]. These cells reflect a normal phenotype of osteoblasts [37,38] and, like them, display the entire di fferentiation sequence and are able to form an extracellular mineralized matrix [39]. All these features contribute to the SaOS-2 cell line being considered as a cellular model to study osteoblast functions and, in particular, processes associated to late osteoblastic di fferentiation stage in human cells, such as the formation of bone nodules by di fferentiated osteoblasts [37,40,41].
