Helen Muir

Summary
Chondrocytes are specialised cells which produce and maintain the extracellular
matrix of cartilage, a tissue that is resilient and pliant. In vivo, it has to withstand
very high compressive loads, and that is explicable in terms of the physico-
chemical properties of cartilage-specific macromolecules and with the movement
of water and ions within the matrix. The functions of the cartilage-specific
collagens, aggrecan (a hydrophilic proteoglycan) and hyaluronan are discussed
within this context. The structures of cartilage collagens and proteoglycans and
their genes are known and a number of informative mutations have been
identified. In particular, collagen fibrillogenesis is a complex process which can
be altered by mutations whose effects fit what is known about collagen molecular
structural functions. In other instances, mutations have indicated new functions
for particular molecular domains. As cartilage provides the template for the
developing skeleton, mutations in genes for cartilage-specific proteins often
produce developmental abnormalities. The search for mutations amongst such
genes in heritable disorders is being actively pursued by many groups, although
mutation and phenotype are not always well correlated, probably because of
compensatory mechanisms. The special nature of the chondrocyte is stressed in
connection with its cell involvement in osteoarthritis, the most widespread
Accepted
disease of diarthrodial joints. 18 August 1995

Introduction surrounding chondrocytes is not uniform, as is evident from

The chondrocyte is a cell whose remarkable properties and
capabilities set it apart from other types of mesenchymal
cell. The rounded, undistinguished appearance (Fig. 1) dis-
guises a highly differentiated cell that is the sole architect of
cartilage, a tissue which provides permanent flexible com-
ponents for the skeleton and which, importantly, also serves
as a temporary template for skeletal structures during devel-
opment. This article will address those aspects of chondro-
cytes that are not shared by other types of cell and will con-
sider those structural molecules that are peculiar to
cartilage, a tissue that has to be both pliant and resilient,
and where the architecture of the extracellular matrix is cru-
cial to function at the molecular and organisational levels.
Indeed, the spatial organisation of different macromolecules
newly introduced microscopic methods such as confocal
microscopy (see Fig. 5).
The extracellular matrix of cartilage is a fibre-reinforced
gel, which is beautifully structured in molecular terms for its
purpose and which is produced by surprisingly few cells.
Indeed, the proportion of cells to matrix is much lower in car-
tilage than in other tissues. For example, in adult human
femoral head cartilage there are only 10,000 cells/mm3().
Furthermore, irrespective of the size of a given animal, there
is an inverse relationship between cell density and cartilage
thickness(*). As cartilage is avascular, its nutrition depends
on diffusion from outside and this may limit the total number
of cells that can be sustained in a given volume(*).However,
chondrocytes are remarkable in being able to exist under
very low oxygen tensions and, accordingly, they metabolise
glucose primarily by glycolysis to produce lactate(2).This
anaerobic pathway is preferentially maintained even under
aerobic conditions(3).Chondrocytes are well adapted by low
oxygen consumption to conditions in cartilage where in
deep layers oxygen tension may be as low as YO(^) com-
pared with 24% in normal atmosphere. Anaerobic metab-
olism provides for energy needs(5)and proteoglycan syn-
thesis and, owing to anaerobic glycolysis, ATP levels are
unaffected by incubation under nitrogen or azide treatment,
whereas iodoacetate reduces ATP levels and lactate pro-
duction markedly@).
Chondrocytes, whose prime function is to produce the
matrix of cartilage, are mesenchymal cells that differentiate
during development, but after growth has ceased there is no
detectable cell division in healthy adult articular cartilage().
Articular chondrocytes are thus normally long-lived cells
that survive as long as their owners. The metabolic state of
arrested cell division breaks down, however, whenever the
integrity of the collagen network is compromised, as hap-
pens in the vicinity of lesions in osteoarthritis where cells
appear to have divided anew, albeit rather slowly.
Although cartilage is not confined to diarthroidial joints,
articular cartilage has received most attention as arthritis
and joint abnormalities cause widespread disability. The
physicochemical properties and biomechanical function of
articular cartilage are considered in this article in relation to
the structure and function of matrix structural proteins, and
also to their molecular biology, where recent work on
mutations has provided new insight into function, as in many
cases natural mutations are found to affect function.
Collagen properties and the biomechanics of cartilage
Articular cartilage has to be resilient to compression, yet pli-
ant and undergo limited deformation in order to distribute
compressive forces more widely in the joint. Within joint car-
tilage compressive forces are transient but can be very high,
and rise from 1-2 atmospheres when unloaded(8)to 100-
200 atmospheres on standing, and cycle between 40-50
atmospheres when walking(g).It is surprising therefore that
articular cartilage is 70-80% water(lO)and it is the water,
ions and proteoglycans within the collagenous network
which endow cartilage with its load-bearing properties
(reviewed by Urban()). Collagen accounts for the major
proportion of the dry weight of articular cartilage (50-90%),
which is highest at the surface and, owing to the intrinsic
tendency of cartilage to swell (see below), collagen is under
constant tension. The physical condition of the collagen fib-
rils is therefore crucially important. Decrease in the tensile
stiffness of osteoarthritic cartilage(12)results in increased
water content and softer cartilage(), with separation of col-
lagen fibres(13).
Collagens are extended extracellular proteins composed
of three polypeptide chains (a-chains), each possessing a
characteristic tripeptide sequence (gly-x-y) that forms a left-
handed helix. Three a-chains in each molecule are twisted
tightly into a right-handed helix to form a rope-like structure
that is stabilised by hydrogen bonds, while peptide bonds
are buried inside the helix (Fig. 2). Glycine, placed at every
third residue of the tripeptide sequence, is small enough to
occupy the crowded interior of the helix, while frequent other
amino acids are proline and hydroxyproline. Collagen pre-
cursors or procollagens are synthesised with large C- and
N-terminal extensions which, among other functions, are
involved in chain assembly necessary for triple helix forma-
tion. These extension pro-peptides are cleaved by specific
procollagen peptidases after secretion but prior to fibril for-
mation. Collagen fibrils are further stabilised by cross links
that involve lysine residues. Fibrillar collagen, which is the
biologically functional form, results from a series of post-
translational modifications,both intra- and extracellular,that
require a number of specific and non-specific enzymes. The
formation of fibrils is an entropy-driven process which is not
fully understood. Fibres grow to a uniform thickness by
accretion of single molecules in solution. Mutations that
affect any step of fibril formation are likely to be detrimental
and usually result in some sort of aberrant phenotype, illus-
trating the crucial importance of correct fibril formation to
proper development of cartilage.
Amongst the many different collagens that are known, fib-
rillar type I1 collagen is specific to cartilage and is a marker of
chondrocyte differentiation. What makes it an essential con-
stituent of cartilage? Type I and type I1 collagen molecules
are rather similar in structure and pyridinoline cross links
between fibrils are formed by both types of collagen. Their
fibril morphology is somewhat different, however. In cartilage
the fibrils of type II collagen are thinner than those of type I
fibrils found in other tissues. Fibrillogenesis of type II and
type I collagens have been compared in vitro under con-
ditions resembling those in vivo, using procollagen pepti-
dase to cleave extension propeptides. Fibrils formed from
type II collagen are thinner than those from type I collagen
Fig. 1. Chondrocyte from pig articular cartilage (metacarpal phalangeal joint);
transmission electron micrograph (~13,500). Fixation was in 2%
glutaraldehyde containing ruthenium hexamine trichloride (RHT), followed by
osmium tetroxide, dehydration in an ethanol series and embedding in Araldite
resin (reproduced from ref. 78, courtesy of Dr R. Young).
and of different morphology. The free energy change is lower
and fibril formation is slower than for type I collagen, so that
the differences are intrinsic to each type of collagen(14).An
important difference is that type II collagen forms cross links
with type IX collagen, a minor collagen also specific to carti-
lage. The cross-linking sites and anti-parallel orientation of
the molecules of each type of collagen have been estab-
lished by partial degradation and peptide analysis(15)(Fig. 2).
This orientation allows cross links to form between different
fibrils, these cross links tying them into an open meshwork of
fibres that permits limited but necessary deformation under
compression, as has been observed when wet cartilage is
compressed(16).Proteolytic attack by stromelysin, for exam-
ple, which cleaves type IX collagen near cross-linking
sites(15),can weaken the collagenous meshwork and allow
cartilage to swell, which is an early event in osteoarthritis.
Collagen in articular cartilage is normally very long-lived(17)
and as a consequence undergoes gradual changes and
decline in tensile strength with age(I8).
Matrix architecture
The matrix of articular cartilage is far from uniform in mor-
phology or composition. The fibrillar network is oriented par-
allel to the surface and gradually becomes essentially per-
pendicular with depth from the surface, compatible with the
existence of fibrillar 'arcades' as suggested by Benning-
hoff(2).Collagen fibres are thinner and more closely packed
towards the surface, where the collagen content is higher
than in deeper layers.
Hyaluronan, another key component of the matrix, is an
essential although minor constituent of cartilage, which is
produced by chondrocytes themselves. Particle exclusion
assays show that chondrocytes have surface receptors for
hyaluronan of the same class of hyaloadherins as CD44(I9),
which are distinct from the HA-binding domains of aggrecan
and link protein of cartilage, since hexasaccharides (HAG)
rather than decasaccharides compete with hyaluronan.
Hyaluronan thus provides a gel to which chondrocytes are
able to attach, as well as providing the central component of
aggregates. It has a general stabilising role as it also associ-
ates with other matrix constituents, which are present in car-
tilage but not exclusive to it, such as versican and type VI
collagen. How relatively few chondrocytes construct the
matrix of cartilage in all its complexity is not known, but the
..,
Fig. 2. Schematic models of type II and type IX collagen
molecules in fibril showing antiparallel orientation and
positions of cross links.
molecular topography of articular cartilage suggests that
mechanical forces have a strong influence.
Chondrocytes encapsulate themselves in basket-like net-
works of fine fibrils of elaborate structure, each termed a
chondron. These may be isolated intact and are found to
contain one or several chondrocytes. They are isolated
by controlled low speed homogenisation of cartilage, after
which most cells within the chondrons remain viable and
incorporate 35S04(20). Chondrons appear to be com-
pression-resistant,fluid-filled bladders that dampen mechani-
cal, osmotic and physico-chemical changes induced by
dynamic loading. Their long axes are oriented parallel to the
lines of force operating in situ. lmrnunolocalisation experi-
ments show that chondrons contain several constituents,
includingtype VI, type I I and type IX collagens. Scanning con-
focal microscopy (Fig. 3) shows type IX collagen mainly in the
outer margins of the chondron capsule(21).That the formation
of chondrons is intrinsic and specific to chondrocytes is
suggested by the appearance of chondron-like structures
surroundingchondrocytesthat have been cultured for several
weeks within alginate beads or in agarose(22).
The function of aggregating proteoglycans and the
chondrocytes' environment
The load bearing function of cartilage depends essentially on
the properties of the large cartilage-specific proteoglycan,
named aggrecan (Mr 1-4x106), which is immobilised in the col-
lagen network. It is a bottle brush structure with a core protein
(Mr 220-225) to which are attached laterally along its length as
many as 100 chondroitin sulphate chains and 30 keratan sul-
phate chains (Fig. 4). Aggrecan belongs to the family of
hyaluronan-binding proteins(23), recently termed hyaload-
herins, which are found in most parts of the body. Aggrecan
was the first one to be identified(24)and its binding properties
described. The globular region that binds hyaluronan (GI in
Fig. 4) is located towards the N-terminal end of the core pro-
tein. It associates optimallywith a minimum length of 10 disac-
charides of hyal~ronan(*~), hence many proteoglycan mol-
ecules can bind to a single chain of hyaluronan with high
affinity (KOapprox. M)(26)to form huge multimolecular
aggregates. Newly secreted aggrecan molecules, however,
have low affinity for hyaluronan and are processedslowly into
a high affinity form, both in vitro and in vim, where processing
Type 1X
b C N ,c
Type II Type II
Fig. 3. Chondron clusters. Scanning confocal
image of chondrosarcoma chondron clusters
labelled with antibody to type IX collagen and
visualised with a FITC-conjugated 2nd antibody
and imaged in two optical sections by scanning
confocal microscopy. Differential phase contrast
image (green) merged with fluorescent image
(orange). The two images are contrasted by
artificial computer generated colours (~1000).
(Reproduced from ref. 79, by kind permission of
the authors and the publisher, Chapman & Hall
Ltd.)
becomes slower with age(27).Such mechanisms, which prob-
ably involve re-arrangement of disulphide bonds, would
ensure that newly secreted molecules can diffuse away from
the cell before becoming immobilised by binding to the small
amounts of hyaluronan in cartilage(28).The importance of
aggregation to retain aggrecan in the matrix is shown by the
appearance of aggrecan molecules that are unable to bind
hyal~ronan(~~) in the medium, both of cartilage organ cultures
and of chondrocytes cultured within alginate beads(22).Aggre-
gates throughout cartilage are further stabilised by interaction
with the so-called link protein, which is a relatively small gly-
coprotein. It binds to aggrecan in stoichiometric proportions
(1:l) and to a further segment of hyaluronan, thereby locking
the three constituents together (Fig. 5 ) so that dissociation is
immeasurably The sites in aggrecan and link protein
that bind hyaluronan are similar but not identical looped struc-
tures. Aggrecan and link protein bind to each other via
immunoglobulin folds to form tertiary complexes with hyaluro-
Such aggregates, consistingof perhaps 100 aggrecan
molecules, are so large that they are immobilised in the col-
lagen network. Since each aggrecan molecule is also highly
sulphated, this creates a fixed negative charge within the net-
work such that inorganic counter ions give rise to high osmotic
pressures within the network. This explains why cartilage has
a tendency to swell, but this is resisted by the collagen network
which is therefore under constant tension, even in unloaded
cartilage. High transient loads in articular cartilage are
reversibly accommodated by changes in osmotic and hydro-
static pressure when fluid is forced from loaded to unloaded
areas, while aggrecan remains immobilised within the col-
lagen network provided it is intact and bound to hyaluronan. It
is on this that the load-bearing function of cartilage
depends( l). The structure of aggregates, however, makes
them vulnerable to proteases that attack the hyaluronan-bind-
ing sites of aggrecan and link protein, while hyaluronan itself is
vulnerable to agents that generate oxygen free radicals. Even
limited degradation of hyaluronan would reduce the size of
aggregates so that they would no longer be so effectively
immobilised within the collagen network.
The Donnan equilibrium ensures that in cartilage in vivo,
chondrocytes exist in a relatively acidic environment(lO)and
at a higher osmolarity (380 mOsm) than other types of cell
(280 mOsm), but they are remarkably resilient cells and are
usually cultured under conditions that are not normal for
them. When chondrocytes are isolated and placed in stan-
dard culture medium their extracellular environment changes
sharply and they swell as they are exposed to abnormally low
osmolarities (250-280 mOsm) and their 35S04and 3H-proline
incorporation rate falls by 90% per cell. This change may be
linked to changes in cell volume as synthesis rates can be
partially restored within 30 minutes by addition of NaCl to
increase osmolarity optimally to that found in vivo, i.e. 350-
400 m O ~ m ( ~ Under
). standard culture conditions chondro-
cytes are also exposed to oxygen tensions much higher than
those in vivo and to a significantly higher pH. Moreover, their
physical environment is constant, whereas in vivo they
experience constantly changing osmotic and hydrostatic
pressures (50-150 atmospheres) to which they respond
directly. Mechanisms of mechanotransductionin these cells
are not yet understood, however(ll),in part becausethe influ-
ence of the matrix is so complex; studies using isolated chon-
drocytes are not entirely appropriate for that reason. Fluctuat-
ing loads stimulate sulphate incorporation, whereas static
loads inhibit it. Stimulation is influenced by frequency, ampli-
tude and wave forms of dynamic c o m p r e ~ s i o n ( ~and * ~ ~is~ )
most pronounced in load-bearing areas(34).Hence there is
significant turnover of proteoglycans in vivo. The mean half
life of cartilage in adult human articular cartilage is about 1000
days, as determined in vitro by isotopic labelling(lO),in con-
trast with the extremely slow turnover of collagen in normal
cartilage estimated by racemizationof aspartic acid(7).
Normal genes of matrix proteins and the influence on
function of some natural mutations
The structures of the genes of the principal structural con-
stituents of cartilage are now established and valuable infor-
mation on function may therefore be deduced from the
effects of natural mutations. In many cases phenotypes may
be explained by structural changes to the gene product.
However, others appear anomalous, suggesting that con-
stituents may function in conjunction with others rather than
in isolation.
Table 1 shows those cartilage matrix proteins where
known mutations affect function.
(1) Aggrecan
The aggrecan gene is highly conserved and is similar in
man(35),rat(36)and mouse(37).It has 18 exons, where exons
3-9 encode the globular GI and G 2 domains. The G I con-
tains the hyaluronan-bindingregion (HABR) (Fig. 4), but the
function of G2 is not known, although it is structurally similar
to G I . One interesting mutant found in mouse cartilage
matrix deficiency (cmd), an autosomal recessive condition,
is caused by a 7 bp deletion in exon 5, which produces a
truncated GI domain that cannot bind hyaluronan to form
stable aggregates(37),although normal levels of link protein
as well as type II collagen are produced. The severe pheno-
type that results in homozygotes which die at birth and have
short limbs and cleft palate, illustrates the function and
importance of proteoglycanaggregation in development. As
aggrecan cannot be immobilised in the collagen network of
this mutant, due to defective G1, the matrix is deficient in
proteoglycans. Heterozygotes, however, are normal in size
and phenotype, presumably because sufficient functional
aggrecan is produced. Indeed, the deficient matrix of
cmdlcmd chondrocytes can be corrected by adding normal
exogenous aggrecan to the cultures(38).
Nanomelia in chickens has a similar phenotype where
cartilage matrix is also deficient in prote~glycan(~~), but it is
caused by an entirely different mutation that affects the C-
terminal region of the aggrecan core protein. A single base
mutation produces a stop codon which eliminates the G 3
globular domain and adjacent regions (see Fig. 4) to pro-
duce a somewhat shortened core protein(40).The mutant
PROTEOGLYCAN
n
GI
-G2
NH2
NH2
ohgosaccharides
Link Protein Keratan sulphate
Fig. 4. Schematic molecular models of aggregating proteoglycan and link protein of cartilage modified from ref. 23.G1, hyaluronan binding region (HABr); G2,
unknown function; G3, intracellular trafficking.
aggrecan accumulates in the ER and is not processed in the
Golgi or secreted, and is removed by some apparently non-
lysosomal mechanism. In contrast, the trafficking of type II
collagen is The biological function of the C-termi-
nal globular region of aggrecan G3, which contains EGF-
like and CRP-like domains, is uncertain. The studies of
nanomelic aggrecan strongly suggest that G 3 has a role in
intracellular translocation.
(2) Link protein
Link protein, whose complete nucleotide sequence for the
coding region has been determined(42),is co-expressed
with aggrecan and type II collagen during chondrogenesis.
It is an essential constituent of cartilage but also occurs in
diverse non-cartilaginous tissues, where link protein
enhances and stabilises the interaction of HA-binding pro-
teoglycans with hyaluronan exactly as in cartilage(43).The
size and amino acid sequence of link protein and the
hyaluronan-binding region of aggrecan are very similar(44),
as are their cDNA sequences. The N-terminal domains
belong to the immunoglobulin superfamily (Ig fold)(23),
which includes CD44, the lymphocyte homing receptor. The
human link protein gene has 5 e ~ o n s (and ~ ~ )the mRNA
sequence coding for the translated protein is invariant
between species so far investigated. Exon 3 codes for the
Ig-fold domain and exon 4 the PTR loop which binds
hyaluronan. The untranslated regions 5' and 3' are encoded
by exons 1 and 5, which vary in size between species. The
translated product exists in three forms due to variation in
glycosylation and a specific protein cleavage, but all three
products are functional. Both parts of the molecule that bind
respectively to hyaluronan and aggrecan, are essential in
stabilising aggregates, and the invariant amino acid compo-
sition suggests that there is no scope for variation without
loss of function. No mutations have so far been identified. If
- G3
Chondroitin sulphate
 (i) Type I1 collagen
Most work has focused on type I I collagen, the principal col-
lagen formed by chondrocytes. It is a homotrimer, whose
rope-like fibrils of low extensibility endow cartilage with
great tensile strength. The type II collagen gene, like that of
type I collagen, is a multiexon gene. Alternative splicing
gives rise to two forms; in type IIA exon 2 is present, which
codes for a cysteine-rich domain in the procollagen
aminopeptide. Exon 2 is absent from the type IIB collagen
gene(46).Transcripts of type IIA are expressed predomi-
nantly in non-chondrogenic tissues, but also in regions of
potential cartilage development(47),while chondrogenic
commitment is marked by expression of type llB mRNA.
Triple helical collagen molecules are stable if folded cor-
rectly, when they form fibrils spontaneously by precise lat-
eral aggregation. Imperfect folding interferes with fibril for-
mation, which in turn affects skeletal development and
normal function. Owing to the complexity of the type II col-
lagen gene there are many possibilities for mutations to
occur, some of which may interfere with fibril formation and
stability to a greater or lesser extent. Attempts have been
made to link rare and even common skeletal diseases or
abnormalities with changes in the gene. Natural or experi-
mental mutations that affect triple helix formation are detri-
Fig. 5. Proteoglycan aggregation. (A) Schematic diagram involving the
interaction of proteoglycan monomers and link protein (1 :1 stoichiometry) mental, depending on where the mutation has occurred and
with a chain of hyaluronan. (6) Electron micrograph of proteoglycan whether the mutant gene product interfereswith the function
aggregate spread and visualised by the Kleinschmidt technique. Each arm of normal endogenous collagen. However, severity
represents one proteoglycan molecule (~60,000). By courtesy of J.A.
Buckwalter.
depends not only on where the mutation in the gene
occurred but also on the relative proportions of normal and
mutant collagens incorporated into fibrils.
they do arise they are likely to produce developmental Mutations in regions encoding the triple helical domain,
abnormalities, perhaps similar to those of cmd mice. In the such as replacement of a glycine codon by a codon for a
absence of functional link protein, aggrecan would not be bulkier amino acid, interfere with triple helix formation and
immobilised as stable aggregates within the collagen net- produce a broad spectrum of phenotypes from mild to
work, which would lead to a deficiency of aggrecan in carti- In other patients, those with Stickler syndrome,
lage. Since link protein also occurs in non-cartilaginous the importance of triple helix formation is shown in another
tissues, particularly during development(43),other wide- way. Here mutations which introduce stop codons result in
spread developmental abnormalities would probably result deletions which interfere with triple helix formation(49).How-
from defective function of link protein due to mutation. ever, in-frame deletions of a complete section of 12 exons
encoding part of the triple helical domain of human type II
(3) Chondrocyte collagens collagen gene did not reduce thermal stability of the mutant
Genetic studies have confirmed earlier ideas concerning collagen, presumably because there was enough of the a-
the function of chondrocyte collagens in relation to struc- chain left to form triple helices(50).Propagation of an inter-
ture. Collagen is crucially important to biomechanical func- nally deleted procollagen gene in an inbred strain of mouse
tion and amongst the variety of collagens identified so far, produced widely variable phenotypes from virtually normal
only types I I , IX, X and XI are restricted to chondrocytes. to lethal(51),due presumably to differences in the proportion
Several other types of collagen common to other kinds of of hybrid to normal molecules.
cell are also present but their function in cartilage is not cer- In contrast with such effects of shortened a-chains, over
tain and they will not be discussed here. expression of normal type I1collagen in transgenic mice also
The genes of chondrocyte collagens have now been disrupted normal fibril assembly and caused various devel-
cloned and new information and insight into naturally occur- opmental abnormalities(52).In this case, even though the col-
ring genetic skeletal diseases has emerged. Manipulationof lagen was normal when the transgene exceeded 40% of the
genes and transgenic experiments have provided direct collagen, abnormally thick branched fibrils were formed, pre-
tests for ideas concerning the function of chondrocyte col- sumably because of an imbalance with molecules that may
lagens at the molecular level. regulate fibril width (see type XI collagen section).
Table 1. Cartilage matrix proteins where known mutations
affect their function
Type II Collagen
A fibril forming collagen, principal collagen of cartilage. intervertebral disc
and vitreous Prime marker of cartilage differentiation.
Homotrimer, length 300 nm.
Function: provides tensile strength and stiffness to cartilage.
Type IX Collagen
Fibril associated collagen with interrupted triple helices (FACIT). Lesser
collagen, covalently attached to type II fibrils. Single chondroitin sulphate
chain attached to a ~ ( l X ) Marker
. of cartilage differentiation.
Heterotrimer, length 200 nm.
Function: gives lateral strength with flexibility to fibrillar
network.
Type XI Collagen
Lesser fibril forming collagen in mixed fibrils of type II and type IX
collagens.
Heterotrimer, length 300 nm?
Function: uncertain.
Type X Collagen
Short chain collagen restricted to hypertrophic mineralising cartilage.
Homotrimer. length 150 nm.
Function: uncertain.
Aggrecan
Large, extremely hydrophilic chondroitin sulphate proteoglycan specific to
cartilage intervertebral disc and vitreous. Occupies large solvent volume;
has about 100 chondroitin sulphate and 30 keratan sulphate chains per
molecule. Forms enormous multimolecular aggregates where many
molecules bind to single hyaluronan chains, which are further stabilised by
interaction with link protein. This huge polyanion is immobilised in the
collagenous network.
Aggrecan molecules, approx. length 350 nm.
Multimolecular aggregates, length up to 15000 nm.
Function: endows cartilage matrix with hydrodynamic and
osmotic properties essential for dynamic load bearing.
Besides large changes in the gene, even a single base
change can affect the behaviour of the resulting collagen.
Normal type II collagen lacks cysteine, but in a patient with
early onset osteoarthritis a single base mutation, which
introduced cysteine in place of arginine, resulted in a pro-
portion of disulphide-bonded chains being formed. In the
tissues of the patient the proportion of mutant to normal
chains was 1:3(53).
Mutations causing disease need not necessarily be in the
coding region, however. In another example of early onset
osteoarthritis, closely linked to the type II collagen gene, no
mutation has been identified in the coding region, but
instead may lie in the promoter region(54).Furthermore,
since collagen biosynthesis is a complex process which
involves a series of co-ordinated actions of several
enzymes, mutations affecting the activity of essential
enzymes might lead indirectly to the formation of abnormal
fibres, by analogy with dermatosparaxis in type I collagen
fibrillogenesis.
Although the importance of type II collagen for normal
development and function is evident from these and other
examples, in a case of achondrogenesis, an extremely rare
congenital disease described by Eyre et cartilaginous
tissues contained no type II collagen detectable by
cyanogen bromide peptide analysis; instead type I collagen
was predominant. Surprisingly in the virtual absence of type
II collagen, the mutant foetus, although grossly abnormal,
survived to be born alive at 30 weeks. Type IX collagen,
another marker of chondrocyte differentiation, was never-
theless detected in tissues in this foetus Further studies of
cells from such cases should throw considerable light on
chondrocyte differentiation, which is normally marked by
expression of type llB collagen.
(ii) Type IX collagen
Type IX collagen is a heterotrimer that forms mixed fibrils
with type II collagen (Fig. 2) via intrafibrillar hydroxypri-
dinium cross links(15).A high proportion of molecules of type
IX collagen possess a single chondroitin sulphate chain(56)
attached to a serine residue of the a 2 IX chain, but the amino
acid sequence surrounding the serine residues that bear
chondroitin sulphate chains differs from that in other proteo-
glycans(57).
Whatever the biological function of type IX collagen is, it
is highly conserved, as shown by nucleotide and amino acid
sequence identity between human and chick a2 IX(58).That
its function is important is shown in transgenic mice in which
a truncated form of a1, IX, gave rise to mild chondrodyspla-
sia and osteoarthritis whose severity correlated with the
higher expression of the transgene in homozygotes com-
pared with heterozyg~tes(~~).
(iii) Type XI collagen
Type XI collagen is another minor heterotrimeric collagen
associated with type II collagen. It forms mixed fibrils with
type II and type IX collagen(60).lmmunoelectron micro-
scopic studies show that it is buried inside type II collagen
fibrils, forming a central core which requires physical(60)or
pepsin(6l)disruption to be exposed and react with antibody.
How the formation of a3 (XI) and a1 (11) is regulated, since
they are products of the same gene, is not clear, but this
may involve different processing as they are produced inde-
pendently. Thus immortalised rat chondrosarcoma cells
synthesised type XI but very little type II collagen(62).Con-
versely, in CHO/CHO mice type II collagen can be formed in
the absence of type XI collagen(63).The genetic defect in the
CHO mouse is a single nucleotide deletion, which intro-
duces a translational stop codon downstream of the
deletion(63)when no detectable type XI is formed. Homozy-
gotes have severe abnormalities in cartilage and die at birth.
Their cartilage is fragile and lacks cohesion with abnormally
thick fibrils, indicating an important function of type XI colla-
gen.
(iv) Type X collagen
In addition to providing permanent flexible skeletal compo-
nents resilient to compression, as discussed above, cartilage
serves during development as a temporary template for
skeletal structures. The formation of bone involves the
process of endochondral ossification when chondrocytes in
the growth plate pass through a number of stages of differen-
tiation before they cease to proliferate, become hypertrophic
and start to synthesise type X collagen, whose a-chains have
non-helical globular domains at each end separated by a
relatively short helical region. Type X collagen is formed
exclusively and transiently at sites of calcification by hyper-
trophic chondrocytes. In sifu hybridisation studies of the
expression of type X collagen in foetal human cartilage show
that it follows hypertrophy(64).The sequence of events in
developing embryonic chick sternae has been followed by in
sifu hybridisation, immunocytochemistryand nuclear run-off
assays(65).Transcription of type X collagen occurred two
days before mineralisation (alizarin red staining)(66). stage can be affected by mutation with complete or partial
Expression of the gene is controlled by multiple up-stream
negative elements that act additively to restrict expression to
hypertrophic ~hondrocytes(6~). Although developmentally
regulated, the function of type X collagen remains an enigma.
It binds calcium in a dose-dependent manner and may there-
fore sequester calcium and release it when it is degraded.
Whether this is its prime function remains to be established,
however.
The relationship between cellular proliferation and
response to repressor activity of the promoter of the type X
collagen gene has been shown in chick embryonic sterna,
where expression of type X collagen is restricted to hyper-
trophic non-proliferating cells(68). Repressor activity was
raised in proliferating cells, which contained high levels of c-
myc transcripts undetectable in post-mitotic hypertrophic
cells. Moreover, constitutive over-expression of c-myc, intro-
duced by retroviral vectors, maintained cultured chondro-
cytes in a proliferative state and blocked their maturation into
hypertrophic cells. Decrease in c-myc expression and prolif-
eration thus appears to be necessary for maturation, but the
absence of proliferationalone does not promote hypertrophy,
as chondrocytesin normal adult cartilage do not divide and do
not express type X collagen. They may be in a state of
arrested maturation, which is disturbed in osteoarthritis, and
at sites where re-initiationof endochondral ossificationoccurs
expression of type X is detectable by in sifuhybridi~ation(~~). pension over agarose) for about 2 weeks for significant type
The structure and organisation of the h ~ m a n ( ~ ~and 9 ~ ) II collagen to be demonstrable. Chondrocytes have thus
mouse genes have been established. Unlike that of type II col-
lagen, the gene has only three exons, where exon 3 encodes
the entire triple-helicaldomain as well as the N- and C-terminal
domains. They are closely homologous, as are their gene
products. The importance of type X collagen in skeletal devel-
opment is illustratedby observationsthat Schmidt type human
metaphyseal chondrodyslplasia is associated with mutations
of type X collagen in the carboxy terminal region which may
impair correct chain association (see ref. 72). In transgenic
mice with a dominant negative mutation in the type X collagen
gene, in-frame deletions in the triple helical domain result in
truncated a-chains.These would be expected to interferewith
the formation of stable mixed triple helices(73) between
mutated and endogenous a-chains, which would be unable to
fold properly into triple helices. This is the same situation as
occurs with truncated type II a-chains, except that in type X
collagen the helical region is much shorter, hence truncation
would affect correct folding more acutely. Northern blot analy-
sis showed that the transgene was expressed and localised
immunochemically in hypertrophic chondrocytes. Postnatally
the mice appeared normal but then developed spondylometa-
physeal dysplasia and compression of the growth plate. Para-
doxically, mice lacking type X collagen (type X null mice)
develop normally; their bones at 3 weeks of age are histologi-
cally normal and immunostaining shows other extracellular
matrix constituentsto be
Fibril formation is a complex sequential process and any
loss of function of the resulting collagen. In the case of minor
collagens, this may illuminate their normal function, not
otherwise easily discerned.
Abnormalities in skeletal development can, of course,
arise from causes other than mutations in matrix genes.
Thus the commonest form of achondroplasia has recently
been shown to be due to mutations in the transmembrane
domain of fibroblast growth factor receptor 3 (FGFR3)(75).
This illustrates how mutations that affect key cell regulatory
factors rather than the main structural proteins themselves
can indirectly, but strongly, affect development.
Concluding remarks
Chondrocytes have received rather little attention, being dif-
ficult to culture, not readily available nor easily charac-
terised, as there are no known specific cell-surface markers.
They alone produce and maintain the extracellular cartilage
matrix in which they are sparsely distributed and isolated
from their neighbours. The accepted indicator of the chon-
drocyte phenotype is the production of type It collagen, but
the amounts formed by proliferating monolayer cultures of
chondrocytes are low. Since attachment to a surface is con-
ducive to proliferation, it is necessary to transfer chondro-
cyte cultures to non-proliferative conditions (such as sus-
acquired a reputation for not maintainingtheir phenotype on
proliferation. However, a study of long-term proliferativecul-
ture of adult human articular chondrocytes suggests that
this view may be mistaken(76).The life-span of human chon-
drocytes was not previously known. They replicate much
more slowly than either human fibroblasts or chondrocytes
of other species. They were found to undergo around 35
population doublings in long-term culture before they
became senescent and ceased to divide. Nevertheless,
they retained expression of type II collagen, albeit at a low
level, even when their morphology had become fibroblas-
On the other hand, type I collagen is often regarded
as signifying loss of phenotype, but it is expressed at low
levels even by chondrocytes in fresh human articular carti-
lage, suggesting that formation of type I collagen does not of
itself indicate loss of phenotype.
Normally in adult cartilage chondrocytes are long-lived and
seldom divide, but the capacity to replicate is retained and re-
appears when the collagenous network of their local matrix is
damaged, as occurs in osteoarthritis.Why this should be and
how the response is initiated is not understood. The response
involves increasedsynthesis of matrix proteins, includingtype
I1 collagen, but unfortunately the fabric of the matrix is not
restored to its former state and its inferior biomechanical prop-
erties lead to progressive deterioration of articular cartilage. It
is this failure that underliesthe early stages of osteoarthritis. If
methods are to be developedto arrest this deterioration, better
understanding of chondrocyte cell biology is required. The
study of chondrocytes is made difficult by the need to consider
transient intra- and extracellular physicochemical changes
brought about by mechanical stress to which chondrocytes in
vivo are subjected, and which affect the immediate and also
the long-term activity of the cell, that may last for many hours
afterwards. The situation is further complicated by spatial het-
erogeneity of the matrix and also of the cells, for which there is
evidence of sub-populations. It is also notable that various
specific macromolecules in cartilage are intimatelyassociated
with each other and yet are synthesised and degraded inde-
pendently,which is difficult to explain.
There are several glycoproteins specific to cartilage
whose functions are unknown, such as cartilage oligomeric
protein (COMP)(77). Some of these proteins may be
involved in adhesion of chondrocytes to each other or to
matrix proteins and so affect the behaviour of the cells. In
lesions of human osteoarthritic cartilage where cells have
replicated, the progeny remain close together in groups with
little matrix between cells, within what appears to be
enlarged chondrons. Such cells have not been charac-
terised to establish whether they are normal. The properties
of the local matrix would not be normal, however, and bio-
mechanical studies of normal and localised diseased carti-
lage would be informative.
It is conceivable that abnormalities may also result from
mutations to cartilage glycoproteins, which could affect car-
tilage elsewhere than in the joints as different types of carti-
lage do not all contain the same glycoproteins.
The prime function of cartilage is physical. Studies of sig-
nal transduction and the effects of growth factors, nutrients,
etc. under different biomechanical conditions are likely to be
very informative. Although technically difficult because
mechanical testing must be performed under sterile con-
ditions for long periods, working with articular cartilage has
the advantage that, by careful dissection, other types of cell
may be excluded from the preparation. Thus new insight
into a tissue essential for locomotion and skeletal develop-
ment of vertebrates should result from the adroit combina-
tion of biomechanics and cell biology.
References
1 Venn, M. and Maroudas, A. (1977). Chemical composition and swelling of
normal and osteoarthritic femoral head cartilage. 1. Chemical composition. Ann.
Rheum. Dis. 36, 121-129.
2 Stockwell, R.A. (1979). Biology of Cartilage Cells. Cambridge, Cambridge
University Press.
3 Marcus, R.E. and Srivastava, V.M.L. (1973). Effect of low oxygen tensions on
glucose metabolising enzymes in cultured articular chondrocytes. Roc. SOC.Exp.
Biol. Med. 143,488-491.
4 Brighton, C.T. and Happenstall, R.B. (1971). Oxygen tensions in zones of
epiphyseal plate metaphysis and diaphysis. J. Bone Joint Surg. 53A, 719-728.
5 Spencer, C.A., Palmer, T.N. and Mason, R.M. (1990). Intermediarymetabolism
in the Swarm rat chondrosarcomachondrocyte. Biochem. J. 265,911-914.
6 Mason, R.M. and Sweeney, C. (1994). The relationship between proteoglycan
synthesis in Swarm chondrocytes and pathways of cellular energy and UDP-sugar
metabolism. Carbohydrate Res. 255,255-270.
7 Mankin, H.J. (1963). Localisation of tritiated thymidine in articular cartilage of
rabbits. 111. Mature articular cartilage. J. Bone Joint Surg. 45A, 529-540.
8 Grushko, G., Schneiderman, R. and Maroudas, A. (1989). Some biochemical
and biophysical parameters for the study of the pathogenesis of osteoarthritis. A
comparison between processes of ageing and degeneration. Connect. Tiss. Res.
19,149-176.
9 Afoke, N.Y.P., Byers, P.D. and Hutton, W.C. (1987). Contact pressures in the
human hip joint. J. Bone Joint Surg. 696, 536-542.
10 Maroudas, A. (1980). Physical chemistry of articular cartilage and the
intervertebral disc. In Joints and Synovial Fluid, vol. II (ed. L. Sokoloff), pp. 239-
291. New York. Academic Press.
11 Urban, J.P.G. (1994). The chondrocyte: a cell under pressure. Br. J.
Rheumatol. 33, 901-908.
12 Kempson, G.E., Muir, H., Pollard, C. and Tuke, M. (1973). The tensile
properties of the cartilage of human femoral condyles related to the content of
collagen and glycosaminoglycans. Biochim. Biophys. Acta 237.606-61 0.
13 Stockwell, R.A., Billingham, M.E.J. and Muir, H. (1983). Ultrastructural
changes in articular cartilage after experimental section of the anterior cruciate
ligament of the dog knee. J. Anat. 136,425-439.
14 Fertala, A,, Sieron, A.L., Hojima, Y., Ganguly, A. and Prockop, D.J. (1994).
Self-assembly into fibrils of collagen II by enzymic cleavage of recombinant
procollagen II. J. Biol. Chem. 269, 11584-11489.
15 Wu, J.J., Woods, P.E. and Eyre, D.R. (1992). Identificationof cross-linkingsites
in bovine cartilage type IX collagen reveals an anti-paralleltype Il-type IX molecular
relationshipand type IX to type II bonding. J. Biol. Chem. 267,23007-23014.
16 Broom, N. (1982). Abnormal softening in articular cartilage: its relation to the
collagen framework. Arthritis Rheum. 25, 1209-1216.
17 Maroudas, A., Palla, G. and Gilav, E. (1992). Racemization of aspartic acid in
human articular cartilage. Connect. Tiss. Res. 28. 161.169.
18 Kempson, G.E. (1991). Age-related changes in tensile properties of human
articular cartilage; comparative study between femoral head of hip joint and talus
of the ankle joint. Biochim. Biophys. Acta 1075,223-230.
19 Knudson, C.B. (1993). Hyaluronan receptor directed assembly of chondrocyte
pericellular matrix. J. Biol. Chem. 120, 825-834.
20 Poole, C.A. (1992).The chondron and its pericellular environment. In Articular
Cartilage and Osteoarfhritis (ed. K.E. Kuettner. R. Schleyerbach, J.G. Peyron and
V.C. Hascall). pp. 201-220. Raven Press, New York.
21 Wotton, S.F., Jeacocke, R.E., Maciewicz, R.A., Wardale, R.J. and Duance,
V. (1991). The application of scanning confocal microscopy in cartilage research.
Histochem. J. 23,328-335.
22 Hauselmann, H.J. et a/. (1994). Phenotypic stability of bovine articular
chondrocytes after long-term culture In alginate beads. J. CellSci. 107, 17-27.
23 Hardingham, T.E. and Fosang, A.J. (1992). Proteoglycans, many forms and
many functions. FASEB J. 6,861-870.
24 Hardingham, T.E. and Muir, H. (1972). Specific interaction of hyaluronic acid
with cartilage proteoglycans. Biochim. Biophys. Acta279,401-405.
25 Hascall, V.C. and Heineghrd, D. (1974). Aggregating cartilage proteoglycans
II. Oligosaccharide competitions of proteoglycan-hyaluronic interaction. J. Biol.
Chem. 249,4242-4249.
26 Nieduszynski,LA., Sheehan, J.K., Phelps, C.F., Hardingham,T.E. and Muir,
H. (1980). Equilibrium binding studies of pig laryngeal cartilage proteoglycanswith
hyaluronateoligosaccharidefractions. Biochem. J. 185, 107-114.
27 Sandy, J.D., ONeill, J.R. and Ratzlaff, L.C. (1989). Acquisition of hyaluronate
binding affinity in vivo by newly synthesised cartilage proteoglycans. Biochem. J.
258.875-880.
28 Hardingham, T.E. and Muir, H. (1974). Hyaluronic acid in cartilage and
proteoglycan aggregation. Biochem. J. 139,565-581.
29 Campbell, M.A., Handley, C.J. and DSouza, S.E. (1989). Turnover of
proteoglycans in articular cartilage cultures. Characterisation of proteoglycans
released in the medium. Biochem. J. 259,21-25.
30 Hardingham, T.E. (1979). The role of link protein in the structure of cartilage
proteoglycan aggregates. Biochem. J. 177,237-247.
31 Urban, J.P.G., Hall, A.C. and Gehl, K.A. (1993). Regulation of matrix
synthesis rates by the ionic and osmotic environment of articular chondrocytes. J.
Cell Physiol. 154,262-270.
32 Sah, R.L., Kim, Y.L., Doong, J.Y.H., Grodzinski, A.J., Plaas, A.J. and
Sandy, J.D. (1989). Biosynthetic responses of cartilage explants to dynamic
compression,J, Orthop. Res. 7,619.
33 Hall, A,, Urban, J.P.G. and Gehl, K. (1991). The effects of hydrostatic
pressure on matrix synthesis in articular cartilage. J. Orthop. Res. 9. 1-10,
34 Parkkinen, J.J., Mammi, M.J., Helminen, H.J. and Tammi, M. (1992). Local
stimulation of proteoglycan synthesis in articular cartilage explants by dynamic
compression in vitro. J. Orthop. Res. 10,610-620.
35 Doege, K.J., Sasaki, M., Kimura, T. and Yamada, Y. (1991). Complete coding
sequence and deduced primary structure of the human large aggregating
proteoglycanaggrecan. J. Eiol. Chem. 266,894-902.
36 Doege, K.J., Sasaki, M., Horigan, E., Hassell, J.R. and Yamada, Y. (1987).
Complete primary sequence of the rat cartilage proteoglycan core protein
deduced from cDNA clones. J. Eiol. Chem. 262, 17757-17767.
37 Watanabe, H. eta/.(1994). Mouse cartilage matrix deficiency (cmd) caused by
a 7 bp deletion in the aggrecan gene. Nature Genetics7, 154-157.
38 Takada, M., Iwata, H., Sazuki, S., Brown, K.S. and Kimata, K. (1980).
Correction of abnormal matrix formed by cmdlcmd chondrocytes in culture by
exogenously added cartilage proteoglycan. J. Cell Biol. 103, 1605-1616.
39 Stirpe, N.S., Argraves, W.S. and Goetinck, P. (1987). Chondrocyte from
cartilage deficient mutant nanomelia greatly reduced levels of proteoglycan core
protein transcripts. Dev. Eiol. 124, 77-81.
40 Li, H., Schwartz, N. and Vertel, M.B. (1993). cDNA cloning of chick cartilage
chondroitin sulphate (aggrecan) core protein and identification of a stop codon in
the aggrecan gene associated with the chondrodystrophy nanomelia. J. BIol.
Chem. 268.23504-2351 1.
41 Vertel, M.B., Walter, L.M., Grier, B., Maine, N. and Goetinck, P.F. (1993).
Nanomelic chondrocytes synthesise but fail to translocate a truncated aggrecan
precursor. J. CellSci. 104,939-948.
42 Dudhia, J. and Hardingham, T.E. (1990). Complete nucleotide sequence for
the coding region of human cartilage link protein. Nucl. Acids Res. 18, 1292.
43 Binette, F., Cravers, J., Kahoussie, B., Haudenschild, D. and Goetinck,
P.F. (1994). Link protein in ubiquitously expressed in non-cartilaginous tissues
where it enhances and stabilises the interaction of proteoglycans with hyaluronic
acid. J. Eiol. Chem.269, 19116-19122
44 Neame, P.J. and Barry, F.P. (1993). The link proteins. Experientia 49, 393-
402.
45 Dudhia, J., Bayliss, M.T. and Hardingham, T.E. (1994). Human link protein
gene structure and transcription pattern in chondrocytes. Biochem. J. 303, 329-
333.
46 Ryan, M.C. and Sandell, L.J. (1990). Differential expression of a cysteine-rich
domain in the amino terminal propeptide of type I1 (cartilage) procollagen by
alternative splicing of mRNA. J. Biol. Chem. 265, 10334-10339.
47 Sandell, L.J., Nalin, A.M. and Reife, R.A. (1994). Alternative splice form of
type II procollagen mRNA (HA) is predominant in skeletal precursors and non-
cartilaginoustissues in early mouse development. Devel. Dynamics199, 129-139.
48 Kokko, J., Ritvaniemi, P., Haatja, L., Kaariainen, H, Kivirikko, K.I. and
Prockop, D.J. (1993). Mutation of type I1 procollagen (Col2Al) that substitutes
aspartate for glycine a1 67 and that causes cataracts and retinal detachment;
evidence for molecular heterogeneity in Wagner's syndrome and Stickler's
syndrome (arthro-ophthalmopathy).Am. J. Hum. Genet. 53,55-61.
49 Ritvaniemi, P., Hyland, T., Ignatius, J., Kivirikko, K.I., Prockop, D.J. and
Ala-Kokko, L. (1993). A fourth example suggests that premature termination
codons in the Col2A1 gene are a common cause of the Stickler syndrome;
analysis of the Go12 A1 gene by denaturing gradient gel electrophoresis.
Genomics17.218-221.
50 Sieron, A.L., Fertala, A., Ala-Kokko, L. and Prockop, D.J. (1993). Deletion of
a large domain in recombinant procollagen II does not alter thermal stability of the
triple helix. J. Biui. Chem. 268.21232-21237.
51 Helminen, H.J. et a/. (1993). Inbred line of transgenic mice expressing an
internally deleted gene for type II procollagen (Coll2Al). Young mice have a
variable phenotype of a chondrodysplasia and older mice have osteoarthritic
changes. J. Clin. Invest. 92, 582-595.
52 Garafolo, S. eta/. (1993). Assembly of cartilage collagen fibrils is disrupted by
over-expression of normal type I I collagen in transgenic mice. Proc. NaN Acad.
So. USA 90.3825-3829.
53 Eyre, D.R.,Weis, M.A. and Moskowitz, R.W. (1991). Cartilage expression of
a type II collagen mutation in an inherited form of osteoarthritic associated with a
mild chondrodysplasia.J. Clin. Invest. 87. 357-362.
54 Vikkula, M. et a/. (1993). Early onset osteoarthritis linked to the type I1
procollagen gene. Detailed clinical phenotype and further analysis of the gene.
Arthritis Rheum. 36,401-409.
55 Eyre, D.R., Upton, M., Shapiro, F.D., Wilkinson, R.H. and Vawter, G.F.
(1986). Non-expression of cartilage type I1 collagen in a case of Langer-Saldino
achondrogenesis. Am. J. Hum. Genef. 39, 52-67.
56 Bruckner, P., Vaughan, L. and Winterhalter, K.H. (1985). Type IX collagen
from sternal cartilage of chicken embryo contains covalently bound
glycosaminoglycan. Roc. Natl Acad. So. USA 82. 2608-2612.
57 Huber, S., Winterhalter, K.H. and Vaughan, L. (1988). Isolation and
sequence analysis of the glycosaminoglycan attachment site of type IX collagen.
J. Eiol. Chem. 263,752-756.
58 Perala, M., Hanninen, M., Hastbacka, J., Elima, K. and Vuorio, E. (1993).
Molecular cloning of the human alpha 2 (IX) collagen cDNA and assignment of the
human Co19A2 gene to chromosome 1. FEES Lett. 319, 177-180.
59 Nakata, K. etal. (1993). Osteoarthritis associated with mild chondrodysplasia
in transgenic mice expressing a1 (IX) collagen chains with central deletion. froc.
NaflAcad. Sci. USA 90,2870-2874.
60 Mendler, M., Eich-Bender, S.G., Vaughan, L, Winterhalter, K.H. and
Bruckner, P. (1989). Cartilage contains mixed fibrils of collagen types 11, IX and
XI. J. CellEiol. 108, 191-197.
61 Petit, B, Ronziere, M.C., Hartman, D.J. and Herbage, D. (1993).
Ultrastructural organisation of type XI collagen in fetal bovine epiphyseal cartilage.
Histochemistry 100,231-239.
62 Oxford, T.J., Doege, K.J., Horton, W.E. and Morris, N. (1994).
Characterisation of type II and type XI collagen synthesis by an immortalised rat
chondrocyte cell line (IRC) having a low level of type II collagen mRNA expression.
Exp. CellRes. 213,28-36.
63 Li, Y. eta/. (1994). A fibrillar collagen gene, coll II a1 , is essential for skeletal
morphogenesis. CeN80,423-430.
64 Reichenberger, E., Aigner, T., von der Mark, K., Stoss, H.V. and Bertling,
W. (1991). In situ hybridisation studies on the expression of type X collagen in fetal
human cartilage. Dev. EIol. 148,562-572.
65lyama, K., Ninomiya, Y., Olsen, B.R., Linsenmayer, T.F,Trelstad, R.L. and
Hayashi, M. (1991). Spatiotemporal pattern of type X collagen gene expression
and collagen deposition in embryonic chick vertebrae undergoing endochondral
ossification. Anat. Rec. 229,462-472.
66 Lu Valle, P., Daniels, K., Hay, E.D. and Olsen, B.R. (1992). Type X collagen is
transcriptionally activated and specifically localised during sternal cartilage
maturation. Matrix 12, 404-413.
67 Lu Valle, P., Iwamoto, M., Fanning, P. and Pacifici, M. (1993). Multiple
negative elements in a gene that encodes for an extracellular matrix protein
collagen X restrict expression to hypertrophic chondrocytes. J. Cell Eiol. 121,
1173-1179.
68 Iwamoto, M. et a/. (1993). Expression and role of c myc in chondrocytes
undergoing endochondral ossification. J. Eiol. Chem. 268, 9645-9652.
69 Hoyland, J.A. et a/. (1991). Distribution of type X collagen mRNA in
osteoarthritic human cartilage. Bone Miner. 15, 151-163.
70 Reichenberger, E.M., Beier, F., Lu Valle, P., Olsen, B.R., von der Mark, K.,
Bertling, W.M. (1992). Genomic organisation and full length cDNA sequence of
human collagen X. FEBS Left. 31 1,305-310.
71 Thomas, J.T. etal. (1992). The human collagen X gene. Complete primary
translated sequence and chromosomal localisation. Eiochem. J. 280, 617-623.
72 Jacenko, 0..Olsen, B.R. and Warman, M.L. (1994). Invited Editorial. Of mice
and men: heritable skeletal disorders. Am. J. Hum. Genet. 54, 163-168.
73 Jacenko, O., Lu Valle, P. and Olsen, B.R. (1993). Spondylometaphyseal
dysplasia in mice carrying a dominant negative mutation in a matrix protein
specific for cartilage to bone transition. Nature365, 56-61.
74 Rosati, R. et a/. (1994). Normal bone growth and development in type X
collagen null mice. Nature Genefics8, 129-135.
75 Shiang, R. etal. (1994). Mutations in the transmembrane domain of FGFR3
cause the most common genetic form of dwarfism. achondroplasia. Cell78. 335-
342.
76 Kolettas, E., Buluwela, L., Bayliss, M.T. and Muir, H. (1995). Expression of
cartilage-specific molecules is retained on long-term culture of human articular
chondrocytes. J. Cel/Sci 108, 1991-1999.
77 Heinegiird, D and Oldberg A. (1993) Glycosylated matrix proteins. In
Connective Tissue and its Heritable Disorders (ed. P.M. Royce and 6 .
Steinmann). pp 189-209. Wiley-Liss, New York.
78 Hunziker, E.B., Hermman, W. and Shenk, R.K. (1982). Improved cartilage
fixation by RHT. J. Ultrastruct. Res. 81,l-2.
79 Wotton, S.F., Jeacock, RE., Maciewicz, R.A., Wardale, R.J. and Duance,
V.C. (1991). The application of scanning confocal microscopy in cartilage
research. Histochem. J. 23,332-335.
Helen Muir is at the Department of Biochemistry, Charing Cross
and Westminster Medical School, Fulham Palace Road, London,
W6 8RF, UK.