Cell Tissue Res (2014) 355:23–33 DOI 10.1007/s00441-013-1750-3

REVIEW

Osteoblast ontogeny and implications for bone pathology: an overview Irina Titorencu & Vasile Pruna & Victor V. Jinga & Maya Simionescu

Received: 13 August 2013 / Accepted: 4 October 2013 / Published online: 29 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Osteoblasts are specialized mesenchyme-derived cells accountable for bone synthesis, remodelling and healing. Differentiation of osteoblasts from mesenchymal stem cells (MSC) towards osteocytes is a multi-step process strictly controlled by various genes, transcription factors and signalling proteins. The aim of this review is to provide an update on the nature of bone-forming osteoblastic cells, highlighting recent data on MSC—osteoblast—osteocyte transformation from a molecular perspective and to discuss osteoblast malfunctions in various bone diseases. We present here the consecutive stages occurring in the differentiation of osteoblasts from MSC, the transcription factors involved and the role of miRNAs in the process. Recent data concerning the pathogenic mechanisms underlying the loss of bone mass and architecture caused by malfunctions in the synthetic activity and metabolism of osteoblasts in osteoporosis, osteogenesis imperfecta, osteoarthritis and rheumatoid arthritis are discussed. The newly acquired knowledge of the ontogeny of osteoblasts will assist in unravelling the abnormalities taking place during their differentiation and will facilitate the prevention and/or treatment of bone diseases by therapy directed against altered molecules and mechanisms.

The authors gratefully acknowledge the financial support of the Romanian National CNCSIS (grant IDEI PCCE code 248/2010) and the Romanian Academy. I. Titorencu : V. Pruna : V. V. Jinga Regenerative Medicine Department, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania M. Simionescu (*) Cellular and Molecular Biology Department, Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Bucharest, Romania e-mail: [email protected]

Keywords Osteoblasts . Bone . Osteoblasts ontogeny . Mesenchymal stem cell . Bone pathology

Introduction Osteoblasts, the cells responsible for bone synthesis and remodelling, are derived from multipotent marrow stromal stem cells, termed mesenchymal stem cells (MSC). During ontogeny, under the influence of specific transcription factors, MSC become committed to the osteoblast lineage and pass through various stages that include determined osteoprogenitor cells, pre-osteoblasts, osteoblasts and ultimately osteocytes (Oreffo et al. 2005). Each cell type has important roles in the bone tissue and the bone marrow niche. Once committed to differentiation towards the osteoblast lineage, the secretory capacity of preosteoblasts and osteoblasts increases, thus participating in bone synthesis and remodelling. A better understanding of the ontogeny of osteoblastic cells, i.e., the conditions and the gene expression associated with each stage of differentiation, is critical for unravelling the mechanisms underlying bone physiology and pathology.

Bone: many functions are carried out within its complex structure Bone is a dynamic, constantly renewing type of hard connective tissue, having the unique capacity to heal and remodel without leaving a scar (Sommerfeldt and Rubin 2001). Among its several major functions, bone provides structural support for the soft tissues of the body, houses the bone marrow and serves as a mineral reservoir with a central role in mineral body homeostasis (Kartsogiannis and Ng 2004; Rodan 1992). As in all connective tissues, the basic constituents of the bone are an extensive matrix (a bonding of multiple

24

fibres and various ions) and diverse cell types that function in bone synthesis and continuous remodelling. The bone matrix consists of an organic component (synthesized by osteoblasts) reinforced by inorganic calcium salt deposits. Type I collagen and an extra-fibrillar matrix (proteoglycans or non-collagenous proteins) constitute the organic component. The collagen fibres comprise about 90 % of the total protein in the bone and are oriented in a preferential direction, conferring the structure of the lamellar bone. The remaining 10 % of the organic matrix is composed of (1) proteoglycans (chondroitin sulphate and heparin sulphate), (2) cell attachment proteins such as fibronectin, thrombospondin, osteopontin and bone sialoprotein (Robey et al. 1989; Somerman et al. 1988), (3) γ-carboxylated (gla) proteins (osteocalcin; Robey et al. 1989) and (4) growth-related proteins, i.e., transforming growth factor-β (TGF-β) and insulin-like growth factors (IGF; Hauschka et al. 1988; Canalis et al. 1989). These anionic complexes have a high ion-binding capacity and play an important role in the calcification process. Bone crystals are deposited within this three-dimensional organic network. They are composed principally of calcium and phosphate, which combine to form spindle- or plate-shaped hydroxyapatite crystals [Ca10[PO4)6(OH)2] that are 30–50 nm long, 15–30 nm wide and 2–10 nm thick (Eppell et al. 2001). Skeletal tissue is formed and remodelled throughout life by the action of two cell lineages: the bone-forming cells (osteoblasts, osteocytes and bone-lining cells) and the boneresorbing cells (osteoclasts) that together with their precursors and associated cells (i.e., endothelial cells, nerve cells) are organized in specialized units, the bone multicellular units (Frost 2001). Tissue obtained by osteoblastic activity is maintained by the action of osteocytes and bone-lining cells, which are terminally differentiated forms of osteoblasts (Robling et al. 2006). The bone-forming cells and bone-resorbing cells have different origins: the former are derived from mesenchymal progenitor cells, whereas osteoclasts are derived from haematopoietic progenitor cells. They proliferate and differentiate in response to osteotropic hormones, cytokines and various other stimuli. Uncovering the mechanisms that control the differentiation of osteoblasts is one of the fundamental areas of bone biology research.

Ontogeny of osteoblasts MSC: the source of osteoblasts, chondrocytes and adipocytes The non-haematopoietic compartment of bone marrow is populated by pluripotent MSC that differentiate into a variety of cells upon the activation of specific signalling transcription pathways. MSC are defined by two key characteristics: (1) the ability to differentiate into osteoblasts, chondrocytes and

Cell Tissue Res (2014) 355:23–33

adipocytes and (2) their limited proliferation capacity, since they enter senescence after a few rounds of populationdoubling in culture (Bonab et al. 2006). The exact location of MSC in the bone marrow is not known but data suggest that they are located in the perivascular spaces as subendothelial cells surrounding the vascular sinusoids (Sacchetti et al. 2007; Arvidson et al. 2011). In addition to bone marrow, MSC are present in many other tissues of the body such as the synovium (Jones et al. 2010), adipose tissue (Lin et al. 2010), skeletal muscle (Bosch et al. 2000), periosteum (Sampaio de Mara et al. 2011), dermis (Vaculik et al. 2012), blood (He et al. 2007), deciduous teeth (Huang et al. 2009), amniotic fluid (Sessarego et al. 2008; Baghaban Eslaminejad et al. 2011) and umbilical cord blood (Bieback and Klüter 2007). MSC can be isolated from the mononuclear non-phagocytic fraction of the bone marrow aspirate; they represent about 0.01 %–0.001 % of the marrow mononuclear population (Pittenger et al. 1999). When expanded in vitro, they form fibroblast-like cell clusters (fibroblast colony forming units, CFU-F), the number of which is dependent on the clonogenic potential of MSC (Jiang et al. 2002; Krampera et al. 2005). The adherent cells express a complex pattern of molecules that include CD105 (SH2), CD73 (SH3 and SH4), CD106 (VCAM-1), CD54 (ICAM-1), CD44, CD90, CD29, STRO-1, CD120a and CD124 (Pittenger et al. 1999; Horwitz et al. 1999; Jiang et al. 2002; Smith et al. 2004; Krampera et al. 2006a). Moreover, they express immune molecules such as HLA class I and II (the latter only upon the effect of interferon-gamma, IFN-γ) and CD119 (IFN-γ receptor; Krampera et al. 2006b). Generally they are negative for haematopoietic markers, such as CD45 and CD34 (Pittenger et al. 1999; Horwitz et al. 1999; Jiang et al. 2002; Smith et al. 2004; Krampera et al. 2006a, 2006b). Osteoblast differentiate from pluripotent MSC The differentiation of MSC into osteoblasts is a complex, highly regulated process that can be defined by four consecutive stages: (1) lineage commitment, (2) proliferation, (3) extracellular matrix maturation and (4) matrix mineralization (Taipaleenmäki et al. 2012). Each stage is characterized by specific subsets of expressed genes and factors regulating the process. As shown in Fig. 1, in stage 1, under the influence of bone morphogenetic protein (BMP), MSC become committed to differentiate into osteoprogenitor cells. During stage 2, the osteoprogenitor cells express Runt-related transcription factor 2 (Runx-2) and collagen type I (Coll I) and synthesize the histones needed for the high rate of proliferation characteristic of this step. MSC-derived osteoprogenitor cells represent an intermediate stage between a stem cell and the differentiated progeny, the osteoblasts. In stage 3, the parathyroid hormone (PTH) induces the cell expression of alkaline phosphatase,

Cell Tissue Res (2014) 355:23–33

25

Fig. 1 The four differentiation stages of mesenchymal stem cells (MSC) into osteoblasts: lineage commitment, proliferation, extracellular matrix maturation and matrix mineralization. Under the influence of bone morphogenetic protein (BMP), MSC become committed to differentiate into osteoprogenitor cells. The parathyroid hormone (PTH) induces the cell expression of alkaline phosphatase, type I collagen and bone sialoprotein II and thereby the passage to the pre-osteoblastic cells. Under the influence of insulin growth factor 1 (IGF1) and prostaglandin E2 (PGE2),

pre-osteoblastic cells become mature osteoblasts expressing osteocalcin, collagenases and bone sialoprotein type I and II. The extracellular matrix then becomes mineralized and the osteoblasts trapped in a cavity within the matrix reach the stage of osteocytes. The microclimate that is provided by the osteoid (blue) favours crystallization. Other mature osteoblasts are not entrenched within the matrix; these are the bone-lining cells (inactive osteoblasts) that cover the entire available bone surface and function as an ion barrier

type I collagen and bone sialoprotein II (BSPII) and thereby the passage to the pre-osteoblastic cells. The latter are involved in the synthesis of growth factors and the matrix components of bone tissue. During stage 4, under the influence of IGF1 and prostaglandin E2 (PGE2), pre-osteoblastic cells become mature osteoblasts expressing osteocalcin, collagenases and bone sialoprotein type I and II (BSP I, BSP II). At this stage, the extracellular matrix becomes mineralized and the osteoblasts trapped in a cavity within the matrix reach the stage of osteocytes. Other mature osteoblasts are not entrenched within the matrix; these are the bone-lining cells (inactive osteoblasts) that cover the entire available bone surface and function as an ion barrier. Indeed, bone marrow contains coexisting cells that exhibit various stages in bone development including MSC, determined osteoprogenitor cells and preo-steoblasts that have started to differentiate but that have not yet begun to synthesize the extracellular matrix (Oreffo et al. 2005). Regulators such as neurotransmitters and peptides, hormones (PTH parathyroid hormone), vitamins (vitamin D, 1, 25(OH)2), growth factors (BMPs, IGF and fibroblasts growth factors, FGFs), transcription factors and mechanical loading are involved in the regulation of the complex and finely tuned

process of osteoblast differentiation (Giustina et al. 2008; Nakashima and De Crombrugghe 2003; Kapinas and Delany 2011). These factors activate specific intracellular pathways that trigger the expression of several osteoblast-specific transcription factors, which together with RNA-binding proteins and microRNAs (miRNAs) act coordinately to control the gene expression networks essential for osteoblast differentiation and function.

Transcription factors involved in differentiation of osteoblasts The factor Runx2, also known as osteoblast specific factor (Osf 2) or core-binding factor α (Cbfa1), is an essential transcription factor for the differentiation of MSC into osteoblasts. It is involved in osteoblast recruitment and the regulation of the expression of several osteoblastic genes, such as COL1A1 (for collagen Col1a1), ALPL (for alkaline phosphatase), BSP (for bone sialoprotein), SPP1 (for osteopontin) and BGLAP (for osteocalcin; Komori et al. 1997; Karsenty et al. 1999; Ducy et al. 1999).

26

Runx2 is important throughout the differentiation of MSC and its level increases gradually over the time course of the process. The expression of Runx2 is necessary and sufficient for the induction of MSC differentiation towards the osteoblastic lineage and for suppressing their differentiation into adipocytes and chondrocytes (Komori 2006; Lian et al. 2004). However, Runx2 is not critical for the maintenance of the expression of major bone matrix protein genes in the mature osteoblasts (Liu et al. 2001; Maruyama et al. 2007). The activity of Runx2 is controlled by transcription factors and by protein-DNA or protein-protein interactions. Oestrogen interacts with Runx2. The oestradiol receptor (ERα) interacts with Runx2 either directly through its DNAbinding domain or indirectly through its N-terminal and ligand-binding domain resulting in a strong repression of Runx2 transcriptional activity (Khalid et al. 2008). Because Runx 2, through its two isoforms (Runx2-II and Runx2-I) has a dose-dependent effect on the differentiation of mesenchymal cells into the osteoblastic lineage, its repression leads to a decreased number of osteoblasts and thus to a low turnover (Zhang et al. 2009). Androgen receptors (AR) interact with Runx2 in the presence of dihydrotestosterone resulting in repression of Runx2 in the osteoblastic cells. Repression is associated with the nuclear co-localization of the two proteins (Runx2 and AR) and does not require the transactivation function of AR. Activated AR prevents RUNX2 binding to DNA or other nuclear components. As in the case of oestrogen receptors, the AR-induced inactivation of Runx2 functions leads to a low production of osteoblasts and has an important role in bone metabolism and in the prevention of high bone turnover (Baniwal et al. 2009). Runx2, c-Fos and c-Jun interact and cooperatively bind the AP-1 and Runt-domain-binding sites in the collagenase-3 promoter resulting in promoter activation (D’Alonzo et al. 2002). Collagenase-3 (MMP-13) is a matrix metalloproteinase synthesized by osteoblasts and cleaves many types of extracellular matrix proteins, thus being operational in bone growth, repair and remodelling (Partridge et al. 1996). In early preosteoblast development, the factors Hoxa2 and Satb2 regulate Runx2 activity (Dobreva et al. 2006), whereas other factors such as Stat1,Sox9, Sox8, Aj18, MEF, Nrf2 and YAP repress Runx2 expression (Kim et al. 2007; Schmidt et al. 2005; Zhou et al. 2006). Osterix (osteoblast-specific transcription factor), a zincfinger transcription factor, is a second transcription factor that is essential for osteoblast differentiation, in vivo bone formation and mineralization (Nakashima et al. 2002). Osterix is believed to act downstream of Runx2 interacting with NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1). Both osterix and Runx2 expression in differentiated osteoblasts are modulated by the intracellular immunophilin-mediated calcineurin/NFATc1 pathway and

Cell Tissue Res (2014) 355:23–33

increase alkaline phosphatase and Col1a1 promoter activity (Varanasi and Datta 2005). Other important transcription factors involved in the differentiation of osteoblasts include: activating transcription factor 4 (ATF4), which is the major determinant of osteoblast function (Karsenty 2008), transcriptional coactivator (TAZ), which functions as a transcriptional coactivator for Runx2 (Cui et al. 2003) and homeodomain proteins Msx (Msh homeobox homolog) Msx1, Msx2, Dlx (distal-less homeobox), Dlx5 and Dlx6, which act at early stages of osteoblast differentiation and during skeletal development (Gordeladze et al. 2010).

MiRNAs and differentiation of osteoblasts Recent progress in the regulation of osteogenesis has highlighted the role of miRNAs, which reportedly target the principal transcription factors and signalling molecules involved in the differentiation and function of osteoblasts. miRNAs are small singlestranded endogenous RNAs (19–25 nucleotide long) that control gene expression by targeting mRNAs at the post-transcriptional level (Vimalraj and Selvamurugan 2012). Depending on the target, miRNA have positive or negative effect on osteoblast lineage development (Table 1). MiRNAs that promote differentiation of osteoblasts A large number of miRNAs have been identified that regulate every step of osteogenesis, from bone development to the maintenance and repair of adult tissue, by regulating the activity of cells involved in the growth, differentiation and mineralization of bone (Lian et al. 2012). Some miRNAs (miR-29b, miR-148b, miR-196a, miR-210, miR-335-5p, miR-2861 and miR-3960) have been reported to exert stimulatory effects by targeting and triggering the down-regulation of various inhibitors of osteoblast differentiation (Hu et al. 2011; Li et al. 2009a; Kapinas et al. 2009). Other miRNAs (miRNA-29 family) have multiple distinct activities at various stages of osteoblast differentiation. Thus, the expression of miRNA-29 is low during the early matrix deposition phase of osteoblastogenesis. Subsequently, the expression increases throughout matrix maturation and the terminal differentiation stage of the osteoblasts. miRNA-29a and c are reported to be involved in the differentiation of osteoblasts by targeting negative regulators of Wnt signalling, Dkk1, Kremen and sFRP2, thus further promoting Wnt activity (Kapinas et al. 2010), whereas miRNA-29b represses protein LZIC, an inhibitor of β-catenin. Some miRNAs are embedded in introns and are expressed together with the genes in which they reside, whereas other miRNAs are clustered and/or are regulated by their own promoters (Lian et al. 2012); as an example, the miRNA23a-27a-24-4 cluster has key regulatory circuits for the

Cell Tissue Res (2014) 355:23–33

27

Table 1 MicroRNAs (miRNAs) involved in the stimulation/inhibition of osteogenesis miRNAs

Target genes

miR-9

MyoD, Mif5

Effects

Promote osteogenesis by down-regulation of muscle transcription factor miR-20a BAMB1, CRIM Promote osteogenesis by increasing BMP signalling miR-27 APC Promote osteogenesis by enhancing Wnt signalling via the repression of APC miR-29b TGFβ3, HDAC4, DUSP2,AcvR2b Promote osteogenesis miR-29c SPARC, Dkk1, Kremen,SFRP2 Promote osteogenesis by Wnt signalling miR-125 Osterix Promotes osteogenesis miR-196a HOXC8 Promotes osteogenesis miR-210 TGFβ, ACVR1b Promote osteogenesis miR-335-5p DKK1 Promotes osteogenesis miR-378 GaINT7, Nephronectin Promote osteogenesis miR-2861 HDAC5 Promotes ostogenesis miR-148b Unknown Promotes ostogenesis let-7a, b c, miR-24, -125b, Unknown Promote ostogenesis through PDGF -138, -320 pathway miR-23a, -27a, -24-2 RUNX2, SatB2, Hoxa10 Suppress osteogenesis miR-26a Smad1 Suppresses osteogenesis miR-29a,b Col1A, Col3A, Col5 Suppress osteogenesis miR-125b ERBB2 Suppress osteogenesis miR-133,-135 RUNX2, SMAD5 Suppress osteogenesis miR-141, -200a Dlx5 Suppresses osteogenesis miR-206 CX43 Suppresses osteogenesis miR-204, -211, -205, -217, -338 RUNX2 Suppresses osteogenesis miR-335 RUNX2 Suppresses osteogenesis miR-363, -95,-let7a BGLAP Suppresses osteogenesis miR-637 Osterix Suppresses osteogenesis

induction of osteogenesis and the regulation of the progression of differentiation. The promoter of the cluster has a regulatory element bound by the Runx family of tissue-specific transcription factors and, as such, the expression of the cluster in osteoblasts is down-regulated by Runx2 (Hassan et al. 2010). Another example is the miR-2861tomiR-3960 cluster, which is induced by the BMP2–Runx2 pathway (Hu et al. 2011). Thus, the miR-23ato27ato24-2 and the miR-2861tomiR-3960 clusters represent central components of two intersecting regulatory loops that are repressed by Runx2. MiRNAs that inhibit differentiation of osteoblasts The balance between bone formation and bone resorption processes is controlled, among others, by various types of miRNAs. In the process of regulation bone remodelling, some miRNAs inhibit osteogenesis through the repression of osteoblastic genes. Thus, the overexpression of miR-206 in osteoblasts inhibits their differentiation (Vimalraj and Selvamurugan 2012). In addition, several miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-211, miR-217

Reference Li et al. 2008 Zhang et al. 2011a, 2011b, 2011c Wang and Xu 2010 Li et al. 2009a Kapinas et al. 2010 Goettsch et al. 2011 Kim et al. 2009 Mizuno et al. 2009 Tome et al. 2011 Kahai et al. 2009 Li et al. 2009b Schoolmeesters et al. 2009 Goff et al. 2008 Hassan et al. 2010 Luzi et al. 2008 Maurer et al. 2010 Mizuno et al. 2008 Li et al. 2008 Li et al. 2008 Inose et al. 2009 Huang et al. 2010 Tome et al. 2011 Octacílio-Silva et al. 2010 Zhang et al. 2011a, 2011b, 2011c

and miR-338) have the same effect by targeting the Runx2 gene and thus decreasing the expression of Runx2 protein (Hassan et al. 2010; Zhang et al. 2011a, 2011b, 2011c). MiR-125b inhibits osteoblastic proliferation by ErbB2 receptor tyrosine kinase inhibition (Li et al. 2008). Some miRNAs are overexpressed in osteo-differentiated MSC and target MSC markers (such as CD44, ITGB1, FLT1, FGF2 and CXCL12) and genes involved in the inhibition of osteogenesis (BMPER, CAMTA1 and GDF6; Vimalraj and Selvamurugan 2012). In addition, the differentiation of osteoblasts can be down-regulated by hsa-miR-27a and hsa-miR-489, which induce the repression of GCA (grancalcin), PEX7 (peroxisomal matrix enzymes) and APL (liver/bone/kidney-specific alkaline phosphatase) proteins that are critical for osteogenesis (see Table 1).

Osteoblasts and the synthesis of bone material Mature osteoblasts are cuboidal cells that are organized in clusters located on the bone surface and that are adapted for

28

a secretory function. Structurally, they display a well-developed rough endoplasmic reticulum with dilated cisternae, a welldeveloped Golgi complex and numerous transport vesicles and vacuoles loaded with fibrillar structures, most probably pro-collagen and proteoglycans (Jayakumar and Di Silvio 2010; Nakamura 2007; Neve et al. 2011). The two main stages in the development of the mature bone are the synthesis of the matrix and the mineralization of the matrix by the deposition of minerals. In the first stage, osteoblasts synthesize and secrete an organic matrix, the osteoid, predominantly formed by collagen type I (Fig. 1). In the process of mineralization, the high activity of alkaline phosphatase induces an increase in the local concentration of calcium and phosphate. In addition, osteoblasts produce osteocalcin, a protein that binds calcium, thereby contributing to the increase in calcium concentration. These processes lead to the local over-saturation of extracellular fluid and, within the microclimate that is provided by the osteoid and that favours crystallization, the formation of crystals results in the mineralization of the organic matrix (Jayakumar and Di Silvio 2010). Two types of crystal are found as a function of location: intrafibrillar and interfibrillar crystals. The intrafibrillar crystals are small and their disposition is directed by collagen fibrils (Bonucci 1992), whereas the interfibrillar crystals are formed on the surface and in-between the collagen fibres (Martin et al. 1998). Osteoblasts also secrete non-collagenous proteins (osteonectin, osteopontin, sialoproteins) that play an important role in bone mineralization, many of these proteins being highly charged (Giachelli and Steitz 2000; Gericke et al. 2005). From osteoblasts to osteocytes and lining cells Osteoblasts play an important role in osteogenesis, both in endochondral and in intramembranous ossification. In both cases, a primary immature bone tissue is formed. This temporary structure has an irregular network of small collagen fibres and is rapidly mineralized. The primary tissue is replaced by secondary bone (found in adults), which is highly organized with large collagen fibrils that form lamellar structures. At this stage, slow matrix mineralization begins with the deposition of calcium salts on collagen fibres, a process in which proteoglycans and non-collagenous proteins with a high affinity for calcium are involved (Bonucci 1992; Junqueira and Carneiro 2008). After the active stage of bone formation, some osteoblasts become osteocytes, buried in the newly formed matrix (Fig. 1). Osteocytes are the terminal stage of osteoblast differentiation; reportedly, they have important roles in sensing mechanical strain and translating the information into biochemical signals that lead to the functional adaptation of bone. In addition,

Cell Tissue Res (2014) 355:23–33

osteocytes function in bone mineralization and phosphate metabolism. They produce signals acting in both bone resorption and bone synthesis through specific factors such as DMP1 (dentin matrix protein 1) and MEPE (matrix extracellular phosphoglycoprotein). These factors are augmented in response to loading, whereas sclerostin increases under conditions of unloading. DMP-1 has been found to promote mineralization and mineral homeostasis, whereas MEPE is an inhibitor of mineralization and sclerostin inhibits the Wnt signalling pathways leading to decreased bone formation (Kogianni and Noble 2007; Bonewald 2007). Other osteoblasts, the lining cells, remain on the bone surface (Fig. 1) and have a different morphology and function: they are flattened in shape and contain few cell organelles (Nakamura 2007). They act as a deposit of osteogenic “determined” precursors and can proliferate and differentiate into active osteoblasts under appropriate conditions. Lining cells also have important roles in haematopoiesis, in bone resorption and remodelling and in controlling the flow of ions between interstitial fluid and bone (Miller et al. 1989).

Osteoblasts in pathology Abnormalities occurring during the differentiation of osteoblasts and/or in their activity generate a number of severe human diseases such as osteoporosis, osteoarthritis, rheumatoid arthritis and osteogenesis imperfecta. Osteoporosis This disease is the result of several pathogenic mechanisms converging in the loss of bone mass and density and an alteration in bone strength and architecture. Osteoporosis might arise because of abnormal bone formation in terms of weight and strength, excessive bone resorption or imbalanced bone formation/resorption during the bone remodelling process (Raisz 2005). In osteoporotic conditions, osteoblasts have a lower proliferation rate than normal and their functions are modified, i.e., they produce a smaller amount of osteocalcin compared with normal cells (Maruotti et al. 2009). Osteoporosis cannot be attributed only to a decreased activity of osteoblasts; it might also be the result of an increased activity of osteoclasts in the remodelling process. An inadequate rate of bone resorption by osteoclast cells and bone replacement by malfunctioning osteoblasts can lead to a decrease in bone density, osteoporosis and a high risk of fracture. An important factor in the development of osteoporosis is oestrogen deficiency both in women and in men (Riggs et al. 1998). Low levels of oestrogens increase the rate of bone remodelling, a process that can be slowed down by oestrogen treatment. Oestrogens act through the oestrogen receptor beta

Cell Tissue Res (2014) 355:23–33

(ERβ) and alpha (ERα), the latter being the main mediator of oestrogen action on bone cells (Windahl et al. 2001; Sims et al. 2002). The receptors are ligand-dependent and have been found both in cultured osteoblast-like cells and in native osteoblasts. In primary osteoblasts, the presence of a 46-kDa isoform of ERα inhibits the proliferation that is induced by the normal ER isoform in human osteosarcoma cells (Denger et al. 2001). ERs act through genomic and non-genomic mechanisms. The genomic mechanism involves the direct binding of ERs to oestrogen response elements (the short DNA sequences within the promoter of a gene) or the interaction between ERs and other transcription factors. The nongenomic mechanisms lead to rapid cellular responses (within minutes) and are attributable to the activation of certain signalling pathways (such as mitogen-activated protein kinase [MAPK] and phosphatidyl-inositol-3-kinase [PI3K]) by ERs located in the cytoplasm or on the plasma membrane (Manolagas et al. 2004). During the differentiation of osteoblasts, ERα and ERβ are expressed at different levels, at least at the mRNA level. In the cultured SV-HFO cell line (human fetal osteoblastic cells immortalized with SV 40 virus), both ERβ mRNA and ERα mRNA have been found to increase until the cells reach confluence; in postconfluent cell culture, only the level of ERβ increass, whereas ERα mRNA remains constant until mineralization (Arts et al. 1997). Osteoclasts (bone-resorbing cells), which are derived from the haematopoetic lineage, require an interaction with osteoblasts for their differentiation. Important roles in this process are played by tumour necrosis factor (TNF) and the TNF receptor superfamily, receptor activator of nuclear factor-kb (RANK), its ligand RANKL and osteoprotegerin (OPG). Osteoblasts produce RANKL, which can be both secreted and membrane-bound (Burgess et al. 1999). RANKL acts as a ligand for the RANK receptor, which is present on the surface of haematopoietic cells. Osteoblasts also produce OPG or osteoclast inhibiting factor, which is a glycoprotein also belonging to the TNF family. After the interaction between RANKL and the RANK receptors of osteoblastic cells, haematopoietic cells differentiate into osteoclasts. When OPG interacts with RANKL, the differentiation of cells from the haematopoietic lineage to osteoclasts is blocked. Stimulators of bone resorption increase RANKL expression in osteoblasts and decrease OPG expression (Suda et al. 1999). In early postmenopausal women, the RANKL level is increased on the surface of bone marrow cells in response to the low oestrogens concentration (Eghbali-Fatourechi et al. 2003). Osteogenesis imperfecta The malfunction in the osteoblast synthesis of extracellular matrix components and the factors involved in its mineralization (osteocalcin, osteonectin and bone sialoproteins) lead to

29

severe manifestations such as those in osteogenesis imperfecta (or Lobstein syndrome), which is a heritable syndrome that affects the quality and quantity of the extracellular matrix, resulting in bone fragility and a high risk of fracture (Byers and Steiner 1992; Kalajzic et al. 2002). Depending on clinical and radiographic features, osteogenesis imperfecta has been classified into four types but, currently, based on detailed radiographic and molecular genetic analyses, the classification has been expanded to seven types (Kozloff et al. 2004). The severity of manifestations ranges from almost asymptomatic (mild risk of fracture) to severe bone fragility with a high risk of fracture, deformities and even prenatal death. Mutation in type I collagen is the major cause (90 %) of osteogenesis imperfecta. As an example, in osteogenesis imperfecta type IV, ∼85 % of cases result from the substitution of glycine by a larger aminoacid (i.e., cysteine, serine, arginine) in the collagen primary structure (Kozloff et al. 2004). A clear correlation exists between the various mutations, their position and associated phenotype. In addition, the low production of collagen might cause osteogenesis imperfecta (Sillence et al. 1979; Rowe 2008). Osteoarthritis This is a chronic inflammatory disease, being a degenerative joint disorder that involves synovial membrane, periarticular bone and degenerated cartilage that is progressively consumed. The hypothesis that subchondral bone has a central role in this disease has been confirmed by several studies that have demonstrated that osteoblasts from subchondral bone adjacent to the affected joint have an abnormal metabolism (El Miedany et al. 2000; Hilal et al. 1998; Lajeunesse and Reboul 2003). In osteoarthritis, altered bone remodelling and Wnt and TGF-β/BMP signalling have been observed (Hopwood et al. 2007). Moreover, in such osteoblasts, an increased level of secreted osteocalcin and alkaline phosphatase activity has been reported (Hilal et al. 1998). As in the case of osteoporosis, the system OPG/RANK/RANKL plays a key role in the remodelling rate of subchondral bone, since the ratio between OPG/RANKL controls the differentiation of haematopoietic lineage into osteoclasts; an abnormal ratio of OPG/RANKL has been reported in the subchondral bone of patients with osteoarthritis (Tat et al. 2008a, 2008b). Rheumatoid arthritis Rheumatoid arthritis is a chronic immune-mediated inflammation of the synovial membrane resulting in the degradation of articular cartilage and periarticular bone. Inability of the bone to recover indicates possible changes in bone cell function under the influence of inflammatory factors, leading to an imbalance between bone synthesis by osteoblasts and bone degradation by osteoclasts. Osteoclasts are the main cells involved in bone loss

30

in rheumatoid arthritis (Pettit et al. 2001; Shealy et al. 2002); however, osteoblast differentiation and function are also abnormal in this disease. During this process, TNF-α produced by inflammatory cells induces the up-regulation of Dickkoph (DKK) proteins and secreted Frizzled-related proteins (sFRP), which are inhibitors of the Wnt signalling pathway, thus compromising osteoblast function and the ensuing bone matrix mineralization (Walsh et al. 2009; Diarra et al. 2007). Conclusions and future perspectives The new knowledge of the biology of the MSC-derived osteoblastic lineage has acted as a guide to novel therapeutic opportunities and approaches to the healing of bone tissue. The fine-tuning of the required factors and the conditions for osteoblast-specific gene expression associated with each stage of differentiation, such as the hormones, cytokines, transcriptional factors and proteins, remain to be investigated. Deciphering these mechanisms will allow us to distinguish when and where a modification occurs and will make possible the prevention and/or treatment of bone diseases by therapy targeted to altered molecules or mechanisms. In the future, promising bone repair strategies will include the utilization of biocompatible three-dimensional scaffolds populated with bone-forming cells (osteoprogenitor/osteoblastic cells) and the utilization of drug-delivery systems with the controlled release of osteoinductive and angiogenic factors. Thereby, regenerative biology resources constitute new alternatives of great expectation in the treatment of bone disorders and trauma.

References Arts J, Kuiper GGJM, Janssen JMMF, Gustafsson J-A, Lowik CWGM, Pols HAP, Van Leeuwen JPTM (1997) Differential expression of estrogen receptors a and b during differentiation of human osteoblast SV-HFO cells. Endocrinology 138:5067–5070 Arvidson K, Abdallah BM, Applegate LA, Baldini N, Cenni E, GomezBarrena E, Granch D, Kassem M, Konttinen YT, Mustafa K, Pioletti DP, Sillat T, Finne-Wistrand A (2011) Bone regeneration and stem cells. J Cell Mol Med 4:718–746 Baghaban Eslaminejad M, Jahangir S, Aghdami N (2011) Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation. Arch Iran Med 14:96–103 Baniwal SK, Khalid O, Sir D, Buchanan G, Coetzee GA, Frenkelb B (2009) Repression of Runx-2 by androgen receptor (AR) in osteoblasts and prostate cancer cells: AR binds Runx-2 and abrogates its recruitment to DNA. Mol Endocrinol 23:1203–1214 Bieback K, Klüter H (2007) Mesenchymal stromal cells from umbilical cord blood. Curr Stem Cell Res Ther 2:310–323 Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B (2006) Aging of mesenchymal stem cell in vitro. BMC Cell Biol 10:7–14 Bonewald LF (2007) Osteocytes as dynamic multifunctional cells. Ann N Y Acad Sci 1116:281–290

Cell Tissue Res (2014) 355:23–33 Bonucci E (1992) Role of collagen fibrils in calcification. In: Bonucci E (ed) Calcification in biological systems. CRC Press, Boca Raton, pp 19–41 Bosch P, Musgrave DS, Lee JY, Cummins J, Shuler T, Ghivizzani TC, Evans T, Robbins TD, Huard J (2000) Osteoprogenitor cells within skeletal muscle. J Orthop Res 18:933–944 Burgess TL, Qian Y, Kaufman S, Ring BD, Van G, Capparelli C, Kelley M, Hsu H, Boyle WJ, Dunstan CR, Hu S, Lacey DLJ (1999) The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527–538 Byers PH, Steiner RD (1992) Osteogenesis imperfecta. Annu Rev Med 43:269–282 Canalis E, McCarthy TL, Centrella M (1989) Growth factors and the skeletal system. J Endocrinol Invest 12:577–584 Cui CB, Cooper LF, Yang X, Karsenty G, Aukhiand I (2003) Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ. Mol Cell Biol 23:1004–1013 D’Alonzo RC, Selvamurugan N, Karsenty G, Partridge NC (2002) Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. J Biol Chem 277:816–822 Denger S, Reid G, Kos M, Flouriot G, Parsch D, Brand H, Korach KS, Sonntag-Buck V, Gannon F (2001) ER α gene expression in human primary osteoblasts: evidence for the expression of two receptor proteins. Mol Endocrinol 15:2064–2077 Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D, Korb A, Smolen J, Hoffmann M, Scheinecker C, van der Heide D, Landewe R, Lacey D, Richards WG, Schett G (2007) Dickkopf-1 is a master regulator of joint remodeling. Nat Med 13:156–163 Dobreva G, Chahrour M, Dautzenberg M, Chirivella L, Kanzler B, Farinas I, Karsenty G, Grosschedl R (2006) SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125:971–986 Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, Amling M, Karsenty G (1999) A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 13: 1025–1036 Eghbali-Fatourechi G, Khosla S, Sanyal A, Boyle WJ, Lacey DL, Riggs BL (2003) Role of RANK ligand in mediating increased bone resorbtion in early postmenopausal women. J Clin Invest 111: 1221–1230 El Miedany YM, Mehanna AN, El Baddini MA (2000) Altered bone mineral metabolism in patients with osteoarthritis. Joint Bone Spine 6:521–527 Eppell SJ, Tong W, Katz JL, Kuhn L, Glimcher MJ (2001) Shape and size of isolated bone mineralites measured using atomic force microscopy. J Orthop Res 19:1027–1034 Frost HMJ (2001) Why should many skeletal scientists and clinicians learn the Utah paradigm of skeletal physiology. J Musculoskelet Neuronal Interact 2:121–130 Gericke A, Qin C, Spevak L, Fujimoto Y, Butler WT, Sorensen ES, Boskey AL (2005) Importance of phosphorylation for osteopontin regulation of biomineralization. Calcif Tissue Int 77:45–54 Giachelli CM, Steitz S (2000) Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol 19:615–622 Giustina A, Mazziotti G, Canalis E (2008) Growth hormone, insulin-like growth factors, and the skeleton. Endocr Rev 29:535–559 Goettsch C, Rauner M, Pacyna N, Hempel U, Bornstein SR, Hofbauer LC (2011) miR-125b regulates calcification of vascular smooth muscle cells. Am J Pathol 179:1594–1600 Goff LA, Boucher S, Ricupero CL, Fenstermacher S, Swerdel M, Chase LG, Adams CC, Chesnut J, Lakshmipathy U, Hart RP (2008) Differentiating human multipotent mesenchymal stromal cells regulate microRNAs: prediction of microRNA regulation by PDGF during osteogenesis. Exp Hematol 36:1354–1369

Cell Tissue Res (2014) 355:23–33 Gordeladze JO, Reseland JE, Duroux-Richard I, Apparailly F, Jorgensen C (2010) From stem cells to bone: phenotype acquisition, stabilization, and tissue engineering in animal models. ILAR J 51:42–61 Hassan MQ, Gordon JAR, Belot MM, Croce CM, van Wijnen AJ, Stein JL, Stein GS, Lian JB (2010) A network connecting Runx2, SATB2, and the miR-23a 27a 24–2 cluster regulates the osteoblast differentiation program. Proc Natl Acad Sci U S A 107:19879–19884 Hauschka PV, Chen TL, Mavrakos AE (1988) Polypeptide growth factors in bone matrix. Ciba Found Symp 136:207–225 He Q, Wan C, Li G (2007) Concise review: multipotent mesenchymal stromal cells in blood. Stem Cells 25:69–77 Hilal G, Martel-Pelletier J, Pelletier JP, Ranger P, Lajeunesse D (1998) Osteoblast-like cells from human subchondral osteoarthritic bone demonstrate an altered phenotype in vitro. Arthritis Rheum 41: 891–899 Hopwood B, Tsykin A, Findlay DM, Fazzalari NL (2007) Microarray gene expression profiling of osteoarthritic bone suggests altered bone remodelling, WNT and transforming growth factor-β/bone morphogenic protein signaling. Arthritis Res Ther 9:R100 Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, Sussman M, Orchard P, Marx JC, Pyeritz RE, Brenner MK (1999) Transplantability and therapeutic effects of bone marrowderived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5:262–264 Hu R, Liu W, Li H, Yang L, Chen C, Xia ZY, Guo LJ, Xie H, Zhou HD, Wu XP, Luo XH (2011) A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation. J Biol Chem 286:12328–12339 Huang GTJ, Gronthos S, Shi S (2009) Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 88:792–806 Huang J, Zhao L, Xing L, Chen D (2010) MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells 28:357–364 Inose H, Ochi H, Kimura A, Fujita K, Xu R, Sato S, Iwasaki M, Sunamura S, Takeuchi Y, Fukumoto S, Saito K, Nakamura T, Siomi H, Ito H, Arai Y, Shinomiya K, Takeda S (2009) A microRNA regulatory mechanism of osteoblast differentiation. Proc Natl Acad Sci U S A 106:20794–20799 Jayakumar P, Di Silvio L (2010) Osteoblasts in bone tissue engineering. Proc Inst Mech Eng H 224:1415–1440 Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, OrtizGonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41–49 Jones E, Churchman SM, English A, Buch MH, Horner EA, Burgoyne CH, Reece R, Kinsey S, Emery P, McGonagle D, Ponchel F (2010) Mesenchymal stem cells in rheumatoid synovium: enumeration and functional assessment in relation to synovial inflammation level. Ann Rheum Dis 69:450–457 Junqueira LC, Carneiro J (2008) Tesutul osos. In: Cuculici GP, Gheorghiu AW (eds) Histologie. Editura Medicala Callisto, Bucharest, pp 134–152 Kahai S, Lee SC, Lee DY, Yang J, Li M, Wang CH, Jiang Z, Zhang Y, Peng C, Yang BB (2009) MicroRNA miR-378 regulates nephronectin expression modulating osteoblast differentiation by targeting GalNT-7. PLoS One 4:e7535 Kalajzic I, Terzic J, Rumboldt Z, Mack K, Naprta A, Ledgard F, Gronowicz G, Clark SH, Rowe DW (2002) Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (OIM) mouse. Endocrinology 143:1594–1601 Kapinas K, Delany AM (2011) MicroRNA biogenesis and regulation of bone remodeling. Arthritis Res Ther 13:220–231

31 Kapinas K, Kessler CB, Delany AM (2009) miR-29 suppression of osteonectin in osteoblasts: regulation during differentiation and by canonical Wnt signalling. J Cell Biochem 108:216–224 Kapinas K, Kessler C, Ricks T, Gronowicz G, Delany AM (2010) miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. J Biol Chem 285:25221–25231 Karsenty G (2008) Transcriptional control of skeletogenesis. Annu Rev Genomics Hum Genet 9:183–196 Karsenty G, Ducy P, Starbuck M, Priemel M, Shen J, Geoffroy V, Amling M (1999) Cbfa1 as a regulator of osteoblast differentiation and function. Bone 25:107–108 Kartsogiannis V, Ng KW (2004) Cell lines and primary cell cultures in the study of bone cell biology. Mol Cell Endocrinol 228:79–102 Khalid O, Baniwal SK, Purcell DJ, Leclerc N, Gabet Y, Stallcup MR, Coetzee GA, Frenkel B (2008) Modulation of Runx-2 activity by estrogen receptor α: implication for osteoporosis and breast cancer. Endocrinology 149:5984–5995 Kim YJ, Kim BG, Lee SJ, Lee HK, Lee SH, Ryoo HM, Cho JY (2007) The suppressive effect of myeloid Elf-1-like factor (MEF) in osteogenic differentiation. J Cell Physiol 211:253–260 Kim YJ, Bae SW, Yu SS, Bae YC, Jung JS (2009) miR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res 24: 816–825 Kogianni G, Noble BS (2007) The biology of osteocytes. Curr Osteoporos Rep 5:81–86 Komori T (2006) Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 99:1233–1239 Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764 Kozloff KM, Carden A, Bergwitz C, Forlino A, Uveges TE, Morris MD, Marini JC, Goldstein SA (2004) Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength. J Bone Miner Res 19: 614–622 Krampera M, Pasini A, Rigo A, Scupoli MT, Tecchio C, Malpeli G, Scarpa A, Dazzi F, Pizzolo G, Vinante F (2005) HB-EGF/HER-1 signalling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multi-lineage differentiation. Blood 106:59–66 Krampera M, Pizzolo G, Aprili G, Franchini M (2006a) Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair. Bone 39:678–683 Krampera M, Cosmi L, Angeli R, Pasini A, Liotta F, Andreini A, Santarlasci V, Mazzinghi B, Pizzolo G, Vinante F, Romagnani P, Maggi E, Romagnani S, Annunziato F (2006b) Role of the IFN-γ in the immunomodulatory activity of human mesenchymal stem cells. Stem Cells 24:386–398 Lajeunesse D, Reboul P (2003) Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol 15:628–633 Li Z, Hassan MQ, Volinia S, van Wijnen AJ, Stein JL, Croce CM, Lian JB, Stein GS (2008) A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A 105:13906–13911 Li H, Xie H, Liu W, Huang B, Tan YF, Liao EY, Xu K, Sheng ZF, Zhou HD, Wu XP, Luo XH (2009a) A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest 119:3666–3677 Li Z, Hassan MQ, Jafferji M, Aqeilan RI, Garzon R, Croce CM, van Wijnen AJ, Stein JL, Stein GS, Lian JB (2009b) Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem 284:15676–15684

32 Lian JB, Javed A, Zaidi SK, Lengner C, Montecino M, van Wijnen AJ, Stein JL, Stein GS (2004) Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. Crit Rev Eukaryot Gene Expr 14:1–41 Lian JB, Stein GS, van Wijnen AJ, Stein JL, Hassan MQ, Gaur T, Zhang Y (2012) MicroRNA control of bone formation and homeostasis. Nat Rev Endocrinol 8:212–227 Lin CS, Xin ZC, Deng CH, Ning H, Lin G, Lue TF (2010) Defining adipose tissue-derived stem cells in tissue and in culture. Histol Histopathol 25:807–815 Liu W, Toyosawa S, Furuichi T, Kanatani N, Yoshida C, Liu Y, Himeno M, Narai S, Yamaguchi A, Komori T (2001) Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol 155:157–166 Luzi E, Marini F, Sala SC, Tognarini I, Galli G, Brandi ML (2008) Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res 23:287–295 Manolagas SC, Kousteni S, Chen JR, Schuller M, Plotkin L, Bellido T (2004) Kinase-mediated transcription, activators of nongenotropic estrogen-like signaling (ANGELS), and osteoporosis: a different perspective on the HRT dilemma. Kidney Int Suppl 91: S41–S49 Martin RB, Burr D, Sharkey N (1998) Skeletal biology. In: Martin RB (ed) Skeletal tissue mechanics. Springer, New York, pp 29–77 Maruotti N, Corrado A, Grano M, Colucci S, Cantatore FP (2009) Normal and osteoporotic human osteoblast behaviour after 1,25dihydroxy-vitamin (D3) stimulation. Rheumatol Int 29:667–672 Maruyama Z, Yoshida CA, Furuichi T, Amizuka N, Ito M, Fukuyama R, Miyazaki T, Kitaura H, Nakamura K, Fujita T, Kanatani N, Moriishi T, Yamana K, Liu W, Kawaguchi H, Nakamura K, Komori T (2007) Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency. Dev Dyn 236:1876–1890 Maurer B, Stanczyk J, Jüngel A, Akhmetshina A, Trenkmann M, Brock M, Kowal-Bielecka O, Gay RE, Michel BA, Distler JH, Gay S, Distler O (2010) MicroRNA-29, a key regulator of collagen expression in systemic sclerosis. Arthritis Rheum 62:1733–1743 Miller SC, de Saint-Georges L, Bowman BM, Jee WS (1989) Bone lining cells: structure and function. Scanning Microsc 3:953–960 Mizuno Y, Yagi K, Tokuzawa Y, Kanesaki-Yatsuka Y, Suda T, Katagiri T, Fukuda T, Maruyama M, Okuda A, Amemiya T, Kondoh Y, Tashiro H, Okazaki Y (2008) miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun 368:267–272 Mizuno Y, Tokuzawa Y, Ninomiya Y, Yagi K, Yatsuka-Kanesaki Y, Suda T, Fukuda T, Katagiri T, Kondoh Y, Amemiya T, Tashiro H, Okazaki Y (2009) miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett 583:2263–2268 Nakamura H (2007) Morphology, functions, and differentiation of bone cells. J Hard Tissue Biol 16:15–22 Nakashima K, De Crombrugghe B (2003) Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet 19:458–466 Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, De Crombrugghe B (2002) The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29 Neve A, Corrado A, Cantatore FP (2011) Osteoblast physiology in normal and pathological conditions. Cell Tissue Res 343:289–302 Octacílio-Silva S, Marques MM, Evangelista AF, Magalhaes DA, Dernowsek JA, Bombonato-Prado KF, Passos GAS (2010) Defining mRNA targets of microRNAs during osteoblastic differentiation of human mesenchymal stem cells by microarray transcriptional interaction networks. Resumos do 56º Congresso Brasileiro de Genética, ISBN 978-85-89109-06-2.1

Cell Tissue Res (2014) 355:23–33 Oreffo ROC, Cooper C, Mason C, Clements M (2005) Mesenchymal stem cells lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev 1:169–178 Partridge NC, Walling HW, Bloch SR, Omura TH, Chan PT, Pearman AT, Chou WY (1996) The regulation and regulatory role of collagenase in bone. Crit Rev Eukaryot Gene Expr 6:15–27 Pettit AR, Ji H, Stechow D, Müller R, Goldring SR, Choi Y, Benoist C, Gravallese EM (2001) TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 5:1689–1699 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 Raisz LA (2005) Pathogenesis of osteoporosis: concepts, conflicts and prospects. J Clin Invest 115:3318–3325 Riggs BL, Khosla S, Melton LJ (1998) A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res 13:763–773 Robey PG, Young MF, Fisher LW, McClain TD (1989) Thrombospondin is an osteoblast derived component of mineralized extracellular matrix. J Cell Biol 108:719–727 Robling AG, Castillo AB, Turner CH (2006) Biomechanical and molecular regulation of bone remodeling. Annu Rev Biomed Eng 8:455–498 Rodan GA (1992) Introduction to bone biology. Bone 13:S3–S6 Rowe DW (2008) Osteogenesis imperfecta. In: Bilezikian JP, Raisz LG, Martin TJ (eds) Principles of bone biology, vol 1. Academic Press, New York, pp 1511–1532 Sacchetti B, Funari A, Michienzi S, Di CS, Piersanti S, Saggio I, Tagliafico E, Ferrari S, Robey PG, Riminucci M, Bianco P (2007) Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131:324–336 Sampaio de Mara C, Sartori AR, Duarte AS, Andrade ALL, Pedro AMC, Coimbra IB (2011) Periosteum as a source of mesenchymal stem cells: the effects of TGF-b3 on chondrogenesis. Clinics (Sao Paulo) 66:487–492 Schmidt K, Schinke T, Haberland M, Priemel M, Schilling AF, Mueldner C, Rueger JM, Sock E, Wegner M, Amling M (2005) The high mobility group transcription factor SOX 8 is a negative regulator of osteoblast differentiation. J Cell Biol 168:899–910 Schoolmeesters A, Eklund T, Leake D, Vermeulen A, Smith Q, Aldred SF, Fedorov Y (2009) Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells. PLoS One 4:e5605 Sessarego N, Parodi A, Podesta M, Benvenuto F, Mogni M, Raviolo V, Lituania M, Kunkl A, Ferlazzo G, Bricarelli FD, Uccelli A, Frassoni F (2008) Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application. Haematologica 93: 339–346 Shealy DJ, Wooley PH, Emmell E, Volk A, Rosenberg A, Treacy G, Wagner CL, Mayton L, Griswold DE, Song XY (2002) Anti-TNF alpha antibody allows healing of joint damage in polyarthritic transgenic mice. Arthritis Res 4:R7 Sillence DO, Senn A, Danks DM (1979) Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 16:101–116 Sims NA, Dupont S, Krust A, Clement-Lacroix P, Minet D, RescheRigon M, Gaillard-Kelly M, Baron R (2002) Deletion of estrogen receptor reveals a regulatory role for estrogen receptors–beta in bone remodeling in females but not in males. Bone 30:18–25 Smith JR, Pochampally R, Perry A, Hsu SC, Prockop DJ (2004) Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells 22:823–831 Somerman MJ, Archer SY, Imm GR, Foster RA (1988) A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro. J Dent Res 67:66–70

Cell Tissue Res (2014) 355:23–33 Sommerfeldt DW, Rubin CT (2001) Biology of bone and how it orchestrates the form and function of the skeleton. Eur Spine J 10:S86–S95 Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin JT (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357 Taipaleenmäki H, Bjerre Hokland L, Chen L, Kauppinen S, Kassem M (2012) Mechanisms in endocrinology: micro-RNAs: targets for enhancing osteoblast differentiation and bone formation. Eur J Endocrinol 166:359–371 Tat SK, Pelletier JP, Lajeunesse D, Fahmi H, Lavigne M, Martel-Pelletier J (2008a) The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells. Clin Exp Rheumatol 26: 295–304 Tat SK, Pelletier JP, Lajeunesse D, Fahmi H, Duval N, Martel-Pelletier J (2008b) Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: influence of osteotropic factors. Bone 43:284–291 Tome M, López-Romero P, Albo C, Sepúlveda JC, Fernández-Gutiérrez B, Dopazo A, Bernad A, González MA (2011) MiR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells. Cell Death Differ 18:985–995 Vaculik C, Schuster C, Bauer W, Iram N, Pfisterer K, Kramer G, Reinisch A, Strunk D, Elbe-Bürger A (2012) Human dermis harbors distinct mesenchymal stromal cell subsets. J Invest Dermatol 132:563–574 Varanasi SS, Datta HK (2005) Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and Osterix in ROS 17/2.8 cells. Bone 36:243–253

33 Vimalraj S, Selvamurugan N (2012) MicroRNAs: synthesis, gene regulation and osteoblast differentiation. Curr Issues Mol Biol 15:7–18 Walsh NC, Reinwald S, Manning CA, Keith CKW, Iwata K, Burr DB, Gravallese EM (2009) Osteoblast function is compromised at sites of focal bone erosion in inflammatory arthritis. J Bone Miner Res 9: 1572–1585 Wang T, Xu Z (2010) miR-27 promotes osteoblast differentiation by modulating Wnt signaling. Biochem Biophys Res Commun 402: 186–189 Windahl SH, Hollberg K, Vidal O, Gustafsson JA, Ohlsson C, Andersson G (2001) Female estrogen receptor B−/− mice are partially protected against age-related trabecular bone loss. J Bone Miner Res 16: 1388–1398 Zhang S, Xiao Z, Luo J, He N, Mahlios J, Quarles LD (2009) Dosedependent effects of Runx2 on bone development. J Bone Miner Res 24:1889–1904 Zhang JF, Fu WM, He ML, Wang H, Wang WM, Yu SC, Bian XW, Zhou J, Lin MC, Lu G, Poon WS, Kung HF (2011a) MiR-637 maintains the balance between adipocytes and osteoblasts by directly targeting Osterix. Mol Biol Cell 22:3955–3961 Zhang JF, Fu WM, He ML, Xie WD, Lv Q, Wan G, Li G, Wang H, Lu G, Hu X, Jiang S, Li JN, Lin MCM, Zhang YO, Kung H (2011b) MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling. RNA Biol 8:1–10 Zhang Y, Xie RL, Croce CM, Stein JL, Lian JB, van Wijnen AJ, Stein GS (2011c) A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2. Proc Natl Acad Sci U S A 108:9863–9868 Zhou G, Zheng Q, Engin F, Munivez E, Chen Y, Sebald E, Krakow D, Lee B (2006) Dominance of SOX 9 function over Runx 2 during skeletogenesis. Proc Natl Acad Sci U S A 103:19004–19009