• Bone is a large but fundamental A-list basic science topic that regularly appears in the part 1 SBA/EMI paper and the part 2 basic science viva examination.

• Bone is a rigid and dynamic organ of specialised connective tissue that is capable of regeneration.
• It is a composite of inorganic and organic matrix, with 10% of its structure consisting of cells. The dynamic state of bone refers to the process of skeletal remodeling.
• There are five main types of bone cells: (1) osteoblasts (2) osteocytes (3) osteoclasts, (4) osteoprogenitor and (5) bone lining cells.
• Classified by gross anatomy and histology.
• Excluding sesamoid bones and ossicles, the adult skeleton contains 206 bones.

Forms the rigid endoskeleton and has three main functions:

• Mechanical, to move, support, and protect the internal organs.
• As a source of erthythrocytes, leukocytes and platelets.
• Storage and regulation of the majority of calcium and phosphate in the body.

• Fetal bone development occurs through endochondral and intramembranous ossification, which replaces primitive fetal cartilage by bone.
• Endochondral ossification: long bones, vertebrae, the pelvis and bones of the skull base.
• Bone structure, initially formed by a solid hyaline cartilage, develops primary and secondary ossification centres.
• The primary ossification centre is the first area to ossify and takes place during fetal development.
• Chondrocytes within the diaphysis of an immature long bone resorb surrounding cartilage, producing cartilage trabeculae that calcify. Mesenchymal stem cells later occupy this region to produce osteoblasts that produce immature woven bone.
• Proximal and distal to the primary ossification centre, these cartilaginous ends are still growing, with resorption and ossification of the cartilage continuing, resulting in the epiphyseal ends; these are sites for secondary ossification.
• Between the shaft and articular cartilage lies the growth plate, which is in continuous growth. The chondrocytes at the diaphyseal end of this plate are gradually replaced by bone.
• Intramembranous ossification: the cranium, maxilla and mandible (flat bones).
• Bone replaces mesenchymal tissue; osteoblasts produce osteoid at multiple sites.

• Four major types of bone based on shape (flat; long; short and irregular); bone can also be classified based on location (appendicular or axial skeleton) and size (long or short).
• Long bones can be subdivided into four elements:

1. The epiphysis is the region covered by cartilage up to the growth plate.
2. The physis (epiphyseal or growth plate) lies between the epiphysis and metaphysis in a growing bone and is the zone of endochondral ossification.
3. The metaphysis is an expanded portion between the epiphysis (or physis in a growing bone).
4. Diaphysis or shaft contains the medullary canal.

Figure 1. Micrograph of cancellous bone

Woven

• Woven (immature) bone is primarily confined to the fetal skeleton and is highly metabolically active.
• Appears in adults during fracture healing or in pathology such as Paget’s disease and osteosarcoma; lamellar bone represents the normal adult skeleton.
• Collagen fibres are randomly arranged, thus producing no lamellae.
• Mechanically less resilient to compressive forces.

Lamellar

• Lamellar (mature) bone is an organised collagen matrix, producing concentric lamellae.
• Subdivided into an outer dense cortical (compact) bone, which represents 80% of the skeleton and has a high matrix mass per unit volume, making it resilient to compressive forces. Lamellae in cortical bone constitute the basic structural unit, or osteon.

Figure 2. Micrograph of a single Haversian system, depicting the central canal surrounded by concentric lamellae of matrix, with empty lacunae (L); canaliculi (C) connect adjacent lacunae.

• Cancellous (trabecular) bone is characteristic to the metaphysis and epiphysis of long bones and has a higher porosity and reduced matrix mass compared to cortical bone.
• Periosteum covers the external surface of bones and it is divided into an outer fibrous layer comprised of fibroblasts and inner cambium (cellular) layer; this inner cellular layer contains mesenchymal stem cells, which produce osteoblasts.
• Endosteum lines the internal bone surface and is covered by a single layer of bone lining cells.

• The osteon, otherwise known as the Harversian system, is the basic structural unit of cortical bone.
• Lies parallel to the long axis of the bone.
• Consists of a central Haversian canal that transmits a neurovascular bundle; surrounding this canal are at least five concentric lamellae of collagen.
• Within each lamella, collagen fibres lie in parallel but perpendicular to those fibres of adjacent lamellae.
• Volkmann’s canals run transversely to the bone’s long axis and permit communication between the outer vessels of the periosteum and the Harversian canals.
• Osteocytes are situated within lacunae between lamellae, communicating with adjacent osteocytes via cytoplasmic processes that travel through canaliculi.
• Cement lines separate osteons.
• The endosteum is the innermost layer of lamellae, with inactive osteoblasts; when active, these cells synthesise osteoid that produces an additional lamellar layer.

• Bone matrix is a composite of organic and inorganic matrix.
• Up to 70% of mature bone consists of an inorganic matrix of calcium hydroxyapatite (Ca₁₀PO₄OH₂) and gives bone its mechanical strength.
• The remaining 30% is organic, represented primarily by type I collagen; ground substance proteoglycans also contribute to this matrix and regulate the concentration of water in bone.

• Osteoblasts are derived from mesenchymal stem cells.
• Osteoclasts are derived from the macrophage-monocyte cell line.

• Osteoblasts are derived locally from mesenchymal stem cells in the bone marrow and periosteum, giving rise to osteoprogenitor cells.
• The events for bone formation require mesenchymal stem cell proliferation, differentiation of osteoprogenitor cells into osteoblast precursors and maturation of this cell to form an organic then inorganic cellular matrix.
• Osteoblasts produce bone by depositing osteoid; an organic unmineralised matrix of predominately type I collagen (triple helix of two α₁chains and one α₁chain).
• Active osteoblasts are large spindle-shaped cells with abundant quantities of rough endoplasmic reticula, reflecting high levels of protein synthesis.
• Hole zones and pores facilitate the primary mineralisation of collagen.
• Osteoblasts surrounded by calcified cell matrix become osteocytes and communicate with neighbouring cells via cytoplasmic processes.

Figure 4. Micrograph of active osteoblasts (Ob) with new osteoid (red) surrounded by mineralized bone (blue).  Some osteoblasts are trapped in mineralised matrix and become osteocytes (Oc).

Figure 5. Micrograph depicting active osteoblasts (Ob) producing osteoid.

• Osteocytes are the most abundant type of bone cell and are trapped within spaces of mineralised matrix called lacunae and these cells are regulated by the hormones calcitonin and parathyroid hormone, which activate or inhibit their function, respectively.
• Canaliculi connect neighbouring lacunae and carry the cytoplasmic processes of osteocytes, transmitting nutrients and intracellular messengers.
• Osteocytes are thus important mediators of calcium and phosphorus metabolism

• Osteoclasts are large multinucleate cells, situated within mineralised depressions known as Howship’s lacunae; ultrastructurally, they are distinguished by the presence of a ruffled border that is opposed to bone. Another phenotypic feature is the presence of calcitonin receptors, where calcitonin binding to these receptors directly inhibits osteoclast activity.
• These activated cells are involved in the resorption of organic and inorganic bone at specific sites, within depressions termed Howship’s lacunae. They are found in large numbers at sites of active bone resorption in the developing skeleton and in pathological states exhibiting osteolysis.
• Osteoclasts are derived from hematopoietic stem cells, from the monocyte/macrophage lineage, a population that also gives rise to monocytes and macrophages.
• Mononuclear osteoclast precursors circulate in the blood and differentiate, through fusion, into mature osteoclastsat sites of remodeling.
• The transcription factor family, nuclear factor κB, is capable of crossing the nuclear membrane and regulating gene expression; activators of this system include receptor activator of nuclear factor κB ligand (RANKL), tissue necrosis factor α (TNFα) and other cytokines.

• Osteoclast differentiation and function, a process termed osteoclastogenesis, are regulated by osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL).
• RANK is expressed on the surface of osteoclast precursors and mature osteoclasts.
• In response to RANK activation, specific genes are expressed (including NFATc1) and the osteoclast undergoes changes to resorb bone. Factors that initiate bone resorption lead to the expression of RANK ligand. Central to remodeling is the process of coupling, in which RANKL expressed by osteoblasts leads to bone resorption by osteoclasts and osteoid synthesis by osteoblasts. OPG acts as a decoy receptor, blocking RANKL binding and subsequent activation of the RANK system, thus inhibiting osteoclast differentiation and bone resorption.

• The osteoclast surface in apposition with the bone has a large surface area, facilitated through the ruffled border of microvilli.
• The osteoclast binds to bone at its ruffled border through integrins, α_vβ₃. Attachment to bone produces a sealing zone between the cell and bone surface.
• Hydrogen ions are exported via the ATP6i complex and proteolytic lysosomal enzymes tartrate-resistant acid phosphatase (TRAP) and pro-cathepsin K (pro-CATK) that resorb bone matrix.
• The osteoclast processes the degradation products, which enter via the ruffled border through endocytosis. These travel to the apical surface andare exocytosed into the extracellular space.
• Osteoclasts are also regulated by specific hormones. Parathyroid hormone is secreted by chief cells of the parathyroid gland during hypocalcaemia and this acts to stimulate the activity of osteoclasts by increasing RANKL expression from increasing RANKL expression from osteoblasts and stromal cells. In contrast, calcitonin is a direct inhibitor of osteoclast activity, causing dissolution of the ruffled border and arrest of bone resorption. It is produced by parafollicular cells of the thyroid in response to states of hypercalcaemia. Sex hormones can also influence bone remodeling, slowing the rate of bone loss.

Figure 7. Mechanism of osteoclastic bone resorption