Characteristics of osteoblasts and their lineage

Morphologically, osteoblasts have a cuboidal shape and they form a dense monolayer of cells at the bone surface. Functionally, osteoblasts are the specialised cells within bone that produce extracellular matrix and also regulate mineralisation⁽Reference Sommerfeldt and Rubin²⁾. More precisely, bone tissue consists of hydroxyapatite crystals and various extracellular matrix proteins (i.e. type I collagen, osteocalcin, osteonectin, osteopontin, bone sialoprotein and proteoglycans) secreted and deposited by mature osteoblasts⁽Reference Bringhurst, Demay, Krane, Kasper, Braunwald and Fauci¹¹⁾.

Osteoblasts are derived from precursors originating in the bone marrow. These precursors are multipotent mesenchymal stem cells that can differentiate into several kinds of tissue-specific cells such as osteoblasts, myoblasts, adipocytes and chondrocytes. Each differentiation pathway can be specifically regulated by transcription factors such as runt-related transcription factor-2 (Runx2)/Osterix, MyoD family, PPARγ and SOX5/6/7, respectively⁽Reference Katagiri and Takahashi¹²⁾.

Regulation of osteoblastogenesis

In the process of osteoblastogenesis, different stages are involved including proliferation, extracellular matrix synthesis, maturation and mineralisation. Each stage is regulated by the coordinated expression of major transcription factors⁽Reference Marie¹⁰⁾. The most important transcription factors controlling bone formation are Runx2, Osterix, β-catenin, activating transcription factor-4, activator protein-1 (AP-1) and CCAAT/enhancer binding proteins (C/EBP). Other transcription factors belonging to homeobox proteins (Msx and Dlx proteins) or to helix-loop-helix proteins (Id and Twist) may play a role in osteoblast development⁽Reference Marie¹⁰⁾. Furthermore, the expression of all these transcription factors is known to be modulated by several hormones (parathyroid hormone, oestrogens, glucocorticoids, 1,25-dihydroxyvitamin D)⁽Reference Strewler¹³⁾ or growth factors (bone morphogenetic protein (BMP), transforming growth factor-β, insulin-like growth factor-1, fibroblast growth factor-2)⁽Reference Baylink, Finkelman and Mohan¹⁴⁾.

Osteoblast differentiation is stimulated by some hormones and local factors which act on signalling pathways in cells within the osteoprogenitor lineage⁽Reference Kalajzic, Staal and Yang¹⁵⁾. Various pathways such as BMP, transforming growth factor-β, insulin-like growth factor, fibroblast growth factor, Hedgehog, WNT (wingless-type MMTV integration site family) and mitogen-activated protein kinase (MAPK) have been implicated⁽Reference Kalajzic, Staal and Yang^15–Reference Huang, Yang and Shao¹⁹⁾ (Fig. 2). Many markers are available and currently used to study the influence of several factors on osteoblast developmental stages or functions. Indeed, alkaline phosphatase (ALP) collagen type I, osteopontin and bone sialoprotein are early markers of the osteoblast phenotype while osteocalcin plays a role in mineralisation⁽Reference Stein and Lian²⁰⁾ (Fig. 3). Cytokines and/or growth factors produced by osteoblasts and other cells have an effect on differentiation and the maturation process through binding to the proteins of bone extracellular matrix. Mature osteoblasts surrounded by bone matrix become osteocytes. They probably function as mechanosensors which regulate the bone response to mechanical stimuli⁽Reference Ehrlich and Lanyon²¹⁾. Activated osteocytes produce signalling molecules such as NO, which could stimulate the activity of osteoblasts and osteoclasts. Intercellular signalling is essential for bone adaptation⁽Reference Burger, Klein-Nulend and Smit²²⁾. Thus, bone cells cooperate together and regulate their own functions.

Fig. 2 Signal-transduction pathways implicated in regulation of osteoblast functions. BMP, bone morphogenetic protein; TGFβ, transforming growth factor-β; IGF-1, insulin-like growth factor-1; FGF, fibroblast growth factor; WNT, wingless-type MMTV integration site family; BMPR, bone morphogenetic protein receptor; TGFβR, transforming growth factor-β receptor; IGFR, insulin-like growth factor receptor; FGFR, fibroblast growth factor receptor; Lrp5/6, LDL-related protein 5/6; SMAD, mothers against decapentaplegic homologue; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; Gli, glioma-associated oncogene; Runx2, runt-related transcription factor-2; AP-1, activator protein-1; ATF4, activating transcription factor-4; TF, transcription factors; Col1, type 1 collagen; ALP, alkaline phosphatase; OPN, osteopontin; BSP, bone sialoprotein; OCN, osteocalcin.

Fig. 3 Main factors produced by osteoblasts and used as markers of different developmental stages (adapted from Stein & Lian⁽Reference Stein and Lian²⁰⁾). ECM, extracellular matrix; AP-1, activator protein-1; Col1, type 1 collagen; ALP, alkaline phosphatase; OPN, osteopontin; OCN, osteocalcin; BSP, bone sialoprotein.

Osteoblasts regulate osteoclastogenesis and osteoblasts play a direct and essential role in the regulation of osteoclast function within the bone microenvironment. Osteoclasts derive from haematopoietic progenitors (monocyte/macrophage lineage) in the bone marrow and they are formed by the fusion of precursor cells⁽Reference Ash, Loutit and Townsend²³⁾. They are responsible for bone resorption and therefore for normal skeletal development (growth and modelling). Macrophage colony-stimulated factor produced by osteoblasts/stromal cells is crucial for osteoclast formation and development⁽Reference Felix, Cecchini and Fleisch^24–Reference Kodama, Nose and Niida²⁶⁾. It has been shown that the osteoprotegerin (OPG)/receptor activator of NF-κB (RANK) ligand (RANKL)/RANK system plays a role in osteoclastogenesis as the dominant and final mediator of this process.

There are many factors that can influence osteoblastogenesis and/or osteoclastogenesis. It is very important to maintain the balance between these cellular processes which correspond with bone formation and bone resorption. An imbalance between these processes could lead to a decrease in bone mineral density and thus to an increase in the risk of osteoporosis during ageing⁽Reference Sambrook and Cooper²⁷⁾.

Cellular models for bone formation

The molecular events that regulate osteoblastic differentiation and function are frequently studied using in vitro cellular models. In order to explore the impact of pharmacological agents or dietary molecules, various osteogenic cell-culture systems can be used: immortal cell lines (for example, murine osteoblastic cells (MC3T3-E1) or fetal human osteoblastic cell line (hFOB)) or genetically atypical cell lines derived from tumours (for example, sarcoma osteogenic cells (SaOS-2) or human osteosarcoma cell line (MG-63)). However, tumour origins and genetic alterations associated with the prolonged culture of cell lines can raise questions about the extent to which they match the normal in vivo physiology. That is why in vitro model systems using primary osteoblast precursor cells derived from either the developing calvaria of neonatal rodents or from the bone marrow (murine bone marrow mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells) should be promoted.

Structure

Phytochemicals are a large group of plant-derived non-nutritive chemical compounds divided into several classes: phenolics (polyphenols), carotenoids, alkaloids, sterols, terpenes and fibre. Phytochemicals produced by plants are used to protect themselves but recent research demonstrates that certain molecules may protect humans against some pathologies such as cancer, cardiovascular diseases and osteoporosis⁽Reference Scalbert, Manach and Morand²⁸⁾.

Polyphenols can be divided into different groups depending on the number of phenol rings they contain and on the structural elements bound to these rings. Considering this, polyphenols have been classified as phenolic acids, flavonoids, stilbenes, tannins, coumarins and lignans. Moreover, among flavonoids, six subclasses exist and share a common structure of two aromatic rings (A and B) bound together by three carbon atoms that form an oxygenated heterocycle (ring C). These are flavones, flavonols, flavanones, isoflavones, flavanols (catechins and proanthocyanidins) and anthocyanidins⁽Reference Manach, Williamson and Morand²⁹⁾.

Metabolism of polyphenols

Knowledge on phenolic metabolites generated in an organism is useful to choose the appropriate molecule for in vitro studies. Biological activities of polyphenols are often examined in cell lines usually treated with aglycones or polyphenol-rich extracts (glycoside forms) at concentrations which elicit an appropriate response⁽Reference Kroon, Clifford and Crozier³⁰⁾. The compounds abundant in plants are not exactly the same as those found in plasma. Neither aglycones (except for green tea catechins) nor forms found in the diet reach the blood and tissue. The compounds, after consumption, are metabolised by the organism and/or microflora enzymes to conjugates of glucuronate or sulfate (with or without methylation across the catechol functional group) during absorption to the bloodstream and/or eventually eliminated⁽Reference Manach, Scalbert and Morand³¹⁾. The circulating forms may possess different biological properties within cells and tissues compared with polyphenol aglycones. Moreover, the maximum plasma concentration of polyphenols from foods is in the range of 0·1–10 μm. As far as possible, conjugates of molecules should be used for in vitro studies in the physiological concentration⁽Reference Williamson, Barron and Shimoi³²⁾ even if they are poorly or not absorbed by cells⁽Reference Serra, Mendes and Bronze³³⁾. Unfortunately, many metabolites are not commercialised due to problems of isolation and synthesis and it is difficult to obtain them for in vitro experiments⁽Reference Williamson, Barron and Shimoi³²⁾. However, some cells (in organisms) could potentially deconjugate the circulating molecules through the activity of enzymes such as β-glucuronidase and be present in cells in the aglycone form⁽Reference O'Leary, Day and Needs³⁴⁾. Hence, this enzyme in human tissues could play a role in the turnover of flavonoids (conjugated/non-conjugated forms). It has also been shown that cells in culture are able to take up aglycone forms of flavonoids such as diosmetin, hesperetin and naringenin and conjugate them⁽Reference Serra, Mendes and Bronze³³⁾. Treatments of cells with conjugated forms are the most representative method to investigate the molecular mechanism of polyphenols. However, at the present time, there are no reported studies that have used conjugated forms in osteoblastic cells. In the present review, concerning nutrition we focused on studies of molecules at physiological doses (up to 10 μm) corresponding to the concentration of polyphenols found in plasma after polyphenol-containing food consumption.

Cellular bioactivities of polyphenols

The chemical structure of compounds is related to their biological activity and thus activation of different signalling pathways could be involved⁽Reference Khlebnikov, Schepetkin and Domina³⁵⁾ in their mode of action.

Flavonoids and more recently their metabolites (sulfates, glucuronides, O-methylated forms) have been shown to act on cells, independently of their antioxidant capacity⁽Reference Williamson, Barron and Shimoi^32, Reference Loke, Proudfoot and McKinley^36–Reference Suri, Taylor and Verity³⁸⁾. They may interact with specific proteins involved in intracellular signalling pathways⁽Reference Schroeter, Boyd and Spencer³⁹⁾. They can selectively act on protein kinase and lipid kinase signalling cascades such as MAPK, protein kinase C, protein kinase B (PKB/AKT) (serine/threonine kinase), tyrosine kinases and phosphoinositide 3-kinase (PI3K) (for a review, see Williams et al. ⁽Reference Williams, Spencer and Rice-Evans⁴⁰⁾). The regulation can occur at transcriptional and/or post-translational levels by the modulation of gene expression and/or phosphorylation of target proteins, respectively. The positive or negative effects of flavonoids depend on both cell type and metabolic disorder. For example, genistein (isoflavone) can inhibit protein tyrosine kinases and thus cause cell cycle arrest and apoptosis in leukaemic cells⁽Reference Spinozzi, Pagliacci and Migliorati⁴¹⁾ whereas it can stimulate osteoblast differentiation⁽Reference Yamaguchi^42, Reference Sugimoto and Yamaguchi⁴³⁾. The flavonoid-stimulated signalling pathways are more precisely described by Williams et al. ⁽Reference Williams, Spencer and Rice-Evans⁴⁰⁾ and Orzechowski et al. ⁽Reference Orzechowski, Ostaszewski and Jank⁴⁴⁾.

Flavonoids also have an impact at the transcriptional level. They may affect the expression of NF-κB and AP-1 composed of c-jun/c-fos complexes which are implicated in the MAPK pathway. Indeed, it has been shown that green tea polyphenols such as ( − )-epigallocatechin-3-gallate (EGCG) can modulate phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 MAP kinase in normal human epidermal keratinocytes⁽Reference Katiyar, Afaq and Azizuddin⁴⁵⁾, which shows that this polyphenol can affect the MAPK signalling pathway⁽Reference Balasubramanian, Efimova and Eckert⁴⁶⁾. In the same way, resveratrol (trans-3,4′,5-trihydroxystilbene), a natural stilbene present abundantly in grape skins and red wine, has cardioprotective effects⁽Reference Bradamante, Barenghi and Villa⁴⁷⁾, cancer-preventive properties⁽Reference Aziz, Kumar and Ahmad⁴⁸⁾ and an impact on bone metabolism⁽Reference King, Bomser and Min⁴⁹⁾. It has been shown that resveratrol could act through ERK1/2 and p38 MAPK signalling⁽Reference Das, Tosaki and Bagchi⁵⁰⁾, similar to flavonoids.

Beside these indirect molecular effects, it is possible that the positive health effects of polyphenols occur through their direct action on response elements in the regulatory regions of target genes⁽Reference Orzechowski, Ostaszewski and Jank⁴⁴⁾. Some phenolic compounds can bind to specific cytoplasmic receptors which can play a role as DNA binding transcription factors to regulate target gene expression. This mechanism is well known for glucocorticoids⁽Reference Linder, Falkner and Srinivasan⁵¹⁾ and oestrogens⁽Reference Klinge⁵²⁾ which bind to glucocorticoid and oestrogen receptors, respectively. These complexes enter the nucleus to stimulate the expression of glucocorticoid- or oestrogen-related genes. Some plant compounds, so-called phyto-oestrogens, can interact with oestrogen receptors. This is the case for soyabean isoflavones (mainly genistein, daidzein and glycitein) as well as flavonoids such as luteolin and naringenin that exhibit oestrogenic properties⁽Reference Kuiper, Lemmen and Carlsson⁵³⁾. These phytochemicals play a role as partial agonists and/or antagonists of oestrogen receptors and can modulate the oestrogen-dependent pathway⁽Reference Bennetau-Pelissero, Latonnelle and Sequeira⁵⁴⁾.

Effect of polyphenols on transcription factors

It is known that macro- and micronutrients may enter cells and then interact with transcription factors which are known to influence target gene expression⁽Reference Muller and Kersten⁶¹⁾. Considering the field of bone metabolism and more specifically osteoblasts, it has been reported that some polyphenols such as EGCG, resveratrol and icariin can modulate some transcription factors which in turn can activate osteoblast-related genes (Table 1).

Table 1 Polyphenol regulation of transcription factors

Runx2, runt-related transcription factor-2; OB, osteoblasts; ↑ , increase; MBMSC, murine bone marrow mesenchymal stem cells; EGCG, ( − )-epigallocatechin-3-gallate; HBMSC, human bone marrow-derived mesenchymal stem cells; SaOS-2, sarcoma osteogenic cells; MC3T3-E1, murine osteoblastic cells; POB, primary osteoblasts; AP-1, activator protein-1; ↓ , decrease.

A story beyond and around bone morphogenetic protein-stimulated pathways

Recent studies have shown that BMP induce osteoblast differentiation and play an important role in osteogenesis during development and in adult life⁽Reference Canalis, Economides and Gazzerro⁸³⁾. Furthermore, BMP stimulate the formation of mineralised bone-like nodules⁽Reference Yamashita, Ishii and Shimoda⁸⁴⁾. These proteins, by an autocrine action, may activate different signalling cascades. BMP regulatory molecules can transmit signals through SMAD-dependent and SMAD-independent pathways, including ERK, JNK, and p38 MAP kinase pathways⁽Reference Miyazono, Maeda and Imamura⁸⁵⁾. Members of the BMP family can bind to various serine/threonine kinase receptors I and II which are necessary for signal transduction⁽Reference Shi and Massague⁸⁶⁾. Moreover, BMP signalling can interact with other pathways such as transforming growth factor-β, Notch, Wnt, signal transducer and activator of transcription (STAT) and MAPK by cross-talk with them⁽Reference Miyazono, Maeda and Imamura⁸⁵⁾. Because BMP stimulate new bone formation⁽Reference Boden, Kang and Sandhu⁸⁷⁾, indicating a clear role in the regulation of osteoblast function, they represent molecular targets used to identify new agents to prevent osteoporosis⁽Reference Mundy⁵⁾. BMP-2 is one of the major proteins implicated, as an important regulator, in bone metabolism.

Regulation of BMP signalling is important for cell functions and BMP activity can be regulated by many mechanisms (intra- and/or extracellular; for a review, see Canalis et al. ⁽Reference Canalis, Economides and Gazzerro⁸³⁾). One mechanism is regulation by extracellular antagonists such as noggin which can bind BMP and thus prevent stimulation of BMP receptors and cell signalling⁽Reference Zimmerman, De Jesús-Escobar and Harland^88, Reference Groppe, Greenwald and Wiater⁸⁹⁾. Noggin selectively blocks BMP function; thus it can be used as a tool in cell biology to inhibit the BMP pathway⁽Reference Gazzerro, Gangji and Canalis⁹⁰⁾.

It has been shown that some phenolic compounds such as coumarin derivatives⁽Reference Kuo, Hsu and Chang^91, Reference Tang, Yang and Chien⁹²⁾, piceatannol (stilbene)⁽Reference Chang, Hsu and Teng⁹³⁾, resveratrol (stilbene)⁽Reference Su, Yang and Zhao⁹⁴⁾ and myricetin (flavonoid)⁽Reference Hsu, Chang and Tsai⁹⁵⁾ increase BMP-2 production in osteoblastic cells (Table 2). Moreover, the ALP activity and osteocalcin production induced by these compounds was inhibited by noggin, suggesting that BMP-2 signalling is involved in osteoblast differentiation. Myricetin (3,3′,4′,4,5,5′,7-hexahydroxyflavone), a flavonoid present in grape juice and red wine, as well as piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene) found in the skins of grapes, rhubarb and cane sugar exhibit potential healthy effects on organisms⁽Reference Wolter, Clausnitzer and Akoglu^96, Reference Yanez, Vicente and Alcaraz⁹⁷⁾. More specifically, myricetin (1–20 μm)⁽Reference Hsu, Chang and Tsai⁹⁵⁾ and piceatannol (0·1–20 μm)⁽Reference Chang, Hsu and Teng⁹³⁾ dose dependently stimulated osteoblast differentiation at various stages and also mineralisation in both human osteoblastic cell line hFOB and human osteosarcoma cell line MG-63. It was demonstrated that myricetin induced phosphorylation of SMAD1/5/8 complex and p38 protein which can be abrogated by noggin pre-treatment. Addition of the specific inhibitor of p38 decreased ALP activity and osteocalcin secretion as well as phosphorylation of p38 in cells treated with myricetin. In this experiment, myricetin through BMP-2 activated not only the SMAD-dependent pathway but also the p38 kinase pathway that has been implicated in differentiation⁽Reference Hsu, Chang and Tsai⁹⁵⁾.

Table 2 Interaction of polyphenols with the bone morphogenetic protein (BMP) pathway

hFOB, fetal human osteoblastic cell line; ↑ , increase; MG-63, human osteosarcoma cell line; SMAD, mothers against decapentaplegic homologue; ERK, extracellular signal-regulated kinase; POB, primary osteoblasts; MC3T3-E1, murine osteoblastic cells.

Coumarin derivates such as imperatorin and bergapten increased BMP-2 gene expression in a time- and dose-dependent manner but not BMP-4 or BMP-7 in primary osteoblasts⁽Reference Tang, Yang and Chien⁹²⁾. On the contrary, icariin (a flavonol glycoside) induced the expression of BMP-4 in osteoblasts⁽Reference Zhao, Ohba and Shinkai⁷³⁾. Coumarin derivatives increased phosphorylation of SMAD1/5/8, p38 and ERK in a time-dependent manner. Pre-treatment with a p38 inhibitor and MAPK or ERK kinase (MEK) inhibitor markedly diminished coumarin-induced p38 and ERK1/2 phosphorylation, as well as BMP-2 gene expression⁽Reference Tang, Yang and Chien⁹²⁾. It has been observed that another coumarin derivate osthole (7-methoxy-8-isopentenoxycoumarin), present in Chinese medicine, used the same mechanisms to induce osteoblast differentiation in hFOB and MG-63 cells⁽Reference Kuo, Hsu and Chang⁹¹⁾. Thus, BMP-2 protein appears to be required for coumarin-mediated maturation of osteoblasts.

Lee et al. have shown that emodin at the range of 2–5 μm accelerated osteoblast differentiation via stimulation of BMP-2 gene expression and the activation of PI3K-AKT/MAP kinases (especially JNK and p38). These effects were abolished by a PI3K inhibitor, suggesting that emodin-induced BMP-2 expression was dependent on PI3K-AKT/MAP kinase cascades⁽Reference Lee, Shin and Min⁸¹⁾.

Results from these studies have shown that polyphenols were able to influence osteoblast differentiation and mineralisation through an increase in BMP-2 production, which in turn could activate phosphorylation of specific proteins (SMAD, p38, ERK1/2). Taken together, polyphenols can affect osteoblast function by the complex network of BMP-related signalling pathways (Table 2).

What about polyphenols and oestrogen receptor signalling?

Both oestrogen α and β receptors can be found in osteoblasts⁽Reference Kousteni, Bellido and Plotkin^98–Reference Manolagas, Kousteni and Jilka¹⁰⁰⁾. Oestrogens can modulate gene expression through genomic and/or non-genomic mechanisms. Genomic transcriptional regulation is achieved through recruitment of oestrogen receptors to the promoter region of the target gene, either directly through interaction with DNA sequences (i.e. oestrogen receptor element) or through protein–protein interaction with other transcriptional factors such as AP-1 and NF-κB⁽Reference Ascenzi, Bocedi and Maria^101, Reference Nilsson, Makela and Treuter¹⁰²⁾. Non-genomic action (called ‘rapid’) can also initiate gene transcription but through oestrogen activation of signal-transduction pathways which are insensitive to inhibitors of transcription and translation⁽Reference Losel, Falkenstein and Feuring¹⁰³⁾. However, it is possible that different oestrogen receptor ligands induce conformational changes in oestrogen receptors, blocking the activation of signalling cascades⁽Reference Watson, Bulayeva and Wozniak¹⁰⁴⁾. The anti-apoptotic effects of oestrogens and oestrogenic compounds on osteoblastic cells are mediated by oestrogen receptor-mediated ERK pathway activation, which is distinct from genomic actions of oestrogen receptors⁽Reference Prouillet, Maziere and Maziere^105–Reference Kono, Oshima and Hoshi¹⁰⁹⁾. The oestrogen receptor–oestrogen complex interacts with Src tyrosine kinase and results in the rapid activation of ERK. ERK in turn is responsible for downstream activation of Elk-1, cAMP responsive element binding protein (CREB) and CCAAT/enhancer binding protein-β, transcription factors involved in anti-apoptotic effects of sex steroids on osteoblasts. Also inactivation by phosphorylation of a pro-apoptotic protein Bad takes place. On the other hand, JNK activity and downstream transcription driven by transcription factor AP-1 are down-regulated via a non-genomic oestrogen receptor-mediated pathway⁽Reference Kousteni, Han and Chen^99, Reference Driggers and Segars¹¹⁰⁾. In general, oestrogen acts on osteoblasts by modulating the activity of cytosolic kinases and subsequent post-transcriptional modification of transcription factors, which results in up- or down-regulation of gene expression⁽Reference Kousteni, Bellido and Plotkin⁹⁸⁾.

Isoflavones such as daidzein and genistein are classified as phyto-oestrogens and are thought to act via the oestrogen receptors to improve bone health. Plant-derived flavonoids and phyto-oestrogens can change oestrogen receptor-α and oestrogen receptor-β activities in different ways and act as oestrogen agonists and/or antagonists depending on tissue type and molecule concentration⁽Reference Kuiper, Lemmen and Carlsson⁵³⁾. Some nutritional flavonoids such as quercetin and naringenin acting as oestrogen-mimetics can activate p38/MAPK in the presence of oestrogen receptor-β⁽Reference Totta, Acconcia and Leone¹¹¹⁾. These molecules have an antioxidant function in plants but they could act on oestrogen receptors as agonists and/or antagonists in the organism⁽Reference Cassidy, Dalais, Gibney, Macdonald and Roche¹¹²⁾. It has been shown that isoflavones have anabolic effects on bone metabolism by stimulating osteoblasts and decreasing osteoclast functions⁽Reference Yamaguchi⁴²⁾. The oestrogenic properties of isoflavones were investigated by Chen et al. ⁽Reference Chen, Garner and Anderson¹¹³⁾ in two hFOB osteoblastic cell lines expressing different levels of oestrogen receptors. The expression and the production of osteoclastogenesis-regulatory cytokines such as IL-6 and OPG were dependent on the level of oestrogen receptors. The human fetal osteoblastic cell lines hFOB1·19 (400 oestrogen receptors/nucleus) and hFOB/ER9 (8000 oestrogen receptors/nucleus) were treated with 10 nm-17β-oestradiol, 0·1–10 nm-genistein or daidzein and/or 10 μm-ICI (inhibitor of oestrogen receptor) for 48 h. IL-6 protein production was decreased by about 30–40 % in hFOB1·19 cells and 40–60 % in hFOB/ER9 cells by isoflavone treatments in comparison with the non-treated control cells. The effect of genistein and daidzein was slightly dose dependent and was greater in cells that were most abundant in oestrogen receptors (hFOB/ER9)⁽Reference Chen, Garner and Anderson¹¹³⁾. Moreover, it has been shown that daidzein could enhance the cell content of oestrogen receptor-β, which seems to be involved in the daidzein action in osteoblasts⁽Reference De Wilde, Lieberherr and Colin⁶⁹⁾.

Concerning other polyphenols, ellagitannins and ellagic acid, mainly found in red fruit, such as raspberries, strawberries, blackcurrants and pomegranate⁽Reference Clifford and Scalbert¹¹⁴⁾, are also oestrogenic but have not been studied as much. It has been shown that inhibitors of oestrogen receptors decreased the activity of ALP and mineralisation induced by ellagic acid in osteoblastic cells⁽Reference Papoutsi, Kassi and Tsiapara¹¹⁵⁾. Indeed, ellagic acid may act as a natural selective oestrogen receptor modulator⁽Reference Papoutsi, Kassi and Tsiapara¹¹⁵⁾ and may act through oestrogen receptors, but the molecular mechanisms are not well known and they need to be further investigated.

Resveratrol enhanced proliferation and osteoblastic differentiation in a dose-dependent manner in human bone marrow-derived mesenchymal stem cells⁽Reference Dai, Li and Quarles⁷¹⁾. This stimulating effect could be induced by an oestrogen receptor-dependent mechanism coupled to ERK1/2 activation. Moreover, ICI completely abolished the resveratrol-induced phosphorylation of ERK1/2 and p38, suggesting that oestrogen receptors were implicated in resveratrol MAPK activation⁽Reference Dai, Li and Quarles⁷¹⁾.

As reported above, some phenolic compounds can act on bone remodelling through the oestrogen receptors as well as via non-oestrogenic mechanisms, for example, resveratrol. This increases the difficulty associated with elucidating the molecular mechanisms of action of a given compound in a specific cell or tissue.

Polyphenols and osteoprotegerin/receptor activator of nuclear factor-κB ligand

Osteoblasts are implicated in the regulation of osteoclastogenesis through the OPG/RANKL/RANK regulatory system. OPG is a member of the TNF receptor family. This protein was characterised as an essential osteoclast differentiation factor expressed by osteoblastic/stromal cells and is involved in cell development⁽Reference Simonet, Lacey and Dunstan^116, Reference Rodan and Martin¹¹⁷⁾. Osteoblasts produce OPG, which acts as a decoy receptor for RANKL and thereby neutralises its function in osteoclastogenesis. Indeed, RANKL activates osteoclastogenesis by binding to RANK while OPG may inhibit the activation of osteoclasts and promote osteoclast apoptosis. Bone homeostasis seems to depend on the local RANKL:OPG ratio⁽Reference Lacey, Timms and Tan^118, Reference Yasuda, Shima and Nakagawa¹¹⁹⁾ and a mechanism involving the RANKL/OPG system is plausible because RANK is present in pre-osteoclastic cells⁽Reference Hsu, Lacey and Dunstan¹²⁰⁾.

Within the polyphenols there is most evidence to show that isoflavones are able to influence OPG/RANKL/RANK regulatory machinery implicated in the osteoblast and osteoclast relationship. Daidzein and 17β-oestradiol at low concentration (1 nm) stimulated osteoblast differentiation and were able to increase OPG secretion into the medium. Daidzein and genistein at physiological concentrations (0·1 and 10 nm) up-regulated OPG gene expression, but RANKL expression was not detectable in human osteoblastic cell lines⁽Reference Chen, Garner and Anderson¹¹³⁾. Moreover, daidzein enhanced RANKL protein secretion and content in the membrane in porcine osteoblasts⁽Reference De Wilde, Lieberherr and Colin⁶⁹⁾. This stimulatory effect was reversed by ICI, suggesting that the RANKL:OPG ratio is dependent on the oestrogen receptor pathway⁽Reference De Wilde, Lieberherr and Colin^69, Reference Chen, Garner and Anderson¹¹³⁾. It is possible that OPG and RANKL secretion induced by daidzein leads to an increase in the binding capacity of OPG to RANKL. In consequence, the amount of RANKL available to induce osteoclast formation and differentiation is reduced⁽Reference De Wilde, Lieberherr and Colin⁶⁹⁾.

However, Pang et al. demonstrated, in mouse primary calvaria osteoblasts, that kaempferol and quercetin dose dependently (5–20 μm) inhibited RANKL-induced expression of osteoclastic differentiation markers, which led to a decrease in RANKL-induced formation of multinucleated osteoclasts⁽Reference Pang, Ricupero and Huang⁷⁹⁾. The relationship between osteoblasts, osteoclasts and phytochemicals remains to be further investigated.

Effects of polyphenol extracts on osteoblasts

The effects of polyphenols on osteoblasts were mostly studied using pure molecules, unfortunately most of the time in aglycone forms. However, individuals consume not isolated molecules but fruit and vegetables, which are rich in many different polyphenols. It could be thus interesting to assess effects of phenolic mixtures on osteoblast function. It is important to take into consideration that polyphenols are metabolised before reaching the target cells.

The only available data with respect to osteoblasts are from Bu et al. who studied the influence of dried plum polyphenol extract at concentrations of 2·5, 5 and 10 μg/ml on MC3T3-E1 cells under normal and inflammatory conditions⁽Reference Bu, Hunt and Smith¹²¹⁾. This extract, non-toxic for osteoblasts, was able to stimulate ALP activity and mineralise nodule formation. The plum extract was not able to alter the expression of the transcription factors Runx2 and Osterix nor genes implicated in osteoclast regulation such as RANKL and OPG. However, this extract could restore Runx2 and Osterix expression in cells that had been pre-treated with TNFα (inflammatory condition) and as a result exhibited reduced Runx2 and Osterix expression. The extract also suppressed the TNFα-induced up-regulation of RANKL expression⁽Reference Bu, Hunt and Smith¹²¹⁾, but OPG expression was not affected by any treatments. These results suggest that consumption of dried plums could modulate both bone formation and resorption but the molecular mechanisms are poorly elucidated.