Recent studies show that changes in the oxidative state and the regulation of redox homeostasis affect bone turnover and remodeling [1
]. 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
]. 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
]. 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
]. 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
]. 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
]. Increased ROS production is responsible for diverting bone precursor cell differentiation toward the formation of other cell types rather than bone tissue cells [15
]. 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
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 management of osteoporosis and bone inflammatory diseases related to oxidative stress [17
]. Indeed, plasma antioxidant content was found diminished in osteoporotic women [11
]. These antioxidant compounds scavenge ROS [1
] and reduce oxidative stress in many diseases including bone diseases and, in particular, osteoporosis [17
]. Many literature data suggest a positive relationship between antioxidant intake and prevention of bone loss often associated with increased bone fractures [18
]. 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
]. Recently, it was shown that diets containing blueberry (BB) prevent osteoporosis in ovariectomized rats [19
]; 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
], with beneficial properties in bone anabolism [17
]. Moreover, recent studies suggest VM as a “functional food” and, as such, of benefit for dietary supplementation [31
]; 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
]. 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 management 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 differentiation and the mineralization process [13
]. These cells reflect a normal phenotype of osteoblasts [37
] and, like them, display the entire differentiation 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 differentiation stage in human cells, such as the formation of bone nodules by differentiated osteoblasts [37
2. Materials and Methods
All common reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), Extrasynthèse (Genay, France), GE Healthcare (Little Chalfont, Great Britain), Santa Cruz Biotechnologies (Santa Cruz, CA, USA), Millipore (Bedford, MA, USA), Abcam (Cambridge, UK), Euroclone (Milan, Italy), Thermo Scientific (Waltham, MA, USA), Bioassay Systems (Hayward, CA, USA), Promega (Madison, WI, USA), and Invitrogen (Carlsbad, CA, USA), unless differently specified in the text.
The following reagents were purchased from Sigma-Aldrich: Ham’s F12 Coon’s modification medium, l-glutamine, dimethyl sulfoxide (DMSO), BSO, trypsin, bovine serum albumin, Tris/HCl, Triton X100, NaCl, NaF, ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA), β-glycerophosphate, human sirtuin type 1 (SIRT1) small interfering RNA (siRNA), Universal Negative Control #1, Alizarin Red S, phenolic reference standards ellagic acid (assay HPLC ≥ 95%) for hydroxybenzoic acids, (+)-catechin hydrate (assay HPLC ≥ 96.0%) for flavan-3-ols), acetonitrile HPLC grade (assay 99.9%), formic acid for (HPLC assay 98–100%), ascorbic acid, dexamethasone, paraformaldehyde, cetylpyridinium chloride, Folin–Ciocalteu reagent, NaCl/Pi.
The following reagents were purchased from Extrasynthèse: anthocyanin reference standard cyanidin 3-glucoside chloride (assay HPLC ≥ 96%), flavonol reference standard quercetin 3-O-glucoside (assay HPLC ≥ 99%), hydroxycinnamic acid reference standard 3-O-caffeoyl quinic acid (chlorogenic acid, assay HPLC ≥ 99%).
The following reagents were purchased from GE Healthcare: penicillin/streptomycin 100× solution, phosphate-buffered saline (PBS), polyvinylidene fluoride (PVDF) membrane, enhanced chemiluminescence (ECL) Western Blotting Detection Reagent kit.
The following reagents were purchased from Santa Cruz Biotechnologies: EX527, Protein A/G PLUS-Agarose, anti-Runt-related transcription factor 2 (RUNX-2), anti-phospho-tyrosine, anti-histone H3.
The following reagents were purchased from Milipore: Milli-Q water, Cytobuster Protein Extraction Reagent.
The following reagents were purchased from Abcam: SIRT1 ELISA kit, anti-histone H3.
The following reagent was purchased from Euroclone: fetal bovine serum South American origin.
The following reagent was purchased from Thermo Scientific: Pierce bicinchoninic acid (BCA) protein assay kit.
The following reagent was purchased from Bioassay Systems: QuantiFluo Alkaline Phosphatase Assay Kit.
The following reagent was purchased from Promega: CellTiter-Glo Luminescent Cell Viability Assay.
The following reagents were purchased from Invitrogen: lipofectamine RNAiMAXTM, 2′,7′-dichlorodihydrofluorescein diacetate.
2.2. Preparation of Blueberry Juice and Determination of Total Soluble Polyphenols
BBs, harvested in August 2018/2019 in Tuscany Apennines and supplied by IL BAGGIOLO S.R.L. (Abetone, Pt, Italy) and DANTI GIAMPIERO S.R.L. (Cutigliano, Pt, Italy), were frozen freshly picked in aliquots of 100 g each and homogenized in a refrigerated Waring Blender to prepare BJ. Insoluble particles were removed by filtration under vacuum and centrifuged at 20,000× g
for 10 min. Aliquots of BJ were stored at −20 °C until use. The total soluble polyphenol (TSP) fraction of BJ was quantified with Folin–Ciocalteu reagent using gallic acid as the standard as described in our previous work [33
] and via the HPLC method reported below. TSP concentration in BJ obtained from 100 g of BB fresh weight was expressed as mg/100 mL ± SD and the values measured by Folin–Ciocalteu assay or HPLC method were 169.5 ± 19.4 and 158.8 ± 12.3, respectively.
2.3. HPLC-PDA-MS Analysis of Phenolic Compounds
The identification of phenolic compounds was performed using a Waters Alliance 2695 coupled online with a Waters 2996 photodiode array detector, and with a Quattro micro mass spectrometry detector with an electrospray interface. Separations were performed on a C18 reversed-phase Gemini Phenomenex (150 × 3 mm, 5 µm particle size) with a mobile phase flow rate of 0.4 mL∙min−1
. The mobile phase consisted of (A) H2
O containing 5% formic acid and (B) MeCN. A gradient elution program was applied as follows: 0–1.0 min held on 8% B, 1.0–16.0 min linear gradient to 15% B, 16.0–28.0 min linear gradient 50% B, 28.0–36.0 min linear gradient to 95% B, then in 1 min to the initial (starting) condition, and held 8 min for re-equilibration. The total run time was 45 min. The sample was diluted 1:10 (v
) with 8% B and 92% A, with an injection volume of 10 µL. Determination of phenolic compounds was performed using two detectors online: a photodiode array UV detector, followed by a single quadrupole mass spectrometry detector. The photodiode array scanned the samples at λmax
270, 320, 360, and 520 nm. The mass spectrometer detector was optimized to the following conditions: capillary voltage 3.20 kV, source block temperature 125 °C, and desolvation temperature 350 °C, operating in electrospray positive mode, detection range 100–1000 Da with total ion count extracting acquisition. The cone voltage was 32 V, the extractor lens was 3 V, and the cone and desolvation gas flows were 20 and 320 L∙h−1
, respectively. Phenolic compound identification in the sample was carried out by comparing UV absorption spectra and mass spectra of each compound with those reported in the literature [42
]. The quantification of polyphenols was calculated using the method of an external standard. Each standard was freshly prepared up to 300 μg/mL concentration and injected three times to obtain its calibration curve. Quantification was obtained as total content of each polyphenol group. Quantification of total constituents of each class of flavonoids was carried out using single anthocyanin, flavonol, and flavan-3-ol standards, namely, cyanidin-3-glucoside, quercetin-3-glucoside, and (+)-catechin equivalents, respectively. The values were expressed as gallic acid equivalent.
2.4. Cell Cultures, Treatments, Osteogenic Differentiation, and Cellular Viability
Osteoblast-like SaOS-2 cells were cultured in Ham’s F12 Coon’s modification medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 72 mg/L penicillin, and 100 mg/mL streptomycin (growth medium, GM), and incubated at 37 °C in a 5% CO2 humidified atmosphere with 20% oxygen. For the experiments of osteogenic differentiation, when SaOS-2 cells reached 70–80% confluence, GM was changed with osteogenic medium (OM) to induce osteogenic differentiation. OM was the growth medium supplemented with 10 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate. Then, 40 μM BSO, or BJ containing 7.5 or 15 μg∙mL−1 TSP, or BJ + BSO, or BJ + 10 μM EX527, or BJ + BSO + 10 μM EX527 were added or not to GM for 24 h (day 1) before exchanging it for OM, containing or not the before mentioned compounds, to stimulate the differentiation process. The OM was refreshed twice a week for the whole study period, BSO was added for only two days after the beginning of differentiation and BJ or EX527 were added to the OM at each change from the beginning of the differentiation process for the whole study period.
Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay, according to the manufacturer’s instructions.
In some experiments, the cells were transiently transfected with 75 nM human SIRT1 siRNA corresponding to two DNA target sequences of human SIRT1 (5′-GUGUCAUGGUUCCUUUGCA[dT][dT]-3′ accession number SASI_Hs01_00153666; 5′-UGCAAAGGAACCAUGACAC[dT][dT]-3′, accession number SASI_Hs01_00153666 6_AS) or scrambled siRNA (Scr siRNA) (Universal Negative Control #1), using lipofectamine RNAiMAXTM, according to the manufacturer’s instructions. The ability of SIRT1 siRNA to silence SIRT1 expression levels of about 50% was checked in control cells transfected for 24 h in GM and for other 48 h in OM. Additionally, 0.008% DMSO was present in experiments with EX527 in all conditions.
2.5. Determination of Intracellular ROS
The cell-permeant probe, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), was added in the culture medium of SaOS-2 seeded in 12-well plates one hour before the end of the various treatments performed as written above. The probe after deacetylation by esterases is rapidly oxidized to a highly fluorescent compound in the presence of ROS. After PBS washing, adherent cells were lysed in radioimmunoprecipitation assay RIPA buffer (50 mM Tris/HCL pH 7.5, 1% Triton X-100, 150 mM NaCl, 100 mM NaF, 2 mM EGTA, phosphatase, and protease inhibitor cocktail), centrifuged at 20,000× g (ALC PK121R, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min, and the intracellular levels of ROS were measured by florescence analysis at 510 nm. The normalization of the data was obtained by using total proteins, and the values were expressed as percentages with respect to the controls.
2.6. Alkaline Phosphatase Activity
SaOS-2 cells seeded in six-well plates during differentiation in the presence or not of the various treatments, as described above, were collected in Cytobuster Protein Extraction Reagent (Milipore, Burlington, MA, USA). After sonication twice on ice and centrifugation at 4 °C for 15 min at 1000× g, alkaline phosphatase (ALP) activity was measured in the supernatants using the QuantiFluo Alkaline Phosphatase Assay Kit following the manufacturer’s instructions. The ALP activity was normalized to protein content for each well, and data were expressed as percentages relative to the control values.
2.7. Western Blot Analysis of RUNX and RUNX-2 Phosphorylation
Western blot analysis was performed in SaOS-2 cells six days after differentiation and treated or not (control) as described above. Whole-cell lysates and nuclear extracts were obtained as previously described in References [13
], respectively. Equal amounts of nuclear proteins were then incubated with antibody against Runt-related transcription factor 2 (RUNX-2) for 1 h at 4 °C. Subsequently, the immune complexes were precipitated using Protein A/G PLUS-Agarose. The immunoprecipitates (200 μg) were mixed with Laemmli buffer for 5 min at 95 °C, subjected to SDS/PAGE, and electrotransferred to a PVDF membrane [13
]. Phospho-RUNX-2 (p-RUNX-2), RUNX-2, histone H3, and β-actin were visualized using antibody anti-phospho-tyrosine proteins, anti-RUNX-2, anti-histone H3, or anti-β-actin, respectively. Antigen–antibody complexes were detected using chemiluminescence ECL Western Blotting Detection Reagent kit. Digital images of the bands were detected by Amersham Imager A600 (GE Healthcare, Chicago, IL, USA).
2.8. Alizarin Red S Assay
The deposition of calcium was measured 12 and 24 days after differentiation in cells treated as described above. Cells were fixed in 4% paraformaldehyde for 15 min after washing twice with NaCl/Pi for a few minutes; subsequently, they were washed another three times with deionized water. Calcium mineral deposits were stained by using 2% Alizarin Red S at pH 7.8 for 2 min and were destained using 10% cetylpyridinium chloride in deionized water for 60 min at 50 °C. The absorbance of Alizarin Red S extracts was measured at 560 nm. Calcium content was evaluated using a standard curve of hydroxyapatite (100 μg/mL in cetylpyridinium chloride solution) and expressed as mg hydroxyapatite (HA) per cm2.
2.9. SIRT1 Expression Assay
SIRT1 levels were measured by using the Human SIRT1 ELISA kit in SaOS-2 cells, seeded in 12-well plates. Cell were solubilized in Cell Extraction Buffer and centrifuged at 18,000× g for 20 min at 4 °C according to manufacturer’s instructions. Data, normalized on total protein content, were expressed as percentages of control levels.
2.10. Protein Assay
Protein concentrations were determined by the bicinchoninic acid solution protein reagent assay using bovine serum albumin as the standard.
2.11. Statistical Analysis
One-way ANOVA analysis with Bonferroni’s multiple comparison test, using GraphPad Prism Software, or Student’s t-test were used to determine the statistical significance. A p-value ≤ 0.05 was considered statistically significant.
This study reports new data on the role of BJ phytochemicals and polyphenolic antioxidants in the activation of osteogenic factors and in the induction of the mineralization process in the presence or not of oxidative stress induced by GSH depletion. In particular, the polyphenolic content of the juice was characterized, and it was shown that BJ performs an important antioxidant action and protects from damage induced by oxidative stress, as well as upregulates factors such as ALP and RUNX-2, related to differentiation and the mineralization process, in normal intracellular redox state conditions. The involvement of SIRT1 in these events was also demonstrated.
In this study, the effect of BJ containing certain amounts of TSP was evaluated in GSH-depleted SaOS-2 cells, an in vitro condition that mimics what happens in vivo in the bone environment in the presence of oxidative stress due to microdamage and/or estrogen deficiency, [4
]. Indeed, GSH is involved in osteoblast and osteoclast differentiation and, together with other thiol antioxidants, it may play a crucial role in estrogen deficiency-associated bone loss [1
]. In fact, some data show that bone loss due to a lack of estrogen is related to the lowered thiol antioxidants in osteoclasts, and this activates osteoclastogenic signals which induce ROS-enhanced expression of cytokines promoting osteoclastic bone resorption [51
We used BJ given that BBs are commercialized in different ways, mainly as fresh or frozen products, also in addition to juices or dry extracts. However, the drying process and treatment with solvents (i.e., for the production of dry extracts) might partially destroy anthocyanins and their antioxidant effects [53
], and the anthocyanins seem to be more stable over time in a juice with acidic pH than in a dry extract [53
]. The results obtained from the qualitative and quantitative analysis of TSP in BJ show that the main polyphenolic component was represented by anthocyanins belonging to the flavonoid family, which were present mainly as glucosides, galactosides, and arabinosides; these data are similar to those found in the literature [42
]. Moreover, TSP content in BJ obtained by HPLC-PDA was very similar to the value obtained with the Folin–Ciocalteu method, indicating that quantification via this spectrometric method is feasible and realistic.
Some studies demonstrated that many BB polyphenols are bioavailable; in fact, after various processes of ingestion, they were found in the plasma [36
]. It was also demonstrated that anthocyanins or cyanidins, after oral administration, can be absorbed in intact form as glycosides and/or aglycones [34
]. Moreover, even if BB polyphenols undergo complex metabolic modifications, their derivatives have the same functional characteristics [34
]. The flavonoids and anthocyanins present in BB have strong antioxidant capacity [24
], and these, along with the other polyphenol compounds and their derivatives, favor the formation of bone mass [18
The data of this study demonstrate that TSPs, together with other phytochemicals contained in BJ, are able to prevent BSO-induced oxidative stress, and the results partly correlate with what was previously obtained with thiol antioxidants, such as GSH and N
-acetyl cysteine, in SaOS-2 cells under similar conditions of oxidative stress [13
]. The BJ antioxidant effect in SaOS-2 cells was obtained with the same concentrations of TSP used in osteocytes in which oxidative stress was induced by starvation [33
], and ROS reduction was achieved by BJ treatment very quickly both in osteocytes and in SaOS-2 cells [13
], similarly to thiol antioxidants [6
]. Therefore, thanks to their antioxidant action, BJ phytocompounds may effectively contribute to preventing and/or eliminating oxidative stress damage present in bone pathologies, particularly in osteoporosis, as reported in the literature for various polyphenols or their derivatives [18
]. Our results support these data; in fact, the initial stimuli that induce osteogenic activity in SaOS-2 cells are sensitive to changes in the oxidative state. Indeed, both ALP and RUNX-2 are important markers related to the activation of the first phase of the osteoblast differentiation process and to the subsequent induction of calcium and matrix deposition by differentiated osteoblasts [13
]. In fact, we observed a strong initial increase of ALP activity that subsequently remained high and then decreased, indicating the achievement of a high degree of differentiation, as also previously observed [13
]. Both ALP activity and RUNX-2 expression and activation significantly decreased in the presence of BSO-induced oxidative stress, as well as the mineralization process. These events are efficiently prevented by BJ antioxidant action.
The results of this study also show that BJ treatment in the presence of normal ROS levels has a remarkable non-redox-regulated osteogenic action that occurs through a significant upregulation of ALP and RUNX-2 activity. It is worthy to note that BJ maintains ALP activity levels higher than those in untreated cells for a long time, and this seems to be related to the activation of the mineralization process that is evident only in the late phase of this event. Finally, the similar and high levels of calcium, obtained in cells treated with BSO + BJ or with BJ alone after 24 days, show that the elimination of the oxidative state in the initial phase of the differentiation allows BJ to perform the long-term activation of this process. Indeed, modulation of ALP and RUNX-2 activity in BJ-treated SaOS-2 cells, in the presence or not of BSO-induced oxidative stress, is similar to that previously observed in these cells treated with thiol antioxidants, even if the effect of the latter on the mineralization process became evident more quickly [13
]. These differences may be due to the different chemical characteristics and action mechanisms of the antioxidants. However, in both studies, thiol and non-thiol antioxidants regulated the osteogenic activity of osteoblasts through mechanisms unrelated to their antioxidant activity. In fact, the activating effect of BJ or of thiol antioxidants on the mineralization process occurred in cells in which there was no alteration in the redox state. It is worthy to note that the BJ effects on osteogenic factors were found using TSP concentrations similar to those used to demonstrate the osteogenic activity of the polyphenolic component of dried plums [56
]. In fact, these antioxidants restore the TNFα-induced suppression of ALP activity and upregulate RUNX-2 expression, influencing mineralized matrix formation under normal and inflammatory conditions [56
]. Moreover, our data agree with the increased expression of RUNX-2 and ALP in pre-osteoblast cells treated with serum from BB-fed rats and with the subsequent increase in osteoblast activity and bone formation [57
]. These data are also related to the ability of BB-rich diets administered in young female rats to prevent bone loss in ovariectomized adult female rats [19
], where the effects of dietary BB appear similar to those of estrogens [57
]. In fact, the polyphenols seem to interact with the estrogen receptors and induce their effects through redox-independent factors and signaling pathways related to the regulation of bone cell activity [36
Moreover, many data suggest that anthocyanins, in addition to their antioxidant activity, can also have other beneficial health effects [20
]. This may agree with the possible involvement of SIRT1 in ALP and RUNX-2 activation, as well as the increase in calcium deposition, due to BJ treatment in the presence or not of a normal redox state. Indeed, SIRT1 levels did not change in the presence of BSO-induced oxidative stress, indicating that SIRT1 expression does not seem to be a redox-regulated mechanism. BJ was effectively able to upregulate SIRT1 expression in BSO-treated and untreated cells, and BJ antioxidant action was partly related to SIRT1 activity. Previously, we similarly demonstrated a significant relationship between BJ phytocompounds and molecular events related to apoptosis and expression of osteoclastogenic factors induced by oxidative stress and SIRT1 activation [33
]. Indeed, dietary BBs increase SIRT1 levels in mammals [49
], and SIRT1 overexpression is also related to the inhibition of osteoclastogenic factors [58
]. Moreover, SIRT1 activity promotes osteogenic differentiation of mesenchymal stem cells and activation of RUNX-2 [46
]. Previously, we also demonstrated that SIRT1 activity contributes, in part, to the BJ protective effect in MSCs against cytotoxicity due to oxidative damage [33
]. Therefore, the possible role of SIRT1 can explain, at the molecular level, the positive action of TSP and/or other phytocompounds contained in BJ on the osteogenic activity and the mineralization process of osteoblasts, although other experiments will have to be performed to validate SIRT1’s involvement in these events.
The activation of osteogenic factors and mineralization due to BJ treatment is also in agreement with the increase in bone mass found in young subjects fed with diets rich in blueberries or fruits rich in antioxidant phytochemicals [17
]. It was also shown that BBs stimulate the growth of bone in growing rats, and this appears to be due to polyphenols and their metabolic derivatives [18
]. Indeed, phenolic acid derivatives present in the diet promote the differentiation of osteoblasts and bone growth in young mice [63
]. Some studies demonstrated that daily consumption of these compounds may be important in increasing the bone mass peak [20
], and this is an independent predictor of increased bone mass in early pubertal children [61