Basic Science in Orthopaedics by fagf

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									    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-α 1 collagen (a major component of osteoid), osteocalcin (in response to 1, 25(OH)2D3 ) and bone
morphogenetic proteins. They initiate mineralization 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
mineralization 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 α 1 and one α 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 mineralization.

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 (BMP1-6) , Platelet-
Derived Growth Factor (PDGF), Colony Stimulating Factors (CSFs), Heparin-Binding Growth Factors
(HBGFs), Tumour Necrosis Factor α (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 mineralization of the matrix. (Mineralization 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 mineralization occurs in gaps in collagen, while secondary
mineralization 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 bone remodelling involves osteoclast resorption of bone, followed by the deposition of osteoid
by osteoblasts.

Bone Circulation

Anatomy

Bones arnes receive blood well-supplied with arteries, receiving 5% of cardiac output under basal
conditions. Long bud 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 vascularized 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 cambium, 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/mineralization

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.
Mineralization 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

Mineralization

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 mineralization. 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 mineralization.

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 mineralization, 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.

Vascularized grafts do not undergo the incorporation process described for nonvascularized 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 calcium (as the phosphate, sulfate, carbonate,
or coralline 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 mineralization, 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 Hormona
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-β 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

Hyperparathyroidism Excess PTH (80%            Osteopaenia, Osteitis        Kidney stone, weight loss,
                       due to adenoma)         fibrosa cystica              hyperreflexia

Familial syndromes     Excess PTH (MEN-        Osteopaenia                  Endocrine or renal
                       I/II, renal)                                         abnormality

Hypocalcaemia

Hypoparathyroidism     Insufficient PTH        Calcified basal ganglia      Neuromuscular irritability,
                       (idiopathic)                                         tetany, cataracts

PHP/Albright           PTH receptor defect     Exostoses, brachydactyly     Short stature, obesity, short
                                                                            metacarpals & metatarsals

Renal                  Chronic renal failure   Rugger jersey spine          Renal abnormalities
osteodystrophy         → ↓ phosphate
                       excretion



Rickets
(Osteomalacia)

Vit D deficient           Insufficient Vit D (from Craniotabes, frontal       Bony deformities (eg. genu
                          diet or malabsorption) bossing, rachitic rosary,    varum/valgum), hypotonia
                                                  pathological fractures

Vit D dependent           Defective renal 1α      Poor mineralisation         Total alopecia
                          hydroxylation

Vit D resistant           ↓ phosphate             Poor mineralisation         Short stature in childhood,
                          reabsorption in renal                               hypotonia
                          tubules

Hypophosphatasia          Low alkaline            Poor mineralisation,        Bone deformities, hypotonia
                          phosphatase             fractures

Osteopaenia

Osteoporosis              ↓ bone mass             Fractures (crush to         Fractures, kyphosis
                                                  vertebra; hip; radius)

Scurvy                    Vit C deficiency →      Thin cortices, poorly       Fatigue, bleeding gums,
                          abnormal collagen       defined trabeculae, corner bruises, joint pain/ effusions
                                                  sign

Osteodense

Paget’s Disease           osteoclast activity +   Areas of sclerosis &        Bone pain, deformity,
                          irregular bone          radiodensity, "picture      fractures, high output cardiac
                          formation               frame" vertebrae            failure

Osteopetrosis             Abnormal osteoclasts Widened metaphysics,           Aplastic anaemia,
                          - cause unclear         bone within bone            hepatosplenomegaly



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 mineralization 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 1α -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
mineralization; 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 north-western 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 mineralization 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 mineralization).

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 mineralization,
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        Bone Scan       MRI      Intraosseous Radiographs      Treatment
                         Examination                                 Pressure

  0         Nil          Normal           Normal         Normal                 Normal          None

   I        Minimal      ↓ int.rotation   Nondiagnostic Early                   Normal          Core
                                                         changes                                decompression
                                                                                                (?)

  II        Moderate     ↓ ROM            Positive       Positive               Porosis/        Strut graft
                                                                                sclerosis

  III       Advanced ↓ ROM                Positive       Positive               Flat/crescent Hemiarthro-
                                                                                sign            plasty

  IV        Severe       Pain             Positive       Positive               Acetabular      Total hip
                                                                                changes         arthroplasty



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 Osteochondrosis (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

A normal joint is designed to carry out a range of movements. Synovial joints have a dense fibrous capsule
which may be reinforced by ligaments and muscles. The joint is lined by synovium and filled with synovial
fluid for nutrition and lubrication of articular tissues. Articular cartilage is composed of connective tissue
which is suited to distributing load and decreasing friction. Aspects of joints discussed here are: Cartilage,
Articular Cartilage, Synovium Meniscus.

Cartilage: Cartilage is a form of connective tissue composed of chondrocytes and a specialised
extracellular matrix. This matrix consists of water, collagen, proteoglycans and other components such as
adhesives and lipids.
There are several different types of cartilage. Articular hyaline cartilage consists of a glassy and
homogenous matrix with lacunae containing chondrocytes. Articular cartilage contains more collagen than
other types of hyaline cartilage. It lines the bones of synovial joints and functions in load distribution and
decreasing friction. The articular cartilage matrix is avascular, aneural and alymphatic relying on the
process of diffusion to provide nutrients for the chondrocytes.

Other types of cartilage include fibrocartilage, elastic cartilage, and physeal cartilage. Fibrocartilage
contains abundant thick bundles of mostly type I collagen which can be seen with the light microscope.
This type of cartilage is found at ligament and tendon insertions into bone, in menisci, intervertebral discs,
the symphysis pubis, temporomandibular and sternoclavicular joints. Fibrocartilage provides good
resistance to shear and compression forces. Elastic cartilage is characterised by the presence of elastic
fibres within the matrix which increase elasticity in tissues such as the external ear and trachea. Physeal
cartilage provides longitudinal growth to immature long bones.

Articular Cartilage: Important properties of articular cartilage include:

    •    Avascular (no blood vessels)
    •    Aneural (no nerve fibers)
    •    Alymphatic (no lymphatic vessels)
    •    Very low friction on cartilage on cartilage motion
    •    Self-renewing (maintenance and restoration of extracellular matrix)
    •    With aging, loss of ability to maintain the extracellular matrix

In regard to chondrocytes:

    •    By cartilage volume, the cells only represent about 1%.
    •    Chondrocytes are synthetic machines producing the extracellular matrix.
             o Intracellular organelles
                           Endoplasmic reticulum
                           Golgi apparatus
    •    Chondrocytes do not have cell-to-cell contact in the extracellular matrix.
    •    With aging, chondrocytes lose their synthetic abilities.
    •    Chondrocytes respond to a number of stimuli:
             o Increase matrix production after sensing degradation of the matrix
             o Sense loads and increase matrix production
             o Respond to growth factors and anabolic stimuli

Articular cartilage has three principal classes of macromolecules:

    •    Collagen – 60%
    •    Proteoglycans – 25% to 35%
    •    Noncollagenous proteins/glycoproteins – 15% to 20%

The three articular cartilage collagens that form cross bands are types II, IX, and XI. Of particular note:

    •    Type XI binds to type II.
    •    Type IX binds to the cross-banded fibrils in the superficial layer.
    •    Type VI attaches to the matrix around the chondrocytes.
    •    Type X is near the calcified layer and is probably involved in mineralization of the calcified layer.

Noncollagenous proteins include:

    •    Decorin and fibromodulin bind to type II collagen and likely stabilize the type II collagen
         network.
Cartilage has a number of distinct zones.

The superficial zone has a number of important characteristics:

    •    Thinnest articular cartilage layer
    •    Two layers:
             o Most superficial – fine collagen fibrils (lamina splendens)
             o Deep layer – flattened fibroblast-like chondrocytes (parallel to joint surface)
    •    Forms a cartilage skin
    •    Important chemical properties:
             o High collagen and low proteoglycan concentration
             o Fibronectin and water concentrations are highest in this zone
    •    Great tensile stiffness and strength
    •    Seals off the cartilage from the immune system

The transitional zone lies between the superficial and middle zones of the articular cartilage.

The following important points should be remembered:

    •    The chondrocytes have a high concentration of synthetic organelles such as rough endoplasmic
         reticulum and Golgi apparatus.
    •    The collagen fibers are larger than in the superficial zone.
    •    The proteoglycan concentration is higher than the superficial zone.

The chondrocytes in the calcified cartilage zone show the least metabolic activity.

In contrast, the chondrocytes of the other areas are very active:

    •    Superficial zone
             o Fine collagen fibrils (lamina splendens)
             o High collagen and low proteoglycan concentration
             o Fibronectin and water concentrations are highest in this zone
    •    Transitional zone
             o The chondrocytes have a high concentration of synthetic organelles such as rough
                  endoplasmic reticulum and Golgi apparatus.
             o The collagen fibers are larger than in the superficial zone.
             o The proteoglycan concentration is higher than the superficial zone.
    •    Middle (radial or deep) zone
             o Largest diameter collagen fibrils
             o Highest proteoglycan content

Other important points:

Interleukin I has the potential to increase expression of matrix metalloproteinases that can dissolve the
extracellular matrix.

Type II collagen fibers resist tensile and shear deformation forces in the articular cartilage.

In contrast, the glycosaminoglycan aggregates resist articular cartilage compression and fluid flow.

Cyclic compressive loads have the ability to stimulate matrix synthesis – aggrecan core protein and the
glycosaminoglycans.

The characteristic findings in osteoarthritis are:

    •    Asymmetric loss of the joint space
    •    Subchondral sclerosis and cysts
    •    Osteophyte formation
Osteoarthritis

As the cartilage degenerates, progressive bone remodelling occurs. The cause of osteoarthritis is
unknown. From a chemical standpoint, one of the earliest findings is a decrease in the proteoglycan and
an increase in the water content. One should remember:
Constant type II collagen content
Decreased proteoglycan concentration and decreased chain length
Increased water content
The decreased proteoglycan content results in increased permeability of the cartilage. A reduction of the
stiffness makes the articular cartilage less able to bear loads.
In the second stage, there is a cellular response – chondrocyte proliferation. Clusters of chondrocytes
producing new matrix are visible.
In this stage, there is nitric oxide and interleukin I production. These are catabolic factors that increase
matrix metalloproteinase activity. Degradative enzymes break down types IX and XI collagen, which may
compromise the stability of the type II collagen framework.
In the last stage of osteoarthritis, there is reduced chondrocyte proliferation and function, which may be
secondary to reduced ability to respond to anabolic factors (down regulation). There may be accumulation
of molecules that bind to the anabolic factors (and keep the factors from the chondrocytes) such as
decorin and insulin-dependent growth factor binding protein.

Articular cartilage: Components

Chondrocytes (5% wet weight)

Chondroblasts which are derived from mesenchymal cells become trapped in lacunae and develop into
chondrocytes. Chondrocytes are important in the control of matrix turnover through production of:


    •    collagen

    •    proteoglycans

    •    enzymes for cartilage metabolism.

Matrix

Water (60-80% wet weight)


    •    Articular cartilage is a highly hydrated material. Water distribution varies, making up 65% of wet
         weight at the deep zone and 80% at the surface.

    •    Weight bearing capacity is made possible through regional changes in water content which allow
         deformation of the cartilage surface in response to stress.

    •    Water provides nutrition and lubrication of cartilage.

    •    Increases in water content lead to:

              o     increased permeability

              o     decreased strength

              o     decreased Young’s modulus

    •
    •    Water content decreases with normal ageing.

    •    Water content increases in osteoarthritis

Collagen (10-20% wet weight) – (image 1)


    •    forms a cartilaginous framework which provides tensile strength.
    •   90-95% is type II collagen with increased Gly, Lys-OH, Pro-OH and hydrogen bonding.

    •   small amounts of types V, VI, IX, X and XI collagen are present.




Proteoglycans (10-15% wet weight) – (image 2)


    •   half life of three months

    •   provide compressive strength

    •   regulate matrix hydration by providing a porous structure to trap and hold water

    •   composed of subunits of glycosaminoglycans (GAG’s - disaccharide polymers)

            •
            o    chondroitin-4-sulfate (decreases with age)

            o    chondroitin-6-sulfate

            o    keratin sulfate (increases with age)

    •   GAG’s are bound to a protein core by sugar bonds to form a proteoglycan aggrecan molecule.

    •   Aggrecan molecules are further stabilised by link proteins which bind them to hyaluronic acid to
        form a proteoglycan aggregate.
Adhesives

Molecular interactions between chondrocytes and collagen fibrils are mediated by fibronectin,
chondronectin and anchorin CII.

Lipids are present in cartilage but their function is unknown.

Collagen molecules and proteoglycans interweave to form cartilage (image 3)




Layers (image 4)

Superficial Gliding Zone


    •    abundant tangentially oriented collagen fibres

    •    low proteoglycan concentration

    •    high water content

    •    discoid flattened cells, parallel to surface

    •    low metabolic activity (proteoglycan synthesis low)
Transitional Zone


    •      thicker fibrils

    •      oblique fibres

    •      cells arranged singly or in pairs

    •      high metabolic activity


Radial Zone


    •      increased collagen size

    •      vertical (radial) orientation of fibres

    •      high proteoglycan concentration

Tidemark


    •      Undulating barrier tangential to the surface which represents a plane of weakness.

Calcified Zone


    •      Hydroxyapatite crystals anchor the cartilage to the subchondral bone.


Metabolism

Collagen

Synthesis of collagen takes place in stages at various intracellular and extracellular sites. Post-
translational modifications occur in the rER and Golgi.
Intracellular Events


    •       mRNA messages are translated into polypeptide chains which are released into the cisternae of
            the rER.

    •       The signal peptide is cleaved.

    •       Lysine and proline residues are hydroxylated.

    •       Hydroxylysine residues are glycosylated.

    •       N-linked sugars are added to the terminal portion of the polypeptide.

    •       Polypeptide chains form a triple helix molecule.

    •       Procollagen is formed through intrachain and interchain disulfide bonds which stabilise the
            polypeptides and determine the shape of the molecule.

    •       Procollagen is packed into secretory granules which move along microtubules to be released into
            the extracellular matrix

Extracellular Events


    •       Uncoiled terminal ends of procollagen are cleaved to form tropocollagen.

    •       Tropocollagen molecules aggregate and lysine and hydroxylysine residues are crosslinked to
            form a collagen fibril.

    •       Fibrils aggregate to form collagen fibres.

Collagen catabolism is poorly understood, but enzymatic processes and mechanical factors may be
involved.

Proteoglycans

The process of proteoglycan synthesis begins with translation of mRNA to form a protein core to which
glycosaminoglycan chains are added. The resultant aggrecan molecules are transported to secretory
vesicles and released into the extracellular matrix. Link proteins and hyaluronate from the cell membrane
bind to the molecules forming proteoglycan aggregates.

Proteoglycan catabolism depends on cleavage of globular domains resulting in non-aggregation of the
resultant fragments.

Regulation of Growth

Cartilage synthesis is regulated by growth factors.

Platelet-Derived Growth Factors (PDGF)


    •       may have a role in healing cartilage lacerations

Transforming Growth Factor Beta (TGF-b )


    •       stimulates proteoglycan synthesis

    •       suppresses synthesis of type II collagen

    •       prevents the degradative action of plasmin and stremolysin through stimulation of formation of
            plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase (TIMP).

Fibroblast Growth Factor (b-FGF)
    •    Stimulates DNA synthesis in articular chondrocytes in adult articular cartilage.

    •    may play a role in repair process.

Insulin-Like Growth Factor-I (IGF-I)


    •    stimulates DNA and cartilage matrix synthesis in both adult articular cartilage and immature
         growth plate cartilage.

Changes With Ageing

With ageing cartilage becomes hypocellular and has decreased elasticity.

Chondrocytes


    •    increase in size

    •    increased lysosomal enzymes

    •    cartilage becomes hypocellular (cells stop reproducing)

Matrix


    •    Proteoglycans

              o    decrease in mass and size - decreased length of chondroitin sulfate chains

              o    change in proportion - increased keratin sulfate

    •
    •    Water content decreases

    •    Protein content increases

Healing of Articular Cartilage

Deep lacerations


    •    extend below the tidemark

    •    heal with fibrocartilage

    •    blunt trauma may cause osteoarthritic changes

Superficial lacerations


    •    above the tidemark

    •    chondrocytes proliferate but do not heal

    •    immobilisation leads to atrophy while continuous passive motion is beneficial to healing

Synovium

The interior surfaces of normal joints (except articular cartilage and menisci) are lined by a synovial
membrane. Synovium is composed of vascularized connective tissue that lacks a basement membrane
and contains two predominant cell types which reflect the function of the tissue.

Type A cells - important in phagocytosis

Type B cells - fibroblast-like cells which produce synovial fluid.
There are other undifferentiated cells that have a reparative role. Type C cells may exist as an
intermediate cell type.

Synovial fluid produced by the synovium, is an ultrafiltrate of blood plasma containing in addition,
hyaluronic acid, proteinase, collagenases and prostaglandins, but no red blood cells, clotting factors, or
haemoglobin.

Function


    •    Nourishment of articular cartilage through diffusion.

    •    Lubrication of the joint space:

Joint lubrication can be divided into two types. It is likely that different types of lubrication become
important in different types of movement.

Boundary lubrication (slippery surfaces) is where in response to a load, the joint surfaces are separated by
a mono/multi molecular layer of low shear strength material (hyaluronate-protein complexes in the synovial
fluid). This allows sliding motion while preventing adhesions or abrasions.

Fluid film lubrication is where fluid separates the joint surfaces. There are several different types.

Hydrodynamic Lubrication


    •    a wedge shaped film of fluid is pulled between two opposing surfaces

    •    a modification of this type is seen in human joints: elasto-hydrodynamic

              o    deformation of the surfaces also occur

Squeeze Film Lubrication


    •    Fluid forms a film after being forced from the articular surfaces subjected to a load.

    •    The viscosity of the fluid increases so that a gel is formed which has an osmotic pressure
         equivalent to the pressure applied.

Weeping (Hydrostatic) Lubrication


    •    A film is formed between the articular surfaces as fluid leaks out of the cartilage.

Boosted Lubrication (fluid entrapment)


    •    Similar to squeeze film lubrication but avoids contact between the articular surfaces by trapping
         high viscosity gel in localised depressions.

Flow characteristics:

Flow is non-Newtonian (the viscosity coefficient µ is not a constant; the fluid is not linearly viscous). Its
viscosity increases as the shear rate increases.

Lubricin, a glycoprotein, is the key lubricating component of synovial fluid. Hyaluronan molecules in the
knee become entangled and behave like an elastic solid during high strain activities (running, jumping).

Meniscus

The meniscus functions to deepen the articular surface of a number of synovial joints. By doing so it
increases the contact area available for load distribution. These joints include:
    •      Acromioclavicular

    •      Sternoclavicular

    •      Glenohumeral

    •      Hip

    •      Knee

We will focus here on the meniscus of the knee joint.

Anatomy

The meniscus is a triangular, semilunar structure. Its peripheral border is attached to the joint capsule. In
the knee, the medial meniscus is semicircular and the lateral meniscus is circular.

Components

The meniscus is composed of fibrocartilage.

Cellular components

These synthesise and maintain the extracellular matrix, and are responsible for anaerobic metabolism.
Cells found are chondrocytes and fibroblasts, which are referred to as fibrochondrocytes.

Fusiform cells:


    •      in lacunae in superficial layer

    •      resemble chondrocytes and fibroblasts

    •      abundant ER and Golgi

Ovoid cells:


    •      surface and middle layer

    •      abundant ER and Golgi

Matrix components

Collagen


    •      primarily type I (55-65% dry weight), also types II, III, V, VI (5-10% dry weight)

    •      Superficial layer - mesh like fibres oriented radially

    •      Surface layer - (deep to superficial) collagen bundles aligned irregularly

    •      Middle layer - (deep) parallel to circumferential fibres

Elastin (0.6% dry weight)

Proteoglycans → (1-3%

Glycoproteins → dry weight)

Adhesive glycoproteins include fibronectin, thrombospondin.

Blood supply

The geniculate arteries supply the menisci. The outer 25% of the menisci are supplied by a
circumferentially arranged plexus, and the remaining 75% receive supply via diffusion.
Tears that occur in the peripheral vascularised region (red zone) will heal via fibrovascular scar formation
by fibrochondrocytes. Tears that occur in the central avascular regions (white zone), however, can't heal.

Nerve supply

The outer two-thirds of the menisci is innervated by type I and type II nerve endings which are
concentrated in the anterior and posterior horns, with few fibres in the meniscal body.

Non-Inflammatory Arthritides: Osteoarthritis Neuropathic Arthropathy Acute Rheumatic Fever
Ochronosis Secondary Pulmonary Hypertrophic Osteoarthropathy

Inflammatory Arthritides: Rheumatoid Arthritis Systemic Lupus Erythematosus Juvenile Rheumatoid
Arthritis Relapsing Polychondritis Spondyloarthropathies Ankylosing Spondylitis Reiter’s Syndrome
Psoriatic Arthritis Enteropathic Arthritis Crystal Deposition Disease Gout Chondrocalcinosis Calcium
Hydroxyaptite Crystal Deposition Disease

Infectious Arthritides Pyogenic Arthritis Tuberculous Arthritis Fungal Arthritis Lyme Disease

Haemorrhagic Arthritides Haemophilic Arthropathy Sickle Cell Disease Pigmented Villonodular Synovitis

Joint Fluid Analysis



Arthroses                                  Analysis


1. Non-inflammatory Arthritides            • 200 WBCs, 25% PMNLs

                                           • Glucose and protein equal serum
                                           values

                                           • Normal viscosity (high)

                                           • Straw colour

                                           • firm mucin clot


2. Inflammatory arthritides                • 2000-75000 WBCs, 50% PMNLs

                                           • Moderately decreased glucose
                                           (25mg/dl

                                           lower than serum glucose)

                                           • Low viscosity

                                           • Yellow-green colour

                                           • Friable mucin clot

                                           • Synovial fluid complement is
                                           decreased

                                           in RA and normal in ankylosing

                                           spondylitis
3. Infectious arthritides                  • > 80000 WBCs, ≥ 75% PMNLs

                                           • Positive gram stain (and cultures)

                                           • Low glucose (> 25mg/dl less than
                                           serum

                                           values)

                                           • Opaque fluid

                                           • Increased synovial lactate




Non-inflammatory Arthritides

Osteoarthritis (OA)

Osteoarthritis (OA) is a degenerative joint disease characterised by progressive loss of articular cartilage
with associated new bone formation and capsular fibrosis. It is the most common form of arthritis. Nearly
everyone who lives long enough will be affected by OA. Its prevalence increases steeply with age and
there may be a genetic predisposition.

OA is classified as primary when it arises without an obvious cause, and secondary when it occurs
following certain predisposing factors (such as previous trauma, congenital deformity, infection or a
metabolic disorder).

Aetiology

On a cellular level, osteoarthritis may be due to failure of chondrocytes to repair damaged cartilage.
Excessive stresses are applied to articular cartilage and there is an inadequate chondrocyte response,
leading to degeneration of the articular cartilage. The disparity between the stress applied and the
chondrocyte response may be due to:


    •    abnormal loads over a small area of cartilage

    •    weakening of the cartilage (not well understood)

    •    abnormal support by subchondral bone
Pathogenesis

Current concepts on the pathogenesis of OA are based on the assumption that whatever the provoking
cause, there is a final common pathway of changes in articular cartilage. It has been suggested that the
initiating event is fatigue fracture of the collagen meshwork, is followed by increased hydration of the
articular cartilage and loss of proteoglycans from the matrix into the synovial fluid. There is some evidence
of increased collagenolytic activity but collagen loss may also be due to mechanical causes.

Alternatively, it has been proposed that the initial lesions are microfractures of the subchondral bone
following repetitive loading. Healing of these microfractures results in significant loss of resilience of the
subchondral bone. A stress gradient develops in the adjacent articular cartilage. As the process evolves
there is fibrillation of the cartilage, and deep clefts appear with reduplication and proliferation of
chondrocytes within them. Proliferative changes also occur simultaneously at the joint margins with
formation of osteophytes. Eventually articular cartilage is lost altogether in areas of maximum mechanical
stress and the underlying bone becomes hardened and eburnated. There may be cyst formation.



Changes in osteoarthritic cartilage:


    1.   Increased water content (in contrast to decreased water content in normal ageing)

    2.   This occurs early, and suggests some weakening of type II collagen

    3.   Changes in proteoglycans

    4.   There are shorter chains and shifts in the concentration of proteoglycans. The chondroitin/keratin
         ratio is decreased.

    5.   Collagen abnormalities

    6.   Disruption is caused by an increase in collagenase and proteoglycan-degrading enzyme
         concentration.

    7.   Proteoglycan binding

    8.   Proteoglycans bind to hyaluronic acid. This results from the action of proteolytic enzymes, due to
         increased prostaglandin E and decreased numbers of link protein.

    9.   Rate of synthesis

    10. Rate of synthesis of DNA, collagen, and proteoglycans are increased

    11. Increased levels of:

    12. Cathepsins B and D, metalloproteinases (collagenase, gelatinase, and stromelysin).

    13. Interleukin-1

    14. Enhances enzyme synthesis and has a catabolic effect (causing cartilage degeneration).

    15. GAGs and polysulfuric acid



Clinical features

Patients usually present after 40-50 years. The knee joint is most commonly affected. Pain is of an
insidious onset, and progresses over months or years. Stiffness and swelling occur in later stages of the
disease. Range of movement is reduced and there may be associated crepitus.
Investigations and Diagnosis

Diagnosis is made largely clinically by the presence of gradual onset of pain after activity, pattern of joint
involvement and lack of significant soft-tissue swelling. Radiology may be useful and joint fluid analysis
helps rule out other causes of arthritis.

Radiology:


    •    osteophytes

    •    narrowing of joint space

    •    subarticular sclerosis and bone cysts



Joints that are commonly involved are the distal and proximal interphalangeal joints, and carpometacarpal
joints of the hand. There is usually superolateral involvement of the hip joint and asymmetric involvement
of the knee.

Microscopic changes:


    •    loss of superficial chondrocytes

    •    cloning of chondrocytes (more than one chondrocyte per lacunae)

    •    replication and breakdown of the tidemark

    •    fissuring

    •    cartilage destruction with eburnation of subchondral pagetoid bone

Treatment


    •    Protection of affected joints from overloading

               o     weight loss

               o     use of walking stick

    •
    •    Exercise of supporting muscles around joints to avoid wasting.

    •    Supportive measures such as pain relief by analgesics or NSAIDs.

    •    Surgical treatment is indicated for patients with persistent symptoms and pain and ranges from
         arthroscopy to arthroplasty.

    •    Realignment osteotomies may be done in younger patients to redistribute weight bearing load at
         the knee to prevent further damage.

    •    Total joint arthroplasties for older patients (over 60) in advanced cases that are resistant to
         conservative treatment.

Neuropathic Arthropathy

Neuropathic arthropathy, also known as Charcot joint, is a rapidly progressive form of osteoarthritis caused
by a disturbance in the sensory (position sense and pain) innervation of a joint. In the upper limbs it is
most commonly associated with syringomyelia, followed next by Hansen’s disease. 25% of patients with
syringomyelia get a Charcot joint. In the lower limbs, common causes include tabes dorsalis, peripheral
neuropathies especially diabetes, cauda equina lesions, and congenital insensitivity to pain.

Clinical features

Include an unstable, swollen, yet painless joint. Effusions may be seen. Patients are typically older, and
may present with haemarthrosis. Radiological changes include advanced destruction on both sides of the
joint, joint distension due to increased amount of fluid, irregular bits of bone embedded in fibrous tissue,
and heterotopic ossification.

Treatment

Involves stabilising the affected joints by limitation of activity and use of appropriate splintage, such as
callipers and casts. Total joint arthroplasty and use of other orthopaedic hardware are contraindicated.



Acute Rheumatic Fever

Acute rheumatic fever is sometimes included in the inflammatory group of arthritides. Although was the
most common cause of childhood arthritis, its prevalence in developed countries has progressively
declined since the advent of antibiotics.

Pathogenesis

Acute Rheumatic Fever is triggered by infection with specific streptococcus.

Molecular Genetics:

I. Chromosome

A. 23 pairs (46) in humans.

B. Located in the nucleus of every cell.

C. Contains minimum of 150,000 genes, however only some genes are expressed in a particular cell
determining its unique qualities.

D. contains deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

II. DNA

A. Located inside the chromosome.

B. Function is to regulate cellular activity by protein synthesis. It is also necessary for cell division, DNA
replication, transcription and production of RNA.

C. Contains 2 sugar molecules formed in double helix. The sugar molecules have one of adenine,
guanine, cytosine, thymine and are linked to one another by hydrogen bonds. Adenine has 2 hydrogen
bonds with thymine and guanine has 3 bonds with cytosine.



III. Nucleotide

A. consists of sugar molecule, phosphate and either adenine, guanine, cytosine and thymine.

B. group of 3 nucleotides (codon) indicates a specific amino acid.

C. due to pairing of adenine-thymine and cytosine-guanine, nucleotide sequences of one strand of DNA
dictate the sequences of the other strand.

IV. Gene
A . parts of DNA (made up of a number of nucleotides).

B. coding a specific protein.

V. Transcription

A. A process where DNA is transcribed to mRNA via RNA polymerase.

B. One of steps in protein synthesis-important in regulation of cell activity.

VI. Translation

A. a process of sequencing amino acids (which are coded by mRNA) in order to build protein.

VII. Regulating DNA

A. gene promoter: initiate transcription.

B. consensus sequences: binding sites for regulatory proteins.

C. gene enhances: binding sites for transcription proteins.

VIII. Biogenetic techniques

A. Restriction enzymes: cut DNA at a location determined by a specific nucleotide sequences. The
resulting fragments are called restriction fragments.

B. Agarose gel electrophoresis: negatively charged DNA fragments are put in a gel and exposed to
electrical field. As a result, there is separate of DNA fragments based on size - the smaller the gragments,
the closer they are to the positive pole of electrical field.

C. DNA ligation: attached human genes to plasmids in order to study the genes.

D. Plasmid vectors: so called for the human genes about to be ligated to plasmids. The products of DNA
ligation is called recombinant plasmid.

E. Transformation: insertion of recombinant plasmid into bacterium which then replicates the recombinant
plasmid hence increases the amount for study.

F. Genomic library: a library of the entire genome, in the form of recombinant DNAs. Achieved by
restricting the genes, which is then ligated with plasmid and transformed into E.coli which amplify the
amount of recombinant genes and the genes are then isolated from the bacterial colony.

G. Transgenic animals: animals with foreign genes in every cells of their body due to insertion and
incorporation into chromosomes of those foreign genes into a single cell embryo.

H. Southern Hybridization: technique to find a particular DNA sequence in mixed DNA.

I. Northern Hybridization: technique to find a particular RNA in mixed RNA.

J. Polymerase Chain Reaction (PCR) amplification: to increase the amount of specific DNA sequence in
vivo by repeatedly synthesizing it. Used to screen DNA for gene mutation.

IX. Effects on orthopaedics

A. screening of genetic diseases such as osteogenesis Imperfecta, Marfan syndrome, chondrodystrophies,
Vitamin D-resistant rickets and familial aortic aneurysm.

B. bone tumours are a result of failure in regulating cell growth due to gene mutation.

Immunology: I. Nonspecific Immune response
A. inflammatory responses following a recognition of a foreign antigen. It consists of Histamine release
(cause vasodilatation) and phagocytic cells that only recognize the antigen as non-self (macrophage,
neutrophil, etc.) and respond by enzymatically digesting the foreign materials.

B. It can be enhanced by complement system or subsided by anti- flammatory medication.

II. Specific immune responses

A. cell mediated and humoral immune responses.

B. antigens: when recognized as foreign, starts an immune response.

C. macrophages: eat the foreign body and presents antigen on its surface to T lymphocytes.

D. T lymphocytes: originate in bone marrow but mature in thymus. It consists of helper T cells, suppressor
T cells and killer T cells. Helper T cells produce cytokines to stimulate B cells, macrophages, killer T cells
and suppressor T cells. Killer T cells also produce cytokines to stimulate suppressor T cells. Suppressor T
cells are then damped down the immune response once the antigens are cleared.

E. B lymphocytes: originate and mature in bone marrow. Differentiate into plasma cells and memory cells
upon stimulation. Plasma cells reduce immunoglobulins (lg) against specific antigens.

F. Immunoglobulins: 5 types-IgA (releases to and acts on mucosal surfaces), lgM (only inside blood
circulation, largest, produced earliest by foetus and plasma cells at first encounter with new antigen), lgG
(commonest, produced after lgM in response to new infection), lgD (acts as a receptor), lgE (attach to mast
cells, involved in allergic reactions).

G. Cytokines:

a) glycoproteins produced by cell in response to foreign antigen.

b) role in regulation of inflammatory and immune reaction.

c) they are interferons, growth factors, colony stimulating factors, interleukins.

H. Complement system

a) 25 proteins.

b) acts in sequence to amplify immune response.

I. Immunogenetics

a) Located on the short arm of chromosome 6 called Human Leukocyte antigens (HLA).

b) make up the specificity of immune recognition.

c) 6 class I loci and 14 class II loci.

J. Transplantation

a) allogenic grafting: tissue transplanted to another non genetically identical of the same species.

b) xenografting: tissues transplanted to another of different species.

c) Graft preparation: a) Reduce cellular response by freezing. b) Eliminate cellular response by freeze-
drying (lyophilisation).

K. Oncology-tumour cells have cell surface antigens that are:

a) identical to the ones on normal cells indicating their origin, however they may lose some of these.

b) maybe identical to those on flat cells.

c) tumour associated.
PREOP. CHECKS
1. Pulmonary complications

A. Blood Gas:


    •   pO2 = 7(FiO2 where pO2 is the normal value for a given FiO2 is the percent spired oxygen and
        pCOs is the value taken from the test result.

    •   Aa gradient = normal pO2 - test result pO2.

    •   Percent physiologic shunt = Aa gradient /20.

B. Thromboembolism:


    •   common problem especially with hip procedures.

    •   Risk factors: a history of thromboembolism, obesity, malignancy, age, congestive heart failure,
        birth control pill use, varicose veins, smoking, general anaesthetics, increased blood viscosity,
        immobilization, paralysis d pregnancy.

1. Deep venous thrombosis (DVT):


    •   venography: 97% accurate (70% for iliac veins).

    •   125
              l-labelled fibrinogen: false positive at operative site.

    •   impedance plethysmography: sensitivity is poor.

    •   duplex ultrasonography (B mode): sensitivity for DVT proximal to bifurcation is 90%.

    •   Doppler imaging: bedside tool.

    •   Prophylaxis such as heparin, coumadin (warfarin), aspirin, dextran, pneumatic compression,
        enoxaparin are important in morbidity and mortality. Coumadin can be reversed with Vitamin K or
        fresh frozen plasma.

    •   Treatment is by heparin then followed by warfarin therapy for the next 3 months. All thigh DVTs
        need treatment, but those below popliteal fossa may not. Patients with preoperative finding of
        DVT require vena cava filter.

2. Pulmonary embolism (PE):


    •   suspected in postoperative patients with acute onset of pleuritic pain, tachypnoea (90%) and
        tachycardia (60%).

    •   ECG shows right bundle branch block, right axis deviation in 25%, may show ST depression or T
        wave inversion lead III.

    •   Chest X ray will hardly show hyperlucency and ABGs may show normal PaOs but this does not
        exclude PE.

    •   V/Q scan may show the site of embolus but otherwise pulmonary angiography is the gold
        standard.

    •   Treatment with heparin (continuous IV infusion) and its efficacy is monitored with partial
        thromboplastin time (PTT) for 7-10 days then followed by oral warfarin for 3 months which is
        monitored using prothrombin time (PT).
3. Coagulation:

a) intrinsic pathway: initiated by factor XII touching the exposed collagen in blood vessels. monitored by
PTT.

b) extrinsic pathway: initiated y presence of thromboplastin in the circulation due to cellular injury,
monitored by PT>.

c) platelet function is measured by bleeding time test. Fibrinolytic system is responsible in dissolving clot
by converting plasminogen to plasmin and plasmin dissolves the clot.

C. Adult Respiratory Distress Syndrome (ARDS):


    •    acute respiratory failure following pulmonary oedema. Initiated by trauma, shock, infection etc.

    •    Aetiologies: pulmonary infection, sepsis, fat embolism, microembolism, aspiration, fluid overload,
         atelectasis, oxygen toxicity, pulmonary contusion, head injury.

    •    manifested as tachypnoea, dyspnoea, hypoxemia, decreased lung compliance. Diagnosis can be
         made using ABGs following a long bone fracture.

    •    50% mortality rate.

    •    Treatment is by ventilation with PEEP, but best avoided by early stabilization of long bone
         fractures.

D. Fat embolism:


    •    24-72 hrs after incidence of long bone fractures in 3-4% of patients.

    •    fatal in 10-15%.

    •    manifested as tachypnoea, tachycardia, mental status changes and upper extremity petechiae.

    •    aetiologies: bone marrow fat (mechanical theory), chylomicron changes due to stress (metabolic
         theory).

    •    Ventilation-perfusion deficit resulted consistent with ARDS.

    •    Treatment is by ventilation with high levels of PEEP, but can be prevented by early stabilization of
         the fracture.

E. Pneumonia:


    •    aspiration pneumonia in patients with decreased mentation, supine position and decreased GI
         motility.

    •    Preventative measures: raising the bed’s head, use of antacids and metoclopramide

    •    Treatment by IV antibiotics and pulmonary toilet.

F. Pulmonary complications of orthopaedic disorders include scoliosis, Marfan syndrome.

II. Non pulmonary complications

A. Nutrition


    •    sufficient nutrition prior to elective surgery, otherwise complications such as wound dehiscence,
         infection, pneumonia and sepsis can occur.
    •    indicators: arm muscle circumference size (best indicator), energy panels, albumin levels,
         transferrin levels.

    •    atrophy of the intestinal mucosa can occur from lack of enteral feeding which then causes
         bacterial translocation. Early elemental feeding through a jejunostomi tube helps multiple trauma
         patients from complications.

    •    Full enteral or parenteral nutrition should be given since energy requirement is elevated during
         stress. Protein supplements are beneficial in patients at risk of developing multi organ failure.

B. Myocardial infarction (MI)


    •    identified by acute chest pain, radiation, EKG changes. Confirmed by elevated cardiac enzymes.

• Risk factors include age, smoking, increased cholesterol, hypertension, aortic stenosis, history of
coronary artery disease, diabetes, family history of heart problems.
C. GI complications


    •    ileus : treated with nasogastric suction and antacids. Common in diabetic with neuropathy.

    •    upper GI bleeding: risk factors include history of ulcers, NSAID use and smoking. Treated by
         lavage, antacids and H2-blockers, vasopressin at left gastric artery may be required for more
         serious cases.

    •    Ogilvie syndrome: can follow total joint replacement surgery. Signified by caecum distention. If
         caecum > 10 cm on abdominal X-ray, decompression thru colonoscopy is required.

D. Decubitus ulcers


    •    risk factors include advanced age, critical illness and neurologic impairment.

    •    usually at sacrum, heels and buttocks.

    •    increase the risk of infections and morbidity. Once there, debridement and sometimes soft tissue
         flaps are required for healing.

    •    prevented by constant changing of position, special mattresses and treatment of systemic illness
         and malnutrition.

E. Urinary tract infections (UTI)


    •    most common nosocomial infections.

    •    resulting in increased risk of infections at joint following TJA.

    •    Preoperatively, should be treated sufficiently. Preoperative catherisation may reduce the rate of
         postoperative UTI. Catheter is removed 24 hrs postoperatively.

F. Prostatic hyperplasia


    •    results in urinary retention and increased risk of UTI.

    •    Should be sought before the surgery based on history, physical examination especially PR and
         urine flow studies < 17 ml/s peak flow rate.

G. Acute tubular necrosis
    •      results in renal failure in trauma patients.

    •      Early treatment includes alkalisation of urine.

H. Genitourinary injury


    •      Retrograde urethrogram is used to find lower urinary tract injury.

    •      Risks increased with the use of NSAID. Should be suspected in patients with displaced anterior
           pelvic fractures.

I. Shock


    •      insufficient perfusion to vital tissues and organs.

    •      Hypovolaemic shock: due to volume loss - resulting in decreased cardiac output (CO), increased
           peripheral vascular resistance (PVR) and venous construction. Treated by Ringer’s lactate fluid,
           then blood transfusion. Massive blood replacement requires fresh frozen plasma and platelets.
           Urine output shows the adequacy of fluid resuscitation. Insufficient fluid resuscitation can result in
           metabolic acidosis.

    •      Cardiogenic shock: due to ineffective pumping - resulting in decreased CO, increased PVR,
           venous dilation.

    •      Vasogenic shock: due to PE or pericardial tamponade - arteriolar constriction, venous dilation.

    •      Neurogenic shock/ septic shock: due to blood pooling- arteriolar, capillary and venous dilation.

J. Compartment syndrome

III. Intraoperative consideration

A. Anaesthesia


    •      local anaesthesia may allow quicker recovery, decreased blood loss and fewer postoperative
           complications.

    •      reduced blood loss with controlled hypotension during surgery - by nitroprussode, nitroglycerine,
           isoflurane.

    •      Patients with neuromuscular disorders (Duchenne’s muscular dystrophy, arthrogryposis,
           osteogenesis imperfecta) should be suspected for malignant hyperthermia (autosomal dominant,
           hypermetabolic disorder of skeletal muscles) following the use of anaesthetics especially
           halothane and succinylcholine. Disease is marked by muscle rigidity, hypermetabolism, masseter
           muscle spasm, increased temperature and acidosis due to defect of cell membrane affecting
           calcium transport. For definitive diagnosis, muscle biopsy is required. Treated by dantrolene,
           electrolytes balance increased urinary output, respiratory support and cooling.

B. Spinal cord monitoring


    •      usually testing the posterior column.

    •      Somatosensory cortical evoked potentials (SCEP) monitors spinal cord by evoking response from
           stimulation of peripheral areas.

    •      Somatosensory spinal evoked potentials (SSEP) are more invasive but more sensitive.
           Preoperative recordings are compared to the ones measured at critical times during surgery.
    •    The wake up test is the standard, done by patients responds in moving the peripheral parts
         according to commands upon lightening of anaesthesia.

C. Torniquet


    •    can result to injury of nerve and muscle underneath it.

    •    prevented by careful application, wide cuffs, lower pressure (200 mmHg in upper extremity and
         250 mmHg or 100-150 mmHg above systolic in lower extremity), double cuffs.

IV. Other problems

A. Pain control


    •    acute implies the presence of potential tissue damage whereas chronic (3-6 months) does not.

    •    Postoperative control of pain can be mediated at the nociceptors, decrease transduction of the
         nerve A and C fibers, dorsal column, spinothalamic tract, thalamus (local prostaglandin inhibitors,
         long acting local anaesthetics); increase modulation of brain stem centres or production of
         endogenous opiates (perispinal and systemic opiates).

B. Transfusion


    •    transfusion reactions

* allergic reaction: most common, occurs at the end of transfusion. Chills, pruritus, erythema and urticaria
results but usually resolved spontaneously. Pretreated by diphenhydramine/Benadryl and hydrocortisone
in patients with history of allergy.

* febrile reaction: occurs after the initial 100-300cc of packed RBCs transfusion. Chills and fevers due to
antibody reaction to foreign WBCs. Treatment is as allergy.

* haemolytic reaction: occurs early in the transfusion. Signified by chills, fever, tachycardia, chest
tightness, flank pain. Treated by stopping the transfusion, IV fluids, appropriate lab studies and monitoring
in intensive care.


    •    transfusion risks

* hepatitis, cytomegalovirus, HTLV-1 and HIV.

* donors deferral of high risk individuals and more effective screening methods decrease the prevalence.


    •    alternative to homologous blood transfusion

* autologous deposition: At least Hb=11 and Hematocrit of 33%. Iron supplement during donation is
routine. Storage of several units of blood prior to elective surgery with anticipated blood loss. Need
adequate times between the donation and surgery.

* cell saver- intraoperative autotransfusion: 400 ml blood loss to recover 250 ml. Can be used for only 4
hours at one time.

* autotransfusion: postoperative drain recuperation is used.

* acute preoperative normovolemic hemodilution: storage of autologous blood (replaced by crystalloids)
preoperatively for immediate use of intra/postoperatively.
* pharmacologic intervention: desmopression (ADH) analogue to increase level of factor VIII, recombinant
erythropoietin to increase erythropoiesis and synthetic erythrocyte substitutes.

* judicious use of blood products: platelet transfusion in massive bleeding or coagulopathies. Fresh frozen
plasma for patients with massive bleeding and abnormal coagulation tests. Cryoprecipitate for haemophilia
and for consumptive coagulopathies as a source of fibrinogen.

C. Heterotopic ossification


    •    seen after THA, in head injured patients and in elbow injuries patients.

    •    Prophylaxis during THA: indomethacin. Diphosphonate does not prevent formation of osteoid
         matrix since the matrix calcifies after discontinuation of medication. Etidronate sodium prevents
         bone resorption at low dose and bone mineralization at high dose.


Functions of the skeleton

Bone, the material that makes vertebrates distinct from other animals, has evolved
over several hundred million years to become a remarkable tissue. Bone is a
material that has the same strength as cast iron, but achieves this while remaining
as light as wood.

The front leg of a horse can withstand the loads generated while this 1500-pound
animal travels at 30 miles per hour. The upper arm is able to keep birds aloft
through entire migrations, sometimes over 10,000 miles without landing. The
antlers of deer, used as weapons in territorial clashes with other deer, undergo
tremendous impacts without fracturing, ready to fight another day.
At some point, unfortunately, forces of impact exceed even
bone's ability to hold up. Falling on the ice, suffering a
collision in a car or a tumble on the ski slopes can cause the
bone to fail. While fractures are disastrous, bone - because it
is a live tissue - almost instantly begins a healing process.
Without question, bone is the ultimate biomaterial. It is light,
strong, can adapt to its functional demands, and repair itself.

Functions of the skeleton

*   Structural support for heart, lungs and marrow
*   Protection for brain, uterus, and other internal organs
*   Attachment sites for muscles allowing movement of limbs
*   Mineral reservoir for calcium and phosphorus
*   Defense against acidosis
*   Trap for some dangerous minerals such as lead
Bone architecture

There are two major kinds
of bone, trabecular
(spongy) and cortical.
Trabecular bone gives
supporting strength to the
ends of the weight-bearing
bone. The cortical (solid)
bone on the outside forms
the shaft of the long bone.

This xray of a femur
shows the thick cortical
bone, and the trabecular
bone which is arranged to
withstand the stresses
from usual standing and
walking. Compressive
stresses are those of the
body weight pushing the
bone down, and tensile
stresses are from the
muscles, pulling the bone
apart.

Photo courtesy of Clint Rubin
Move mouse over image for labels
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larger view




Magnified view of a cut
surface of bone, showing
the cortical bone and
trabecular bone
surrounded by marrow
tissue (M). This is from
the iliac crest, part of the
pelvic bone. The actual
width is about 1 cm.
Further magnification
demonstrates the
organization of the cortical
bone into Haversian
systems, consisting of
concentric layers of bone
and a central canal which
supplies blood. The small
black dots are spaces that
contain osteocytes. The
boundaries between
Haversian systems are the
cement lines.




Mineral reservoir

In addition to its mechanical functions, the bone is a reservoir for minerals (a
"metabolic" function). The bone stores 99% of the body's calcium and 85% of the
phosphorus. It is very important to keep the blood level of calcium within a narrow
range. If blood calcium gets too high or too low, the muscles and nerves will not
function. In times of need, for example, during pregnancy, calcium can be removed
from the bones. This process is carefully regulated by hormones and is discussed
more completely in the section about hormones.
Bone material properties

Components of bone:

* The organic matrix is composed primarily of the protein collagen which provides
flexibility. 10% of adult bone mass is collagen. This is discussed further in the
section on collagen and bone matrix
* The mineral component is composed of hydroxyapatite, which is an insoluble salt
of calcium and phosphorus. About 65% of adult bone mass is hydroxyapatite.
* Bone also contains small amounts of magnesium, sodium, and bicarbonate.
* Water comprises approximately 25% of adult bone mass.




Changes with aging

This graph shows values for bone
mineral density at the hip in
Caucasian men and women and
African-American men and
women. With aging, bone density
decreases in all groups. This
inevitable bone loss is frequently
the cause of osteoporosis




This image shows trabecular bone
structure in the lower spine of a
young adult compared to an
osteoporotic elderly adult.




                                     Young adult   Elderly Adult with osteoporosis
Bone Cells
          There are two categories of bone cells. Osteoclasts are in the first category. They
          resorb (dissolve) the bone. The other category is the osteoblast family, which
          consists of osteoblasts that form bone, osteocytes that help maintain bone, and
          lining cells that cover the surface of the bone.

          Osteoclasts


* . . . are large cells with many nuclei.
* . . . share lineage with blood cells (especially macrophages).
* Precursors circulate in the blood and bone marrow.
* Mature osteoclasts are formed from fusion of the precursors.
* This happens when RANK receptors on the osteoclast precursors are activated by the RANK-ligand
which was secreted by osteoblasts.
* Osteoprotegerin (OPG) is a factor in the marrow which also binds RANK-ligand, so it can help to
regulate the osteoclast activation.
* Osteoclasts resorb the bone. They form sealed compartments next to the bone surface and secrete
acids and enzymes which degrade the bone. The edge next to the bone is called the ruffled border.
* After they finish resorbing bone, they undergo apoptosis (programmed cell death, sometimes called
'cell suicide'). This process is regulated by proteins from other cells.
           Osteoblasts


*. . . are cuboidal and columnar in shape with a central nucleus
found on the bone surface.
*Gap junctions with neighbouring osteoblasts allow cells to
communicate with each other.
*They come from bone marrow precursor cells. These precursors
are capable of turning into either osteoblasts or fat cells, and
various factors determine which kind of cells will be made. One of
the factors is called Cbfa 1, which will cause the cell to
differentiate into an osteoblast.
*The job of osteoblasts is to make the proteins that will form the
organic matrix of bone and to control mineralization of the bone
*They have receptors for hormones such as vitamin D,
oestrogen, and parathyroid hormone.
*They secrete factors that activate osteoclasts (RANK-ligand) and
other factors which communicate with other cells.
*They secrete PHEX, a protein that helps to regulate the amount
of phosphate excreted by the kidney.
*When the team of osteoblasts has finished making new bone,
some become surrounded with matrix and differentiate into
osteocytes. Others will remain on the surface of the new bone
and differentiate into lining cells. The rest undergo apoptosis (cell
suicide) and disintegrate.
         Osteocytes



* . . . live inside the bone and have long branches
which allow them to contact each other as well as
the lining cells on the bone surface.
* . . . are in a perfect position to sense any
mechanical strain on the bone.
* . . . can secrete growth factors which activate the
lining cells or stimulate the osteoblasts.
* Their exact role is still under investigation, but
probably the osteocytes direct bone remodelling to
accomodate mechanical strain and repair fatigue
damage.




         Lining cells

         * . . . are former osteoblasts which have become flat and pancake-shaped.
         * . . . line the entire surface of the bone.
         * . . . are responsible for immediate release of calcium from the bone if the blood
         calcium is too low.
         * . . . protect the bone from chemicals in the blood which dissolve crystals (such as
         pyrophosphate).
         * . . . have receptors for hormones and factors that initiate bone remodelling.


         Bone cell origins

         This diagram summarizes the origins and fates of the bone cells. Mesenchymal
         refers to cells which were deep within the embryo during early development; some
         of them remain in the bone marrow but do not form blood cells. The haematopoietic
         cells form the liquid part of the bone marrow, and some of them circulate with the
         blood.
Local regulators

Bone cells produce molecules (usually proteins) that communicate with other cells.
These molecules are called growth factors and cytokines. They act on nearby cells,
and thus are considered local regulators. These factors control cell division
(proliferation), differentiation, and survival.

Growth factors
Bone morphogenetic proteins (BMPs): BMPs are produced in the bone or bone
marrow. They bind to BMP receptors that are on mesenchymal stem cells within the
bone marrow. This causes the cells to produce Cbfa 1, which is a factor that
activates the DNA so proteins can be made -- a process known as gene
transcription. When Cbfa 1 activates the genes, the cells differentiate into mature
osteoblasts. Without Cbfa 1, the cells would turn into fat cells instead!

Insulin-like growth factors (IGFs): These growth factors are produced by
osteoblastic cells in response to several bone active hormones, such as parathyroid
hormone and estrogens, or BMPs. IGFs accumulate in the bone matrix and are
released during the process of bone remodelling by osteoclasts. IGFs stimulate
osteoblastic cell replication -- in other words, they cause the osteoblasts to divide,
forming new cells. They may also induce differentiation.

Cytokines
Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) family of
cytokines: These factors are produced by osteoblastic cells in response to systemic
hormones or other cytokines. IL-6 can cause:
* Bone marrow stem cells to differentiate into pre-osteoclasts
* Changes in proliferation and differentiation of osteoblasts
* Inhibition of apoptosis of osteoblasts

RANKL (RANK-ligand) is a cytokine that stays on the surface of osteoblast-related
cells. The cells make RANKL in response to systemic hormones (such as
1,25dihydroxyvitamin D3) and cytokines (such as IL-6). Cell contact between
RANKL-expressing osteoblastic cells and RANK-expressing osteoclast precursors
induces osteoclast development, as shown in the animation in the osteoclast
section.


Apoptosis
The mature bone is always remodelling: the old bone is resorbed and replaced with
new bone. A team of osteoblasts and osteoclasts move along the bone, dissolving
and rebuilding. What happens to the cells when they have finished rebuilding an
area of bone? The osteoclasts and most of the osteoblasts undergo a process called
apoptosis, or cell suicide. They are not killed. There is no lack of oxygen or
nutrients. There are no toxic materials. Instead, there are genes in the cell which
can be activated, causing the cell to disintegrate. These genes (of course) are
carefully regulated within the cell. The factors that regulate apoptosis are currently
under investigation. Some are related to estrogens, or to interleukins. Medications
which could modify apoptosis have the potential for treating or preventing
osteoporosis.
            Collagen and Bone Matrix




Mature bone is composed of proteins and minerals. Approximately 60% the weight of the bone is
mineral, mainly calcium and phosphate. The rest is water and matrix, which is formed before the
mineral is deposited, and can be considered the scaffolding for the bone. About 90% of the matrix
proteins are collagen, which is the most abundant protein in the body. Collagen is very strong and
forms bone, cartilage, skin, and tendons.

How you would construct a strong but light material from only the following 3 ingredients: short
threads, plaster of Paris, and glue? The best way would be to twist or braid the thread into strings,
then glue the strings into ropes, then lay the ropes in a pattern and pour the plaster of Paris over
them, the way concrete is poured over steel rebar.

Collagen is formed as chains (short pieces of thread) which twist into triple helices (strings). These
line up and are bonded together into ropes (fibrils). The fibrils then are arranged in layers, and
mineral crystals will deposit between the layers.
Below is a scanning electron image (B) that shows bone collagen fibrils in both longitudinal and cross
sections. The back-scattered electron image (C) shows the regular patterns of collagen in layers in
bone, which is why normal bone is called lamellar bone.
The following table gives information about some of the other bone matrix proteins that seem most
important. For some of these, the gene has been deleted in special populations of mice (called
"knockout mice") to try to understand the function of the protein.



           NAME                                       COMMENTS                              Effect of "knockout"

                            Relatively abundant, may help regulate osteoblast
Fibronectin                                                                                 Lethal
                            differentiation
Osteonectin                 "Bone connector" may regulate mineralization                    Osteoporosis
Thrombospondin              May inhibit bone cell precursors                                Dense bones
Osteocalcin                 Binds calcium                                                   Bones seem normal
                                                                                            Normal bones but
Matrix-gla-protein          Inhibits mineralization                                         calcified blood
                                                                                            vessels



Other matrix proteins


                  SIBLINGS - small integrin-binding ligand, N-linked glycoprotein family
Bone sialoprotein           Binds to integrins, may assist cancer cells                     -
                                                                                            Resistance to PTH
                            Increases angiogenesis (makes new blood vessels) which
Osteopontin                                                                                 and removal of
                            enhances bone resorption in some situations
                                                                                            ovaries
Matrix extracellular
                            May induce a bone disease called osteomalacia                   -
protein

                           Proteoglycans - proteins with many attached sugars

Biglycan                    Function uncertain                                              Osteopenia



              Parathyroid hormone

              Parathyroid Hormone (PTH) is a peptide hormone produced by the parathyroid
              glands. It binds to receptors in the bone and kidney. A decrease in serum calcium
              concentration and an increase in serum phosphorous concentration stimulate PTH
              secretion. PTH also:

              * stimulates osteoclastic bone resorption indirectly to release calcium from bone.
              * stimulates bone formation that is coupled to bone resorption.
              * increases renal tubular reabsorption of calcium.
              * stimulates the renal production of 1,25 dihydroxyvitamin D to increase calcium
              absorption from the intestine.
              * enhances renal phosphate and bicarbonate excretion.
Calcitonin

Calcitonin is a peptide hormone produced by cells within the thyroid gland.
Calcitonin secretion is stimulated by high blood calcium concentrations, and it acts
as a physiologic antagonist to PTH. Osteoclasts have receptors for calcitonin, but
the effects are transient. Calcitonin also:

* inhibits osteoclast resorption
* delays calcium absorption from the intestine
* increases calcium urinary excretion
Vitamin D

1,25 dihydroxyvitamin D is an active hormone which is produced by the kidney,
under the control of PTH, from precursors of dietary vitamin D intake and UV skin-
production of vitamin D. It is not really a vitamin, but the name was given many
years ago, before anybody knew the function of this molecule. Vitamin D receptors
are present in bone, kidney, intestines, and other cells. The chemical name is 1,25-
dihydroxy-cholecalciferol, and it:

* promotes gastrointestinal absorption of calcium and phosphorus.
* is necessary for bone mineralization.
* stimulates bone resorption when given in high doses.
Gonadal steroids

Gonadal steroids are produced by the ovaries and testes and are very important in
maintaining bone balance. They are also important in normal growth and
development and in the development of peak bone mass. The mechanism of action
is unclear but receptors for oestrogen and androgen are found in bone.

Estrogens:

*   are the principal circulating sex steroids in females.
*   are also necessary for bone strength in males.
*   help regulate the rates of bone formation and bone resorption.
*   decrease after menopause, contributing to development of osteoporosis.

Androgens (such as testosterone):

* are necessary for bone




Growth hormone

Growth Hormone (GH) is a growth promoting hormone produced by the pituitary
gland. It is "anabolic", which means it stimulates bone formation. Growth hormone
also:

* stimulates the production of insulin-like growth factor 1 (IGF-1) by the skeleton.
* is important in stimulating longitudinal growth.
* Can increase bone mass when given to adults.
Thyroid hormone

Thyroid hormone is produced by the thyroid gland. Bone cells have receptors for
thyroid. This hormone also:

* is necessary for growth and maturation of the skeleton.
* causes increased osteoclastic bone resorption and osteoporosis when levels are
too high.
Glucocorticoids

Glucocorticoid (also called cortisol) is produced by the adrenal gland. Bone cells
have receptors for glucocorticoid. This hormone is absolutely essential for life, but
excess levels cause multiple deleterious effects on the skeleton. This steroid
hormone:

*   decreases calcium absorption from the intestines.
*   inhibits bone formation.
*   increases bone resorption.
*   increases renal calcium excretion.
*   decreases sex steroid production.




Summary of the effects of hormones on skeletal metabolism

Increase Bone resorption
Parathyroid hormone
Glucocorticoids
Thyroid Hormone
Vitamin D metabolites in high doses

Decrease Bone Resorption
Calcitonin
Gonadal steroids

Increase Bone Formation
Growth hormone
Vitamin D metabolites
Gonadal steroids

Decrease Bone Formation
Glucocorticoids
The D Stitch: An Efficient Method to Facilitate Suture Removal

Harkeerat Dhillon, MD, FRCS, MS (ORTH); Vaneet Randhawa, MD. ORTHOPEDICS    January 09
The “D stitch” method is a simplified and improved method of suture removal that has
shown promising results. In any surgical procedure involving finely placed sutures,
suture removal becomes an intricate and tedious task. The “D stitch” is a means of
facilitating this technique, reducing both the time necessary for suture removal as well
as reducing discomfort to the patient.

Materials and Methods
In the development of the D stitch, a 4-0 nylon mattress suture and 2-0 nylon (D stitch)
suture were typically used. Twenty-five patients were assessed by medical staff at the
time of suture removal. This assessment took into account not only the level of
difficulty in suture removal, but also the anxiety felt by the patient. On a difficulty
scale, with 0/5 being very difficult and 5/5 being very easy, use of the D stitch revealed
11 scores of 5/5, 5 scores of 4/5, and 1 score of 3/5. Standard suture removal, however,
used here as the “control,” yielded 1 case of 0/5, 2 cases of 1/5, and 5 cases of 2/5.




    Figure 1: Application of initial mattress suture (A, B).


Surgical Technique
At the time of wound closure, an initial mattress suture (or continuous,
plain, transverse suture) is placed (Figure 1), in this case, using a 4-0 nylon
suture. Before tying the knot, the D stitch is introduced using a 2-0 nylon
suture that is placed along the incision, taking care that the 2-0 thread is
situated between the two 4-0 threads of the mattress suture (Figure 2A).
The rest of the incision is closed by repeating the above process with each
suture, but care must be taken to ensure that the 2-0 nylon thread, used here
as the D stitch, is positioned adjacent to the incision line between the suture
knots (Figure 2B). On completion of the final suture, both ends of the D
stitch are brought together and tied in a simple knot (Figure 2C), thus
forming a “D” shape (Figure 2C). At the time of suture removal, the D
stitch (Figure 2C) is cut and easily pulled out from beneath the incision-line
sutures, thus leaving a gap between the suture knots and underlying
incision line. This gap facilitates the removal of finely placed sutures by
allowing the tip of the suture cutting scissor blades to be introduced into
this gap.
Figure 2: Introduction of the “D stitch” between the threads of the incision-line suture (A, B), followed by
approximating and knotting of the D stitch and removal of sutures by cutting the D stitch, arrow (C).




     Figure 3: D stitch over a nonlinear incision (A, B).


Principles of Bone Healing

Our contemporary understanding of bone healing has evolved due to knowledge gleaned from a
continuous interaction between basic laboratory investigations and clinical observations
following procedures to augment healing of fractures, osseous defects, and unstable joints. The
stages of bone healing parallel the early stages of bone development. The bone healing
process is greatly influenced by a variety of systemic and local factors. A thorough
understanding of the basic science of bone healing as well as the many factors that can affect it
is critical to the management of a variety of musculoskeletal disorders. In particular, the evolving
management of spinal disorders can greatly benefit from the advancement of our understanding
of the principles of bone healing.

Introduction

Bone is a dynamic biological tissue composed of metabolically active cells that are integrated
into a rigid framework. The healing potential of bone, whether in a fracture or fusion model, is
influenced by a variety of biochemical, biomechanical, cellular, hormonal, and pathological
mechanisms. A continuously occurring state of bone deposition, resorption, and remodelling
facilitates the healing process.

The success of many spine operations depends on the restoration of long-term spinal stability.
Whereas spinal instrumentation devices may provide temporary support, a solid osseous union
must be achieved to provide permanent stability. The failure of fusion to occur may result in the
fatigue and failure of supporting instrumentation and persistence or worsening of symptoms.
Understanding the basic biological and physiological principles of bone transplantation and
healing will aid the spine surgeon in selecting the most effective techniques to achieve
successful fusions. In this paper the anatomical, histological and biological features of this
process will be reviewed.

one Anatomy and Histology

The cellular components of bone consist of osteogenic precursor cells, osteoblasts, osteoclasts,
osteocytes, and the hematopoietic elements of bone marrow.[10,22] Osteoprogenitor cells are
present on all nonresorptive bone surfaces, and they make up the deep layer of the periosteum,
which invests the outer surface of bone, and the endosteum, which lines the internal medullary
surfaces. The periosteum is a tough, vascular layer of connective tissue that covers the bone
but not its articulating surfaces. The thick outer layer, termed the "fibrous layer," consists of
irregular, dense connective tissue. A thinner, poorly defined inner layer called the "osteogenic
layer" is made up of osteogenic cells. The endosteum is a single layer of osteogenic cells
lacking a fibrous component.

Osteoblasts are mature, metabolically active, bone-forming cells. They secrete osteoid, the
unmineralized organic matrix that subsequently undergoes mineralization, giving the bone its
strength and rigidity. As their bone-forming activity nears completion, some osteoblasts are
converted into osteocytes whereas others remain on the periosteal or endosteal surfaces of
bone as lining cells. Osteoblasts also play a role in the activation of bone resorption by
osteoclasts.

Osteocytes are mature osteoblasts trapped within the bone matrix. From each osteocyte a
network of cytoplasmic processes extends through cylindrical canaliculi to blood vessels and
other osteocytes. These cells are involved in the control of extracellular concentration of calcium
and phosphorus, as well as in adaptive remodelling behaviour via cell-to-cell interactions in
response to local environment.

Osteoclasts are multinucleated, bone-resorbing cells controlled by hormonal and cellular
mechanisms. These cells function in groups termed "cutting cones" that attach to bare bone
surfaces and, by releasing hydrolytic enzymes, dissolve the inorganic and organic matrices of
bone and calcified cartilage. This process results in the formation of shallow erosive pits on the
bone surface called Howship lacunae.[12]

There are three primary types of bone: woven bone, cortical bone, and cancellous
bone.[10,22] Woven bone is found during embryonic development, during fracture healing (callus
formation), and in some pathological states such as hyperparathyroidism and Paget
disease.[22] It is composed of randomly arranged collagen bundles and irregularly shaped
vascular spaces lined with osteoblasts. Woven bone is normally remodeled and replaced with
cortical or cancellous bone.

Cortical bone, also called compact or lamellar bone, is remodeled from woven bone by means
of vascular channels that invade the embryonic bone from its periosteal and endosteal surfaces.
It forms the internal and external tables of flat bones and the external surfaces of long bones.
The primary structural unit of cortical bone is an osteon, also known as a haversian system.
Osteons consist of cylindrical shaped lamellar bone that surrounds longitudinally oriented
vascular channels called haversian canals. Horizontally oriented canals (Volkmann canals)
connect adjacent osteons. The mechanical strength of cortical bone depends on the tight
packing of the osteons.

Cancellous bone (trabecular bone) lies between cortical bone surfaces and consists of a
network of honeycombed interstices containing hematopoietic elements and bony trabeculae.
The trabeculae are predominantly oriented perpendicular to external forces to provide structural
support. Cancellous bone is continually undergoing remodelling on the internal endosteal
surfaces.

Bone Biochemistry

Bone is composed of organic and inorganic elements. By weight, bone is approximately 20%
water.[22] The weight of dry bone is made up of inorganic calcium phosphate (65-70% of the
weight) and an organic matrix of fibrous protein and collagen (30-35% of the weight).[10,19,21,22]

Osteoid is the unmineralized organic matrix secreted by osteoblasts. It is composed of 90%
type I collagen and 10% ground substance, which consists of noncollagenous proteins,
glycoproteins, proteoglycans, peptides, carbohydrates, and lipids.[20,22] The mineralization of
osteoid by inorganic mineral salts provides bone with its strength and rigidity.

The inorganic content of bone consists primarily of calcium phosphate and calcium carbonate,
with small quantities of magnesium, fluoride, and sodium. The mineral crystals form
hydroxyapatite, which precipitates in an orderly arrangement around the collagen fibers of the
osteoid. The initial calcification of osteoid typically occurs within a few days of secretion but is
completed over the course of several months.

Regulators of Bone Metabolism

Bone metabolism is under constant regulation by a host of hormonal and local factors. Three of
the calcitropic hormones that most affect bone metabolism are parathyroid hormone, vitamin D,
and calcitonin. Parathyroid hormone increases the flow of calcium into the calcium pool and
maintains the body's extracellular calcium levels at a relatively constant level. Osteoblasts are
the only bone cells that have parathyroid hormone receptors. This hormone can induce
cytoskeletal changes in osteoblasts. Vitamin D stimulates intestinal and renal calcium-binding
proteins and facilitates active calcium transport. Calcitonin is secreted by the parafollicular cells
of the thyroid gland in response to an acutely rising plasma calcium level. Calcitonin serves to
inhibit calcium-dependent cellular metabolic activity.

Bone metabolism is also affected by a series of proteins, or growth factors, released from
platelets, macrophages, and fibroblasts. These proteins cause healing bone to vascularize,
solidify, incorporate, and function mechanically. They can induce mesenchymal-derived cells,
such as monocytes and fibroblasts, to migrate, proliferate, and differentiate into bone cells. The
proteins that enhance bone healing include the BMPs, insulin-like growth factors, transforming
growth factors, platelet derived growth factor, and fibroblast growth factor among others.[18,32]

The most well known of these proteins are the BMPs, a family of glycoproteins derived from
bone matrix. Bone morphogenetic proteins induce mesenchymal cells to differentiate into bone
cells. Although typically present in only minute quantities in the body, several BMPs have been
synthesized using recombinant DNA technology and are currently undergoing clinical trials to
assess their potential to facilitate bone fusion in humans.[26-28]

Other proteins influence bone healing in different ways. Transforming growth factor-regulates
angiogenesis, bone formation, extracellular matrix synthesis, and controls cell-mediated
activities. Osteonectin, fibronectin, osteonectin, and osteocalcin promote cell attachment,
facilitate cell migration, and activate cells.
Physiology of Bone Repair and Fusion

The use of a bone graft for purposes of achieving arthrodesis is affected by each of the
aforementioned anatomical, histological, and biochemical principles. Additionally, several
physiological properties of bone grafts directly affect the success or failure of graft incorporation.
These properties are osteogenesis, osteoinduction, and osteoconduction.[20]

Osteogenesis is the ability of the graft to produce new bone, and this process is dependent on
the presence of live bone cells in the graft. Osteogenic graft materials contain viable cells with
the ability to form bone (osteoprogenitor cells) or the potential to differentiate into bone-forming
cells (inducible osteogenic precursor cells). These cells, which participate in the early stages of
the healing process to unite the graft with the host bone, must be protected during the grafting
procedure to ensure viability. Osteogenesis is a property found only in fresh autogenous bone
and in bone marrow cells, although the authors of radiolabeling studies of graft cells have
shown that very few of these transplanted cells survive.[19]

Osteoconduction is the physical property of the graft to serve as a scaffold for viable bone
healing. Osteoconduction allows for the ingrowth of neovasculature and the infiltration of
osteogenic precursor cells into the graft site. Osteoconductive properties are found in
cancellous autografts and allografts, demineralized bone matrix, hydroxyapatite, collagen, and
calcium phosphate.[19]

Osteoinduction is the ability of graft material to induce stem cells to differentiate into mature
bone cells. This process is typically associated with the presence of bone growth factors within
the graft material or as a supplement to the bone graft. Bone morphogenic proteins and
demineralized bone matrix are the principal osteoinductive materials. To a much lesser degree,
autograft and allograft bone also have some osteoinductive properties.

 Process of bone graft incorporation in a spinal fusion model is similar to the bone healing
process that occurs in fractured long bones.[4] Fracture healing restores the tissue to its original
physical and mechanical properties and is influenced by a variety of systemic and local factors.
Healing occurs in three distinct but overlapping stages: 1) the early inflammatory stage; 2) the
repair stage; and 3) the late remodelling stage.[9,13]

In the inflammatory stage, a hematoma develops within the fracture site during the first few
hours and days. Inflammatory cells (macrophages, monocytes, lymphocytes, and
polymorphonuclear cells) and fibroblasts infiltrate the bone under prostaglandin mediation. This
results in the formation of granulation tissue, ingrowth of vascular tissue, and migration of
mesenchymal cells. The primary nutrient and oxygen supply of this early process is provided by
the exposed cancellous bone and muscle. The use of anti-inflammatory or cytotoxic medication
during this 1st week may alter the inflammatory response and inhibit bone healing.

During the repair stage, fibroblasts begin to lay down a stroma that helps support vascular
ingrowth. It is during this stage that the presence of nicotine in the system can inhibit this
capillary ingrowth.[11,23-25] A significantly decreased union rate had been consistently
demonstrated in tobacco abusers.[2,3,6]

As vascular ingrowth progresses, a collagen matrix is laid down while osteoid is secreted and
subsequently mineralized, which leads to the formation of a soft callus around the repair site. In
terms of resistance to movement, this callus is very weak in the first 4 to 6 weeks of the healing
process and requires adequate protection in the form of bracing or internal fixation. Eventually,
the callus ossifies, forming a bridge of woven bone between the fracture fragments.
Alternatively, if proper immobilization is not used, ossification of the callus may not occur, and
an unstable fibrous union may develop instead.

Fracture healing is completed during the remodelling stage in which the healing bone is
restored to its original shape, structure, and mechanical strength. Remodelling of the bone
occurs slowly over months to years and is facilitated by mechanical stress placed on the bone.
As the fracture site is exposed to an axial loading force, bone is generally laid down where it is
needed and resorbed from where it is not needed. Adequate strength is typically achieved in 3
to 6 months.

Although the physiological stages of bone repair in the spinal fusion model are similar to those
that occur in long bone fractures, there are some differences. Unlike long bone fractures, bone
grafts are used in spinal fusion procedures. During the spinal fusion healing process, bone
grafts are incorporated by an integrated process in which old necrotic bone is slowly resorbed
and simultaneously replaced with new viable bone. This incorporation process is termed
"creeping substitution."[17,20] Primitive mesenchymal cells differentiate into osteoblasts that
deposit osteoid around cores of necrotic bone. This process of bone deposition and remodelling
eventually results in the replacement of necrotic bone within the graft.

The most critical period of bone healing is the first 1 to 2 weeks in which inflammation and
revascularization occur. The incorporation and remodelling of a bone graft require that
mesenchymal cells have vascular access to the graft to differentiate into osteoblasts and
osteoclasts. A variety of systemic factors can inhibit bone healing, including cigarette smoking,
malnutrition, diabetes, rheumatoid arthritis, and osteoporosis. In particular, during the 1st week
of bone healing, steroid medications, cytotoxic agents, and nonsteroidal anti-inflammatory
medications can have harmful effects. Irradiation of the fusion site within the first 2 to 3 weeks
can inhibit cell proliferation and induce an acute vasculitis that significantly compromises bone
healing (SE Emery, unpublished data).

Bone grafts are also strongly influenced by local mechanical forces during the remodelling
stage. The density, geometry, thickness, and trabecular orientation of bone can change
depending on the mechanical demands of the graft. In 1892, Wolff first popularized the concept
of structural adaptation of bone, noting that bone placed under compressive or tensile stress is
remodeled. Bone is formed where stresses require its presence and resorbed where stresses
do not require it.[22,31] This serves to optimize the structural strength of the graft. Conversely, if
the graft is significantly shielded from mechanical stresses, as in the case of rigid spinal
implants, excessive bone resorption can potentially occur and result in a weakening of the graft.
This potential disadvantage of instrumentation needs to be balanced with the beneficial effects
that spinal fixation has on the fusion process.

Bone Grafts

The two types of bone grafts frequently used in spinal fusion are autografts and allografts.
Autograft bone is transplanted from another part of the recipient's body. Allograft bone is
transplanted from genetically nonidentical members of the same species. Both types of bone
grafts are commonly used in spine surgery.

The ideal bone graft should be: 1) osteoinductive and conductive; 2) biomechanically stable; 3)
disease free; and 4) contain minimal antigenic factors. These features are all present with
autograft bone. The disadvantages of autografts include the need for a separate incision for
harvesting, increased operating time and blood loss, the risk of donor-site complications, and
the frequent insufficient quantity of bone graft. The advantage of allograft bone is that it avoids
the morbidity associated with donor-site complications and is readily available in the desired
configuration and quantity. The disadvantages of allograft include delayed vascular penetration,
slow bone formation, accelerated bone resorption, and delayed or incomplete graft
incorporation. In general, allograft bone has a higher incidence of non-union or delayed union
than autograft. Allografts are osteoconductive but are only weakly osteoinductive. Although
transmission of infection and lack of histocompatibility are potential problems with allograft
bone, improved tissue-banking standards have greatly reduced their incidence.

Bone grafts can also be classified according to their structural anatomy: cortical or cancellous.
Cortical bone has fewer osteoblasts and osteocytes, less surface area per unit weight, and
contributes a barrier to vascular ingrowth and remodelling compared with cancellous bone. The
advantage of cortical bone is its superior structural strength.

The initial remodelling response to cortical bone is resorptive as osteoclastic activity
predominates. Cortical grafts progressively weaken with time because of this bone resorption as
well as slow, incomplete remodelling. Conversely, cancellous bone becomes progressively
stronger because of its ability to induce early, rapid, new bone formation.

When selecting a bone graft, the spine surgeon needs to consider the specific structural and
biological demands that will be placed on the graft. If the graft is placed anteriorly in a
compressive mode, cortical bone, either autogenic or allogenic, will be required. If placed
posteriorly as a graft under tension with lower demands for structural support but also a lower
probability of early vascular ingrowth, a cancellous autograft is preferred.

Conclusions

An understanding of the basic science of bone healing is critical to the consistent success of
spinal fusion surgery. Although great advances have been made in the field of spinal
instrumentation, it is only a solid osseous union that will ensure long-term spinal stability.
Selection of the most appropriate bone graft material as well as careful attention to the
principles of bone healing can greatly facilitate the potential for clinical success.

								
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