I. Histology of Bone

Types

Normal bone is lamellar - highly organised in mineralised plates, relatively hypocellular, and stress-oriented. It can be cortical or cancellous.

Cortical bone makes up 80% of the skeleton, and is found in the outer shell of bone. It is composed of tightly-packed osteons or Haversian systems, made up of small concentric lamellar cylinders surrounding a central vascular channel, connected by Haversian (Volkmann’s) canals. These canals contain capillaries, arterioles, venules, nerves and possibly lymphatics. Lying between these osteons are interstitial lamellae. Fibrils often connect lamellae but do not cross cement lines, which form the outer border of osteons. The intraosseous circulation provides nutrition. Cortical bone has a slow turnover rate, a relatively high Young’s modulus, and a high resistance to bending and torsion.

Cancellous bone is less dense and more elastic than cortical bone, has a smaller Young’s modulus, and a higher turnover rate. It is organised in trabecular struts, with lamellae running parallel to the trabeculae. It is found in the epiphyseal and metaphyseal regions of long bones and throughout the interior of short bones.

Immature or pathologic bone is woven and more random, with more osteocytes than lamellar bone. It is the product of rapid bone formation, resulting in an irregular, disorganised pattern of collagen orientation and osteocyte distribution. It is found in embryonic and foetal development, and in healthy adults at ligament and tendon insertions. It also occurs in response to bony injury and dramatic changes in mechanical stimulation. It provides a temporary mechanical adjunct to allow bone to maintain or return quickly to its role as a structural support.

Cellular Biology

Osteoblasts form osteoid, the nonmineralised component of bone matrix. They differentiate from mesenchymal progenitor cells, and contain extensive endoplasmic reticulum with multiple cisternae, well-developed Golgi bodies, and numerous ribosomes and mitochondria, allowing for their abundant synthesis and secretion of matrix. Their differentiation in vivo is stimulated by various cytokines such as the interleukins, insulin-derived growth factor (IDGF) and platelet-derived growth factor (PDGF). They produce pro-a 1 collagen (a major component of osteoid), osteocalcin (in response to 1,25(OH)₂D₃ ) and bone morphogenetic proteins. They initiate mineralisation of osteoid material, possibly by modulating electrolyte fluxes between the extracellular fluid volume and osseous fluid. Osteoblasts are connected by numerous gap junctions, facilitating electrical/chemical communication between cells.

Bone-lining cells are narrow, flattened cells which differentiate from osteoblasts but have fewer active organelles than osteoblasts. They envelop quiescent bone surfaces including endosteal, periosteal and intracortical surfaces. Their function is to encase the bone surface and moderate site-specific mineralisation or resorption on activation by PTH.

Osteocytes maintain bone, and comprise 90% of all cells in the mature skeleton. They originate as osteoblasts which have been trapped within osteoid formed by surrounding osteoblasts, forming a lacuna. They have a single nucleus, and an increased nucleus:cytoplasm ratio. Osteocytes are smaller in size and have fewer numbers of organelles than osteoblasts, therefore are not as active in matrix production. They maintain the cytoplasmic extensions of the osteoblasts, creating a large canalicular system - essentially a "syncytium". This system may transport biophysical data to cells within and at the surface of bone. Osteocytes play a role in controlling the extracellular concentration of calcium and phosphate - they are directly stimulated by calcitonin and inhibited by PTH.

Osteoclasts act in opposition to osteoblasts, and their role is to resorb bone. Thus bone formation and resorption are coupled. They are multinucleated, irregularly-shaped giant cells which arise from haematopoietic cell lines (monocyte progenitors form giant cells by fusion). Recognition and adherence to the bone surface is mediated via intracellular contractile proteins attached to integrins - this leads to the formation of an apical clear zone and ruffled border (thus increasing surface area). Vacuolar proton-ATPase pumps then localise to this ruffled border and act with intracellular carbonic anhydrase II to lower the pH of the extracellular bone compartment, thus forming a resorption pit, or Howship’s Lacuna. The lowered pH increases the solubility of hydroxyapatite crystals, and the exposed organic matrix is then digested by lysosomal enzymes. Osteoclasts have specific calcitonin receptors, which are induced by 1,25(OH)₂D₃ , PTH and TNF.

Osteoprogenitor cells develop into osteoblasts. They are localised mesenchymal cells lining Haversian canals, endosteum and periosteum, pending the stimulus to differentiate into osteoblasts.

Matrix

Matrix is made up of organic components (40% dry weight in mature bone) and inorganic components (60% dry weight).

1. Organic Components

a. Collagen

Collagen is composed mainly of type I collagen, and provides bone’s tensile strength, comprising 90% of bone matrix. Collagen is composed of a triple helix of tropocollagen (two a ₁ and one a ₂ chains). Bone collagen molecules align themselves head to tail longitudinally, and with a quarter stagger laterally, to produce a collagen fibril. "Hole zones" (gaps) in the collagen fibril are located between the ends of molecules, while "pores" are located between the sides of parallel molecules - calcification is thought to occur within hole zones and pores. Cross-linking leads to decreased solubility and increased tensile strength.

b. Proteoglycans

Proteoglycans contribute to the compressive strength of bone. Their function is unclear, but they are thought to play a role in the reservation of space for bone development, the binding and availability of local growth factors, and the deposition and structuring of collagen fibrils. Inhibit mineralisation.

c. Osteocalcin

Osteocalcin is produced by osteoblasts and makes up 10-20% of the collagenous protein of bone. It attracts osteoclasts, therefore its function is associated with bone remodelling. Increased synthesis is induced 1,25(OH)₂D₃ and inhibited by PTH. Levels in urine and serum are elevated in Paget’s Disease, renal osteodystrophy and hyperparathyroidism. Bone which is deficient in osteocalcin does not undergo resorption in vivo and is associated with premature closure of epiphyseal growth plates.

d. Osteonectin

Osteonectin is secreted by platelets, osteoblasts and osteoclasts. It is thought to play a role in the regulation of calcium or the organisation of material within the matrix, as it binds collagen, has a high affinity for both calcium and hydroxyapatite, and localises to crystal-producing matrix vesicles.

e. Osteopontin

Osteopontin mediates the attachment of cells to bone matrix, similar to integrins. It contains the Arg-Gly-Asp (RGD) amino acid sequence, which is preferentially recognised by cell surface integrin molecules.

f. Growth Factors and Cytokines

These occur in small amounts in bone matrix. They include Transforming Growth Factor b (TGF-b ), Insulin-like Growth Factor (IGF), Interleukins (IL-1, IL-6), Bone Morphogenic Proteins (BMP_1-6) , Platelet-Derived Growth Factor (PDGF), Colony Stimulating Factors (CSFs), Heparin-Binding Growth Factors (HBGFs), Tumour Necrosis Factor a (TNF-a ), Prostaglandins (PGs) and Leukotrienes.

i) Transforming Growth Factor b

TGF-b is one of the most prevalent growth factors found in bone matrix. It is released during bone absorption, and enhances osteoblast activity (via elevated collagen synthesis), increases the bone apposition rate, and inhibits the differentiation of osteoclasts. Its activity is regulated by its conversion into an active peptide, which, in turn, is controlled by PTH.

ii) Insulin-like Growth Factor

In bone tissue, IGF-1 and IGF-2 are produced by fibroblasts and osteoblasts. Synthesis of IGF-1 is enhanced by PTH and PGE₂ , and diminished by cortisol. IGF-1 increases bone apposition rates by increasing preosteoblast cell replication and osteoblastic collagen synthesis, and decreasing bone resorption. Overall, IGF seems to play a role in the maintenance of normal bone mass.

iii) Interleukins

IL-1 is a powerful stimulant of bone resorption. It is mitogenic for osteoclast precursors, and it promotes the proliferation and differentiation of committed precursors. Its action is potentiated by TNF-a , and it acts synergistically with PTH and PTH-related peptide.

IL-6 is mainly responsible for the acute-phase protein response, and plays a major role as a paracrine growth factor in myeloma. It potentiates the bone-resorbing effects of IL-1 and TNF-a by stimulating early osteoclast lineage mitogenesis. Its synthesis is regulated by PTH, IL-1 and 1,25(OH)₂D₃, and is performed by osteoblasts.

iv) Bone Morphogenic Proteins

BMPs are members of the TGF-b superfamily of growth factors. They act on progenitor cells to induce differentiation into osteoblasts and chondroblasts. They are responsible for ectopic bone formation by certain tumour cells, epithelial cells and demineralised bone. BMPs appear to be stored with bone matrix and released with the resorptive activity that often follows injury.

2. inorganic components

a. Calcium Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] Know this formula.

Calcium hydroxyapatite provides the compressive strength of bone. It makes up most of the inorganic matrix, and is responsible for mineralisation of the matrix. (Mineralisation is the transformation of hydroxyapatite from a soluble to a solid form, starting at multiple nucleation sites and then spreading by accretion, or crystal growth) Primary mineralisation occurs in gaps in collagen, while secondary mineralisation occurs at the periphery.

b. Osteocalcium Phosphate (Brushite)

Osteocalcium phosphate comprises the remainder of inorganic matrix.

Bone Remodelling

Bone remodelling is affected by mechanical function, according to Wolff’s Law, which attempts to predict bone adaptation in the face of an altered loading environment.

Generally, remodelling occurs in response to stress, and responds to piezoelectric charges (compression causes negative potential, which stimulates osteoblast activity & bone formation; tension causes positive potential, leading to osteoclast stimulation). Bone is dynamic - coordinated osteoblast and osteoclast activity results in continuous remodelling of both cortical and cancellous bone throughout life.

Cortical bone remodelling occurs by osteoclasts which tunnel through to the bone forming "cutting cones", followed by sheets of osteoblasts which deposit osteoid in lamellae.

Cancellous bstone remodelling involves osteoclast resorption of bone, followed by the deposition of osteoid by osteoblas.

Bone Circulation

Anatomy

Bones arnes receive blooe well-supplied with arteries, receiving 5% of cardiac output under basal conditions. Long bod from periosteal arteries, nutrient arteries, and metaphyseal and epiphyseal arteries.

Periosteal arteries enter the body of a bone at various points and supply the outer third of the cortex of the diaphysis. This is a low pressure system.

Nutrient arteries are branches of major systemic arteries, and pass obliquely through the diaphyseal cortex to reach the medullary canal. Here they divide into longitudinally directed branches which supply at least the inner two-thirds of mature diaphyseal cortex. This is a high pressure system.

Metaphyseal and epiphyseal arteries supply the ends of bone, and arise mainly from the periarticular vascular plexus. In growing bones they supply growth plates, so significant disruptions of blood flow disturb bone growth.

Physiology

Direction of flow

In mature bone, arterial blood flows centrifugally from the high pressure nutrient arteries to the low pressure periosteal arteries. If a displaced fracture causes interruption of the nutrient artery system, the flow reverses as the periosteal system now predominates, so blood flow becomes centripetal.

In developing bone, arterial flow is centripetal, because the periosteum is highly vascularised and is the major component of blood flow in bone.

In mature bone, venous flow is centripetal - cortical capillaries drain to venous sinusoids, which then drain to emissary veins.

Fluid compartments of bone are as follows: extravascular 65%, lacunar 6%, Haversian 6%, red blood cells 3%, other 20%.

As with other tissues and organs, hypoxia, such as at high altitude, causes an increase in blood flow to bone, as does hypercapnia and sympathectomy.

Fracture Healing

After a bony injury, blood flow to the site initially decreases due to disruption of vascular structures. Blood flow then gradually increases over the following hours and days, peaking at around 2 weeks. By 3-5 months, flow has returned to normal.

Fracture healing is largely reliant on bone blood flow - reaming of bone devascularises the central 50-80% of cortex, and thus is associated with most delayed vascularisation of all types of fixation.

Regulation

Blood flow to bone is regulated by humeral, metabolic and autonomic signals. The osseous vessels express various vasoactive receptors which may be exploited in the future by pharmacological agents for the treatment of bone diseases related to circulatory disturbances (eg. osteonecrosis, fracture nonunions).

Tissues surrounding bone

Periosteum is a dense connective tissue membrane which covers bone. It is composed of an outer fibrous layer, which is contiguous with joint capsules, and an inner, or cambrium, layer which is loose, more vascular, and contains osteoblasts (if bone formation is in progress on the surface) and osteoblast precursors. If bone formation is not occurring, the outer layer is the main component of periosteum, and cells in the inner layer are sparse.

Bone marrow

Red marrow is the tissue in which blood cells develop, and is 40% water, 40% fat and 20% protein. In later stages of growth, and in the adult, when the rate of blood cell formation has decreased, red marrow slowly changes to yellow marrow.

Yellow marrow is made up mostly of fat cells (80% fat, 15% water, 5% protein). Under the appropriate stimulus, yellow marrow can revert to red marrow.

Enchondral bone formation/mineralisation

Cartilage Model

Human bones are mostly preformed from hyaline cartilage, some from condensed mesenchyme, usually at 6 weeks. This model is gradually invaded by vascular buds, which bring in osteoprogenitor cells that differentiate into osteoblasts and form primary centres of ossification at around 8 weeks. The cartilage model grows through appositional growth (new bone is applied to the surface of existing bone leading to an increase in width of bone) and interstitial growth (growth and replacement by bone of deeper layers of epiphyseal growth plate, pushing the epiphysis and its overlying articular cartilage away from the metaphysis and diaphysis - leads to increased length of bone). Ossification thus spreads to replace the cartilage model. Marrow is formed by the resorption of the central cancellous bone and invasion of myeloid precursor cells, brought in by capillary buds. Secondary centres of ossification develop at the ends of bone, to form epiphyseal centres of ossification, which allow increase in length until the bone’s adult dimensions are attained. During the developmental stage, the epiphyses enjoy a rich arterial supply composed of an epiphyseal artery, metaphyseal arteries, nutrient arteries and perichondral arteries.

Physis

In immature long bones there are 2 growth plates: 1) horizontal (the physis), and 2) spherical (allowing the growth of the epiphysis; it has the same arrangement as the physis but is less organised).Note this .Physeal cartilage is classified into zones according to growth and function.

Reserve zone - Here there is no evidence of cellular proliferation or active matrix production. There is decreased oxygen tension. Cells here store lipids, glycogen and proteoglycan aggregates for later growth. Therefore diseases such as lysosomal storage diseases (Gaucher’s) can affect this zone.

Proliferative zone - The cartilage cells undergo division and actively produce matrix, and longitudinal growth occurs with chondrocytes forming columns. The oxygen tension here is increased, and there is also increased proteoglycan in the surrounding matrix which inhibits calcification. Defects in this zone (affecting chondrocyte proliferation and column formation) occur in achondroplasia.

Hypertrophic zone - This may be subdivided into 3 zones: maturation, degeneration and provisional calcification. Here the cartilage cells are greatly enlarged (up to 5 times normal size), they have clear cytoplasm as a result of the glycogen accumulated, and the matrix is compressed into linear bands between the columns of hypertrophied cells. The cartilage cells accumulate calcium in mitochondria, then die, releasing calcium from matrix vesicles. Sinusoidal vessels bring osteoblasts, which use the cartilage as a template for bone formation.

Metaphysis

Here osteoblasts from progenitor cells accumulate on cartilage bars formed by physeal expansion. Mineralisation of primary spongiosa (calcified cartilage bars) occurs, forming woven bone which is remodelled to form secondary spongiosa and a "cutback zone" at the metaphysis. Cortical bone is formed when physeal and intramembranous bone are remodelled in response to stress along the periphery of growing long bones.

Periphery of the Physis

This has 2 main components:

a) Groove of Ranvier - allow chondrocytes to travel to the periphery of the growth plate, resulting in lateral growth

b) Perichondrial Ring of LaCroix - dense fibrous tissue which anchors and supports the physis

Mineralisation

Collagen hole zones (between ends of molecules) are seeded with calcium hydroxyapatite crystals, through branching and accretion.

Hormone and Growth Factor Effects on the Growth Plate

Hormones and growth factors affect the growth plate either directly or indirectly, through their effects on chondrocytes and matrix mineralisation. Some factors are produced and act within the growth plate, while others are produced at a distant site.

Intramembranous Ossification

The flat bones of the skull, the mandible, and the clavicle ossify at least partly by intramembranous ossification. This occurs without a cartilage model, and occurs by aggregation of layers of connective tissue cells at the site of future bone formation, and their differentiation into osteoblasts. The osteoblasts then form a centre of ossification which expands by appositional growth.

ii. bone injury and repair

General Principles

Bony response to injury consists of overlapping phases of inflammation, repair (soft callus then hard callus), and remodelling. Fracture healing is affected by systemic factors such as age, hormones and nutrition, and local factors such as degree of local trauma, type of bone affected, and infection.

Inflammation (Haemorrhage/Granulation Tissue-minutes/hours)

This begins immediately after the fracture, and is characterised by bleeding from the fracture site and surrounding tissues, causing haematoma formation, accompanied by oedema and pain. Lysosomal enzymes are released and tissue necrosis occurs - osteoclasts and macrophages remove necrotic bone and tissue debris from the fracture site. This is followed by the stimulation of proliferation of reparative cells such as osteoblasts and endothelial cells.

Repair (Immature Callus/Mature Callus-weeks/months)

Within 2 weeks, primary callus response occurs. If the bone ends are not in apposition to one another, soft (bridging) callus is formed around and between the fragments, reducing their mobility. This soft callus contains fibroblasts, proliferating osteoblasts and often chondroblasts, embedded in a matrix rich in collagen and glycoprotein, into which new blood vessels grow. Hard (medullary) callus supplements the bridging callus - the soft callus is gradually converted into woven bone, mainly by enchondral ossification. This stage is reached about 3 or 4 weeks after injury and continues until firm bony union occurs (around 2 or 3 months later for most adult bones).

The amount of callus formation is indirectly proportional to the degree of immobilisation of the fracture.

Remodelling (years)

This stage overlap with hard callus formation and may continue for up to 7 years. It involves the gradual conversion of the woven bone of the hard callus to lamellar bone. It is considered complete when the site of the fracture can no longer be identified either structurally or functionally. It allows the restoration of bone to its normal configuration and shape, according to the stresses placed on it (Wolff’s Law).

Growth Factors of Bone

Bone Morphogenic Protein (BMP)

BMP is osteoinductive - it acts on progenitor cells to induce differentiation into osteoblasts and chondroblasts. The target of BMP is the undifferentiated perivascular mesenchymal cell. BMP may be the main signal regulating skeletal formation and repair - it is known to induce bone formation de novo, following the same pathways as enchondral ossification.

Transforming Growth Factor b

TGF-b induces mesenchymal cell production of type II collagen and proteoglycans. It also enhances osteoblast activity, via increased collagen synthesis, as well as increasing the bone apposition rate and inhibiting the differentiation of osteoclasts. It is thought to regulate cartilage and bone formation in fracture callus.

Insulin-like Growth Factor II

IGF-II stimulates type I collagen, cellular proliferation and cartilage matrix synthesis.

Platelet-Derived Growth Factor

PDGF serves as a local cytokine regulator, attracting inflammatory cells to the fracture site.

Hormonal Effects on Fracture Healing

Fracture healing is increased by: growth hormone (by increasing callus volume), thyroid hormone/parathyroid hormone (by bone remodelling), and possibly also by calcitonin (mechanism unknown). Cortisone however is known to decrease fracture healing by decreasing callus proliferation.

Electricity and Fracture Healing

Stress-generated potentials act as signals which regulate cellular activity. Examples include piezoelectric effect and streaming potentials.

Streaming potentials arise when electrically charged fluid is forced over a tissue with a set charge.

Piezoelectric effect refers to the displacement of charges in tissues which occur as a result of mechanical forces. Compression generates -ve charges and so bone healing.

Transmembrane potentials are generated by cellular metabolism.

Bone produces small electric potentials on its surface when an appropriate mechanical stress is exerted. It has been suggested that bone remodelling as a response to mechanical stress is mediated by these electric potentials, which then activate osteoclasts and osteoblasts. Therefore, devices have been invented with the aim of stimulating fracture repair by altering a variety of cellular activities of cartilage and bone cells. First used by Dwyer in Sydney.

Various types of electrical stimulation have been used:

Direct Current (DC) stimulates an inflammatory-like response.

Alternating Current (AC) causes changes in cAMP accumulation, increases collagen synthesis, increases DNA synthesis and increases mineralisation.

Pulsed Electromagnetic Fields (PEMF) initiate calcification of fibrocartilage, but cannot induce the calcification of fibrous tissue.

Bone Grafting

Bone grafts provide a passive framework for host osteoblasts and osteoclasts (osteoconduction), and may provide active signals to the host response capable of influencing the process (osteoinduction). Autografts (tissue from the same individual), allografts (tissue transferred between members of the same species; donors must be screened for potential transmissible diseases) or xenografts (tissues transferred between species) can be used.

Cancellous grafts are commonly used due to their porous nature, allowing rapid revascularisation, followed soon after by osteoblastic activity and mineralisation, and later remodelling ("creeping substitution"). The incorporation process in cancellous bone is relatively rapid and complete compared to cortical grafts. They are used for grafting nonunions or cavitary defects.

Cortical grafts are used to repair structural defects(stronger)and have a slower turnover. Revascularisation is slower than in cancellous grafts. Slow remodelling of Haversian systems is followed by a vigorous osteoclastic response, weakening the graft, then deposition of new bone, restoring strength.

Osteoarticular allografts are often used in tumour surgery - these grafts are immunogenic, therefore they are usually subjected to long-term preservation, such as freezing or lyophilisation. This process destroys the viability of many of the chondrocytes. Tissue-matched fresh osteochondral grafts produce minimal immunogenic rejection and are well incorporated.

Vascularised grafts do not undergo the incorporation process described for nonvascularised grafts. Instead they unite to the recipient-site skeleton by a process similar to fracture repair, and allow more rapid union with the preservation of most cells.

Bone grafts may be:

• fresh
• fresh frozen - less immunogenic than fresh
• freeze-dried - loses its structural integrity; least immunogenic
• in bone matrix gelatin

There are 5 recognised stages of graft healing: (as in fracture healing)

1) Inflammation - necrotic debris stimulated chemotaxis

2) Osteoblast differentiation - osteoblasts differentiate from precursors

3) Osteoinduction - osteoblasts and osteoclasts function

4) Osteoconduction - new bone is formed over the graft tissue

5) Remodelling - a process continuing for years

Synthetic bone grafts are now in use, and are composed of either calcium (as the phosphate, sulfate, carbonate, or corralline hydroxyapatite - thermoexchange process used to convert calcium carbonate skeleton to calcium phosphate), silicon (as silicate) or aluminium (as the oxide). Most recent is tantalum metallic mesh.

iii. conditions of bone mineralisation, bone mineral density, and bone viability

Normal Bone Metabolism

Calcium

Calcium exists in 3 forms in the body:

1) as hydrated phosphate in the skeleton.

Over 99% of the body’s calcium is stored here.

2) in the extracellular fluid.

This accounts for less than 1% of total body calcium. The concentration here is maintained at a constant level, even at the expense of calcium in bone. Its function here is in the excitability of nerves and muscles, including the heart; in blood clotting; in membrane permeability; and in the activity of various enzymes.

3) inside cells.

There is a low cytosolic calcium level which is tightly controlled. It plays a role in the functions of many enzymes.

Absorption occurs mostly by an active transport system in the small intestine, which is Vitamin K dependent, and also by passive diffusion in the jejunum. It is 98% resorbed in the kidney, and is excreted in the faeces and urine.

Calcium requirements are 800mg/day for Australian adults, and 1000mg/day for females over the age of 50. Pregnant women require 1500mg/day in the 3rd trimester, and lactating women need 2000mg/day.

There is a 700mg calcium turnover in and out of bone on a daily basis.

Hypocalcaemia can lead to tetany, somnolence and areflexia, while hypercalcaemia can cause hyperreflexia and convulsions.

Phosphate

Phosphate not only plays a role in bone mineral, but also acts as a metabolite and buffer, and participates in enzyme systems. Around 85% of the body’s phosphate stores are in bone.

Plasma phosphate is mostly unbound, and is resorbed in the proximal tubules of the kidney.

The recommended daily intake is 1000-1500mg, and dietary intake is usually sufficient.

Parathyroid Hormone

PTH is a polypeptide chain secreted by the chief cells of the parathyroid glands, and its function is in the control of calcium ion concentration in the extracellular fluid. This is achieved by control of: a) calcium absorption from the gut; b) calcium excretion by the kidneys; and c) calcium release from bones. Decreased calcium levels in the extracellular fluid stimulate PTH release, which then acts at the intestine, kidneys and bone.

Vitamin D₃

Vitamin D₃ is a naturally-occurring steroid that is derived from UV irradiation of 7-dehydrocholesterol. It is absorbed from the small intestine only when fat digestion and absorption are normal. In the liver it is hydroxylated to 25-OH D₃, then it is further hydroxylated in the kidney either to 1,25(OH)₂D₃ (the active form), or to 24,25(OH)₂D₃ (an inactive metabolite). The active form has effects at the kidney, intestine and bone.

Calcitonin

Calcitonin is a large polypeptide secreted by the clear cells of the thyroid gland. Secretion is stimulated by increased calcium levels in the extracellular milieu. It decreases calcium concentration in the extracellular fluid by its effects at the intestine, kidney and bone (where it promotes calcium deposition). It may also play a role in fracture healing, and the treatment of osteoporosis.

Other Hormones

Oestrogen causes increased osteoblastic activity and inhibition of bone resorption. Deficiency, as in menopause, leads to decreased osteoblastic activity in the bones, decreased bone matrix, and decreased deposition of bone calcium and phosphate, thus causing osteoporosis.

Corticosteroids increase resorption and impede fracture healing (¯ binding proteins ® ¯ gut absorption of calcium; ¯ bone formation through inhibition of collagen synthesis)

Thyroid hormones enhance osteoclastic bone resorption, leading to osteoporosis.

Growth hormone increases gut calcium absorption more than its increase in urinary excretion, leading to a positive calcium balance.

Growth factors such as PDGF and TGF-b play a role in bone and cartilage repair.

Interaction

Calcium and phosphate metabolism are influenced by various hormones and also by the levels of the metabolites themselves. Regulation of plasma levels is controlled in part by feedback mechanisms.

It appears that peak bone mass occurs between the ages of 16 and 25 years, with higher peak bone mass in males and African-Americans. After this peak, bone is lost at a rate of 0.3-0.5% per year, with higher rates (2-3%) for untreated women during the 6th to tenth years after menopause.

Conditions of Bone Mineralisation

PHP/Albright

PTH receptor defect

Exostoses, brachydactyly

Chronic renal failure ® ¯ phosphate excretion

Defective renal 1a hydroxylation

Hypercalcaemia

This can lead to such symptoms as lethargy, polyuria, constipation, disorientation, hyperreflexia, kidney stones, psychosis, and cardiac arrhythmias.

Differential diagnoses:

1) Primary Hyperparathyroidism - There is overproduction of PTH, due to a parathyroid adenoma in 80% of cases. Elevated levels of PTH enhance urinary loss of phosphate (leading to hypophosphataemia) and bicarbonate (causing mild hyperchloremic metabolic acidosis. Osteoclastic stimulation results, causing enhanced bone resorption - this raises serum calcium and increases calcium excretion. Laboratory findings include elevated serum calcium, PTH, and urinary phosphate, and decreased serum phosphate. In long-standing hyperparathyroidism, one may see bony changes such as localised or generalised osteopaenia, osteitis fibrosa cystica (fibrous replacement of bone marrow), "brown tumour" (increased giant cells, RBC extravasation, haemosiderin staining and fibrous tissue haemosiderin, caused by subcortical bone resorption in the jaw) or chondrocalcinosis. Histological findings may include osteoblasts and osteoclasts active on both sides of trabeculae, areas of destruction, and wide osteoid seams. Surgical parathyroidectomy is curative.

2) Familial syndromes - Multiple Endocrine Neoplasia types I and II may have associated pituitary adenomas. Familial Hypercalciuric Hypercalcaemia, caused by poor renal clearance of calcium, may also result in hypercalcaemia.

3) Other causes of hypercalcaemia include malignancy (most common; include multiple myeloma and lymphomas), Addison’s Disease, hyperthyroidism, tuberculosis, Paget’s Disease, steroid administration and kidney disease.

Hypocalcaemia

This occurs due to deficiency or inadequate function of PTH or Vitamin D. Clinical features may include neuromuscular irritability (positive Chvostek’s or Trousseau’s signs, seizures, tetany), lenticular cataracts, fungal infections of the nails, pancreatic calcification and prolonged Q interval on ECG.

Differential diagnoses:

1) Primary Hypoparathyroidism - This can be defined as an absolute or relative deficiency or inadequacy of PTH function. Diminished PTH action is associated with hypocalcaemia and hyperphosphataemia (urinary excretion is not enhanced due to lack of PTH). Skull radiographs may reveal basal ganglion calcification.

2) Pseudohypoparathyroidism (PHP) - This is a rare genetic disorder (X-linked dominant) causing a syndrome of PTH resistance due to blocking of PTH action at the cellular level. PTH levels are normal or elevated, with hypocalcaemia and hyperphosphataemia. There is a characteristic short stature, bony abnormalities (such as shortening of the metacarpals and metatarsals), brachydactyly, exostoses, obesity, and intellectual impairment.

3) Renal Osteodystrophy - This is a complex bone disorder in patients who have chronic renal failure. Renal impairment leads to an inability to excrete phosphate, with a compensatory decrease in serum calcium, which is usually adjusted by PTH (which increases urinary excretion of phosphate). However phosphate cannot be secreted, resulting in symptoms similar to hypoparathyroidism. Thus blood levels of PTH are greatly elevated. It is commonly associated with long-term haemodialysis. Radiographs may exhibit a "rugger jersey spine" appearance, associated with sclerosis in the region immediately beneath the vertebral endplates.

4) Rickets (Osteomalacia in adults) - In rickets there is inadequate mineralisation of growing bone, causing changes in the physis (increased width and disorientation) and bone (cortical thinning and bowing). Osteomalacia is the adult equivalent of rickets. Rickets and osteomalacia have various causes, outlined below:

a) Vitamin D Deficiency Rickets

This is rarely seen on the basis of dietary deficiency in developed countries, due to the fortification of dairy products with Vitamin D. It may be seen in children of Asian immigrants, in premature infants on prolonged total parenteral nutrition, in patients with dietary peculiarities, and those with malabsorption (sprue). A lack of exposure to sunlight can play a role in perpetuating rickets.

In the Vitamin D deficient state, there is a reduction in the absorption of calcium and phosphate, leading to a compensatory secondary hyperparathyroidism. An initial increase in bone resorption is able to maintain normal serum calcium levels. Continued Vitamin D deficiency exacerbates the secondary hyperparathyroidism, causing a loss of phosphate and decreased ability to maintain serum calcium levels at normal. Laboratory findings include low Vitamin D levels, low normal calcium, low phosphate, and elevated PTH.

Clinical features may include delayed closure of the fontanelles in the first year of life, leading to widened cranial sutures. There may be thickening of the skull (frontal bossing) and flattening of the occiput (craniotabes). An enlarged costochondral junction (rachitic rosary), posterior displacement of the sternum, and Harrison’s sulcus may be evident in the thorax. Bony deformities may appear on weight-bearing, such as genu varum or genu valgum, coxa vara, or lordosis of the spine. There may also be weakness and hypotonia of the muscles (causing a waddling gait), retarded bone growth (due to a defect in the hypotrophic zone with widened osteoid seams and physeal cupping), hypoplastic dental enamel, and a tendency to pathologic fractures.

Treatment involves giving 5000 units of Vitamin D (as ergocalciferol) daily, and supplemental calcium in early stages of treatment (up to 3 g daily), to avoid hypocalcaemic symptoms.

b) Hereditary Vitamin D-Dependent Rickets

This rare condition is autosomal recessive, and is unresponsive to the usual physiological replacement doses of Vitamin D. It may be due to a defect in renal 1a -hydroxylation of 25-hydroxy Vitamin D , resulting in low levels of, or defective 1,25-(OH)₂D₃. Thus there is impaired calcium absorption, and there are typical features of low serum calcium, secondary hyperparathyroidism, and clinical features of Vitamin D deficiency (except the clinical features may be more florid, and may include total baldness). The metabolic abnormalities require treatment with high levels of Vitamin D - usual doses range from 20,000 to 100,000 units of Vitamin D daily, followed by a maintenance dosage of a 1,25-(OH)₂D₃ analogue.

c) Familial Hypophosphataemic Rickets (Vitamin D-Resistant Rickets)

This is an X-linked disorder characterised by hypophosphataemia due to decreased renal tubular reabsorption of phosphate. There may also be abnormal intestinal absorption of phosphate. Sufferers have a normal GFR and an impaired Vitamin D₃ response. Clinical features are similar to those of other forms of rickets. Treatment requires phosphate replacements (1-4 g daily) and Vitamin D₃.

d) Hypophosphataemia

This is an autosomal recessive disorder and may present in its more severe form as childhood rickets, and is less severe in adults. It is characterised by low alkaline phosphatase (an enzyme required for bone mineralisation; it hydrolyses pyrophosphate to give inorganic phosphate - this step is thought to be required for initiating apatite formation in the matrix). Features resemble those of rickets. There is no satisfactory treatment, but phosphate therapy has been somewhat successful.

Conditions of Bone Mineral Density

Bone mass regulation depends on the relative rates of deposition and resorption of bone.

Osteopaenia

a) Osteoporosis

Osteoporosis is characterised by diminution in bone mass, usually associated with loss of oestrogen in postmenopausal females. It leads to a tendency to fracture, commonly at sites of a large volume of trabecular bone, such as the vertebral body, proximal femur, or distal radius.

Risk factors are Caucasian descent and northwestern European background, early menopause, alcohol consumption, chronic smoking, patients on phenytoin (which impairs Vitamin D metabolism), low Vitamin D and low calcium diets, and breastfeeding. Lack of exercise may be an important influence on bone mass, especially during the growth phase.

Cancellous bone is affected more severely. Patients may present with kyphosis or fracture of a vertebra (particularly crush fracture of T11-L1), hip or distal radius.

Osteoporosis has been divided into two classifications:

i) Type I Osteoporosis (Postmenopausal) - Mainly affects trabecular bone. Fractures of vertebra and distal radius are often seen.

ii) Type II Osteoporosis (Age-related) - Occurs in patients aged over 75 years. It affects both cortical and trabecular bone, and is associated with poor calcium absorption. Fractures of hip and pelvis are common features.

Initial laboratory tests should include serum and urine calcium, serum protein, inorganic phosphates, alkaline phosphatase and full blood count. Results of these are usually unremarkable in osteoporosis, but may help to eliminate hyperthyroidism, hyperparathyroidism, malignancy , Cushing’s syndrome and haematologic disorders as differential diagnoses. Plain radiographs are usually of no help, unless there is greater than 30% bone loss. Special studies may be done, including bone biopsy (to distinguish between osteoporosis and osteomalacia), single and dual photon absorptiometry, quantitative computed tomography, and dual-energy X-ray absorptiometry (DEXA - most accurate and involves less radiation).

Histologically, there is reduced bone mass and characteristic thinning of trabeculae, decreased size of osteons, and widened marrow and Haversian spaces.

Treatment includes physical activity to prevent further bone loss, calcium supplements (more effective in Type II osteoporosis), oestrogen-progesterone therapy (for Type I osteoporosis - most effective if started within 6 years of menopause) and fluoride (inhibits bone resorption, and increases trabecular thickness and volume). Intramuscular calcimar (salmon calcitonin) injections have yielded some successful results in preventing bone loss in postmenopausal osteoporosis, but the effects may be transient and side-effects may occur.

For patients at high risk of osteoporosis, the preventive regimen includes increased weight-bearing physical activity, dietary calcium intake of 1000-1500mg daily, and, if necessary, use of oestrogen therapy at menopause.

Idiopathic Transient Osteoporosis of the Hip is an uncommon disorder, occurring most often in the third trimester of pregnancy. The patient complains of groin pain, limited range of movement, and localised osteopaenia, and the diagnosis is one of exclusion. Treatment involves analgesia and limited weight-bearing.

b) Osteomalacia

This may be considered clinically and histologically to be the adult counterpart of rickets. There is a defect in mineralisation leading to a disproportionately large amount of unmineralised osteoid.

It is caused by Vitamin D deficiency, such as from deficient diet, malabsorption, hepatic or pancreatic disease, gastric bypass surgery, renal osteodystrophy, or various drugs (Phenytoin - induces enzyme systems which enhance Vitamin D degeneration; aluminium-containing phosphate-binding antacids - aluminium deposits in bone prevent mineralisation).

Radiological appearance may be normal, or may show Looser’s zones (aka pseudofractures - radiolucencies occurring as bands at right angles to the cortex; occur at points of stress, and may become complete fractures), biconcave vertebral bodies, or trefoil pelvis. Diagnosis is by biopsy, which give histological appearance of increased width of unmineralised bone, blurred or discontinuous mineralisation, and increased osteoid width.

Treatment involves giving large doses of Vitamin D, and careful monitoring of serum calcium and phosphate (may decrease initially due to rapid remineralisation of bone).

c) Scurvy

Ascorbic acid is an essential nutrient required for the repair and growth of collagen, and for iron absorption. Deficiency leads to defects in collagen growth and repair, and impaired hydroxylation of collagen peptides. Dietary sources include rapidly growing fresh fruits and vegetables.

Vitamin C deficiency may manifest as generalised fatigue, ecchymosis, gum bleeding, perifollicular haemorrhage, joint pain and effusions, and iron deficiency.

Radiographs may show thin cortices, poorly-defined trabeculae, and metaphyseal clefts ("corner sign"). Histologically there may be granulation tissue replacing primary trabeculae, generalised subperiosteal haemorrhage, and widened zone of provisional calcification in the physis.

Treatment is Vitamin C in doses of 100mg t.d.s. - this usually replenishes tissue stores within a week.

d) Osteogenesis Imperfecta

This is a heterogeneous group of disorders characterised by extreme bone fragility and multiple fractures. It is caused by abnormal collagen synthesis (failure of cross-linking), mainly due to a mutation in the genes producing type I collagen.

e) Marrow Packing Disorders

Osteopaenia can result from myeloma, leukaemia, and other disorders.

Increased Osteodensity

a) Paget’s Disease

This is characterised by uncoordinated bone resorption and formation, ultimately leading to skeletal deformity (such as enlarged skull, bowing of long bones of the legs). There are three stages: 1) initial osteolytic phase, 2) mixed stage of osteolysis and osteogenesis, 3) burned-out sclerotic phase. Complications include sarcomatous change in an involved bone, and high-output cardiac failure due to the increased vascularity of the subcutaneous tissue overlying the involved bones.

b) Osteopetrosis (Marble Bone Disease)

This term refers to any of a group of disorders causing increased sclerosis of bones and, in the most severe autosomal recessive form, obliteration of the medullary canal in the long bones (due to decreased osteoclast and chondroclast function), widened metaphyses, "bone within a bone" appearance, hepatosplenomegaly and aplastic anaemia. Pathological fractures are common. This disorder may be the result of an immune abnormality (thymic defect). The autosomal dominant form ("Albers-Schönberg Disease") usually shows generalised osteosclerosis (including the "rugger jersey spine" appearance) most apparent in the skull.

Histologically, osteoclasts lack the normal clear zone and ruffled border, and the marrow spaces are filled with necrotic calcified cartilage.

In childhood, bone marrow transplantation of osteoclast precursors may be life-saving. Treatment may involve high doses of calcitriol, with or without steroids.

c) Osteopoikilosis (Spotted Bone Disease)

This is an autosomal dominant disorder characterised by numerous symmetrical islands of deep cortical bone in the medullary cavity and cancellous bone of long bones, pelvis and scapula. This is usually asymptomatic and there is no observed association with malignant change.

Conditions of Bone Viability

Osteonecrosis

Osteonecrosis, or bone death, occurs as a result of either impaired blood supply (eg. due to trauma) or severe marrow and bone cell damage. The hip joint is commonly affected, causing eventual collapse and flattening of the femoral head. Other susceptible sites include the femoral condyles, head of humerus, capitulum, scaphoid, lunate and talus.

It is associated with steroids and heavy alcohol consumption (both causing fatty infiltration ® capillary compression), and also with blood dyscrasias (such as Sickle-cell Disease), decompression sickness (Caisson Disease), vasculitis, excessive radiation therapy, and Gaucher’s Disease (abnormal accumulation of glucocerebride in the reticuloendothelial system causes pressure on bone sinusoids, thus necrosis).

a) Aetiology

The aetiology of osteonecrosis is uncertain, but various factors have been implicated. These include vascular insults, and the enlargement of space-occupying marrow fat cells, causing ischaemia of adjacent tissues.

b) Pathologic Changes

There are four overlapping stages:

1) Bone death without structural change - Within 24 hours after infarction there is autolysis of osteocytes and necrosis of marrow.

2) Repair and early structural failure - Inflammation occurs, with a vascular reaction. New bone is laid down upon the dead trabeculae (visible on X-ray as increased bone density).

3) Major structural failure - A process of "creeping substitution" occurs, with resorption of necrotic trabeculae and remodelling. The bone is weakest during this phase, and collapse (crescent sign) and fragmentation may occur.

4) Articular destruction - Cartilage, deriving nourishment from synovial fluid, is preserved even in advanced osteonecrosis. However, severe distortion of the surface eventually results in cartilage destruction.

c) Evaluation

Detailed history-taking and physical examination (eg.¯ range of movement, pain, stiffness) are obvious first lines of evaluation. Other joints should be examined in order for early diagnosis of the disease process. In 50% of cases of idiopathic osteonecrosis, and in 80% of cases of steroid-induced osteonecrosis, disease is bilateral.

Diagnosis is aided by MRI and bone scans, as well as radiography which shows distinctively increased bone density due to reactive new bone formation in the surrounding viable tissue. Femoral head pressure measurements may also be done - pressure greater than 30 mm Hg, or an increase of over 10 mm Hg with injection of 5ml of saline is considered abnormal.

d) Treatment should aim to eliminate the cause if possible. Nontraumatic osteonecrosis of the proximal humerus and femoral condyle may show spontaneous improvement. In stages 1 and 2, weight-relief, splintage and surgical decompression of the bone may prevent bone collapse. If in stage 3 (ie. bone collapse has occurred), realignment osteotomy to shift stress to an undamaged area may relieve pain and prevent further bony injury. If in stage 4, treatment is the same as for osteoarthritis.

e) Ficat’s classification of osteonecrosis of the hip

Stage

Pain

Physical Examination

Bone Scan

MRI

Intraosseous Pressure

Radiographs

Treatment

0

­

I

­

II

III

­

IV

­

Osteochondrosis

This refers to any of a group of disorders of one or more ossification centres in children, characterised by degeneration or aseptic necrosis followed by reossification. It may occur at traction apophyses in children and may be associated with trauma, inflammation of the joint capsule, or vascular insult/secondary thrombosis. It is pathologically similar to osteonecrosis in the adult.

Common osteochondroses (OC) include:

• Legg-Calvé-Perthes disease - OC of femoral head
• Osgood-Schlatter disease - OC of tibial tuberosity
• Sinding-Larsen-Johansen syndrome - OC of inferior patella
• Sever’s disease - OC of calcaneus
• Köhler’s disease - OC of tarsal navicular
• Freiberg’s infarction - OC of metatarsal head