Bone mass through the lifespan

BACKGROUND

Bone mass through the lifespan

Bones provide mechanical support for joints, tendons and ligaments, protect vital organs, and act as a reservoir for calcium and phosphate in the preservation of normal mineral homeostasis. Bone is a complex and dynamic tissue that undergoes renewal and repair throughout life as the result of bone remodelling. Most diseases of bone result from abnormalities in bone remodelling that compromise the architecture, structure and mechanical strength of bone, leading to clinical symptoms such as pain, deformity, fracture and abnormalities of calcium and phosphate homeostasis.

Peak bone mass is attained between the age of 20–25 and levels remain relatively static until the age of 45 when bone density starts to fall (Figure 2). Although bone density falls with increasing age in both genders there is an accelerated phase of more rapid bone loss in women after the age of 50, due to the effects of oestrogen deficiency at the menopause, which causes relative uncoupling of bone resorption and bone formation. The progressive increase in bone loss with age is accompanied by an increased risk of osteoporosis-related fracture (Figure 3). Although loss of BMD contributes to this, fall-related risk factors also play an important role, especially in the case of hip fractures. Several factors influence peak bone mass and bone loss. Genetic factors account for 60–85% of variance in bone mass and the strong association between body weight and peak bone mass may partly result from shared genetic influences. Hormonal influences are also important. Longitudinal growth and normal skeletal development depend critically on growth hormone, thyroid hormone, oestrogen and androgens, and defects in any of these impairs skeletal development and peak bone mass. Other environmental factors affecting peak bone mass are intake of calcium and other nutrients, and weight-bearing exercise.

Growth of bone

Composition

Bones develop in the embryo through two main processes.

The main organic component of the bone matrix is type I collagen, a fibrillar protein comprising two collagen α1(I) peptide chains and one α2(I) chain, wound together in a triple helix. Collagen is synthesized as a propeptide, but following its secretion from the osteoblast, the amino and carboxyl terminal fragments are cleaved by proteolytic enzymes in the extracellular space. The triple helical domains that remain then self-assemble in a staggered configuration, forming collagen fibrils. Subsequently, individual collagen molecules within these fibrils become linked at each end by specialized covalent bonds called pyridinium cross-links, which contribute to the tensile strength of bone. When bone is broken down, these cross-links are released into the extracellular fluid; their levels can be measured in serum and urine to provide biochemical markers of bone resorption. When bone is formed rapidly (e.g. Paget’s disease, bone metastases), the collagen fibrils are laid down in a disorderly manner giving rise to ‘woven bone’, which is mechanically weak and susceptible to pathological fracture. Bone matrix also contains small amounts of other collagens, and non-collagenous proteins and glycoproteins. Some (e.g. osteocalcin) are specific to bone; others (e.g. osteopontin, fibronectin, growth factors) are also found in other connective tissues. Noncollagenous bone proteins mediate attachment of bone cells to the matrix and regulate bone cell activity during remodelling. This particularly applies to growth factors (e.g. transforming growth factor β) that are buried in the bone matrix. These factors are thought to be released from the bone matrix during bone resorption, and act as coupling factors between resorption and formation. Mineralization of bone occurs on the organic framework, conferring mechanical rigidity to complement the tensile strength and elasticity of collagen. Bone mineral is mainly calcium and phosphate, in the form of hydroxyapatite (Ca10(PO4)6(OH2)) crystals. Defects in bone mineralization occur in osteomalacia, which is most commonly caused by vitamin D deficiency and presents with bone pain and fractures.

Stuart H Ralston

• Flat bones such as the calvariae of the skull, the mandible and the maxilla develop by intramembranous ossification, in which primitive mesenchymal fibroblasts differentiate into bone cells. • Most other bones, including the long bones of the limbs, the ribs, and the vertebrae, develop by endochondral ossification. A cartilage template is invaded by vascular tissue containing osteoprogenitor cells, then removed and replaced by bone, which extends from centres of ossification situated in the middle and at the ends of the developing bone. A thin remnant of cartilage remains at each end of the bone during childhood; this is termed the ‘growth plate’ or ‘epiphyseal plate’ (Figure 1). Skeletal growth depends on division of cartilage cells (chondrocytes) within the growth plate. This occurs in the proliferative zone near the end of the bone, and newly formed chondrocytes migrate towards the centre of the bone, where they become enlarged in the hypertrophic zone. The hypertrophic chondrocytes then die and the surrounding matrix calcifies, before being removed by osteoclasts and replaced by mature bone. During puberty, the rise in circulating levels of sex hormones causes cell division in the growth plate to cease. This causes the cartilage remnant to disappear as the epiphyses fuse and longitudinal bone growth stops.

Peak bone mass Bone mass increases during childhood at a rate commensurate with longitudinal growth and there is accelerated phase of bone gain in adolescents, coinciding with the pubertal growth spurt.

Professor Stuart H Ralston MD FRCP FMedSci FRSE is Head of the School of Molecular and Clinical Medicine and ARC Professor of Rheumatology at the Molecular Medicine Centre, Western General Hospital.

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BACKGROUND

Anatomy and microanatomy of bone Osteoblasts Osteocytes

Osteoclasts Trabecular bone Bone Growth plate

Calcified zone Hypertrophic zone

Blood vessels

Proliferative zone

Collagen lamellae

Cortical bone

Osteocytes Haversian system There are two main structural types of bone • Cortical bone is dense, has a low surface area and forms an envelope around the marrow cavity. It is formed from Haversian systems, which are concentric lamellae of bone tissue surrounding a central canal containing blood vessels. • Trabecular or cancellous bone (‘spongy bone’) has a lower density and much higher surface area. It fills the centre of long bones, flat bones and vertebrae, and is an interconnecting meshwork of bony trabeculae separated by spaces filled with bone marrow. Most bone (80%) is cortical, but the distribution varies. For example, the distal portion of the long bones, the vertebral bodies and the calcaneus are predominantly trabecular, whereas cortical bone predominates in the shafts of the long bones and the femoral neck. These differences are relevant clinically, because trabecular bone is remodelled more rapidly than cortical bone as a result of its high surface area. Thus, bone is lost more rapidly from sites rich in trabecular bone under conditions of increased bone turnover.

1

Bone remodelling

Bone mineral density of lumbar spine in women1 1.4

Peak bone mass

Menopause

1.2

Bone mineral density (g/cm2)

It has been estimated that about 10% of the adult skeleton is being remodelled (Figure 4) at any one time. Remodelling begins with the attraction of osteoclast precursors in peripheral blood to the site that is to be resorbed. The triggering mechanisms are unclear, but it has been speculated that chemotactic factors released from areas of skeletal microdamage may have a role. Once present in the bone microenvironment, osteoclast precursors start to differentiate and fuse to form multinucleated osteoclasts. This occurs in response to activation of a cell-surface receptor called RANK (receptor activator of nuclear factor kappa B) by its ligand RANK ligand (RANKL), which is expressed on osteoblasts and bone marrow stromal cells. This interaction is inhibited by a soluble protein homologous to RANK, osteoprotegerin (OPG). OPG is a ‘decoy’ receptor, inhibiting osteoclast activity by binding RANKL. RANK and OPG belong to the tumour necrosis factor (TNF) receptor superfamily of molecules, whereas RANKL is a member of the TNF superfamily. Other essential molecules include: • PU.1 (transcription factor expressed in early haemopoietic precursors; differentiation of haemopoietic stem cells in the myeloid lineage) • c-fos (transcription factor necessary for differentiation of myeloid progenitors to osteoclasts)

1.0

Old age

0.8

0.6

0.4

10

20

30

40

50

60

70

80

90

Age (years) 1

Mean bone mineral density (two standard deviations)

2

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BACKGROUND

Incidence of osteoporotic fractures

Hip Spine

3000

3000

Men

Fractures / 100,000 person-years

Women

2000

2000

1000

1000

40

50

60

70

80

40

90

Wrist

50

60

70

80

90

Age

Age

Source: Boon N A et al. Davidson’s Principles and Practice of Medicine (20th Edition). Churchill Livingstone, 2006.

3

• macrophage colony-stimulating factor (released from stromal cells; osteoclast and macrophage differentiation) • TRAF6 (transduces differentiation signals from RANK receptor to Nuclear factor kappa B) • Nuclear factor kappa B and NFATc1 (transcription factors acti-

Bone remodelling cycle

vated by the RANK signalling pathway). Mature osteoclasts attach to the bone surface by forming a tight sealing zone. They then resorb bone by secreting hydrochloric acid and proteolytic enzymes into the space underneath the sealing zone, through a specialized membrane called the ruffled border.

Bone lining cells

Microdamage

Mineralization Quiescence Osteoclasts

Osteocytes

Formation

Resorption

Osteoblasts Osteoid

Reversal

4

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BACKGROUND

Formation of the ruffled border depends on the protein c-src, a signalling molecule involved in cytoskeletal organization. Osteoclasts contain specialized proton and chloride pumps responsible for secretion of hydrochloric acid into the extracellular space. Mutations in the genes encoding components of these pumps (TCIRG1 and CIC-7) cause osteopetrosis by impairing the ability of osteoclasts to secrete acid. Normally, the acid dissolves hydroxyapatite and allows proteolytic enzymes access to degrade components of bone matrix. Cathepsin K is an important proteolytic enzyme expressed by osteoclasts that is largely responsible for degradation of bone collagen. Inactivating mutations in its gene cause a type of osteopetrosis (pycnodysostosis) characterized by defective bone resorption and failure of matrix degradation. When bone resorption is complete, osteoclasts move away from the bone surface and undergo programmed cell death (apoptosis), in the reversal phase that heralds the start of bone formation. Bone formation begins with the attraction of osteoblast precursors to the site that has undergone resorption. These cells are derived from mesenchymal precursors in bone marrow stroma. The transcription factor Cbfa1 is essential for differentiation of progenitor cells into osteoblast precursors and another transcription factor Osterix is required for osteoblast precursors differentiation into mature osteoblasts. Osterix activates expression of several osteoblast-specific genes (e.g. osteocalcin, type I collagen, alkaline phosphatase), giving rise to the mature osteoblast phenotype. Other factors including bone morphogenic proteins, (BMPs) are also thought to promote bone formation by encouraging proliferation and differentiation of osteoblast precursors to mature osteoblasts. Bone formation is also critically dependent on Wnt signalling through the lipoprotein receptor related protein 5 and lipoprotein receptor related protein 6 receptors (LRP5/LRP6) and recent work has identified a novel protein termed sclerostin (SOST) that appears to inhibit bone formation by inhibiting Wnt signalling. Mature osteoblasts lay down uncalcified bone matrix (osteoid) onto the bone surface. After about 10 days, this calcifies, forming mature mineralized bone. Alkaline phosphatase (produced by osteoblasts) promotes mineralization by degrading pyrophosphate (a naturally occurring inhibitor of mineralization that is present in extracellular fluid). It is released from normal osteoblasts and is used clinically as a biochemical measure of bone formation. Osteocytes – during bone formation, some osteoblasts become trapped in the bone matrix and differentiate into osteocytes, which interconnect with one another and with cells on the bone surface by long cytoplasmic processes through canaliculi in the bone matrix. Osteocytes are the most abundant cells in bone, and produce signalling molecules (e.g. prostaglandins, nitric oxide) in response to mechanical loading. It is thought that osteocytes sense the effects of mechanical strain on the skeleton.

1,25-dihydroxyvitamin D3) act together to increase bone remodelling, allowing skeletal calcium to be mobilized for the maintenance of plasma calcium homeostasis. Bone remodelling is increased by other hormones (e.g. thyroid hormone, growth hormone), but suppressed by oestrogen, androgens and calcitonin. Many of the factors that regulate bone remodelling do so by modulating local expression of cytokines and other regulatory factors in the bone microenvironment. For example, sex hormone deficiency causes increased production of bone-resorbing cytokines (e.g. IL-1, IL-6) in the bone microenvironment, and this may contribute to post-menopausal bone loss. These cytokines, along with growth factors and calciotropic hormones such as PTH, have been shown to regulate local expression of RANKL and OPG by bone cells, thereby regulating bone resorption. Calciotropic hormones also regulate expression of components of the LRP5 signalling pathway, thereby providing a pathway for local regulation of bone formation.

Bone remodelling and bone disease Most bone diseases result from abnormal bone remodelling. • Osteoporosis arises because the amount of bone resorbed with age exceeds that formed during growth. It appears to result partly from genetic and environmental determinants of peak bone mass, and partly from menopausal loss of oestrogen, which increases bone turnover by uncoupling resorption and formation. • Paget’s disease of bone is also characterized by increased bone turnover, but the abnormalities are focal, affecting specific bones. In some cases of early-onset familial Paget’s disease and familial expansile osteolysis, activating mutations of the gene which encodes RANK have been found to be responsible whereas the rare syndrome of Juvenile Paget’s disease is caused by mutations of the gene which encodes OPG. Mutations in the sequestosome 1 gene (SQSTM1) are the most important cause of Paget’s disease and these have been found in about 40% of families with the disease. The SQSTM1 gene product plays a key role in NFκB signalling. • Bone metastases are caused by focal increases in osteoclastic bone resorption, stimulated by osteoclast-activating factors released by tumour cells (e.g. IL-1, TNF, PTH-related protein). Bone diseases may also result from impaired bone remodelling. The best example is osteopetrosis, in which osteoclasts are unable to resorb bone normally, resulting in greatly increased bone mass, nerve compression syndromes and bone marrow failure caused by replacement of the bone marrow cavity with bone. 

Regulation of bone remodelling Bone remodelling is regulated by circulating hormones and local regulatory factors. Mechanical loading increases bone formation and suppresses bone resorption, whereas remodelling is increased by inflammatory cytokines such as interleukin-1 (IL-1) and TNF. Local or systemic release of these factors appears to be partly responsible for bone loss in inflammatory diseases (e.g. rheumatoid arthritis). Calciotropic hormones (e.g. parathyroid hormone (PTH),

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