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Reza Omid, MD
Basic Science Update
Molecular Biology

Bone Morphogenetic Proteins
o Subfamily of TGF-β superfamily of growth factors
o BMP-2 through BMP-18 (BMP-1 is not a true BMP)
o BMP-1 is a metalloprotease
o BMP-2 is a potent stimulator of bone formation and strongly induces
chondrogenic differentiation of mesenchymal cells
o BMP-3 is an antagonist to other BMP
o BMP-5, BMP-6 and BMP-7 are effective osteoinductive agents
o BMP-6 and BMP-7 are located in hypertrophic cartilage and promote
endochondral calcification pathway.
o BMP-12 involved in tenodon and ligament healing
o BMP is also critically involved in apoptosis (web space development)
o BMP-2 approved for open tibia fracture
o BMP-7 (aka OP1) approved for tibia non-union
o Bind cell surface protein complexes (receptors) composed of type 1 and 2
which are serinethreonine kinases
o Triggers SMAD proteins and activation of other kinases (Erk)
o Events culminate in cell nuclei where Runx2 is activated

Transforming Growth Factor-β
o Produced by many cells including osteoblasts, chondrocytes and tumor
cells and stimulates proliferation of cells of mesenchymal origin except for
normal epithelial cells
o Binding leads to activation of SMADs.
o Implicated in metastatic bone cancer, carpal tunnel syn and arthritis.
o In cancer, TGF- β stimulates cancer cell growth, angiogenesis and inhibits
host immune response.
o In Marfan’s syndrome, involved in pathogenesis of aortic dilatation

Parathyroid Hormone & Parathyroid Hormone Related Peptide (PTHrP)
o PTH consists of 84 amino acids (PTH1-84)
o PTHrP is 34 amino acids and can activate PTH receptors as well
o PTH causes bone resorption by binding to osteoblasts which then activate
osteoclasts via RANKL.
o PTH acts on kidneys to promote calcium reabsorption.
o When PTHrP is given subcutaneously and once a day has an anabolic
effect on bone but can only be given for 2 years because associated with
sarcoma formation.
o Activation of PTH receptors results in activation of protein kinase A,
phospholipase C and protein kinase C.
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
Indian Hedgehog
o Critical factor for chondrocyte and osteoblast differentiation during
prenatal bone formation.
o Secreted from prehypertrophic chondrocytes (within the proliferative
zone) and promotes chondrocyte proliferation and differentiation into
hypertrophic chondrocytes.
o Interacts with perichondral cells that subsequently produce PTHrP. PTHrP
then provides a negative feedback signal to prehypertrophic chondrocytes
undergoing hypertrophic differentiation and maintain a pool of
proliferating chondrocytes in the growth plate during skeletal immaturity.
o Mutations of Ihh associated with brachydactyly.
o Normal articular cartilage does not have active Ihh signaling.
o Important in endochondral bone formation.
o Osteophytes are formed during pathologic endochondral ossification and
so Ihh may be used as a diagnostic marker of OA. Ihh blockers may also
be a potential target for OA.

Insulin Like Growth Factor
o IGF-1 deficiency is associated with dwarfism because of the ineffective
relaying of signals between growth hormone and growth plates.
o Aberrant activation of IGF signaling is seen in osteosarcomas.
Fibroblast Growth Factor
o Mutations in FGF-1 & FGF-2 associated with craniosynostosis
o FGFR2 mutation associated with Aperts syndrome
o FGFR3 signaling normally suppresses chondrocyte proliferation.
o Gain of function mutation of FGFR3 causes achondroplasia
o FGF-23 involved in tumor induced osteomalacia (FGF-23 inhibits
phosphate reasborption in proximal tubules and inhibits 1α-hydroxylase,
resulting in decreased 1,25-dihydroxyvitamin D3 levels).


Vascular Endothelial Growth Factor
o Stimulates angiogenesis, which is important in endochondral bone
formation and bone regeneration.
o Produced by endothelial cells, bone marrow stromal cells and osteoblasts.

Low Density Lipoprotein Receptor-Related Protein-5 and Wnts
o LRP5 is linked to Wnt signaling
o LRP5 is a coreceptor of the Wnt signaling pathways
o Gain of function mutations in LRP5 results in high bone mass and strong
bones resistant to fractures.
o Loss of function mutations in LRP5 results in osteoporosis-pseudoglioma
syndrome.
o Wnt signaling positively regulates bone mass by promoting the
differentiation of mesenchymal stem cells into osteoblasts. Wnt pathway
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Reza Omid, MD
activation is essential for peak bone mass acquisition during development.
Wnt is also reactivated during fracture repair.
Wnt inhibits osteoclastogenesis by increasing osteoprotegerin (OPG)
expression in osteoblasts (which is a decoy receptor for RANKL).
Wnt signaling is inhibited by sclerostin secreted by osteocytes.
Wnt (mammalian homologue of wingless in drosophila) is very similar to
BMP but uses a different signaling pathway.
Wnt binds to receptors frizzled and LDLR5 and LDLR6 and activates βcatenin which go to the nucleus to regulate gene expression:
 Canonical Pathway: prevents degradation of β-catenin.
 Suppresses bone resportion by upregulating osteoprotegerin
 Noncanonical Pathway: does not involve β-catenin. Involves
mitogen-activated protein kinase (MAPK).
Sclerostin (Scl) and Dickkopf related peptide (DKK-1) are antagonists of
Wnt.
Multiple myeloma suppresses bone formation by overproduction of DKK-1
Neutralizing antibodies to Scl and Dkk-1 in primate studies stimulates
fracture healing.

Sclerostin
o Predominantly secreted by osteocytes
o Negative regulator of osteoblastic bone formation.
o Sclerostin secretion is increased by PTH and decreased by calcitonin.
o Sclerostin (Scl) and Dickkopf related peptide (DKK-1) are antagonists of
Wnt.

Tumor Necrosis Factor
o Pro-inflammatory cytokine
o Regulates osteoclast differentiation and bone resorption
o Works synergistically with RANKL to stimulate osteoclastogenesis
o Can stimulate osteoclast differentiation in the presence of macrophage
colony-stimulating factor (M-CSF) independent of RANKL.
o DMARD meds that block TNF-α:
 Adalimumab (monoclonal antibody that binds TNF-α)
 Infliximab (chimeric human-murine monoclonal antibody)
 Entanercept (recombinant fusion protein that competes with
receptor)

Interleukins
o Interleukin-1 is proinflammatory and promotes gene expression in
chondrocytes and induces bone resorption. IL-1 enhances TNF-induces
osteoclastogenesis in bone marrow stromal cells and the formation of
multinucleated osteoclasts. IL-1 implicated in pathologic conditions such
osteoporosis and inflammatory joint disease.
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o Interleukin-6 is proinflammatory and important for B cell maturation. IL-6
is produced by osteoblastic cells, bone marrow stromal cells and synovial
cells that have been stimulated with IL-1 and TNF. IL-6 activates
osteoclasts indirectly.
o Interleukin-10 opposes pro-inflammatory cytokines and stimulates
humoral and cytotoxic immune responses. IL-10 attenuates localized
inflammation, such as in RA and infections of the bone.
o Interleukin-17 is proinflammatory and inhibitors for RA are being tested.
o DMARD medications that block interleukins:
 Anakinra (binds IL-1 receptors)
 Canakinumab (monoclonal IgG against IL-1β)
 Tocilizumab (monoclonal Ab against IL-6)

Interferon-γ
o Produced by T-cells and NK cells and responsible for cell-mediated
immunity.
o Regulates osteoclast function and formation

Receptor Activator of Nuclear Factor-κB Ligand
o RANKL belongs to TNF superfamily and is produced by osteoblasts
o Potent stimulator of osteoclast formation and differentiation
o Mice lacking RANKL expression have severe osteopetrosis
o RANKL binds to receptors on monocyte/macrophage which leads to
osteoclast precursor cell fusion.
o RANKL is a target for osteoporosis meds and for giant cell tumors.

Macrophage Colony-Stimulating Factor
o M-CSF modulates macrophage formation and osteoclast formation
o M-CSF enhances RANK expression.
o TNF can stimulate osteoclast differentiation in the presence of M-CSF
independent of RANKL.

Osteoprotegerin
o RANKL decoy receptor
o Blocks osteoclast formation and hence osteolysis
o OPG knockout mice are severely osteoporotic and OPG overexpression
causes osteopetrosis.

Runt-Related Transcription Factor 2
o Runx-2 is a key transcription factor for osteoblast differentiation
o Directs the differentiation of mesenchymal cells to the osteoblast lineage,
leading to formation of immature osteoblasts.
o Controls expression of major osteoblast genes (osteopotonin, osteocalcin,
type 1 collagen α-chain.
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
Osterix
o Osx is a zinc finger transcription factor that is expressed exclusively in
osteoblasts and acts downstream of Runx2 during osteoblast
differentiation.
o Osx is required for the differentiation of preosteoblasts to mature
osteoblasts.
o Osx inhibits Wnt signaling during bone formation and forms a
transcriptional complex with NFATc1 (Nuclear factor of activated T cells
c1, see below).

Twist 1
o Belongs to basic helix-loop-helix transcription factors
o Potent inhibitor of osteoblast differentiation, possibly through repressive
effects on Runx2 expression and activity.
o Possible role in etiology of osteoarthritis.

Muscle Segment Homeobox
o Msx2 is a transcription factor involved in osteoblast differentiation.

Distal-less Homeobox
o Dlx5 & Dlx6 transcription factors are expressed during early stages of
osteoblast differentiation.

Sex-Determining Region Y Box-9
o SOX-9 is a transcription factor that promotes chondrocytic differentiation
o Enhacing expression of essential elements of chondrocytic phenotypes
such as type II collage.

c-Fos
o Member of the activator protein-1 (AP-1) family of transcription factors
o Key regulator of osteoclast differentiation and bone remodeling

Nascent Polypeptide Associated Complex and Coactivator alpha
o aNAC is a transcription factor that is activated by the phosphorylation
activity of integrin-linked kinase.
o aNAC is present in differentiated osteoblasts and acts in combination with
AP-1 to potentiate osteocalcin gene transcription.

Myogenic Regulator Factor
o MRF is a basic helix-loop-helix transcription factor that regulates
expression of genes involved in skeletal myogenesis.
o Increases the expression of myogenin (MYOG).
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
Myocyte Enhancer Factor 2
o MEF2 is involved in skeletal myogenesis by increasing the expression of
myogenin (MYOG).
o Acts synergistically with MRF to increase myogenin expression

Nuclear Factor of Activated T cells c1
o NFATc1 is crucial transcription factor for RANKL mediated
osteoclastogenesis.
o Regulates osteoclast specific genes such as TRAP & cathepsinK.

Nuclear Factor Kappa-Light Chain Enhancer of Activated B Cells
o NF-κB is a group of transcription factors implicated in immune response
and osteoclast differentiation.
o NF-κB is activated by RANKL in the early stages of osteoclast formation
and is important in the differentiation and survival of this cell.
o Activation of NF-κB occurs by two pathways:
 Classic pathway: inhibitor of κB (IκB) kinase (IKK) complex
degrades IκB, leading to activation of NF-κB.
 Alternative pathway: NF-κB-induced kinase (NIK) and IKKα mediate
the processing of p100 to generate p52. This leads to activation of
NFκB.

Peroxisome Proliferator-Activated Receptor γ
o PPARγ is important for glucose metabolism & adipogenesis in the bone
marrow.
o Activation of PPARγ leads to differentiation of mesenchymal progenitor
cells towards the adipogenic lineage.

C/EBP
o In addition to PPARγ, the CCAAT-enhancer binding proteins (C/EBP
transcription factor family) regulate differentiation of mesenchymal
progenitor cells into adipocytes.
o C/EBPγ & C/EBPβ are expressed early in the adipogenic process
o C/EBPα is expressed later
o C/EBPα maintains the expression of PPARγ and other adipocyte proteins.

Scleraxis
o Scx is a basic helix-loop-helix transcription factor that regulates tendon
and ligament formation.
o Regulates transcription of aggrecan and collagen genes.
o A commonly used marker for tendon and ligament progenitors.
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
Dentin Matrix Protein-1
o Member of SIBLING (small integrin binding ligand N-linked glycoprotein)
family of proteins (other members include osteopontin, bone sialoprotein
and matrix extracellular phosphoglycoprotein).
o Crucial for proper mineralization of bones
o Important in osteocyte maturation and phosphate metabolism (mutations
cause hypophosphatemia)

Platelet-Rich Plasma
o PRP is platelet enriched fluid portion of blood that is separate from RBCs.
o Plasma consists of 55% whole blood and contains water, electrolytes,
proteins and hormones. Cellular component of blood (45% of the
volume) contains RBCs, neutrophils and platelets.
o Platelets and neutrophils form the “buffy coat layer”.
o Platelets have two types of granules. The alpha granules contain
numerous growth factors and cytokines (PDGF, TGF, IGF, VEGF, IL-8).
o PRP is contains roughly 5-10 times the platelet concentration of normal
blood.
o Numerous preparations are found including with or without neutrophils,
with or without activators. Optimal preparation is unknown.

Mesenchymal Stem Cells
o MSC are adult somatic stem cells that can differentiate into chondrocytes,
osteoblasts, fibroblasts, tenocytes and adipocytes.
o Have the ability to secrete bioactive factors (VEGF, bFGF) that are
important in establishing a regenerative microenvironment after injury.
o MSC can be isolated from virtually any tissue, including deciduous teeth,
periosteum, bone marrow, adipose tissue, skin and peripheral blood.
o Numbers of MSC declines with age.
o STRO1 is a marker specific to MSC.

Adult Somatic Stem Cells
o Undifferentiated cells in the body that are capable of self-renewal and
multipotency.
o They can divide indefinitely without differentiation and also capable of
generating cells from various cell types.
o There are mesenchymal stem cells (MSC), hematopoetic stem cells and
neural stem cells.
o Tumorigenic risk is low with adult stem cells, however there is only a
limited quantity of cells that can be obtained.
o Adult stem cells can generate cell types specific to the tissue in which they
reside and therefore have a restricted differentiation capacity.
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
Embryonic Stem Cells
o ES cells are pluripotent, capable of self-renewal, and harvested from the
inner cell mass (ICM) of a blastocyst.
o Pluripotency is the ability to differentiate into any of the three germ
layers: mesoderm, endoderm or ectoderm. This plasticity is unlike adult
stem cells, which generally are only multipotent within the lineage.
o Under the right conditions, ES cells can replicate and stay
undifferentiated, thereby propagating more stem cells.
o ES cells may suffer graft-versus host response and could lead to teratoma
formation.
o Sox-2 is an essential factor in self-renewal in undifferentiated ES cells.
Other essential genes are Oct3/4, c-Myc and Klf4.
o Nanog is a transcription factor that promotes pluripotency of ES cells

Fetal Stem Cells
o Stem cells isolated from the organs of aborted fetuses, umbilical cord
blood, or amniotic fluid.
o Can grow faster and senesce later than adult stem cells and have
differentiation capacity similar to embryonic stem cells.
o May suffer graft-versus host response

Protein Induced Pluripotent Stem Cells (pIPS) & iPS Cells
o Protein-induced pluripotent stem cells (pIPS) and iPS are artificially
derived from the pluripotent stem cells.
o Differentiated adult cells are taken from a host and induced into stem cells
by viral transfection of stem cell-associated genes such as Sox-2, Oct3/4,
c-Myc and Klf4.

Somatic Cell Nuclear Transfer
o SCNT is used for the creation of totipotent cells that can be used in
therapeutic cloning.
o The nucleus from a desired somatic cell type is extracted intact and placed
into a denucleated egg cell. After electrical shock, the denucleated ovum
and somatic cell nucleus fuse, at which point the host egg cell reprograms
the nucleus and starts to divide.
o Dolly was cloned this way.
Science of Bones
Bone Histology

Lamellar Bone: normal bone, stress oriented
o
Less osteocytes and turnover than woven bone
o
2 subtypes of lamellar bone:
 Cortical: aka compact bone
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makes up 80% of skeleton

mainly diaphyseal

composed of tightly packed osteons or haversian systems
connected by haversian (or volkman) canals

slow turnover

high Youngs module (E)
 Cancellous: aka spongy bone or trabecular bone

mainly metaphyseal/epiphyseal, vertebral body, calcaneus

less dense and more remodeling according to lines of stress

high turnover

more elastic and smaller Youngs module (E)
*Cement lines are found in cortical bone indicating where bone resorption has
stopped and new bone formation has begun. Cement lines define the outer border
of an osteon


Woven Bone: immature/pathologic bone, NOT stress oriented
o
More osteocytes and turnover than lamellar bone
o
Weaker and more flexible than lamellar bone
o
Immature bone found in embryonic skeleton & fracture callus
o
Pathologic bone found in osteogenic sarcoma and fibrous dysplasia
Cell Types Found in Bone
1. Osteoblasts: form bone
a. Derived from undifferentiated mesenchymal cells (osteoprogenitor cells)
found in periosteum, endosteum and haversian canals
b. Produce bone so produce:
i. alkaline phosphatase
ii. type 1 collagen
iii. osteocalcin
c. more Golgi, ER and mitochondria than other cells
d. 2 subtypes:
i. more differentiated, activated cells that line bone surfaces
ii. less active “resting” cells which maintain the ionic mileu of bone.
Disruption of the lining cell layer activated these cells
e. Respond to several factors:
i. PTH (releases RANKL to stimulate osteoclastic activity)
ii. Vitamin D (stimulates matrix and alkaline phosphatase synthesis
and production of bone-specific proteins such as osteocalcin
which then upregulates osteoblasts)
iii. Glucocorticoids (decreases bone production)
iv. Prostaglandins (increase cAMP to cause increased resorption)
v. Estrogen (anabolic and anticatabolic to increase bone production)
vi. PDGF
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Cbfα-1:RUNX2 is the main activator of osteoblasts. Cbfα1:RUNX2 is also critical during the process of chondrocyte
hypertrophy and terminal differentiation and mice without Cbfα1:RUNX2 have an absence of hypertrophic chondrocytes in some
growth plates
1. Mutations of Cbfα-1:RUNX2 result in cleidocranial
dysplasia (abnormal/absent clavicles with face)
f. Certain antiseptic agents are toxic to cultured osteoblasts including:
i. Hydrogen peroxide
ii. Betadine
iii. Bacitracin is less toxic
2. Osteoclasts: resorb bone
a. Multinucleated irregularly shaped giant cells from hematopoetic tissues
(monocyte progenitors form giant cells by fusion)
b. Possess ruffled “brush” borders (which are plasma membrane
enfoldings that increase surface area and are important for bone
resorption) and surrounding clear zone
c. RANK ligand (from osteoblasts) binds to osteoclasts on RANK to increase
bone resorption. This mechanism is inhibited by osteoprotegerin
binding to RANK-L and preventing interaction with osteoclasts and induces
apoptosis.
d. Synthesize TRAP (tartrate-resistant acid phosphate)
e. Bind to bone via integrin (vitronectin) and produce H+ ions (via carbonic
anhydrase) to decrease the pH and increase solubility of hydroxyapatite
crystals [Ca10(PO4)6(OH)2] and organic matrix is removed by digestion.
f. Have calcitonin receptors but NOT PTH receptors
g. Responsible for bone resorption in metastasis and multiple myeloma and
total joint osteolysis.
h. IL-1 is a potent stimulator of osteoclastic bone resportion and has been
found in the membranes surrounding loose total joint implants.
i. IL-10 suppresses osteoclast formation
j. Bisphosphonates inhibit osteoclast resportion by preventing ruffed borders
from forming and inducing apoptosis
3. Osteocytes: maintain bone
a. 90% of cells found in mature bone
b. are former osteoblasts trapped in newly formed matrix (which they help
preserve)
c. high nucleus to cytoplasm ration with long interconnecting cytoplasmic
processes
d. important for control of extracellular calcium and phosphate concentration
e. directly stimulated by calcitonin
f. inhibited by PTH
4. Osteoprogenitor cells: become osteoblasts
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Line Haversian canals, endosteum and periosteum
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Await stimulus to differentiate into osteoblasts
5. Lining cells: narrow flattened cells that form an envelope around bone

Wnt-β catenin Pathway
-Wnt (mammalian homologue of wingless in drosophila) is very similar to BMP (essential
for osteogenesis) but uses a different signaling pathway
-Wnt binds to receptors frizzled and LDLR5 and LDLR6 and activates β catenin
which go to the nucleus to regulate gene expression
-Sclerostin and Dickkopf (DKK-1) are antagonists of Wnt
-BMP uses smad to cause gene expression
Bone Matrix
-Composed of organic (40%) and inorganic component (60%)
-Organic component is composed of:
1. primarily type I collagen
2. proteoglycans (provides compressive strength)
3. non-collagenous matrix proteins (osteocalcin, osteonectin, osteopontin)
4. growth factors and cytokines
-Inorganic component is composed of:
1. calcium hydroxyapatite (provides main compressive strength)
2. osteocalcium phosphate (brushite)
-Collagen is layed down in an arrangement that forms hole zones (which are gaps
between two consecutive fibrils and pores which are gaps between two side to side
fibrils. Mineral deposition (calcification) occurs within the hole zones and
pores. Cross linking decreases collagen solubility and increases its tensile strength.
-Osteocalcin is the most abundant non-collagenous matrix protein. It is inhibited by
PTH and stimulated by vitamin D. Osteocalcin levels can be measured in the serum or
urine as a marker of bone turnover. Increased levels of osteocalcin can be seen in
Paget’s disesase, Renal osteodystrophy, Hyper-PTH
-Osteonectin (aka SPARC) is secreted by platelets and osteoblasts and regulates
calcium and organizing the mineral in bone matrix
-Osteopontin is a cell binding protein (similar role to integrins)
Bone Physiology
-Wolff’s Law: bone remodels in response to mechanical stress. Increased stress
causes increased bone gain and less stress results in loss of bone (disuse osteopenia).
Bone loss seen with paraplegia/quadriplegia occurs throughout the skeleton (except the
skull) for about 16 months and then levels off at 2/3 of the original bone mass.
-Bone remodels in response to electrical changes. Compressive side is electronegative
and stimulates osteoblasts. Tension side is electropositive and stimulates osteoclasts.
-Hueter-Volkmann Law: bone remodeling occurs in small packets of cells known as
basic multicellular units (BMU), modulated by systemic hormones and local cytokines.
Mechanical factors can influence longitudinal growth, bone remodeling and fracture
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repair. Compressive forces inhibit growth and tensile forces stimulate growth. This law
is seen in the progression of scoliosis and Blount’s disease.
Bone Remodeling
-Cortical bone remodeling occurs by osteoclastic tunneling (cutting cones), followed by
layering of osteoblasts and successive deposition of layers of lamellae (after the cement
line has been laid down) until the tunnel size has narrowed to the diameter of the
osteonal central canal. The head of the cutting cone is made up of osteoclasts, which
bore holes through hard cortical bone. Behind osteoclasts are capillaries, followed by
osteoblasts that lay down osteoid to fill the resorption cavity.
-Cancellous bone remodeling occurs by osteoclastic resorption, followed by osteoblasts
laying down new bone.
Bone Blood Supply
-Bone receives 5-10% of cardiac output.
-Long bones receive blood from 3 sources:
1. Nutrient artery: gives rise to the medullary blood supply and supplies
the inner 2/3. This supply is disrupted by fracture and reaming
a. High pressure system
2. Metaphyseal-Epiphyseal system: arises from periarticular vascular
plexus (ex: genicular arteries)
3. Periosteal system: supplies the outer 1/3
a. Low pressure system
-Blood flow is normally centrifugal (inside to outside) in adults due to high pressure of
the nutrient artery and low pressure of the periosteal system. In a completely displaced
fracture with disruption of the endosteal (nutrient) system, the periosteum
predominates and flow becomes centripetal (outside to inside). Blood flow in immature
bones is centripetal due to the very well developed periosteum.
-Venous flow in mature bones is centripetal (outside to inside) from sinusoids to
emissary veins
-Hypoxia, hypercapnia, and sympathectomy increases blood flow to the bone
-Regional acceleratory phenomenon is the response of blood flow to a fracture and
is initially decreased due to vascular disruption at the fracture site and within hours to
days, blood flow increases and peaks at 2 weeks and returns to normal between 3-5
months.
Enchondral Ossification
-Bone replaces a cartilage model (but cartilage is NOT converted to bone)
1. Undifferentiated cells secrete a cartilaginous matrix and differentiates into
chondrocytes
2. Matrix mineralizes and is invades by vascular buds that bring osteoprogenitor
cells into the matrix
3. Osteoclasts resorb calcified cartilage and the osteoblasts form bone
-Examples of enchondral ossification:
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Embronic long bones

Longitudinal growth (physis)

Fracture callus (relative stability)

Bone formed via use of BDM
-Achondroplasia is a disease of abnormal enchondral ossification

Intramembranous Ossification
-Bone formation without a cartilage model
1. Undifferentiated mesenchymal cells aggregate into layers (or membranes)
2. These cells differentiate into osteoblasts and deposite organic matrix that
mineralizes to form bone
-Examples of intramembranous ossification:

Embryonic flat bone formation (pelvis, clavicle, vault of skull)

Bone formation during distraction osteogenesis

Blastema bone (occurs in young children with amputations)

Bone formed with absolute stability
-Cleidocranial dystosis is a disease of abnormal intramembranous ossification
Appositional Ossification
-Osteoblasts align themselves on existing bone surface and lay down new bone
-Examples of appositional ossification:

Periosteal bone enlargement (width)

Bone formation phase of bone remodeling
-Diseases of abnormal appositional ossification:

Melorheostosis

Paget’s disease

Caffey’s disease (infantile hyperostosis)
Physeal Zones
1. Epiphysis
2. Reserve zone: cells store lipids, glycogen and PG aggregates for later growth and
matrix production
-Decreased oxygen tension
o
Affected by lysosomal storage diseases (Gaucher),
Pseudoachondroplasia, Kneist syndrome, Gaucher disease,
Diastrophic dysplasia (Reserve Place Kicker has Good Distance)
3. Proliferation zone: longitudinal growth occurs with stacking of chodrocytes (top
cell is the dividing “mother” cell)
-Increased oxygen tension
-Increased PG in surrounding matrix inhibiting calcification
-Cellular proliferation and matrix production occurs in this zone
o
Affected by Gigantism and Achondroplasia (GPA)
4. Hypertrophic zone: chondrocytes increase 5 times in size and accumulate calcium
in their mitochondria and then die to release calcium.
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-Decreased oxygen tension
-rate of chondrocyted maturation is regulated by systemic hormones and local
growth factors. PTHrP inhibits chondrocyte maturation. Indian hedgehog is
produced by growth plate chondrovytes and regulates expression of PTHrP
-Composed of 3 sub-zones:
 Maturation zone
 Degenerative zone
 Zone of provisional calcification
-This zone widens in Rickets
-Encondromas originate here
o
Affected by Rickets, SCFE, Mucopolysaccaridoses, Osteomalacia,
Rickets, Encondroma, SCFE, Physeal fractures (MORE Sex Please)
Metaphysis:
 Primary spongiosa
 Secondary spongiosa
o
Affected by SCFE with endocrinopathy
Diaphysis
Bone Injury
-Fracture healing is a continuum proceeding from inflammation through repair (soft
callus followed by hard callus) and ending in remodeling
-Most important factor in bone healing is blood supply
-Head injury can increase the osteogenic response to fracture (HO)
-Nicotine increases time to fracture healing and increases the risk of non-union (tibia)
and decreases strength of the facture callus. Also it increases the risk of
pseudoarthrosis after a lumbar fusion by up to 500%
-NSAIDs also adversely affect fracture repair and lumbar spine fusions
-Inflammation phase starts with bleeding from the fracture site and surrounding soft
tissue creating a hematoma which provides a source of hematopoietic cells capable of
secreting growth factors. Fibroblasts, mesenchymal cells and osteoprogenitor cells are
present at the fracture site. Granulation tissue forms at the fracture ends
-Repair phase is when the primary (soft) callus forms at about 2 weeks and at this
stage shortening does not occur but angular deformity can still occur. During callus
formation, type 2 collagen is expressed early followed by type 1 collagen.
-Remodeling phase begins during the middle of the repair phase and continues long
after the fracture has clinically healed (up to 7 years). Fracture healing is complete
when there is repopulation of the marrow space.
-Cortisone decreases callus proliferation
-Thyroid and PTH increase bone remodeling
-Growth hormone increases callus volume
-Low-intensity pulsed ultrasound accelerates fracture healing and increases the
mechanical strength of callus, including torque and stiffness. The proposed mechanism
is that cells responsible for fracture healing respond favorably to the mechanical energy
transmitted by the ultrasound signal.
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Allograft
-Fresh allograft is most immunogenic and most osteoinductive
-Fresh-Frozen is less immunogenic but still preserves BMP
-Freeze-Dried allograft is least immunogenic but looses structural integrity and
depletes BMP. Purely osteoconductive (crutons)
Distraction Osteogenesis
-Use of distraction to stimulate formation of bone. Under optimal stable conditions,
bone forms via intramembranous ossification (unstable conditions form enchondral
ossification or even pseudoarthrosis).
-Histologic phases:
1. Latency phase: 5-7 days
2. Distraction phase: 1mm/day (divided into 0.25mm per 6 hours) or 1 inch per
month.
3. Consolidation phase: twice as long as the distraction phase
-Uses for distaction osteogenesis:
o Limb lengthening
o Hypertrophic non-union
o Deformity correction via differential lengthening (Taylor spatial frame for
Blounts disease)
o Segmental bone loss via bone transport
-Conditions that promote optimal bone formation during distraction osteogenesis:
o Low energy corticotomy/osteotomy (corticotomy is better because leaves
medullary canal intact)
o Minimal soft tissue stripping at corticotomy site
o Stable ex-fix construct
o Latency period of 5-7 days (no lengthening done during this time)
o Distraction done at 0.25mm/6 hours
o Neutral fixation interval (no distraction done during consolidation phase)
o Normal physiologic use of the extremity including weight bearing
Bone Metabolism
-Bone serves as a reservoir for >99% of the body’s calcium. Plasma calcium (<1%) is
about equally free and bound (to albumin). Calcium is absorbed in the duodenum by
active transport (requires ATP, calcium binding proteins and Vitamin D3) and passive
diffusion in the jejunum. The kidney reabsorbes 98% of the calcium (60% in the
proximal tubule). The primary homeostatic regulator of serum calcium are PTH and
vitamin D3. Most people have a positive calcium balance during the first 3 decades and
a negative balance during their 4th decade and after that. 400 mg of calcium is
released from bone daily.
-Dietary requirement of elementary calcium is:
o Children <10: 600 mg/d
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o Age 10-25: 1300 mg/d
o Age 25-65: 750 mg/d
o Pregnancy: 1500 mg/d
o Lactating: 2000 mg/d
o Fractures: 1500 mg/d
o Post-menopausal: 1500 mg/d
o Elderly >65: 1200 mg/d
-8oz of milk has 250mg of elementary calcium
-Phosphate is 85% in the bone and is mostly unbound in the serum. Resorbed by the
proximal tubules. Dietary intake of phosphate is usually adequate (100-1500 mg/d)
-Parathyroid hormone is a peptide secreted from the chief cells of the parathyroid
glands. PTH directly activates osteoblasts and modulates renal phosphate filtration.
Decreased calcium levels in the extracellular fluid stimulates β2 cells to release PTH.
PTH acts on the intestines, kidney and bone and causes bone resportion to increase
serum calcium levels.
-Vitamin D is a naturally occurring steroid activated by UV sunlight and from dietary
intake. Vitamin D helps actively absorb calcium from the duodenum and also at the
kidney. Phenytoin (as well as most other anticonvulsant meds) causes impaired
metabolism of vitamin D. Primary function of vitamin D is to increase the calcium and
phosphate absorption by the intestines. Daily intake of vitamin D should be 800 mg/d.
-Vitamin D metabolism is shown below:
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-Calcitonin is a peptide produced by clear cells of the thyroid gland. Increased serum
calcium levels cause secretion of calcitonin (controlled by β2 receptors). Calcitonin
decreases osteoclast number and activity and leads to a decrease in serum calcium.
-Markers of bone resorption:
1. Urinary hydroxyproline
2. Pyridoline cross-links
-Serum alkaline phosphatase is a marker of bone formation
Osteomalacia


Description
o Failure of mineralization of bone due to multiple etiologies (see table
below).
o Rickets is osteomalacia in children
o Lack of vitamin D is the underlying factor
 Poor nutritional intake
 Lack of sunlight
 Renal or Liver disease
History & Physical
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Reza Omid, MD
o Most common symptom is pain, sometimes localized, more often bilateral
and symmetrical; often initially vague but gradually becomes severe.
o May be proximal muscle weakness
Etiology
-Seen commonly in chronic alcoholics
 Pathophysiology
o The total amount of bone is normal (unlike osteoporosis) but there is a
lack of mineralization
o Serum calcium is low normal, phosphate is very low and alkaline
phosphatase is very high
o Serum vitamin 25-OH-D is markedly depressed, while 1,25(OH) levels may
be initially normal, although they too eventually fall.
o PTH levels tend to rise considerably, resulting in relative preservation of
the serum calcium (at the expense of the bones)
 Imaging
o Radiographs reveal generalized osteopenia
 Classically multiple bilateral and symmetrical cortical lucent areas in
the ribs, scapula, pelvis and femoral neck. These lucent areas
represent stress fractures and typically lie perpendicular to the long
axis of the bone (sometimes referred to as Looser’s zones or
Milkman’s lines)
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


Reza Omid, MD
The axial skeleton is more often affected clinically than the
peripheral skeleton
Pathology
o Microscopic examination (transiliac bone biopsy) is required for diagnosis
and must reveal at least 10% of total bone volume constituted by nonmineralized matrix (osteoid) and widened osteoid seams are seen.
Treatment
o Large doses of vitamin D
Primary Hyperparathyroidism
-Overproduction of PTH from an hypertrophy/adenoma/carcinoma which leads to
increased serum calcium and decreased serum phosphate.
-Leads to osteopenia, osteitis fibrosa cystica (fibrous replacement of marrow), brown
tumors (giant cells, RBC and hemosiderin) and chondrocalcinosis
-Radiographic findings include deformed osteopenic bones, fractures, areas of
radiolucency (at tufts of phalanges, distal clavicle, vertebral end plates), soft tissue
calcifications
Albright Hereditary Osteodystrophy
-Form of pseudohypoparathryroidism (lack of effect of PTH at target cells, PTH
levels normal to high) associated with:
1. Short 1st/4th/5th metacarpals and metatarsals
2. Brachydactyly
3. Exostosis
4. Obesity
5. Diminished intelligence
-Pseudo- pseudohypoparathryroidism is a normocalcemic disorder which is
phenotypically similar to pseudohypoparathryroidism however there is a normal
response to PTH.
Renal Osteodystrophy (Secondary Hyperparathyroidism)
-Abnormal bone metabolism in patients with chronic renal disease.
-Glomerular damage causes phosphate retention and hyperphosphatemia, inhibits
vitamin D production and decreased GI absorption of calcium and also increased
phosphate which leads to ectopic calcification (basal ganglia)
-Tubular damage causes loss of α-hydroxylase activity so decreased vitamin D
production which decreases GI absorption of calcium leading to hypocalcemia and
secondary hyperparathyroidism. Phosphate retention also occurs which causes a rise in
PTH as well. The elevated PTH then causes osteitis fibrosa, ectopic calcifications and
osteomalacia (osteopenia & fragility fractures).
-Traditionally, patients on dialysis were also having worsening of their osteomalacia due
to the high aluminum levels in the dialysis fluid. The aluminum competed with other
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ions to prevent proper formation of the hydroxyapatitie crystals. Now days the dialysis
fluid is not made with aluminum so this is not so much of a problem.
-Treatment of renal osteodystrophy is to give phosphate binders as well as high levels
of vitamin D
Rickets
-In children the equivalent to osteomalacia in adults
-Failure of mineralization leading to changes in the physis in the zone of provisional
calcification (increased width and disorientation of the physis)
-Cortical thinning and bowing
-Several subtypes of Rickets:
1. Nutritional Rickets
a. Vitamin D deficiency: rare except for in Asians, premature babies and
patients with sprue (malabsorption). Low calcium and phosphate,
increased PTH, low vitamin D levels.
i. Rachitic rosary
ii. Bowing of the knees
iii. Codfish vertebrae
iv. Coxa vara
v. Milkman’s fracture (Looser’s lines)
vi. Growth retardation
vii. Muscle hypotonia
-Treatment is 5000 IUD Vitamin D
b. Calcium deficiency
c. Phosphate deficiency
2. Hereditary Vitamin D-dependent Rickets
-Similar to vitamin D deficiency rickets but worse
-Patients may have total baldness
a. Type I: defect in renal 25-OH Vitamin D 1α-hydroxylase enzyme
b. Type II: defect in intracellular receptor for 1,25 (OH)2 vitamin D
3. Familial Hypophosphatemic Rickets
-aka phosphate diabetes
-X-linked dominant
-Due to impaired renal tubular reabsorption of phosphate
-Vitamin D resistant rickets (impaired vitamin D response)
-Normal GFR
-Treatment is phosphate replacement and high dose vitamin D
Osteoporosis
-Quantitative (not qualitative) defect of bone
-2.5 SD below the peak bone mass of a 25 year old
-Vertebral body fracture is the most common
-Lab values are normal, need DEXA scan
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-Z-scores give results for age controlled
-T-scores gives results for young normal adults
-Must rule out hyperthyroidism, hype-PTH, Cushings and malignancy
-Risk Factors: sedentary, thin Caucasian women of norther European descent, smokers,
heavy drinkers, dilantin use, poor diet, breast feeders, family history, premature
menopause.
-History of 2 osteoporotic vertebral fractures is the strongest predictor of subsequent
vertebral fractures in post-menopausal women.
-3 major types of osteoporosis:
1. Senile (age-related): affects trabecular bone and cortical bone
a. Related to poor calcium absorption
b. Hip fractures and Pelvis fractures
2. Post-menopausal (high turnover): affects trabecular bone primarily
a. Vertebral body and distal radius fractures
b. Distal radius fractures
3. Glucocorticoid induced (low turnover):
-Treatment for Osteoporosis:
1. Diet and adequate calcium and vitamin D
2. Weight bearing exercise
3. Estrogen therapy evaluation (works best when started within 6 years of
menopause)
4. Bisphosphonates (Fosamax): bind farnesyl disphosphate synthase and cause
apoptosis in osteoclasts.. Can also be used for OI, Padgets, Osteopetrosis and
metastasis
5. Calcitonin
6. Tamoxifen
7. SERMs
8. Anabolic: PTH (Forteo) is PTH given intermittently to stimulate osteoblasts but
can only give for 2 years, may cause osteosarcoma
Idiopathic Transient Osteoporosis of the Hip
-Uncommon disorder of exclusion
-Most commonly seen in 3rd trimester of pregnancy but also occurs in men (middle age)
-Present with groin pain, limited ROM and localized osteopenia (without history of
trauma)
-MRI is highly sensitive but not specific and must rule out infection, osteonecrosis and
neoplasm.
-Self-limited disease and resolves within 6-8 months with restricted WB and pain
control.
Osteochondroses
-Osteochondrosis is a disease causing degenerative changes in the ossification centers
of the epiphysis of bones, particularly during periods of rapid growth in children. The
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process can continue to AVN. AKA traction apophysitis. May or may not be associated
with trauma, inflammation of joint capsule, or vascular insult/secondary thrombosis.
-Examples of osteochondroses:
1. Van Neck’s Disease: ischiopubic synchrondosis
2. Legg-Calve-Perthese Disease: femoral head
3. Osgood-Schlatter Disease: tibial tuberosity
4. Sinding-Larsen-Johansson Syndrome: inferior patella
5. Blouts Disease: proximal tibial epiphysis
6. Sever’s Disease: calcaneus
7. Kohler’s Disease: navicular
8. Friedberg’s Infraction: 2nd metatarsal head
9. Scheuermanns Disease: discovertebral junction
10. Panner’s Disease: capitellum
11. Thiemanns Disease: phalanges of the hand
12. Kienbock’s Disease: lunate
13. Iselin’s Disease: 5th metatarsal base
14. Preiser’s Disease: scaphoid
Growth and Development of the Skeleton
Limb Development
-The appendicular skeleton forms between the 4th and 8th weeks of gestation. The limb
bud begins as an out-pouching from the lateral body wall and initially consists of both
ectodermal and mesodermal layers.
-This initial outgrowth appears to be under the control of the FGF family
because FGF-1, FGF-2 and FGF-4 all are able to induce the formation of
ectopic limb buds.
-The progressive enlargement of the limb bud results from biomechanical signaling
between the layer of ectodermal cells at the distal layer, apical ectodermal ridge
(AER), and the rapidly proliferating mesodermal cells, progress zone, adjacent tot his
ectodermal layer.
-The apical ectodermal ridge (AER) is to promote the outgrowth of the limb
bud in the proximal distal direction by maintaining the mesodermal cells in an
undifferentiated state.
-The role of the progress zone is to secrete a factor that maintains the AER.
If the AER is experimentally removed, the cells immediately subjacent to the AER
undergo massivce cell death, and the limb formation is truncated at a variable proximaldistal level, depending on the timing of the AER removal. The severe transverse
phocomelic birth defects caused by thalidomide exposure during the 1st trimester of
pregnancy have been postulated to involve damage to the progress zone.
-Surgical replacement of the AER or implantation of a bead loaded with FGF-4 prevents
this cell death and restores the normal proximal-distal axis of limb formation. The
proximal-distal axis of limb formation is therefore under control of the FGF family.
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-As the limb bud enlarges, its constituent cells acquire a positional identity with respect
to each of the three axes (proximal-distal, anterior-posterior, dorsal-ventral) that
ultimately specifies cell fate and sculpts the mesenchymal condensations that form the
precursors of the skeletal elements of the limb. This positional identity is acquired as
cells pass through the progress zone beneath the AER.
-Cells located in the zone of polarizing activity (ZPA) in the posterior aspect
of the limb bud direct both the number and type of digits formed. This
organizing activity of the ZPA has been traced to the product of the Sonic hedgehog
(Shh) gene. Shh controls the formation of digits by activating the expression of
homeobox (HOX) genes.
-Limb formation along the dorsal-ventral axis of the limb bud is under the
control of a member of the Wnt gene family, Wnt-7a. This regulatory molecule
is expressed in a restricted area on the dorsal aspect of the limb bud and specifies
dorsal-ventral patterning. Misexpression of Wnt-7a on the ventral aspect of the limb
bud in mice results in digits that flex in the dorsal direction and extend in the ventral
direction.
-During the 6th week of development, mesenchymal condensations within the limb bud,
which serve as models for skeletal development, undergo chondrogenesis as the cells
begin the synthesize a cartilage extracellular matrix. This transformation into
cartilage tissue is regulated by the activity of members of the SOX gene
family, specifically SOX5, SOX6 and SOX9.
-Mutations of SOX9 have been linked to camptomelic dysplasia ().
-Three dimentional regulatory axis of limb bud pattern formation:
1. Proximal-Distal: FGF
2. Dorsal-Ventral: Wnt-7a
3. Anterior-Posterior: Shh
Science of Joints
Articular Cartilage
-Articular cartilage is a resilient load bearing tissue that forms the articulating surface of
joints. It provides a surfaces with low friction and absorbs mechanical shock and
spreads the load onto subchondral bone.
-Contains no nerves, blood vessels or lymphatics
-Collagen of articular cartilage provides tensile strength
-Proteoglycans of articular cartilage provides compressive strength
-Thickest cartilage is under the patella
-4 Zones of Articular Cartilage:
1. Superficial zone: resists shear force, 10-20%
a. Lamina splendens (unique superficial layer that consists of fibrils and little
or no PG and no cells
b. Ellipsoidal chondrocyte layer
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-High water content
-Low PG content
-Acts as a barrier against movement of molecules between the synovium and the
articular cartilage
-Damage to this zone may contribute to development of OA. Also damage to
this zone could release molecules into the synovial fluid and stimulate a immune
response.
2. Middle (Intermediate) zone: resists compressive forces, 40-60%
-Rounded chondrocytes spread out
-Most metabolically active
-Randomly arranged collagen fibers
3. Deep zone: resists compressive forces, 30%
-Chondrocytes in columns
-Low water content
-High PG content
-Largest collagen fibers, fibers arranged vertical (perpendicular to surface)
*Tidemark separates the deep zone from the calcified zone. It resists shear
forces.
The number of tidemarks increases with age as the tissue is remodeled.
4. Calcified zone:
-seperates the articular cartilage from the subchondral bone
-anchors the cartilage to the bone (equivalent of Sharpy’s fibers with ligaments)
-Articular Cartilage Composition:
1. Water: 65-80% of total weight
a. Resists compression
2. Type II collagen: 10-20% of total weight (50% of dry weight)
a. Provides tensile properties and acts as a framework to immobilize ECM &
PG
3. Proteoglycan Aggragate: 5-10% of total weight
a. GAG’s contain carboxyl & sulfate groups (keratin and chondroitin sulfate).
GAG’s are negatively charged and bind water (act like a sponge) and the
negative charges repel each other maximizing volume.
4. Chondrocytes: 2% of total weight
a. Produce all the ECM macromolecules and degradative enzymes.
b. Chondrocytes are responsible for formation of all ECM macromolecules
and maintenance of articular cartilage. They are derived from
mesenchymal cells and are metabolically active (anaerobic). There
numbers decrease with age. Chondrocytes respond to environmental
stimuli. Exposure of IL-1 may lead to degredation of ECM.
-Articular cartilage nutrition is via diffusion and requires joint mobilization to allow for
movement of the synovial fluid over the articular surface.
Collagen
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-At least 15 types of collagen, all triple helix structures composed of 3 α-chain
polypeptides with large quantities of glycine and praline. Also contain hydroxyproline,
hydroxylysine and glycosylated hydroxylysine.
-Hydroxylation of collagen requires vitamin C
1. Type 1 Collagen: most common type of collagen
a. Found in bone, tendon, ligament, meniscus, annulus fibrosus of discs,
skin, pubic symphysis, articular discs (AC/SC joints)
b. Any site where fibrocartilage is found (microfracture)
2. Type 2 Collagen:
a. Found in articular (hyaline) cartilage, nucleus pulposus of discs.
b. Early fracture callus
3. Type 3 Collagen: Found in skin and blood vessels
4. Type 4 Collagen: Found in basement membranes
5. Type 6 Collagen:
a. Minor component of normal articular cartilage
b. Increases significantly in early OA
6. Type 9 Collagen: Adhesive cartilages which link together with Type 11 collagen
and other type 2
7. Type 10 Collagen:
a. Found only near calcified cartilage (calcified zone of articular cartilage and
hypertrophic zone of growth plate)
b. Produced by hypertrophic chondrocytes during enchondral ossification
seen in:
i. Fracture callus
ii. Growth plate
iii. HO formation
iv. Calcifying cartilaginous tumors
c. Genetic defect of type 10 collagen seen in Schmids’s metaphyseal
chondroplasia
8. Type 11 Collagen: More adhesive than type 9
9. Type 12 Collagen: Found in tendons
-Proteoglycans (PG) are complex macromolecules that consist of a protein core with
covalently bound GAG chains aka proteoglycan aggregate. Half life of PG are 3 months.
PG produce cartilages porous structure and trap and hold water (regulate and retain
water in the matrix)
-95% of proteoglycans are chondroitin and keratin sulfate. The sulfate molecules
are the negative charge that attracts positive cations (Na+& K+) and increase osmotic
pressure to “inflate” the elastic meshwork with water for compression properties.
Proteoglycan aggregate are made up of a hyaluronic acid with several aggregan
molecules bond to it. Each aggrecan molecule has several regions: chondroitin rich
region and keratin rich region. Aggregate size decreases with age.
-Glycosaminoglycans are long chain disaccharide units that make up PG once bound
to hyaluronic acid. Types of GAG:
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1.
2.
3.
4.
Reza Omid, MD
Chondroitin Sulfate (most prevalent, found in articular cartilage, concentrated
at ends of aggrecan molecules)
a. 4-isomer decreases with age
b. 4-isomer increases with OA
c. 6-isomer remains constant with age
d. total chondroitin decreases with age
Keratin Sulfate (articular cartilage, concentrated in middle of aggrecan
molecules)
a. Increases with age
b. Decreases with OA
Dermatan Sulfate
Hyaluronate (HA)
a. HA is unique because it is not sulfated like the other GAG and not
covalently bound to a protein core so not part of a PG
-Glucosamine and Chondroitin Sulfate supplements are over the counter
chondroprotective agents. Glucosamine stimulates chodrocytes and synoviocyte
activites. Chondroitin sulfate inhibits degredative enzymes and prevent sfibrin
thrombus formation in periarticular tissue. These supplements improve pain, joint line
tenderness, ROM and walking speed but no studies show that they affect mechanical
properties or biomechanical consistency of articular cartilage.
-Cartilage Oligometric Protein (COMP) is a protein concentrated primarily in the
chondrocyte territorial matrix. COMP appears to be present mainly in articular cartilage
and binds to chondrocytes and serves as a serum marker for cartilage turnover and of
progression of cartilage degeneration in patients OA/RA or any inflammatory arthritis.
COMP can also be used for monitoring efficiency of treatment.
-Creep is a time-dependent deformation of a viscoelastic tissue (articular cartilage) due
to a constant load). Rate of creep is governed by the rate at which fluid may be forced
out from the tissue, which in turn, is governed by the permeability and stiffness of the
matrix. The tissue will creep until an equilibrium value is reached.
-Mechanosignal transduction occurs via integrins in chodrocytes as pressure or
deformation is transmitted to the cell to increase mRNA expression.
Synovium
-Composed of 2 distinct layers:
1. Intimal lining: in direct contact with IA cavity and responsible for production of
hyaluronic acid
a. Loosely organized and avascular layer 1-2 cells layers thick
2. Sublining: lies beneath the intimal lining and is acellular and contains scattered
blood vessels, fat cells and fibroblasts.
-Cell types present in the synovium:
1. Type A cells: macrophage like (phagocytosis)
2. Type B cells: fibroblast like (produce synovial fluid)
-Synovial fluid consists of HA, lubricin, proteinase, collagenase and PG. It is an
ultrafiltrate of plasma and contains no RBC, clotting factors or hemoglobin
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Meniscus
-Deepens the articular surface of synovial joints and found in the:
1. AC joint
2. SC joint
3. Glenohumeral joint
4. Hip joint
5. Knee joint
-The meniscus broadens the contact area and distributes the load. More elastic and
less permeable than cartilage.
-Composed of type 1 collagen (fibrocartilage)
-Medial meniscus of the knee is semicircular (C-shaped)
-Lateral meniscus of the knee is circular
-2 meniscofemoral ligaments run from the posterior horn of the lateral meniscus to
the medial femoral condyle. Anterior to the PCL is the ligament of Humphrey and
posterior to the PCL is the ligament of Wrisberg.
-2 cell types found within meniscus:
1. Fusiform cells in the superficial zone
2. Fibrochondrocytes (ovoid/polygonal cells) found in the remainder of the
tissue. These cells are responsible for meniscal healing in the peripheral zone.
Meniscal tears with a rim width <4mm have the best healing characteristics
-Meniscus collagen fiber orientation is principally circumferential. A few radial fibers act
as “ties” to provide rigidity and help resist longitudinal splitting. 3 framework layers are
found in the menisci:
1. Superficial layer: fine fibrils in meshlike matrix
2. Surface layer: irregularly aligned bundles
3. Middle layer: circumferential large coarse fibrils which are main force resisting
fibers
-Meniscus motion (excursion is AP displacement) occurs with knee range of motion of
0°-120°:
o
Medial meniscus excursion is 5.1 ± 0.96
o
Lateral meniscus excursion is 11.2 ± 3.27
-Limited peripheral blood supply to the meniscus originates from the lateral and medial
genicular arteries (both inferior and superior). It penetrates 10-30% of the medial
meniscus and 10-25% of the lateral meniscus. The medial inner 2/3 of the meniscus
derives nutrition from the synovial fluid via mechanical pumping.
-Anterior and posterior horns of the menisci are most richly innervated (particularly the
posterior horn). During extremes of flexion/extension, the horns of the menisci become
taut and this increases tension then activates the mechanoreceptors. 3 types of
mechanoreceptors found:
1. Ruffini endings
2. Golgi tendon organs
3. Pacinian corpuscles
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-Following ACL transaction, the meniscus undergoes alterations in ECM, including
increased water content and decreased GAG concentration. With age, meniscus
undergo discoloration and increased deposition of calcium pyrophosphate dehydrate
crystals, but water content does not change with age (unlike articular cartilage).
However there is an increased chondroitin 6 to chondroitin 4 ratio with age (similar to
articular cartilage).
Synovial Fluid
-Synovial fluid id an acellular plasma ultrafiltrate that lubricates the joint. Its high
viscosity gives it important mechanical properties and is related to large amounts of
polymerized hyaluronic acid.
-Should be sent for gram stain, culture, WBC with differential, and crystal analysis
-Dye should be injected only after fluid is obtained from the joint because the
bactericidal effect of iodinated contrast material can cause a false negative culture
result.
-Non-inflammatory synovial fluid contains 200 WBC, 25% PMN, glucose and
protein values equal to serum values and volume <3.5 cc.
-Inflammatory synovial fluid contains 2,000-75,000 WBC, 50% PMN, glucose levels
moderately decreased and volume >3.5 cc. Inflammation decreases viscosity of
synovial fluid so it decreases the string sign length.
-Infectious synovial fluid contains >50,000-80,000 WBC, 75% PMN, glucose levels
significantly decreased and protein levels increased. Inflammation decreases viscosity
of synovial fluid so it decreases the string sign length.
Science of Muscluar Tissue
-Myotendon junction is the weakest link in the muscle. It is often the site of tears,
especially with eccentric contractions (muscle lengthens while contracting)
-Sarcomere is composed of thick (myosin) and thin (actin) filaments intricately
arranged to allow the fibers to slide past each other. The sarcomere is arranged into
bands and lines:
o H-band: contains myosin only
o I-band: contains actin only
o A-band: contains actin and myosin
o M-line: interconnecting site of myosin
o Z-line: anchors the thin (actin) filaments
-Neuromuscular junction: stimulus for a muscle contraction comes from the brain
and travels down the spine to the muscle. The motor endplate (specialized synapse
formed between the nerve and muscle) releases ACh from presynaptic vesicles. The
Ach then binds to a postsynaptic receptors to cause depolarization of the SR which then
releases calcium into the muscle cytoplasm. Calcium binds to troponin (on thin
filaments) causing them to change the position of topomyosin (also on the thin
filaments) and expose the actin filaments. Actin-myosin cross bridging forms and with
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ATP breakdown of ATP the thick and thin filaments slide past one another causing
contraction of the muscle.
-Botunlinum A toxin blocks ACh release presynaptically
-Non-depolarizing drugs (curare, pancuronium and vecuronium) competitively
bind to ACh recepts to block impulse transmission (long term paralytic)
-Depolarizing drugs (succinylcholine) binds to ACh receptors to cause temporary
depolarization of muscle membrane (short term paralytic)
-Anticholinesterases (neostigmine, edrophonium) act at the autonomic ganglia,
prevents breakdown of ACh to enhance its effects. These meds are reversal agents for
non-depolarizing drugs. These meds have muscarinic effects (bronchospasm,
bradycardia, bronchorrhea)
3 Types of Muscle Contraction:
1. Isotonic Contraction:
-muscle tension is constant throughout the ROM.
-measure of dynamic strength
-free weight exercise is an example
-has several phases:
i. Concentric contraction: the muscle shortens during contraction
ii. Eccentric contraction: the muscle lengthens during contraction
2. Isometric Contraction:
-muscle tension is generated but length remains unchanged.
-a measure of static (not dynamic) strength
-increases strength at a specific joint angle at which the training is being
affected
-benefits seem to occur during the early stages of training or rehab
-optimal effects occur at maximal contraction and long enough duration to
recruit as many fibers as possible and best when done several times per
day
-example is wall sits
3. Isokinetic Contraction:
-muscle tension is generated as the muscle maximally contracts at a
constant velocity over a full ROM
-best for maximizing strength and are a measure of dynamic strength
-effective at slow speeds (≤60°/sec)
-cybex machines (exerts a force that equals that exerted by the person)
i. Concentric contraction phase
ii. Eccentric contraction phase
-Eccentric contraction is a phase of muscle contraction, not a type of muscle
contraction. The muscle lengthens during the contraction (internal force is less
than the external force). Eccentric contractions have the greatest potential
for high muscle tension and muscle injury. Example is a “negative”. It is
questionable if it is more effective than other forms of training, some say no.
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-Concentric contraction is a phase of muscle contraction, not a type of muscle
contraction. The muscle shortens during the contraction (internal force exceeds
the external force).
-Plyometrics are a special type of exercise. It is implosion training in which the
muscles are loaded suddenly and forcefully stretched before they can contract.
It is very effective at power generation. Like eccentric loading, it has been
shown to increase muscle soreness and have an increased risk of injury.
Examples are catching and throwing a medicine ball, jumping down from a box
and jumping back up. The stretch stores elastic energy which increases the
force of the concentric contraction.
-Closed-chain exercises include loading an extremity with the most distal
segment stabilized or not moving (on the ground). This allows muscular cocontraction around a joint, which minimizes joint shear (places less stress on the
ACL). Stationary bike is an open chain exercise and is harmful to an ACL
reconstruction.
-Type 1 Muscle Fibers:
o Slow twitch
o Oxidative (red) fibers (“slow red ox”)
o Aerobic therefore more mitochondria and high capillary density
o Good for endurance sports
o First muscles lost without rehabilitation
o Low anaerobic capacity
-Type IIA Muscle Fibers:
o Fast twitch
o Glycolytic (white) and oxidative fibers (FOG “fast oxidative glycolytic”)
o Not as prevalent in humans (seen in humbingbirds)
o Good for strength
-Type IIB Muscle Fibers:
o Fast twitch
o Glycolytic (white) only (unlike IIA)
o Anaerobic
o Quick contractions with larger and stronger motor units
o Good for sprinters and strength atheletes
o Strength training causes hypertrophy of these fibers
ATP-CP System
-aka Phosphogen system
-meets the metabolic requirement for intense muscle activity that lasts up to 20 sec,
such as sprinting a 100-200 meter dash.
-converts stored carbohydrates from within the muscle fiber itself to energy
-does not use oxygen
-does not produce lactate
-energy is derived from the high energy phosphate bonds of ATP
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-creatine supplements can increase the amount of work that is produced in 1st few maxeffort anaerobic trials but does not increase peak force production
Lactic Anaerobic System
-meets the metabolic requirement for intense muscle activity that lasts 20-120 sec, such
as a 400 meter dash.
-involves hydrolysis of on glucose molecule to ultimately produce lactic acid plus energy
converting 2 molecules of ADP to 2 molecules of ATP
Aerobic System
-when oxygen is available, the aerobic system replenishes ATP through oxidative
phosphorylation and the Krebs cycle. It uses glucose or fatty acids to produce ATP.
Meets the metabolic requirements for episodes of longer duration and lower-intensity
muscle activities.
-Aerobic exercise in a healthy adult is recommended for 3-5 days per week for 20-60
min per session. Training should be at 60-90% of max heart rate. Max heart rate is
approximately 220 bpm – age. Aerobic conditioning has proved effective in lowering
the incidence of back injury in workers and in helping elderly remain ambulatory. A
significant decline in aerobic fitness occurs after just 2 weeks of no training (detraining)
Basic Science of Sports Medicine
-Unhealthy weigth loss due to fluid and food restriction (seen in wrestlers, fighters and
jockeys) is associated with:
1. reduced cardiac output
2. increased heart rate
3. smaller stroke volume
4. lower oxygen consumption
5. decreased renal blood flow
6. electrolyte loss (hypokalemia)
-Fluid replacement regimen for a competitive athlete is to replace enough water to
maintain pre-practive weight and maintain a normal diet. Fluid carbohydrate and
electrolyte replacement is most effective when the osmolarity of the replacement fluid is
<10% (glucose polymers minimize osmolarity). Fluid absorption by the gut is enhanced
by solutions of low osmolarity
-Female athlete triad:
1. Amenorrhea: from decreased percentage of body fat and changes in the
hypothalamic-pituitary axis.
2. Osteoporosis: decreased estrogen levels cause bone loss and amenorrhea
3. Anorexia: loss of appetite and desire to lose weight
-Initial management is increasing weight, decreasing exercise and possibly
administration of cyclic estrogens or progesterones.
-Muscle strain is the most common sports injury seen. Most muscle strains occur at
the myotendinous junction in muscles that cross 2 joints (such as the hamstring or
gastrocnemius) and have increased type 2 fibers. Muscle strains occur most commonly
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at the myotendinous junction often during a rapid (high velocity) eccentric contraction
(eccentric contraction develops the highest forces observed in skeletal muscle). Muscle
tears heal with dense scarring. Surgical repair of clean lacerations in the midbelly of
skeletal muscle usually results in minimal regeneration of muscle fibers distally, scar
formation at the laceration and recovery of approximately half of the muscle strength.
-Muscle activation via stretching allows twice the energy absorption prior to failure.
Bouncing types of stretches are deleterious.
-Muscle soreness that apprears 24-48 hours after strenuous exercise is termed delayed
muscle soreness. It is an overuse injury commonly experienced. It is more common
with eccentric contractions. It is associated with changes in the I-band. Torn tissue,
muscle spasm and connective tissue damage may cause muscle soreness.
-Muscle strength gains during the 1st 10 days of rehabilitation are due to improved
neural firing patterns. Later strength gains are due to increases in ROM, muscle fiber
size, muscle repair and tendon repair. Trunk extensors are stronger than trunk flexors.
-Immobilization of injured muscle changes the number of sarcomeres at the
musculotendinous junction and accelerates granulation tissue reponse in the injured
muscle. Immobilization in lengthened positions decreases contractures and maintain
strength. Atrophy can result from disuse and altered nervous system recruitment.
Electrical stimulation (US therapy) can help offset these effects.
4)
Science of Nervous Tissue
CNS Injuries
-Patients may continue to improve up to 6 months after a stroke and up to 18 months
after traumatic brain injury. Most common mechanism of spinal cord injury in adults is
MVA. If <3hrs from injury: 30mg/kg for 1st hour then 5.4mg/kg/hr for the next 23
hours. If injry is between 3-8 hours: 30mg/kg for 1st hour then 5.4mg/kg/hr for the
next 47 hours. This regimen is associated with improved root function and the level of
injury, although improvement of spinal cord function may or may not be seen.
Regimen is not indicated for GSW, brachial plexus injuries and nerve root deficits.
-Concussion is a jarring injury to the brain that results in disturbance to some degree
of cerebral function. 3 grades of severity:
1. Grade 1 (mild): no loss of conciousness, no retrograde amnesia
o
Return to play as soon as asymptomatic
2. Grade 2 (moderate): + loss of conciousness <5min, + retrograde amnesia
(there is always some permanent loss of memory regarding the injury itself),
confusion and disorientation resolved rapidly
o
Return to play afte asymptomatic for 1 week
o
Repeat episode requires long term suspension
3. Grade 3 (severe): prolonged unconsciousness, permanent retrograde amnesia,
confusion persists
o
Long term suspension is required
-Meissner’s corpuscle:
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Mechanoreceptor with Aβ fiber type
o
Rapidly adapting
o
Moving 2 point discrimination
o
Well equipped for analysis of motion
-Merkel’s receptor:
o
Mechanoreceptor with Aβ fiber type
o
Slowly adapting
o
Static 2 point discrimination
o
Responds to a small area of skin pressure
-Pacinian corpuscle:
o
Mechanoreceptor with Aβ fiber type
o
Rapidly adapting
o
Flutter
-Ruffini’s corpuscle:
o
Mechanoreceptor with Aβ fiber type
o
Slowly adapting
o
Vibration
o
Peripheral Nerve Morphology
-Axon coated with a fibrous tissue called endoneurium, group into nerve bundles called
fascicles, which are covered with a connective tissue called perineurium. Peripheral
nerves are composed of one (mono), a few (oligo) or several (poly) fascicles and
surrounding areolar connective tissue (epineurium) enclosed within an epineurial
sheath. Perineurium which surrounds the bundles of fascicles within a peripheral nerve
is the primary structure responsible for tensile strength. Epineurium is thicker but has
less tensile strength and may function more to resist compressive forces.
-Blood supply to nerves is from an extrinsic blood supply from vessels that run in loose
connective tissue surrounding the nerve trunk. Intrinsic blood supply is from vascular
plexus in the epineurium, perineurium and endoneurium (with interconnections
between these three plexus)
-Peripheral nerve injury leads to death of the distal axons and Wallerian degeneration
(of myelin) which extends distal to the somatosensory receptor. Proximal axonal
budding occurs (after 1 month delay) and leads to regeneration at rate of about
1mm/day (possibly 3-5mm/day in children). Pain is the first modality to return
-Nerve regeneration is influenced by:
1. Contact guidance: attraction to basal lamina of the Schwann cell
2. Neurotrophism: factors enhancing growth
3. Neurotropism: preferential attraction towards nerves rather than other tissues
-Upper Motor Neuron Signs (brain or spinal cord):
o
Decreased strength
o
Increased tone
o
Hyperreflexia
o
Babinski
o
Hoffman
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Clonus
-Lower Motor Neuron Signs (nerve injuries):
o
Decreased strength
o
Loss of tone
o
Loss of reflexes
o
Fasciculations
o
Atrophy of muscles
-Types of Nerve Injuries:
o
Neuropraxia: reversible coduction block characterized by local ischemia and
selective demyelination of axon sheath
 Most common
 Best prognosis
o
Axonotmesis: more severe injury with disruption of the axon and myelin
sheath but leaving the epineurium intact
o
Neurotmesis: complete nerve division with disruption of the endoneurium
 Worst prognosis
-Football “stingers” or “burners” are neuropraxia from a brachial plexus stretch
injury. Nerve stretching can affect function as 8% elongation can diminish a nerve’s
microcirculation and 15% elongation disrupts axons. A + histamine response implies
that there is an intact reflex arc indicating that the lesion is proximal to the ganglion
(pre-ganglionic)
-It is crucial to properly align nevre ends during surgical repair in order to maximize
potential for functional recovery. Types of nerve repair include:
1. Direct muscular neurontization: insert nerve stump into the muscle belly
2. Epineural repair: primary repair of outer connective tissue
a. Used for smaller nerves (digital nerves)
b. Remember “kissing but no sex”
3. Grouped fascicular repair: reapproximation of individual fascicles under a
microscope.
a. No significant improvement in results over epineural repair has been
documented
b. Used for larger nerves.
o
5)
Science of Connective Tissue
-Tendons insert into bone via four transitional tissues (for force dissipation):
1. Tendon
2. Fibrocartilage
3. Mineralized fibrocartilage (Sharpy’s fibers)
4. Bone
-Consists of fibroblasts arranged in parallel rows in fascicles of type 1 collagen (85%)
with surrounding loose areolar tissue called the peritenon. Tendons attach muscle to
bone (via Sharpey’s fibers). Immobilization leads to increased tendon strength at the
expense of ROM but decreases tendon strength at the tendon-bone interface. Tendon
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repairs are weakest at 7-10 days and regain maximum strength at 6 months. Two
types of tendons are found in the body:
1. Paratenon-covered tendons
2. Sheathed tendons
-Paratenon tendons are vascularized tendons. They have many vessels supplying a
rich capillary system. They heal better than sheathed tendons. Examples include the
patellar tendon and Achilles tendon
-Sheathed tendons have a mesotenon (vincula) which carries a vessel that supplies
only one tendon segment. Avascular areas receive nutrition via diffusion from
vascularized segments. Examples include flexor tendons of the hand.
-Ligaments stabilize joints (connect bone to bone) and are composed of type 1
collagen (70%). Fibers are more variable than tendons and have higher elastin
content. They have uniform microvascularity (unlike tendons) and receives its supply at
the insertion site. They also possess mechanoreceptors and free nerve endings. They
insert onto bone via direct or indirect insertion. Early healing is via type 3 collagen
which is converted to type 1 collagen. Mobilization increases strength of the ligaments.
-Ligament sprains include 3 grades:
1. Grade 1 Sprain: ligament is stretched but remains intact and results in no
instability
2. Grade 2 Sprain: partial tears that lead to mild instability
3. Grade 3 Sprain: complete tears that result in significant joint instability
-Rupture of sequential series of collagen fiber bundles distributed throughout the body
of the ligament (as opposed to localized to one specific area) is most common
mechanism of ligament failure. Ligaments doe not plastically deform. Midsubstance
tears are common in adults. Avulsion injuries (with small piece of bone) are more
common in children. Ligament avulsion typically occurs between the unmineralized and
mineralized fibrocartilage layers. Local injections of corticosteroids at a site of injured
ligament is detrimental to the healing process.
-Fibrillin is a component of elastic fiber system. Fibrillin metabolism defect has been
demonstrated in some patients with adolescent idiopathic scoliosis as well as Marfan’s.
-Magic angle of MRI occurs when a tendon or ligament is oriented near 55° to the
magnetic field, T1 images may appear to have increased signal and create false
appearance of pathology. Most commonly seen in the shoulder, knee and ankle
-Intervertebral discs of the spine contains various neuropepties including substance
P, calcitonin gene-related peptide, vasoactive intestinal peptide (VIP) and c-flanking
peptide of neuropeptide (CPON). They are composed of 2 components:
1. Central Nucleus Pulposus: hydrated gel with compressibility
i. High GAG and low collagen
ii. Type 2 collagen
2. Annulus Fibrosis: increased tensile strength
i. High collagen and low GAG
ii. Type 1 collagen
iii. Superficial layer contains nerve fibers
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