CHAPTER 32
Connective Tissues
A nimals use different proportions of matrix macromol-
ecules to construct connective tissues with a range of
mechanical properties to support their organs. Bone is a
stiff, hard solid; blood vessel walls are flexible and elastic;
and the vitreous body of the eye is a watery gel. Plant
and fungal cell walls are functionally similar to the animal
extracellular matrix but are composed of completely dif-
ferent molecules. This chapter begins with a discussion
of simple connective tissues then concentrates on carti-
lage, bone, development of the skeleton, and the mecha-
nisms that repair wounds, finishing with a discussion of
the plant cell wall.
Loose Connective Tissue
Loose connective tissue consists of a sparse extracellular
matrix of hyaluronan and proteoglycans supported
by a few collagen fibrils and elastic fibrils. In addition
to fibroblasts, the cell population is heterogeneous,
including both indigenous and emigrant connective
tissue cells (see Fig. 28.1). The loose connective tissue
underlying the epithelium in the gastrointestinal tract is
a good example of this heterogeneity (Fig. 32.1A), with
lymphocytes, plasma cells, macrophages, eosinophils,
neutrophils, and mast cells, as well as fibroblasts and
occasional fat cells (see Chapter 28 for details on these
cells). This variety of defensive cells is appropriate for a
location near the lumen of the intestine, which contains
microorganisms and potentially toxic materials from the
outside world. Loose connective tissue is also found in
and around other organs. In the optically transparent
vitreous body of the eye, fibroblasts produce a highly
hydrated gel of hyaluronan and proteoglycans, sup-
ported by a loose network of type II collagen. Few defen-
sive cells are required, as the interior of the eye is sterile.
Dense Connective Tissue
Collagen fibers, with or without elastic fibers, make up
the bulk of dense connective tissue (Fig. 32.1B). Sparse
A B
Columnar
epithelium
Transitional
epithelium
LOOSE CT
DENSE CT
FIGURE 32.1 CONNECTIVE TISSUES. A, Loose connective
tissue (CT) underlying the columnar epithelium of the small intestine.
Light micrograph of a section stained with Masson trichrome stain.
B, Dense connective tissue (CT) underlying transitional epithelium in
the wall of the ureter. Light micrograph of a section stained with
hematoxylin-eosin. (Courtesy D.W. Fawcett, Harvard Medical School,
Boston, MA.)
fibroblasts are present to manufacture extracellular
matrix. Other connective tissue cells are even rarer, as
these tissues are not usually exposed to microorganisms.
Collagen fibers can be arranged precisely, as in tendons
or cornea (see Fig. 29.3), or less so, as in the wall of the
intestine or the skin. Tendons consist nearly exclusively
of type I collagen fibers, all aligned along the length of
the tendon to provide the tensile strength that is required
to transmit forces from muscle to bone. The cornea that
forms the transparent front surface of the eye is also well
organized into orthogonal layers of collagen fibrils.
Dense connective tissues can also be elastic. For
example, the walls of arteries (see Fig. 29.8) and the
dermal layer of skin consist of both collagen and elastic
fibers. Energy from each heartbeat stretches the elastic
fibers in the walls of arteries. Recoil of these elastic fibers
propels blood between heartbeats and affects the blood
pressure.
555
556 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

Approximately one in 5000 humans inherits a muta-
tion in a gene for fibrillar collagens type I, type III, or
type V, which causes a range of connective tissue defects
called Ehlers-Danlos syndrome. Most affected indi-
viduals have thin skin and lax joints. Severe mutations
lead to rupture of arteries, bowel, or uterus, often with
fatal consequences. Ehlers-Danlos syndrome illustrates
the importance of these collagens with regard to the
integrity of the affected tissues. Inheritance is dominant,
as these collagens consist of trimers of three identical
subunits. Given one mutant gene, only one in eight
( ) procollagen molecules is normal.
Cartilage
Cartilage (Fig. 32.2) is tough, resilient connective tissue
that performs a variety of mechanical roles. It covers the
articular surfaces of joints and supports the trachea,
other large airways, the nose, and ears. Cartilage also
forms the entire skeleton of sharks and the embryonic
precursors of many bones in higher vertebrates. The
mechanical properties of cartilage are attributable to
abundant extracellular matrix consisting of fine collagen
fibrils and high concentrations of glycosaminoglycans
and proteoglycans (Fig. 32.3).
Chondrocytes synthesize and secrete macromole-
cules for the cartilage matrix, which eventually sur-
rounds them completely. Chondrocytes replenish the
matrix as the macromolecules turn over slowly, but their
ability to remodel and repair the matrix is limited. No
blood vessels penetrate cartilage, owing to production
of several inhibitors of endothelial cell growth by chon-
drocytes. Thus, all nutrients must diffuse into cartilage
from the nearest blood vessel in the perichondrium, a
dense capsule of fibrous connective tissue that covers
the surface of cartilage. This capsule contains mesenchy-
mal stem cells (see Box 41.2 and Fig. 28.1) that are
capable of differentiating into chondrocytes.
A meshwork of type II collagen fibrils, accounting
for approximately 25% of the dry mass, fills the extracel-
lular matrix. These slender collagen fibrils are hard to
see even in electron micrographs but are quite stable,
with lifetimes estimated to be many years. Fibrils tend to
line up parallel to surfaces but otherwise are arranged
randomly. Minor collagen type IX crosslinks type II col-
lagen fibrils and collagen type XI binds to the surface of
type II fibrils. Expression of type X collagen is restricted
to cartilage that is undergoing conversion to bone. The
matrix contains several minor adhesive proteins, and
other proteins inhibit invasion of blood vessels.
Glycosaminoglycans, including hyaluronan, constitute
the second major class of matrix macromolecules. Mol-
ecules of the proteoglycan aggrecan attach to a hyaluro-
nan backbone like the bristles of a test tube brush,
forming so-called megacomplexes (see Fig. 29.13).
Aggrecan also binds type II collagen. Highly charged

A B. Chondrocyte
Epithelium
Perichondrium Chondrocytes
ER

C. Matrix
Type II collagen

FIGURE 32.2 CARTILAGE AND CHONDROCYTES. A, Light micrograph of a section of hyaline cartilage in the wall of the respiratory tree
stained with periodic acidSchiff stain and Alcian blue. The cartilage capsule of dense connective tissue (perichondrium) and the columnar epi-
thelium lining the respiratory passage are at the top. Inset, Light micrograph of hyaline cartilage stained with toluidine blue. The proteoglycans
in the matrix stain pink. The rough endoplasmic reticulum stains blue. Shrinkage during fixation and embedding creates the artifactual cavity or
lacuna around each cell. B, Electron micrograph of a thin section of hyaline cartilage showing chondrocytes embedded in dense extracellular
matrix. C, Electron micrograph of cartilage matrix at high magnification. This specimen was rapidly frozen and prepared by freeze-substitution to
avoid collapse of the proteoglycans during dehydration and embedding. ER, endoplasmic reticulum. (A, Courtesy D.W. Fawcett and E.D. Hay,
Harvard Medical School, Boston, MA. B, Courtesy of E.D. Hay, Harvard Medical School, Boston, MA. C, Courtesy E.B. Hunziker, M. Mller
Institute, University of Bern, Switzerland.)

A features of both dense connective tissue (an abundance
+ +
Water Uncapped bottle Capped bottle full of
compresses water resists compression
B. Hyaluronan megacomplex trapped by
collagen attracts water
Aggrecan
Hyaluronan
Type II
collagen
FIGURE 32.3 MACROMOLECULAR STRUCTURE AND
MECHANICAL PROPERTIES OF HYALINE CARTILAGE MATRIX.
A, Hydrostatic model of the mechanical properties of cartilage. Water
trapped in the extracellular matrix resists compression. Neither water
alone (in beaker) nor a pliable container (uncapped plastic bottle)
resists compression. However, if water fills a capped bottle, it resists
compression. B, In the cartilage matrix, flexible strands of type II col-
lagen trap proteoglycans, which attract large amounts of water.
Trapped water resists compression because its container, the
network of collagen fibrils, does not stretch.
glycosaminoglycans fill the extracellular space and attract
water, the most abundant component of the matrix.
A hydrostatic mechanism allows cartilage to resist
deformation (Fig. 32.3). Collagen fibrils provide tensile
strength (ie, resistance to stretching) but do not resist
compression or bending. Glycosaminoglycans strongly
attract water, resulting in an internal swelling pressure
that pushes outward against collagen fibrils aligned paral-
lel to the surface of the cartilage. The force of internal
hydrostatic swelling pressure balances the force pro-
duced by tension on the collagen fibrils. Remarkably, this
internally stressed material can resist strong external
forces such as those on the articular surfaces of joints. A
macroscopic analog is a thin-walled plastic bottle filled
with water. One can stand on the bottle provided that it
is sealed, whereas neither the empty bottle nor the water
could separately support any weight.
Specialized Forms of Cartilage
Hyaline cartilage provides mechanical support for the
respiratory tree, nose, articular surfaces, and developing
bones. Elastic cartilage has abundant elastic fibers in
addition to collagen, making the matrix much more
elastic than hyaline cartilage. Elastic cartilage supports
structures subjected to frequent deformation, including
CHAPTER 32 n Connective Tissues 557
the larynx, epiglottis, and external ear. Fibrocartilage has
of thick collagen fibers) and cartilage (a prominent gly-
cosaminoglycan matrix). It is tough and deformable,
appropriate for its role in intervertebral disks and inser-
tions of tendons.
Differentiation and Growth of Cartilage
Cartilage grows by expansion of the extracellular matrix
either from within or on the surface. For surface growth,
mesenchymal cells in the perichondrium differentiate
into chondrocytes that synthesize and secrete matrix
materials. For internal growth, chondrocytes trapped in
the matrix divide and manufacture additional matrix,
which is sufficiently deformable to allow for internal
expansion. Cartilage has a limited capacity to repair
damage, but stem cell transplants can help some patients.
Many growth factors and their receptors cooperate to
influence the differentiation of precursor cells into chon-
drocytes, the proliferation of chondrocytes, and the pro-
duction of cartilage matrix molecules. These include
Indian hedgehog (Ihh), members of transforming growth
factor- family (TGF- and bone morphogenetic proteins
[BMPs]), multiple fibroblast growth factors (FGFs), para-
thyroid hormonerelated protein (PTHrP), and insulin-
like growth factors (IGF-1 and IGF-2). Chondrocytes
produce some of these growth factors (TGF-, FGFs, and
IGFs). During development, adjacent tissues can induce
cartilage formation by secreting TGF- and FGF. SOX9 is
the key transcription factor mediating expression of
cartilage-specific genes.
Diseases of Cartilage
Cartilage fails in common human diseases, including
arthritis and ruptured intervertebral disks. Osteoarthritis,
degeneration of cartilage on joint surfaces, is very
common in older people and has a complex genetic
component attributable to variations in many genes.
Rarely, human diseases are caused by mutations in single
genes for cartilage proteins, growth factors or growth
factor receptors (Appendix 32.1). For example, more
than 25 different mutations of the human gene for type
II collagen cause disorders of cartilage, ranging in sever-
ity from death in utero to dwarfism or osteoarthritis.
Mutations in genes for minor cartilage-associated colla-
gens cause a variety of symptoms, including degenera-
tive joint disease. A premature stop codon in chicken
aggrecan causes lethal skeletal malformations.
Bone
For most vertebrates, bones provide mechanical support
and serve as a storage site for calcium. The great strength
and light weight of bones are attributable both to the
mechanical properties of the extracellular matrix and
to efficient overall design, including tubular form and
558 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

A. Gross-cut section C. H&E stained

Spongy
bone
Compact
bone
Blood vessels in
Haversian canal

Osteocyte in lacunae

Compact
bone D. Dry bone

B. Histological section Blood

vessels
Circumferential
lamellae Haversian
lamellae Interstitial
lamellae

Trabeculae

E. Osteocyte

Volkmann's
canals
Sharpey's
fibers
Calcified Collagen
Haversian matrix fibrils
canal Spongy
bone Filopodia in
cannaliculus
Compact
bone
Gap junctions
between cells
Marrow
cavity

FIGURE 32.4 ORGANIZATION OF LONG BONES. A, Longitudinal section of a shoulder joint of a dried bone specimen. Struts of trabecular
spongy bone reinforce compact bone in the cortex. B, A wedge of long bone. Circumferential lamellae form the outer layer just beneath the
periosteum (blue) covering the surface. Osteons (Haversian systems) consist of concentric lamellae of calcified matrix and osteocytes arranged
around a channel containing one or two capillaries or venules. Interstitial lamellae are fragments of osteons that remain after remodeling (Fig.
32.10). Radial vascular channels connect longitudinal vascular channels to the medullary cavity or periosteum. C, Light micrograph of a cross
section stained with hematoxylin and eosin (H&E) showing circumferential lamellae on the left and two Haversian canals. D, Light micrograph of
a cross section of dried bone showing a central interstitial lamella surrounded by three osteons. Narrow canaliculi connect the lacunae housing
osteocytes. E, An osteocyte surrounded by calcified matrix and extending filopodia into canaliculi. (Micrographs courtesy D.W. Fawcett, Harvard
Medical School, Boston, MA.)

lamination (Fig. 32.4). A superficial layer of compact
bone surrounds a central cavity that is filled with marrow,
fat, or both and is supported by struts of bone arranged
precisely along lines of mechanical stress. External sur-
faces of bones are covered either by dense connective
tissue, called periosteum, or by cartilage at joint sur-
faces. Two cell types make bone matrix: osteoblasts cov-
ering the internal surfaces and osteocytes embedded in
the bone. A third cell type, called the osteoclast, degrades
bone, recycling matrix components. Blood vessels
 CHAPTER 32 n Connective Tissues 559

penetrate compact bone through a network of channels
to supply the central cavity. Although bone is durable
and strong, continuous remodeling makes bone much
more dynamic than it appears.
Extracellular Matrix of Bone
Bone is a composite material consisting of type I collagen
fibrils (providing tensile strength) embedded in a matrix
of calcium phosphate crystals (providing rigidity) (Fig.
32.4E). The calcium-phosphate crystals are similar to
hydroxyapatite [Ca10(PO4)6(OH)2] and make up about
two-thirds of the dry weight of bone. Macroscopic
analogs of the bone matrix are concrete reinforced
by steel rods and fiberglass consisting of a brittle plastic
reinforced by glass fibers. Each of these composites is
stronger than its separate components. Simple extrac-
tion experiments illustrate the contributions of the two
components of bone. After removal of calcium phos-
phate with a calcium chelator, bone is so rubbery that it
bends easily. After destruction of collagen by heating,
bone is hard but brittle.
Fibrils of type I collagen, the dominant organic com-
ponent of the matrix (Table 32.1), are arranged in sheets
or a meshwork. Covalent crosslinks between the colla-
gen molecules in fibrils make them inextensible. The
matrix also contains more than 100 minor proteins,
including growth factors, proteins that promote hydroxy-
apatite deposition and adhesive glycoproteins, but few
proteoglycans.
Cells that make bone lay down type I collagen as the
substrate for crystallization of calcium phosphate. Some
calcium phosphate crystallizes directly in the collagen
matrix. Other crystals form in small matrix vesicles that
bud from the plasma membranes of osteoblasts and use
pumps and carriers to concentrate calcium and phos-
phate. After being released from these vesicles, tiny crys-
tals associate with collagen fibrils. The crystals grow and
eventually fill spaces between the collagen molecules
within the fibrils.
Bone Cells
Overview
A balance among the activities of osteoblasts, osteocytes,
and osteoclasts forms, grows, and maintains bones.
Osteoblasts and osteocytes produce extracellular matrix
and establish conditions for its calcification. Osteoclasts
resorb and remodel bone. An imbalance of these oppos-
ing cellular activities causes human diseases.
Properties of Osteoblasts
A monolayer of osteoblasts on the surface of growing
bone tissue uses a well-developed secretory pathway to
synthesize and secrete the organic components of the
matrix (Fig. 32.5). Osteoblasts also act as endocrine cells,
secreting growth factors that control the differentiation
of osteoclasts (Fig. 32.6), as well as cells in other organs.
They also help to form the niche in the bone marrow for
hematopoietic stem cells (see Fig. 41.4).
Regulation of Osteoblast Development
Osteoblasts arise from the same mesenchymal stem cells
that give rise to fibroblasts and chondrocytes (see Fig.
28.1). Growth factors control the differentiation from
mesenchymal cells. They include Ihh, a subset of BMPs

TABLE 32.1 Bone Proteins

Name Content Functions A
Bone Minor Transforming growth factor
morphogenic (TGF)- homologs; cartilage Osteoblasts
proteins stimulation and bone Bone Calcified
cartilage
development and repair
Collagen type I 90% Forms fibrils in the bone matrix
B
Osteocalcin 1%2% Network of aspartic acid and
-carboxylated glutamic acid side
chains bind hydroxyapatite; Secretory ER
promotes calcification; attracts vesicle
osteoclasts and osteoblasts Type I Golgi Calcified
collagen matrix
Osteonectin 2% Synthesized in developing and
regenerating bone; binds
collagen and hydroxyapatite; may
nucleate hydroxyapatite
crystallization in bone matrix
Osteopontin Minor Arginine-glycine-aspartic acid
(RGD) sequence; binds osteoclast FIGURE 32.5 OSTEOBLASTS. A, Light micrograph of a section
integrins to bone surface of forming bone stained with toluidine blue. Osteoblasts with abun-
Proteoglycans Minor Decorin, biglycan, osteoadherin; dant, blue-stained, rough endoplasmic reticulum lay down bone matrix
may bind TGF- (light green) on the surface of calcified cartilage (light pink). B, Drawing
of osteoblasts. ER, endoplasmic reticulum. (A, Courtesy R. Dintzis
Sialoproteins 2% RGD sequence; binds osteoclast
and from the work of D. Walker, Johns Hopkins Medical School,
integrins to bone surface
Baltimore, MD.)
560 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

A. Osteoclast genesis
Osteoclast RANKL binding to RANK stimulates
Monocytes precursors precursors to fuse and differentiate
into a multinucleated osteoclast

RANK OPG
blocks
M-CSF RANKL
RANKL
Wnt
Supporting cells, Sclerostin Proteins
osteoblasts Osteocyte not to scale

B. Bone remodeling C. Osteoclast

Bone

Osteoclast

Calcified H+ ATPase and

cartilage Cl channels
Cathepsin-K secrete HCl
secreted Sealing
zone
Bone Sealing
zone Ruffled
membrane

Osteoclast

FIGURE 32.6 OSTEOCLASTS. A, Formation of a multinucleated osteoclast by fusion of monocytes stimulated by the receptor activator of
nuclear factor B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and other factors. B, Light micrograph of a section of forming
bone stained with toluidine blue showing two osteoclasts degrading bone and calcified cartilage. C, An osteoclast attached to the bone matrix
by a sealing zone, forming a resorption cavity (pink). The cell pumps H+ and secretes lysosomal enzymes into this cavity to resorb the surface of
the matrix. ATPase, adenosine triphosphatase; OPG, osteoprotegerin (OPG); RANK, receptor activator of nuclear factor B. (B, Courtesy R.
Dintzis and from the work of D. Walker, Johns Hopkins Medical School, Baltimore, MD.)

(see Fig. 24.7), and some Wnts (see Fig. 30.7). Humans
with loss-of-function mutations in a Wnt coreceptor have
few osteoblasts and low bone density, whereas loss-of-
function mutations in a BMP competitor have the oppo-
site effect. On the other hand, osteocytes secrete a
protein called sclerostin that blocks the Wnt corecep-
tor (Fig. 32.6), so loss-of-function mutations of sclerostin
strengthen Wnt signals and promote bone formation.
Inside osteoblasts, Runx2/Cbfa1 is the master tran-
scription factor controlling the expression of genes that
are required to make bone matrix. Runx2/Cbfa1 is part
of a network of transcription factors and microRNAs
with positive and negative influences on osteoblast
differentiation and function. Mouse embryos lacking
Runx2/Cbfa1 have no osteoblasts or osteoclasts. They
make a cartilage skeleton that never transforms to bone.
Humans and mice with just one active Runx2/Cbfa1
gene lack collarbones and experience a delay in the
fusion of joints between skull bones. This syndrome is
the most common human skeletal defect.
Osteocyte Properties
Once an osteoblast has enclosed itself within bone
matrix, it is called an osteocyte. Long-lived, metaboli-
cally active osteocytes are connected to each other by
many long, slender filopodia that run through narrow
channels in the matrix (Fig. 32.4DE). Gap junctions
between the processes of osteocytes provide a con-
tinuous network of intercellular communication that
stretches from cells adjacent to blood vessels to the most
deeply embedded osteocyte.
Osteocytes can either lay down or resorb matrix in
their immediate vicinity. Circulating hormones influence
the activity of osteoblasts and osteocytes. In response to
the calcium concentration in blood, parathyroid glands
secrete parathyroid hormone, which stimulates osteo-
cytes to mobilize calcium from the surrounding matrix.
This feedback loop maintains a constant concentration
of calcium in the blood.
Osteoclast Properties
Osteoclasts form by fusion of blood monocytes and
resorb bone, as required for growth and remodeling.
Osteoclasts are multinucleated giant cells specialized
for bone resorption (Fig. 32.6). They attach like a suction
cup to the surface of bone. Interactions of a plasma
membrane integrin (V3) with bone matrix proteins
(osteopontin and sialoprotein) help to create a leakproof
compartment on the bone surface. Osteoclasts amplify
the plasma membrane lining this closed space, forming

a ruffled border composed of microvilli enriched with
H+-transporting V-type rotary adenosine triphosphatase
(ATPase) pumps (see Fig. 14.6) and chloride channels
(see Fig. 16.13). The combined activities of the H+ pump
and chloride channels allow the cell to secrete hydro-
chloric acid into the sealed extracellular compartment
on the bone surface. This closed space acts like an extra-
cellular lysosome: Acid dissolves calcium phosphate
crystals, and secreted proteolytic enzymes, including
cathepsin K, digest collagen and other organic com-
ponents. Degradation products are taken up by endocy-
tosis and transported across the cell in vesicles for
secretion on the free surface. Amino acids are reused,
but collagen crosslinking groups are not, so they are
excreted in the urine, where their concentration is a
measure of bone turnover.
Osteoclast Formation
Bone marrow supporting cells, osteoblasts, and activated
T lymphocytes produce two proteins that stimulate
blood monocytes to fuse and differentiate into multinu-
cleated osteoclasts (Fig. 32.6). These key factors are
macrophage colony-stimulating factor (M-CSF) and
RANKL (receptor activator of nuclear factor kappa B
[NF-B] ligand, also called osteoprotegerin ligand [OPGL]
or tumor necrosis factorrelated activation-induced cyto-
kine [TRANCE]). Both factors are produced locally in
bone marrow as transmembrane proteins with the
growth factor domain on the cell surface. These proteins
control differentiation through binding to their recep-
tors on monocytes by either direct cell-to-cell contact or
release of the active domain by proteolytic cleavage.
First, M-CSF activates a cytokine receptor (see Fig. 24.6)
on monocytes, stimulating a JAK (just another kinase)
kinasesignal transducer and activator of transcription
(JAK-STAT) pathway (see Fig. 27.9) and turning on
expression of genes required for the monocyte to dif-
ferentiate into a preosteoclast. An important change is
the expression of a receptor called RANK (receptor for
activation of NF-B, a member of the tumor necrosis
factor [TNF] receptor family; see Fig. 24.9). Once this
receptor is expressed, RANKL can activate preosteo-
clasts through the transcription factor NF-B (see Fig.
10.21C) to express the proteins required for cell fusion
and further differentiation into an osteoclast. Mice
that lack RANKL form no osteoclasts, so bone resorp-
tion fails.
Other growth factors, including TNF itself, contribute
to this process by acting directly on osteoclasts, but
many stimulators of osteoclast differentiation (eg, para-
thyroid hormone, vitamin D, leptin) act indirectly
by stimulating supporting cells to make RANKL. For
example, leptin, a satiety hormone secreted by fat cells,
acts on neurons of the hypothalamus in the brain that
regulate not only appetite but also bone metabo-
lism indirectly via the sympathetic nervous system.
CHAPTER 32 n Connective Tissues 561
Norepinephrine released by sympathetic nerves acti-
vates osteoblasts to secrete RANKL. This explains why
animals and people that lack leptin or its receptor not
only are obese but also have dense bones. Osteoclast
growth factors RANKL, TNF, and interleukin-1 mediate
excess bone resorption at sites of chronic inflammation
in rheumatoid arthritis and gum diseases.
Differentiation of osteoclasts is subject to negative
regulation by a soluble decoy receptor for RANKL
called OPG (osteoprotegerin), which binds RANKL and
competes for activation of RANK (Fig. 32.6). Estrogens
inhibit osteoclast differentiation by stimulating osteo-
blasts to produce OPG, so circulating OPG declines in
parallel with estrogen levels after menopause. The result-
ing increase in osteoclasts contributes to bone loss
(osteoporosis) in older women.
Formation and Growth of the Skeleton
Both genetic and environmental information direct for-
mation of the skeleton. Genetic information predomi-
nates in the master plan and initial development of
skeletal tissues, as the size and shape of bones are
characteristic for each species. Subsequently, environ-
mental information is important in remodeling of the
skeleton in response to use. Mutations in genes for struc-
tural and informational molecules have provided valu-
able clues about the genetic blueprint for the skeleton
(Appendix 32.1).
Genetic information is read out on at least two levels.
First, master genetic regulatorsincluding transcription
factors encoded by HOX (homeobox) and PAX (paired
box) genesspecify the developmental fate of each
embryonic segment. Homeoboxes are DNA sequences
that encode a family of 60-residue protein domains that
bind DNA (see Fig. 15.14). The human genome contains
39 HOX genes arrayed in four linear arrays, similar to
those in other animals. HOX genes were discovered in
flies as a result of mutations that cause homeotic con-
version, whereby the fate of one segment is converted
into another, such as the substitution of a leg for an
antenna. The same thing happens in vertebrates: Mouse
embryos express Hoxd-4 in the second cervical (neck)
vertebra and more posterior segments. Mutation of
Hoxd-4 results in the second cervical vertebra taking on
some of the features of the first cervical vertebra. Muta-
tions in other HOX genes cause congenital malforma-
tions in humans. The pathways from HOX genes to
determinants of three-dimensional architecture are still
incompletely understood.
Both systematically circulating and locally secreted
growth factors control the proliferation and differentia-
tion of the cells of cartilage and bone. Mutations in these
factors and their receptors also cause surprisingly spe-
cific human skeletal malformations (Appendix 32.1). Cir-
culating growth hormone produced by the pituitary
562 SECTION VIII n Cellular Adhesion and the Extracellular Matrix
gland is a major determinant of skeletal size. Individuals
deficient in growth hormone are short in stature. Locally
produced growth factors, including BMPs and FGFs and
their receptors, control the development and growth of
cartilage and bone during embryogenesis, in addition to
stimulating repair after fractures. FGF receptors are tyro-
sine kinases (see Fig. 24.4). BMPs are related in structure
and mechanism to TGF- and are expressed in tissues
other than bone and cartilage (see Fig. 24.7). BMPs are
part of a system of positive and negative factors that
regulates formation of cartilage, bone, and joints. For
example, a BMP called GDF-5 specifies the position of
joints, but joints form only if noggin protein, an inhibitor
of other BMPs, is present.
Embryonic Bone Formation
Bone always forms by replacement of preexisting con-
nective tissue. During embryonic development, flat
bones, such as the skull and shoulder blades, form from
neural crest cell precursors in loose connective tissue
(Fig. 32.7). Somehow, information in the genome is read
out as the three-dimensional pattern of a skull. Growth
factors, vitamins (eg, retinoic acid), and local matrix
molecules, such as glycosaminoglycans, all influence the
differentiation of these cells into osteoblasts at specific
locations in connective tissue. Osteoblasts lay down
struts of bone matrix in the loose connective tissue. As
new bone is laid down on the surface of these bone
spicules, some osteoblasts are trapped and become
osteocytes.
During embryonic and postnatal development, genetic
information precisely controls changes in the size and
proportions of flat bones. For example, for the skull to
increase in size both externally and internally, osteo-
clasts on the outer surface lay down new bone at
the same rate as osteoclasts resorb old bone inside
(Fig. 32.8A). These cellular activities are carefully coor-
dinated to change the proportions of the skull as the
individual matures.
A B
Mesenchymal
cells
FIGURE 32.7 BONE FORMATION BY INTRAMEMBRANOUS OSSIFICATION. A, Light micrograph of a section of forming bone stained
with hematoxylin and eosin. Calcified bone matrix is maroon. B, Interpretive drawings. Connective tissue mesenchymal cells differentiate into
osteoblasts, which lay down bone matrix (blue). Osteoblasts become trapped as the matrix grows. (A, Courtesy D.W. Fawcett, Harvard Medical
School, Boston, MA.)
Long bones, such as the humerus, begin as cartilage
models that are replaced by bone (Fig. 32.9). Multiple,
genetically programmed factors induce clusters of mes-
enchymal cells at specific locations to differentiate into
chondrocytes that secrete type II collagen and glycos-
aminoglycans. This produces a miniature cartilaginous
version of the adult bone.
Bone replaces this cartilage precursor in a series of
steps that are coordinated locally by production of
growth factors. Perichondrial cells and proliferating
chondrocytes secrete PTHrP, which promotes chondro-
cyte division and growth. In supporting roles, BMPs
promote and FGFs inhibit the growth and differentiation
of chondrocytes by acting upon populations of cells that
express particular receptors for these molecules (Appen-
dix 32.1). Active FGF receptors stimulate STAT transcrip-
tion factors (see Fig. 27.9) and/or mitogen-activated
protein (MAP) kinase pathways (see Fig. 27.6). More
mature chondrocytes produce Ihh, which directs the
terminal differentiation of neighboring chondrocytes.
For a long bone to maintain its shape as it grows in
size, deposition and removal of bone tissue must be
highly selective. For the shaft to grow in diameter, new
bone is laid down on the outer surface by osteoblasts at
the same time as old bone is removed inside by osteo-
clasts (Figs. 32.8B and 32.9).
Bones grow longer as a result of interstitial growth of
cartilage in the epiphyseal plate and its continual
replacement by bone. Chondrocytes contribute to the
elongation of bones in two ways: chondrocytes continu-
ously proliferate in one zone and then rapidly increase
in mass and swell in the adjacent zone next to forming
bone (Fig. 32.9B). The hypertrophic chondrocytes
secrete type X collagen and use matrix metalloprotein-
ases to resorb some of their surrounding matrix. They
also direct the calcification of the cartilage matrix before
undergoing apoptosis or differentiating into osteoblasts.
Osteoblasts lay down bone matrix on the surface of the
cavities in the calcified cartilage. Hypertrophic cartilage
Osteocyte
Bone
Osteoclast
Osteoblast
Blood
vessel
 CHAPTER 32 n Connective Tissues 563

1
A Hyaline cartilage B Proliferating
Bone collar Cartilage chondrocytes
eroded
Primary
ossification 2
center Hypertrophic
Periosteal chondrocytes
bud invades

Periosteal bud 3
blood vessel Calcified
Medullary
cavity formed cartilage
New apoptosis
spongy
bone
Medullary
cavity Epiphyses
erode
Secondary
ossification
center
4
Epiphyseal Invasion of
Epiphyses blood vessel cartilage
ossify with bone
deposition
Articular
cartilage

Epiphyseal Epiphyseal
plate (cartilage) plate (ossified)

FIGURE 32.8 FORMATION OF A LONG BONE BY REPLACEMENT OF CARTILAGE. A, The shaft grows in diameter as osteoblasts lay
down bone (tan) on the outer surface of the primary collar of bone and osteoclasts remove bone from the inner surface to form and maintain
the marrow cavity. The bone grows in length by interstitial expansion of the cartilage in the epiphyseal plate and its replacement by bone. B, Light
micrograph of a section of an epiphyseal plate stained with toluidine blue. Cartilage growth, differentiation, and replacement by bone occur in
several zones. Proliferation of chondrocytes and their production of matrix (pink) containing type II collagen are solely responsible for the longi-
tudinal growth of the bone (1). Hypertrophic chondrocytes enlarge and make type X collagen, as well as matrix metalloproteinases that resorb
some of the surrounding matrix (2). Chondrocytes die by apoptosis (see Chapter 46), and the matrix calcifies (3). Blood vessels and osteoblasts
move into spaces vacated by chondrocytes and lay down bone (blue) on the surface of calcified cartilage (4). (Micrograph courtesy R. Dintzis
and from the work of D. Walker, Johns Hopkins Medical School, Baltimore, MD.)

A. Skull growth B. Long bone growth

Osteoclast
Growth
plate
25 years
Bone
6 years deposition
(osteoblasts)
Newborn
7 month Bone
fetus removal
(osteoclasts)

FIGURE 32.9 BONE GROWTH. A, Light micrograph of a section of skull stained with Mallorys trichrome stain and an interpretive drawing.
The skull expands during fetal development and growth to adulthood as osteoblasts lay down new bone on the outer surface (blue) and osteo-
clasts resorb bone (pink) on the inner surface. B, Long bones grow entirely by expansion of cartilage in the epiphyseal plate and its replacement
by bone (tan), followed by resorption (pink). (A, Courtesy D.W. Fawcett, Harvard Medical School, Boston, MA.)
564 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

ceases to make the factors that inhibit endothelial cell
growth, allowing FGF-2, TGF-, and vascular endothe-
lium growth factor to attract capillaries as part of the
transformation of cartilage to bone.
Growth of long bones stops at puberty, when high
concentrations of estrogen and testosterone stop prolif-
eration of epiphyseal chondrocytes so that bone replaces
this cartilage. This closure of the epiphyses throughout
the body occurs over several years in a predictable order,
so one can judge the maturity of a child by examining
epiphyses by radiographic studies. Genetic variations
in this process of maturation give rise to differences in
stature. Metabolic and endocrine disorders can also
affect the timing of epiphyseal closure.
Bone Remodeling
Bone is amazingly dynamic and is remodeled continu-
ously in response to stresses. Bone cells and matrix turn
over every few years. Reorganization of bone requires
two carefully coordinated steps: breakdown of preexist-
ing bone by osteoclasts and replacement with new bone
by osteoblasts. More than 100 years ago, Wolff realized
that the strength of a bone depends on use. For example,
bones of the racquet arm of tennis players are more
robust than the bones of their other arm. Thus, mechani-
cal forces on the bones must generate modulatory signals
that control remodeling. Sensory nerves are involved in
some way, but most research has focused on how cells
detect fluid flow through canaliculi.
Primary cilia have been implicated in both the differ-
entiation of bone cells and their responses to mechanical
forces. Part of their function must be in hedgehog signal-
ing (see Chapter 38), but they probably also sense fluid
flowing through canaliculi as a result of mechanical force
on the bone. Accordingly, some mutations in genes for
components of the intraflagellar transport machinery
(see Fig. 38.18) cause severe skeletal defects.
Formation of the cylindrical units of long bones called
osteons is a good example of well-coordinated remodel-
ing. The process involves two steps (Fig. 32.10). First,
osteoclasts resorb preexisting bone to form long,
cylindrical, resorption channels in the same way that a
plumbers snake clears debris from drain pipes. The
second step is slower, as osteoblasts take weeks to fill in
these channels by depositing concentric layers of lamel-
lar bone against the walls. They lay down matrix at a rate
of about 1 m of thickness per day until bone completely
surrounds the blood vessels trapped in the middle of the
newly formed osteon. Because resorption channels cut
randomly through the bone, fragments of older osteons
are left behind during the remodeling of mature bone.
These fragments are called interstitial lamellae.

A B Forming C D
Cutting resorption
cone cavity
Osteoclast

Reversal
zone Resorption
cavity
Blood
vessel
Fibroblast
Time

Osteoblasts
Forming
Closing Haversian
cone system

Quiescent
osteoblast
Completed
Haversian
system

FIGURE 32.10 BONE REMODELING. AB, Longitudinal and cross sections of a time line illustrating the formation of an osteon. Osteoclasts
cut a cylindrical channel through bone. Osteoblasts follow, laying down bone on the surface of the channel until the matrix surrounds the central
blood vessel of the newly formed osteon. C, Steps in the formation of a new osteon. Parts of older osteons are left behind as interstitial lamellae.
D, Microradiograph of a cross section of a long bone, illustrating the range of ages of the structures. A section of bone is placed on x-ray film,
exposed to x-rays, developed, and examined by light microscopy. Older parts of the bone, such as the interstitial lamellae, are more heavily calci-
fied and therefore absorb more of the x-rays, appearing lighter. Newly formed osteons appear the darkest, as they are the least calcified. Vascular
spaces are empty and fully exposed by the x-rays. (A, Modified from Parfitt AM. The action of parathyroid hormone on bone. Metabolism.
1976;25:809844. D, Courtesy D.W. Fawcett, Harvard Medical School, Boston, MA.)

Resorption may release growth and differentiation
factors from the mineralized matrix that provide a local
stimulus for the next round of bone formation by new
osteoblasts.
Bone Diseases
Osteoporosis, a thinning of bones, is common in elderly
people as a result of an imbalance of bone resorption
over renewal. In the United States, osteoporosis results
in 1.5 million painful fractures, costing nearly $20 billion
annually. Almost half of women suffer from such a frac-
ture at some time in their lives, typically as estrogen
levels decline after menopause. Osteoporosis also occurs
at reduced gravitational forces during space flight. The
pathogenesis is not understood, but both behavioral (eg,
inactivity, poor nutrition, smoking) and multiple genetic
factors have modest effects. Among many genetic factors,
one might be naturally occurring variants of the nuclear
receptor for vitamin D. This receptor is a transcription
factor required for vitamin D to stimulate intestinal
calcium uptake and calcification of bone. Variations in
the genes for type I collagen or bone growth factors may
also contribute.
Two strategies are used to treat osteoporosis. The first
is to reduce bone resorption using with bisphosphonates
(pyrophosphate mimics) or injection of either OPG or
antibodies to RANKL, which interfere with osteoclast
formation. New inhibitors of cathepsin K are also being
tested. The other approach is to promote bone forma-
tion with vitamin D, estrogen, calcium, or strontium, but
these measures are only partially effective. More prom-
ising is injection of an analog of parathyroid hormone
or antibodies to sclerostin, which promote osteoblast
activity.
Osteopetrosis is failure of bone resorption, leading
to an imbalance of renewal over resorption. This rare
disease of osteoclasts is fatal in humans, owing to bone
marrow failure. Recessive mutations in the gene for the
V-type proton-ATPase pump (60%), two genes for a chlo-
ride channel (15%), and two genes for proteins involved
with the secretory pathway account for most human
cases. Naturally occurring or engineered mutations in
the genes for essentially any protein required for osteo-
clast differentiation or function cause osteopetrosis in
mice. Osteoclasts are present in bone but fail to function
properly. The disease can be cured in humans and mice
by transplantation of bone marrow stem cells to replace
defective osteoclast precursors, an early example of
stem cell therapy. If the mutation is in the gene encod-
ing RANKL, replacement of this growth factor cures
the disease.
Osteogenesis imperfecta is the name of a variety of
congenital fragile bone syndromes. Severely affected
fetuses die in utero from multiple broken bones. Mildly
affected individuals are born but suffer multiple fractures
resulting in skeletal deformities. All of the patients have
CHAPTER 32 n Connective Tissues 565
mutations in the gene for type I collagen. Some are dele-
tions or insertions, which may be mild. Most patients
with severe disease have point mutations leading to
replacement of a glycine by a larger amino acid. This
prevents the zipper-like folding of the collagen triple
helix (see Fig. 29.1), even if only one chain is defective
per molecule. This poisons assembly and accounts for
the dominant phenotype. No one knows why these
mutations in type I collagen do not affect other tissues,
such as skin, which are rich in type I collagen.
Repair of Wounds and Fractures
Healing of minor skin wounds is a familiar occurrence
that illustrates the mechanisms controlling the assembly
of connective tissue. Repair of connective tissue in the
dermis underlying the epithelium proceeds in three
stages: formation of a blood clot, assembly of provisional
connective tissue, and remodeling of the connective
tissue (Fig. 32.11).
Tissue damage ruptures blood vessels, releasing
blood that clots to stem the hemorrhage and fill the
damaged area. Thrombin, a proteolytic enzyme in
blood plasma, drives the clotting reaction by cleaving
the plasma protein fibrinogen to form fibrin. Fibrin
spontaneously polymerizes and is crosslinked to itself
and to plasma fibronectin. This provisional extracel-
lular matrix of fibrin and fibronectin provides physical
integrity for the clot and an environment for wound
repair. Thrombin also activates seven-helix receptors on
platelets (see Fig. 30.14), stimulating them to secrete
matrix molecules (thrombospondin, fibrinogen, fibro-
nectin, and von Willebrand factor) and growth factors
(platelet-derived growth factor [PDGF], TGF-, and TGF-
) that initiate the cellular events required to complete
wound repair.
Chemotactic factors attract phagocytes from the
blood into the wound. These factors include PDGF, che-
mokines, peptides cleaved from fibrinogen by thrombin,
and peptides from any contaminating bacteria. Neutro-
phils arrive first from the nearby blood vessels, having
attached to activated endothelial cells (see Fig. 30.13)
and migrated into the connective tissue and clot. They
ingest any bacteria. Then monocytes (using a similar
mechanism) migrate into the clot and clear foreign mate-
rial and any dead neutrophils. The environment in a
wound promotes transformation of monocytes into mac-
rophages (see Fig. 28.6), which synthesize and secrete
cytokines and growth factors that mediate the cellular
events that complete the repair process. In this way,
platelets, monocytes, and fibroblasts form a relay, passing
information from one cell to the next.
During the next phase of repair, macrophages, fibro-
blasts, and capillary endothelial cells migrate into the
fibrin clot and reestablish the connective tissue. Endothe-
lial cells form capillary loops that allow blood to flow and
566 SECTION VIII n Cellular Adhesion and the Extracellular Matrix
A TISSUE. A, Wounding removes some tissue and damages blood
Wound in connective tissue
Blood
B Clot of fibrin and
fibronectin forms
Platelets secrete
PDGF and TGF-
Peptides released from
fibrin attract neutrophils
and monocytes
C Neutrophils ingest bacteria
Monocytes differentiate
into macrophages
Macrophages secrete
cytokines
Cytokines attract capillaries
and fibroblasts
D metalloproteinases (see Fig. 29.19) and assembly of more
Fibroblasts secrete
collagen III and
hyaluronan, which
replace the fibrin coat
E cytoskeleton of skin epithelial cells are replaced with
Provisional matrix is
replaced by collagen I
to provide oxygen. Initially, the endothelial cells are
attracted by growth factors released by platelets, but
macrophages and dissolution of fibrin provide a more
sustained supply of chemoattractants and growth factors.
Integrin receptors for fibronectin allow fibroblasts to
FIGURE 32.11 REPAIR OF A WOUND IN CONNECTIVE
vessels, releasing blood into the defect. B, Blood forms a clot of fibrin
and fibronectin, releasing fibrin peptides, and platelets secrete platelet-
derived growth factor (PDGF) and transforming growth factor (TGF)-,
all of which attract neutrophils and monocytes. C, Neutrophils ingest
any bacteria. Monocytes clean up debris and differentiate into macro-
phages, which secrete cytokines, attracting fibroblasts and blood
vessels. D, Fibroblasts secrete type III collagen and hyaluronan, which,
in turn, replace the fibrin clot. E, Fibroblasts remodel the provisional
connective tissue with type I collagen, and blood vessels grow back
into the new tissue.
migrate into the clot. They secrete more fibronectin as
they move. Within the clot, PDGF and TGF- from
macrophages stimulate fibroblasts to secrete type III col-
lagen, hyaluronan, SPARC (secreted protein acidic and
rich in cysteine), and tenascin. Initially, this loose con-
nective tissue is disorganized and weak. Hyaluronan
predominates transiently, but after about five days, it is
gradually replaced by proteoglycans and type I collagen.
Two events complete the repair of the matrix. First,
fibroblasts differentiate into (smooth musclelike) myo-
fibroblasts, which contract the collagen matrix, closing
the edges of the wound. This step is particularly impor-
tant for large wounds. Second, fibroblasts remodel the
provisional connective tissue to restore its original
architecture with nearly normal physical strength. This
requires resorption of provisional collagen fibrils by
robust type I collagen fibrils.
While fibroblasts repair the connective tissue, the epi-
thelium bordering the wound spreads by cell division
and migration to cover the defect. This process of migra-
tion is initiated within hours of wounding. Both the loss
of contacts with neighboring cells at the edge of the
wound and the release of growth factors in the wound
are thought to transform the static epithelial cells into
migrating cells. Keratin filaments that predominate in the
actin filaments. Hemidesmosomes that anchor the skin
epithelial cells to the basal lamina are lost, and the cells
migrate over the surface of the underlying matrix, which
consists initially of fibrin and fibronectin and later of
collagen. As they go, epithelial cells lay down a new
basal lamina. Depending on the size of the defect, pro-
liferation of epithelial cells might be required to com-
plete coverage of the surface. When it is covered, the
cells begin to differentiate into stratified epithelium.
Many parallels exist between repair of a fractured
bone and repair of a skin wound. Blood escapes from
damaged blood vessels and clots at the fracture site.
PDGF released by platelets stimulates mesenchymal
cells to proliferate in the surrounding tissue. These
 CHAPTER 32 n Connective Tissues 567

cells migrate into the clot along with blood vessels and
macrophages. Stimulated by growth factors released ini-
tially by platelets and in a more sustained fashion by
macrophages, mesenchymal cells differentiate into chon-
drocytes and osteoblasts that recapitulate the develop-
ment of new bone to fill in the defect. Although the bone
that is initially produced to join the fractured ends is
poorly organized, fractures are mechanically stable
within approximately 6 weeks. The fibrin clot is con-
verted directly into bone if the broken bone is immobi-
lized. A cartilage intermediate may form first if the
fracture is allowed to move. Over a period of months,
remodeling reestablishes the normal pattern of the bone.
With time, remodeling can even straighten out bones
that are mildly bent at fracture sites.
In all of these examples, wound healing is coordi-
nated by a variety of growth factors and cytokines
and is supported by the environment provided by the
extracellular matrix. For example, PDGF from platelets
stimulates the proliferation of fibroblasts and attracts
them to the fibrin clot at the site of a wound. TGF-
inhibits fibroblast proliferation but stimulates fibroblasts
to make matrix molecules. The actions of cytokines
and growth factors depend on the local environment
in the matrix. In a fibrin clot, TGF- binds to its recep
tor on cells rather than the matrix. In the normal con-
nective tissue matrix, TGF- binds to proteoglycans in
preference to its cell surface receptors, limiting its
effects. In a fibrin/fibronectin clot, cellular fibronectin
receptors bind the matrix, stimulating production of
matrix metalloproteinases that are appropriate for
remodeling the matrix. In normal connective tissue with
less fibronectin, cells produce less metalloproteinase.
The mechanisms that mediate physiological wound
repair can also contribute to disease. For example, PDGF
that is released from activated platelets in clots at the
sites of wounds initiates the cellular events that are
required for repair. On the other hand, when the endo-
thelium lining of large arteries is damaged, binding to the
exposed basal lamina activates platelets. This stimulates
them to release PDGF, which promotes proliferation of
fibroblasts and smooth muscle cells in the artery wall, an
early step in the development of arteriosclerosis.
Plant Cell Wall
The cell walls of land plants are composite materials
consisting of cellulose, other polysaccharides, and glyco-
proteins (Figs. 32.12 and 32.13). Wood and cotton are
two familiar examples of cell wall material that is left
behind after plant cells have died. Like the extracellular
matrix of animals, plant cell walls not only provide
mechanical support but also may influence develop-
ment. Because of these robust cell walls, plant cells are

A B C Microfibrils

CYTOPLASM
OF CELL 1

CELL
WALL

MIDDLE
LAMELLA

CELL
WALL

Microtubules
CYTOPLASM CYTOPLASM
OF CELL 2

FIGURE 32.12 PLANT CELL WALL. A, Confocal fluorescence micrograph of an Arabidopsis leaf with cell walls stained by the periodic
acidSchiff reaction using Acriflavine as the Schiff reagent. BC, Electron micrographs of thin sections of cell walls in the root-like appendages
of the parasitic weed dodder. B, Two cells are separated by an electron-translucent cell wall consisting of cellulose, xyloglycan, and pectins.
The darker area between the two cell walls is the middle lamella, which contains a high concentration of pectins. C, At high magnification,
an oblique section through the plasma membrane and cell wall shows cellulose microfibrils aligned roughly parallel to cortical microtubules
inside the plasma membrane. (A, Courtesy Steven E. Ruzin, University of California, Berkeley. BC, Courtesy K.C. Vaughn, U.S. Department of
Agriculture, Stoneville, MD.)
568 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

A C CYTOPLASM ECM

Golgi vesicle
with matrix
glycans

B. Cellulose synthase Cellulose synthase

monomer complex with 6
enzyme trimers
Active
site

Microfibrils of
Cellulose Matrix ~18 cellulose
glycans polymers

FIGURE 32.13 CELL WALL SYNTHESIS. A, Confocal fluorescence micrograph of an Arabidopsis hypocotyl epidermal cell expressing tubulin
tagged with cyan fluorescent protein (CFP, shown in magenta) and cellulose synthase CESA6 tagged with yellow fluorescent protein (YFP, shown
in green). This is a superimposition of five successive images taken at 10-second intervals to show green particles of CESA aligned with the
magenta microtubules. B, Ribbon diagram and space-filling module of the crystal structure of a bacterial cellulose synthase showing the eight
transmembrane helices, the glycosyltransferase domain in the cytoplasm between helices 4 and 5, and the growing cellulose polymer (white and
red) threading across the membrane. C, Schematic showing the biosynthesis of the cell wall. ECM, extracellular matrix. (A, Courtesy R. Gutierrez,
J. Lindeboom, and D. Erhardt, Stanford University. For reference, see Paredez AR, Somerville CR, Ehrhardt DW. Visualization of cellulose synthase
demonstrates functional association with microtubules. Science. 2006;312:14911495. B, For reference, see Protein Data Bank [www.rcsb.org]
file 4HG6 and Morgan JL, Strumillo J, Zimmer J. Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature.
2013;493:181186. C, Modified from Cosgrove DJ. Loosening of plant cell walls by expansins. Nature. 2000;407:321326.)

not motile. Therefore the morphology of plants is estab-
lished by the orientation of the cell divisions that occur
during their development. Two types of forces act on
cell walls. Internally, the vacuole of the plant cell applies
a high turgor pressure on the order of one atmosphere.
Cell walls also resist a variety of external mechanical
forces that tend to deform the cell.
The main constituent of cell walls is cellulose, the
most abundant biopolymer on earth. It is a long,
unbranched polymer of glucose (see Fig. 3.25). Cellulose
polymers associate laterally into 5- to 7-nm bundles
called microfibrils (Fig. 32.13C). Two types of
branched polysaccharideshemicelluloses and
pectinsassociate with cellulose microfibrils along
with many proteins. Plasma membrane enzymes synthe-
size cellulose, while the other components come from
the secretory pathway and associate with cellulose
outside the cell. Products of more than 1000 genes are
thought to participate in cell wall synthesis.
Plants inherited their genes for cellulose synthases
from bacteria. Arabidopsis has genes for approximately
10 different cellulose synthases. These enzymes consist
of eight transmembrane helices with a cytoplasmic
-glycosyltransferase domain similar to hyaluronan syn-
thase and chitin synthase (Fig. 32.13B). The active site
is exposed to the cytoplasm to provide access to uridine
diphosphate (UDP)-glucose that supplies the glucose
added to the polymer. The transmembrane helices
form a channel for the cellulose polymer across the
membrane. These transmembrane enzymes form a
rosette of six particles that are visible by electron
microscopy, each particle likely to consist of three
enzyme subunits.
Outside the cell the cellulose polymers assemble into
linear crystals called microfibrils. The number of poly-
mers per microfibril was long thought to be 36, but 18
is now the accepted number. Hydrogen bonds constrain
the glucose units to face in alternate directions in planar
ribbons (Fig. 3.25A). These ribbons self-assemble later-
ally into planar crystalline sheets, which stack vertically
into paracrystalline bundles that are held together by
C-HO hydrogen bonds.
Cellulose synthesis moves the rosettes of cellulose
synthase in the plane of the plasma membrane along
paths defined by cytoplasmic microtubules (Fig. 32.13A).
Typically, cytoplasmic microtubules, the tracks of cel-
lulose synthases, and the cellulose microfibrils are all
aligned like barrel hoops perpendicular to the axis of
cellular growth to allow for directed (or anisotropic)
expansion of the cell wall. Cellulose synthesis continues
without microtubules but it is not so well organized.
Newly synthesized microfibrils are deposited between
the cell surface and older cell wall components.
A large number of glycosyltransferases and other
enzymes in the Golgi apparatus synthesize pectins and
hemicelluloses, which are transported in vesicles to the

surface for secretion. Pectins are acidic polysaccharides
that form a gel between microfibrils and play important
roles in cell wall expansion during growth and develop-
ment. Hemicelluloses are branched polysaccharides that
coat microfibrils but are less important than pectins.
Primary cell walls, laid down at the time of cellular
growth and expansion, mature with the addition of gly-
coproteins and organic molecules, such as lignins (poly-
mers of phenylpropanoid alcohols and acids), which
contribute to the integrity of the secondary cell wall.
Covalent and noncovalent bonds are thought to link cel-
lulose and these other matrix molecules. The great
strength and flexibility of tree branches illustrate the
remarkable mechanical properties of mature cell walls.
Cellulose microfibrils are flexible and have a tensile
strength greater than that of steel, so they do not stretch.
For a plant tissue to expand, microfibrils and bonds
among wall components must rearrange, processes facil-
itated in plants and bacteria by matrix proteins called
expansins. The mechanism is still being investigated,
but expansins may break noncovalent links between the
polymers transiently, allowing turgor pressure to expand
the volume of the cell. Genetic defects in expansins
inhibit the growth of plant tissues and the ripening of
some fruits, such as tomatoes. Expansins in grass pollen
are one allergen responsible for hay fever. Localized
chemical modifications of pectins can also loosen the
cell wall and contribute to expansion of plant cells.
Little is known about the molecular basis of plant
cells adhering to their cell walls. By virtue of their phy
sical connection with their product, cellulose synthases
offer one means of attachment. Other plasma membrane
proteins, including a family of serine/threonine kinases
and some proteins with glycosylphosphatidylinositol
anchors, may contribute to adhesion by binding
cell walls. Integrins are conspicuously missing from
plant cells.
CHAPTER 32 n Connective Tissues 569
ACKNOWLEDGMENT
We thank David Ehrhardt for his suggestions on revisions
of this chapter.
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570 SECTION VIII n Cellular Adhesion and the Extracellular Matrix

APPENDIX 32.1

Genetic Defects of Cartilage and Bone

Protein Species Mutation Phenotype
Growth Factors
BMP-4 Human Overexpression Fibrodysplasia progressiva; ectopic bone formation
BMP-5 Mouse Null Defective ears and sternum (short ear mutation)
CSF-1 Mouse Null Osteopetrosis; reduced osteoclasts (osteopetrotic mutation)
GDF-5 (TGF- family) Mouse Null Reduced size of long bones; no joints (brachypodism mutation)
Growth hormone Human Null Reduced size of bones
OPG (osteoprotegerin) Human Null Recessive juvenile Paget disease with excess bone remodeling
PTHrP Human Null Reduced chondrocyte growth; epiphyseal plates fused at birth
RANKL Mouse Null Osteopetrosis; no osteoclasts
Sclerostin Human Loss of function Dense bones, van Buchem disease
Wnt1 Human Loss of function Osteoporosis
Signal Transduction Components
c-Src Mouse Null Osteopetrosis; osteoclasts fail to attach to or degrade bone
Connexin 43 Human Point mutations Dominant oculodentodigital dysplasia
FGF receptor 1 Human Point mutation Pfeiffer syndrome; cranial synostosis; long bone defects
FGF receptor 2 Human Point mutation Jackson-Weiss syndrome; cranial synostosis; long bone defects
FGF receptor 2 Human Point mutation Crouzon disease; cranial synostosis
FGF receptor 3 Human Point mutation Gain-of-function mutation; achondroplasia; short, wide bones
LRP5 Wnt coreceptor Human Loss of function Osteoporosis-pseudoglioma syndrome
Transcription Factors
c-fos Mouse Null Osteopetrosis; no osteoclasts
hoxa-2 Mouse Null Deletion of the second branchial arch; duplication of first branchial arch
hoxd-13 Mouse Null Deletion fourth sacral derivatives; duplication third sacral derivatives
msx-1 Mouse Null Cleft palate
msx-2 Mouse Null Craniosynostosis (fusion of skull bones)
Runx2/Cbfa-1 Human +/ Dominant skeletal defects (cleidocranial dysplasia)
Mouse Null No osteoblasts or bone
SOX9 Human Point mutations Dominant cartilage and skeletal defects (campomelic dysplasia)
Collagen and Other Structural Components of Cartilage and Bone
Aggrecan Mouse Missense Recessive cartilage deficiency; dwarfism; cleft palate
Cathepsin-K Mouse Deletion Osteopetrosis
CLC7 Human Point mutations Osteopetrosis
COL1 Human Missense, Dominant osteogenesis imperfecta; fragile bones
deletions
COL2 Human Nonsense Dominant Stickler syndrome; chondrodysplasia, eye defects
Human Point mutations Dominant chondrodysplasia and osteoarthritis of variable severity
COL9A2 Human Splicing Defective cartilage with degeneration of knee joint
mutation
COL10A1 Human Point mutations Dominant Schmid metaphyseal chondrodysplasia with short bones
COL11A2 Human Exon skipping Dominant Stickler syndrome; chondrodysplasia, eye defects
Human Point mutation Recessive severe chondrodysplasia; deafness; cleft palate
Lysyl hydroxylase Human Point mutation Bruck disease; fragile bones
Perlecan Mouse Deletion Recessive defects in cartilage and bone formation
Proton ATPase Human Point mutations Osteopetrosis
Sulfate transporter Human DTDST gene Recessive cartilage defects; short limbs; joint deformation

ATPase, adenosine triphosphatase; BMP, bone morphogenetic protein; CSF, colony stimulating factor; FGF, fibroblast growth factor; PTHrP, parathyroid
hormonerelated protein; RANKL, receptor activator of nuclear factor B ligand; TGF, transforming growth factor.