Bone Tissue: macro- and microstructure
Bone is a specialized type of connective tissue that together with cartilage makes up the skeletal system. Flat and long bones are the two main types of bones that are derived from two distinct types of development: intramembranous and endochondral ossification, respectively.
Macroscopically a long bone is usually made up of an epiphysis that is the wider extremity of long bone and a shaft, or diaphysis. The metaphysis is the part between the diaphysis and epiphysis with the epiphysial plate (cartilage) responsible for the longitudinal growth of bone, found in between the metaphysis and epiphysis.
On cross section, the outer layer of bone is made up of dense cortical bone that gives strength while the inner part consists of trabecular bone. Cortical bone constitutes about 85% of the total bone tissue, of which 80 – 90% is calcified. It is mostly found in the long bones of the appendicular skeleton, with its basic component being the osteon or Haversian system. Cells found in cortical bone, and to a lesser extent also those in trabecular, are under the influence of systemic hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (Baron et al., 1999).
Trabecular bone consists of a network of trabeculae that apart from giving strength to the bone itself, have a number of metabolic functions such as mineral homeostasis and are also filled with haematopoietic cells. Trabecular bone makes up about 15% of the skeleton and only 15 – 25% of it is calcified. This type of bone is found mainly in the vertebrae and other areas of the axial skeleton. The association of trabecular architecture with bone strength might be an important predictor of fracture risk that is independent of bone mineral density (BMD) (Aaron et al., 2000). Trabecular BMD is regulated by oestrogen mainly mediated by oestrogen receptor α (ER-α), through the activation of a number of genes including transforming growth factor-β (TGF-β) (Lindberg et al., 2002).
The outer layer of bone is known as the periosteum while the endosteum is the inner layer. Osteoprogenitor cells are found lining the inner cellular layer, which under appropriate stimuli will eventually become osteoblasts.
Osteoblasts are bone forming cells of mesenchymal origin that differentiate from their progenitors when activated by a series of transcriptional factors including Cbfa1/RUNX2 (Ducy et al., 1999), Fra-1 (Jochum et al., 2000) and osterix (Osx) (Nakashima et al., 2002). Under the microscope they appear as cuboidal cells with a round nucleus having a strongly basophilic cytoplasm, with a prominent Golgi apparatus. Bone morphogenic protein (BMP)-2 was observed to activate the initiation of transcription of a number of factors including osterix that is essential for the differentiation of osteoblasts (Lee et al., 2003). Osteoblastic cell differentiation was also observed to be stimulated by the activation of the transmembrane protein notch (Tezuka et al., 2002).
One of the mechanisms by which BMPs regulate osteoblast differentiation is by their interactions with Wnt proteins secreted by osteoblasts themselves in an autocrine loop (Rawadi et al., 2003). Wnt proteins in turn bind to frizzled receptors and the lipoprotein receptor-related protein (LRP)-5 as a co-receptor on the cell surface. This interaction results in the binding of intracellular axin protein to LRP-5 preventing the degradation of β-catenin that in turn results in the activation of transcription (Mao et al., 2001). Wnt induces the expression of BMPs including BMP-2 that enhances osteoblast differentiation and bone formation through BMP receptors on the cell surface. This pathway is controlled by sclerostin, encoded by the SOST gene, which acts as an antagonist to block both BMP and Wnt signalling (Winkler et al., 2005). Mutations in the sclerostin (SOST) gene are responsible for sclerosteosis (MIM 269500), a condition characterised by progressive skeletal overgrowth (Brunkow et al., 2001). Another inhibitor of BMPs is noggin, which binds directly to BMP-2 and BMP-4 preventing their binding to the BMP receptor. Over-expression of noggin in mice resulted in severe osteoporosis due to inhibition of osteoblast differentiation (Wu et al., 2003). BMP-2 was also found to be essential for fracture healing and the healing process did not even start in mice lacking it, even in the presence BMP-4 and BMP-7. These mice also had increased levels of BMP receptors showing that they were primed for stimulation to occur (Tsuji et al., 2006).
Another important protein that plays an important role in osteoblast differentiation is four and half LIM protein 2 (FHL2). This molecule is present in both nucleus and cytosol and is known to activate transcription of AP-1, androgen receptor and β-catenin (Wei et al., 2003). FHL2 deficient mice exhibited greater bone loss when compared to wild-type and FHL2 expressing cells had increased expression of osteoblast differentiation markers (Lai et al., 2006).
Osteoclasts are large multinucleated cells of the macrophage lineage found on bone surfaces where bone resorption is going to take place and beneath the periosteum. Commitment and differentiation of these cells from the heamatopoietic lineage to become fully mature osteoclasts takes place in two sequential stages (Arai et al., 1999). Osteoclast precursors initially express on their surfaces c-Fms followed by receptor activator of nuclear factor-κβ (RANK), the expression of the latter being stimulated by macrophage colony stimulating factor (M-CSF). At this stage osteoclast precursors can still differentiate into a macrophage in the absence of appropriate stimulation by RANK ligand (RANKL).
Figure 1. Multinucleated osteoclasts after 8 days stimulation of murine RAW264.7
macrophages with 10ng/ml M-CSF and 50ng/ml RANKL (Wright's stain seen under X100
Osteocytes and Bone Lining Cells
The osteocyte is a differentiated osteoblast, found enclosed within bone matrix that it produced. These cells have numerous cellular extensions from their plasma membrane, which are important for communication with other osteocytes and osteoblasts found on the bone surface (Lian et al., 1999).
Bone lining cells are flat cells lying on bone surfaces that develop from osteoblasts. These cells are thought to be involved in the removal of collagen left over by osteoclasts in resorptive pits, and in the deposition of a thin layer of collagenous matrix before the initiation of bone formation by mature osteoblasts (Everts et al., 2002).
The Extracellular Matrix
Type I collagen is the most abundant and ubiquitously expressed of the collagen superfamily of proteins that are the most abundant proteins in the human body. Type I collagen is a triple helical molecule made up of two identical α1(I) chains and a structurally similar α2 (I) chain. The collagen molecule is tightly coiled with every third amino acid being glycine, which is usually followed by proline. Collagen undergoes a series of post-translational modifications, among which are cross-linking and hydroxylation of proline and lysine residues, catalyzed by various enzymes such as isomerases and hydroxylases (Myllyharju & Kivirikko, 2004).
Covalent cross-links are formed between molecules while hydrogen bonding keeps the three strands together. Procollagen secreted by the osteoblast contains peptide extensions at both the N-terminal and carboxyl ends. Mature type I collagen molecules are assembled into collagen fibrils, which become interconnected by the formation of pyridinoline and deoxypyridinoline cross-links. These cross-links are released into the circulation during resorption, where they are further metabolized by the liver and finally excreted in urine.
Non Collagenous Proteins
Non collagenous proteins make up to 15% of the total bone protein component and are made up of endogenous and exogenous proteins. Most exogenous proteins are derived from plasma and include albumin while endogenous proteins are those synthesized by bone cells. Among the proteins synthesized by bone cells one can include osteonectin, osteopontin, fibronectin and osteocalcin (Lian et al., 1999).
Osteocalcin is a forty nine residue polypeptide, also known as bone γ-carboxyglutamic acid (Gla) protein that is secreted by osteoblasts. Carboxylation of specific glutamyl residues and calcium binding leads to conformational changes and increases its affinity to hydroxyapatite (Lee et al., 2000; Nishimoto et al., 2003). Osteocalcin also plays an important role in glucose metabolism, where decreased β-cell proliferation, glucose intolerance and insulin resistance were observed in mice lacking it (Lee et al., 2007).
Dentin matrix acidic phosphoprotein (DMP)-1 is an extracellular matrix protein present in mineralised bone matrix and dentin, having multiple calcium binding domains. DMP-1 together with other proteins might play an important role in bone mineralisation and phosphate homeostasis. In pre-osteoblast cells, DMP-1 found in the cytoplasm translocates to the nucleus where it initiates transcription of osteoblast specific genes such as osteocalcin. When the osteoblast matures, phosphorylated DMP-1 is secreted and helps in the mineralisation process while at the same time it might be also responsible for the down regulation of transcription of osteoblast genes and promoting osteocyte differentiation (Feng et al., 2006).
Bone is a dynamic organ that is constantly changing throughout life. Besides having a protective role for vital organs serving as a frame to the body, bone also has important metabolic roles especially in the metabolism of calcium. The integrity of the changing skeleton is maintained by the finely tuned balance between bone formation and resorption, which is tightly controlled by various cytokines, hormones and mechanical forces (Mundy, 1999).
The sequence of events in remodelling starts with the activation of osteoblasts that induce osteoclast differentiation from precursors followed by a resorption phase. Upon stimulation osteoblasts and stromal cells become activated and express RANKL on their surfaces (Bord et al., 2003). This will lead to the differentiation and activation of osteoclasts from their precursors and to bone resorption (Rousselle & Heymann, 2002). Activation and differentiation is controlled by receptor-activator of nuclear factor – κβ (RANK) found on osteoclast precursors and its interactions with RANK ligand (RANKL) on osteoblast / stromal cells (Hofbauer et al., 2000). These interactions are controlled by a decoy receptor known as osteoprotegerin (OPG) that is also synthesized by activated osteoblasts, which thus have an antiresorptive action. The synthesis of OPG is known to be stimulated by oestrogen (Hofbauer et al., 1999) and so the decrease in oestrogen levels brought about by the onset of menopause results in decreased synthesis of OPG and so increased osteoclast activity.
Systemic hormones including PTH and 1,25-dihydroxyvitaminD3 can either directly regulate osteoclasts or else through activation of osteoblasts and stromal cells (Suda et al., 1997). Osteoblasts express RANKL on their surface and secrete M-CSF and OPG upon activation by hormones such as oestrogen (Bord et al., 2003) (Figure 2). Binding of RANKL to RANK present on osteoclast precursors together with that of M-CSF to c-Fms will eventually result in the differentiation and fusion of osteoclast precursors resulting in a mature activated cell. The binding of RANKL to RANK initiates a signal transduction cascade that results in the expression of a number of genes including cathepsin K, calcitonin receptor and tartrate resistant acid phosphatase (TRAP) (Boyle et al., 2003). A very important molecule in the initiation of this signal transduction cascade is the tumour necrosis factor receptor associated factor (TRAF)-6 that binds to the cytoplasmic domain of RANK initiating at least three signalling cascades including that of nuclear factor (NF)-κβ and mitogen activated protein kinase (MAPK) activation that results in the stimulation of transcriptional factor AP-1 (Teitelbaum, 2004). On the other hand, TRAF6 is controlled by its interaction with four and a half LIM domain 2 (FHL2), where FHL2 deficient osteoclasts were observed to have hyper-resorptive activity (Bai et al., 2005). Osteoclast activation is not only controlled by RANKL and OPG but also by interferons (IFN). Nuclear factor of activated T-cells (NFAT)-c1 is a major transcriptional factor involved in osteoclastogenesis following stimulation by RANKL through different pathways involving TRAF6, c-Fos and calcium signalling (Takanayagi, 2005).
Figure 2. Osteoclast regulation by systemic hormones
Kobayashi et al (2000) described a mechanism by which tumour necrosis factor (TNF)-α and interleukin 1 stimulate osteoclastogenesis independently of the RANKL/OPG pathway. This mechanism might play a very important role in inflammatory disorders that result in an increased bone resorption.
The activated osteoclast undergoes a series of structural changes including the formation of the ruffled border and the expression of a number of integrins that help in its attachment to the bone surface (Teitelbaum, 2000). The osteoclast will then start secreting a number of proteases, hydrogen ions and chloride that creates an acidic environment in the extracellular space, resulting in solubilisation and digestion of bone matrix (Rousselle & Heymann, 2002). Activated osteoclasts are controlled by OPG produced from osteoblastic/stromal cells that bind RANKL and promote osteoclast apoptosis (Boyle et al., 2003).
Following bone resorption osteoclasts will undergo apoptosis and osteoblasts are recruited to the resorptive pit in order to start producing new bone. This coupling mechanism between osteoclast and osteoblast has been poorly understood. Osteoblast recruitment is mediated by cytokines that are released during bone resorption such as TGF-β (Erlebacher et al., 1998). Everts and co-workers described how bone lining cells might be key intermediates in the initiation of bone formation (Everts et al., 2002). It was shown that these cells together with matrix metalloproteinases are essential for the digestion of collagen fibrils left behind in the Howship’s lacunae following resorption. Bone lining cells are responsible for the deposition of a thin layer of collagenous matrix along the lacunae that is an obligatory step linking bone resorption with formation (Everts et al., 2002).
Osteoblasts will then attach to bone and start secreting both collagenous and non-collagenous proteins, leading to the production of new matrix. It will take about three months for the osteoblasts to completely refill the resorptive cavity. The mature osteoblast secretes a number of proteins that are important for the structural organisation of bone mineralization as well as for the control of bone remodelling (Udagawa et al., 2000). The activity of osteoblasts is tightly controlled by both systemic hormones and a number of paracrine factors, one of which is platelet-derived growth factor BB (PDGF-BB) secreted by osteoclasts (Kubota et al., 2002). PDGF-BB was also shown to induce the production of osteoprotegerin (OPG) acting as a negative feedback of osteoclastogenesis. When osteoclasts decrease in number the secretion of PDGF-BB also stops and in turn osteoblast precursors differentiate to start forming new bone.
Osteoblasts express steroid receptors for parathyroid hormone (PTH) on their plasma membrane and for oestrogens and 1,25-dihydroxyvitaminD3 within their nuclei (Baron, 1999), showing that osteoblasts are affected by these hormones (Strewler, 2001). It was shown that 1,25-dihydroxyvitaminD3 enhanced the activity of γ-glutamyl carboxylase in osteoblasts and in turn the carboxylation of osteocalcin (Miyake et al., 2001). Also the cholesterol biosynthetic pathway was observed to play a very important role in the development of marrow stromal cells into functional osteoblasts and their ability to form mineralized matrix (Parhami et al., 2002).
The active osteoblast mainly secretes collagen and other matrix proteins such as osteocalcin, where core binding factor alpha (Cbfa)-1 also regulates the transcription of these proteins (Ducy et al., 1999). Leptin and its receptor are expressed in human osteoblasts and were shown to be involved in the stimulation of mineralization. Leptin stimulates the differentiation of osteoblasts from marrow stromal cells and inhibits that of adipocytes (Reseland et al., 2001). This mechanism is different from that of systemic leptin produced by adipocytes where it was observed to inhibit bone formation through a hypothalamic relay (Ducy et al., 2000; Elefteriou et al., 2005). Another important glycoprotein secreted by osteoblasts is OPG which is involved in the regulation of osteoclastogenesis and the expression of which is also controlled by Cbfa1 (Thirunavukkarasu et al., 2000).
Vitamin D and Calcium Metabolism
Vitamin D is a secosteroid hormone, which can be obtained from the diet or derived from sunlight from the precursor molecule 7-dehydrocholesterol followed by two hydroxylation reactions in the liver and kidney (Haussler et al., 1998). From the diet vitamin D can be obtained from various foods we eat like fish, cereals, milk and other foods that are fortified with vitamins. The adequate daily intake varies according to age group and also to daily time exposure to sunlight.
One of the most important biological functions of vitamin D involves mineral homeostasis, where together with other endocrine hormones it is involved in calcium metabolism. The hormone 1,25-dihydroxyvitamin D3 stimulates intestinal calcium absorption and resorption of calcium and phosphates from bone and kidneys in order to ensure enough minerals for bone mineralization (Haussler et al., 1998). When the level of calcium in the blood decreases, the parathyroid glands are stimulated to secrete PTH which will activate renal 1α-hydroxylase that catalyzes the second hydroxylation reaction of vitamin D3 (Juppner et al., 1999).
The active metabolite, 1,25-dihydroxyvitamin D3 will increase the entry of calcium through the plasma membrane of absorptive cells, as well as enhancing its transport through the cytoplasm and finally into the circulation (Holick, 1999). The activity of vitamin D is mediated by binding to its nuclear receptor known as the vitamin D receptor (VDR), and in turn initiating the expression of responsive genes (Haussler et al., 1998). The level of calcium absorption from the intestine can also be affected by other factors that might have a negative effect on bone, such as a very high or low protein diet (Kerstetter et al., 2003).