07-Expression and distribution of cartilage matrix macromolecules in Avian tibial dyschondroplasi

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Avian Pathology
ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: www.tandfonline.com/journals/cavp20
Expression and distribution of cartilage matrix
macromolecules in Avian tibial dyschondroplasia
Chris Tselepis, Judith A. Hoyland, Ruth E. Barber, Barry H. Thorp & Alvin P. L.
Kwan
To cite this article: Chris Tselepis, Judith A. Hoyland, Ruth E. Barber, Barry H. Thorp & Alvin P.
L. Kwan (1996) Expression and distribution of cartilage matrix macromolecules in Avian tibial
dyschondroplasia, Avian Pathology, 25:2, 305-324, DOI: 10.1080/03079459608419143
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Avian Pathology (1996) 25, 305-324
Expression and distribution of cartilage matrix
macromolecules in avian tibial dyschondroplasia
CHRIS TSELEPIS1, JUDITH A. HOYLAND1, RUTH E. BARBER2,
BARRY H. THORP3 & ALVIN P. L. KWAN4,5
1 School of Biological Sciences, University of Manchester, 2.205 Stopford Building,
Oxford Road, Manchester M13 9PT, 2Chiroscience Ltd, Cambridge Science Park,
Milton Road, Cambridge CB4 4WE, 3Roslin Institute, Roslin, Midlothian,
Scotland, and 4School of Molecular and Medical Biosciences, University
of Wales College of Cardiff, Museum Avenue, PO Box 911, Cardiff
CF1 3US, UK
SUMMARY
Tibial dyschondroplasia (TD) is a disorder of endochondral ossification character-
ized by the presence of an avascular, non-mineralized cartilage lesion extending
from the growth plate into the metaphysis. Cells within the TD growth plate
fail to differentiate to full hypertrophy, and instead appear to maintain a
'pre-hypertrophic' or 'transitional' status. The synthesis and distribution of
aggrecan, biglycan, decorin, and collagen types II and X in the growth plates
of normal and tibial dyschondroplastic chickens have been investigated using
in situ hybridization and immunolocalization. Marked reductions in the amount
of aggrecan and biglycan core protein mRNAs were observed in the tibial dys-
chondroplastic lesion by in situ hybridization. Reduction in mRNA production
seemed to be specific to the extracellular matrix components since total mRNA
expression showed no significant difference between normal and dyschondro-
plastic cartilage. In addition, expression of collagen type II and decorin did
not differ significantly between normal and TD cartilage. Distribution of aggrecan
biglycan, decorin, type II and X collagens were examined using immunohisto-
chemistry. Normal hypertrophic cartilage showed a strong matrix labelling
for aggrecan and biglycan. Type X collagen in the normal hypertrophic cartilage
showed strong pericellular and matrix distribution, whereas in TD cartilage
labelling for aggrecan, biglycan and collagen X was located intracellularly with
a very low level of signal in the matrix. In contrast, collagen type II was found
to be present throughout the extracellular matrix of both the normal growth
plate and the TD lesion, suggesting that the differences observed in aggrecan,
biglycan and type X collagen distribution are specific to these proteins and
not a general disturbance of matrix macromolecular metabolism. The reduced
deposition of these macromolecules may have implications in normal and patho-
logical bone development.
Received 20 July 1995; Accepted 30 October 1995.
5To whom correspondence should be addressed.
0307-9457/96/020305-20 © 1996 Houghton Trust Ltd
306 C. TSELEPIS ETAL.
INTRODUCTION
Endochondral ossification, the process by which long bones elongate, takes place
in the epiphyseal growth plate. Chondrocytes within the growth plate follow a
stringently controlled differentiation pathway of proliferation, maturation and
hypertrophy, which culminates in mineralization of the extracellular matrix, and
the invasion of blood vessels and osteogenic cells from the bone marrow (for
review, see Poole, 1991). Each stage of this process is accompanied, not only by
changes in chondrocyte morphology, but by variations in the biosynthetic activity
of the cells and, consequently, by alterations in the composition of their extracel-
lular matrix. For example, type X collagen is exclusively expressed by hyper-
trophic chondrocytes and appears to be implicated in the process of
mineralization (Kwan et al., 1986; Schmid & Iinsenmayer, 1987). The cartilage
proteoglycans are a major component of the growth plate extracellular matrix
interacting with collagen, cell membranes and tissue specific proteins, and play a
fundamental role within the processes of endochondral ossification. However, the
complex organization of the extracellular matrix (ECM) in the growth plate and
the structure-function relationship is still not fully understood. Changes in mol-
ecular composition within the cartilage matrix may contribute significantly to
normal and pathological skeletal development. As part of the study to investigate
the involvement of the changes in the composition of the ECM in endochondral
ossification, we utilized a model of aberrant bone formation, namely avian tibial
dyschondroplasia (TD).
Avian TD is an abnormality of the growth plate commonly seen in the proximal
tibiotarsi of chickens and turkeys. This disease is characterized by an accumula-
tion of avascular cartilage (lesion) extending from the growth plate into the
metaphysis (Leach & Nesheim, 1965). The gross manifestation of the disease is
lameness, bowing (Nairn & Watson, 1972; Sauveur & Mongin, 1978; Rowland,
1988) and fractures (Riddell eta!., 1971; Meinecke et al., 1980) of the tibiotarsus.
Under the microscope the lesion appears to be a mass of transitional chondro-
cytes which have failed to undergo full hypertrophy (Poulos, 1978). The cartilage
is composed of smaller ovoid lacunae and contains more matrix than normal
hypertrophie cartilage (Thorp, 1992). Furthermore, it has recently been shown
(Farquharson et al., 1992) that the lesion is not a result of increased cell
proliferation or decreased résorption, but that the aetiology of the disease is
related to the control of chondrocyte differentiation, mineralization and vascular-
ization (Loveridge et al., 1993). In broiler chickens, the incidence of lameness
from tibial dyschondroplasia varies from less than 1% up to 40% (Siller, 1970;
Prasad et al., 1972; Friedman, 1977), whereas subclinical lesions can affect up to
60% of the birds (Riddell et al., 1971; Poulos, 1978; Riddell, 1981; Cruickshank
& Sim, 1986). A high incidence of TD can also be induced by a dietary imbalance
of calcium and phosphorus (Leach & Nesheim, 1965; Edwards & Veltmann,
1983; Riddell & Pass, 1987).
Avian TD is a good model for the study of the biology of aberrant bone
formation because of the high frequency of lesions, ease of induction and a
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 0 7
similar pathology to that found in other species. In this study, we have utilized
avian TD as a disease model by which to investigate the relationship between
synthesis and distribution of the matrix macromolecules within the growth plate
and their putativeroles in endochondral ossification. The synthesis and distri-
bution of the three main cartilage proteoglycans, aggrecan, biglycan and decorin,
and collagen types II and X between normal hypertrophie and TD cartilage were
examined and compared, with the ultimate aim of gaining a better insight into
their roles in the processes of endochondral ossification, and to understand more
about the biochemistry of the initiation and pathological development of avian
tibial dyschondroplasia.
MATERIALS AND METHODS
Production of chickens with tibial dyschondroplasia
Experiments were conducted using groups of 15 broiler chicks (Cobb strain)
reared under two different dietary regimes in compartments of electrically heated
tier brooders. Food and water was available ad libitum and diets based on wheat
and soya bean meals, were either a control diet calculated to contain 12 g/kg Ca
and 7.6 g/kg P, or a TD-inducing diet, calculated to contain 7.5g/kg Ca and 7.6
g/kg P (Rennie et al., 1993). At 3 weeks of age all the birds in each group were
killed by cervical dislocation. The right and left proximal tibiotarsi were dissected
and sectioned. Most of the tissue was then prepared for in situ hybridization and
immunolocalization. In addition small samples were processed for histological
examination. Growth plates were classified as normal or dyschondroplastic on the
basis of gross and histopathology. Dyschondroplasia was considered to be present
when the proximal tibiotarsal metaphysis contained a plug of avascular physeal
cartilage comprised of transitional chondrocytes (Leach & Nesheim, 1965;
Hargest et al., 1985). The dyschondroplastic growth plates selected for further
study contained 'plugs' of moderate size, typically 4 X 6 mm.
Tissue preparation
Ten normal and 10 tibial dyschondroplastic growth plates were initially fixed for
24 h in 10% (v/v) neutral buffered formalin and then decalcified in 20% (w/v)
EDTA (pH 7.2) until radiologically decalcified (10 to 14 days). Following
décalcification, the tissue was processed and embedded in paraffin wax, and
sections of 7 /¿m thickness were mounted on to silanated slides (Rentrop et al.,
1986).
In situ hybridization
Probes
Human decorin (820 bp fragment within the coding region of human decorin
cDNA) and biglycan (360 bp fragment of the 3' untranslated region from the
308 . C. TSELEPIS ETAL.
human biglycan cDNA) were inserts in pBlue SK". Both probes were kindly
donated by Dr M. Young, NIH. Bovine aggrecan cDNA (2 kb) was a gift from
Dr S. Manning (Department of Anatomy & Developmental Biology, University
College, London) and was subsequently cloned into pBlueSK". Riboprobes of
aggrecan, decorin and biglycan were synthesized using the Boehringer Mannheim
SP6 T7 transcription kit and [32P]aCTP. All synthesized riboprobes were further
hydrolysed to 200 bp fragments before use. Human type II collagen (phcarl, 224
bp) was supplied by Dr J. Hoyland. The [35S]adCTP-labelled type II collagen
cDNA probe was synthesized using a random prime labelling kit (Amersham
International, UK).
Hybridization using cDNA probes
The prehybridization treatments were as described previously by Hoyland et al.
(1991). Briefly sections were immersed sequentially in 0.2 N HC1 for 20 min;
0.2 X SSC [SSC (standard saline citrate) = 0.15 M NaCl and 0.015 M sodium
citrate] for 10 min; 5 /ig/ml of proteinase K in 50 mM Tris-HCl, pH 7.5 for 1 h
at 37°C; 0.2% (w/v) glycine in phosphate-buffered saline (PBS) for 2 min; and
0.4% (w/v) paraformaldehyde in PBS, pH 7 for 20 min and finally in freshly
prepared 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine pH 8 for 20 min.
Following proteinase treatment, serial sections were reacted with 1 mg/ml of
RNAase A in 0.5 X SSC for 30 min at 37°C. All sections were then prehybridized
for 1 h at 37°C in 50% formamide, 1 mg/ml of bovine serum albumin, 0.02%
(w/v) Ficoll, 0.02% (w/v) polyvinyl pyrrolidone, 0.6 M NaCl, 0.2 mg/ml of
sheared salmon sperm DNA, 10 mM Tris (pH 7.4), 0.5 mM EDTA 10 mM
dithiothreitol (DTT) and 10% (w/v) dextran sulphate. Hybridization with
heat-denatured 35S-labelled probes (100 ng/ml prehybridization mixture)
was carried out at 37°C overnight in prehybridization solution. Aliquots of
50 fú were applied to each slide and covered with siliconized coverslips. After
hybridization the tissue sections were washed with a series of high stringency
washes: twice for 5 min in 0.5 X SSC containing 1 mM EDTA and 10 mM DTT;
twice for 5 min in 5 X SSC containing 1 mM EDTA; 15 min in 5 mM Tris
(pH 7.5) containing 50% formamide, 0.15 M NaCl and 0.5 mM EDTA;
four times for 5 min in 0.5 X SSC at 49°C, followed by 5 min at room tempera-
ture in 0.5 X SSC. Slides were then dehydrated in 95% ethanol and air dried.
Autoradiography was performed with Ilford K5 emulsion melted at 40°C and
diluted with an equal volume of distilled water. The slides were exposed at 4°C
for 14 days, and then developed in Kodak D-19 developer and counterstained
with haematoxylin and eosin.
For detection of total message, a synthetic oligo-dT probe of 25-30 nucleotides
(Pharmacia LKB) was 3'-labelled with digoxigenin-11-UTP using terminal de-
oxynucleotidyl transferase (TdT) (Lewis et al., 1985). The corresponding sense
probes were used as controls.
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 0 9
Hybridization using riboprobes
The initial prehybridization treatments were similar to those described for cDNA
probes, with the exception that 4% (w/v) of paraformaldehyde for 5 min was
used. All sections were then prehybridized at 50°C for 1 h with prehybridization
buffer containing 50% formamide, 0.3 M NaCl, 20 mM Tris (pH 8), 5 mM
EDTA (pH 8), 10 mM DTT, 1 X Denharts, 1 mg/ml tRNA. Hybridization with
heat-denatured 32P-labelled probe and 10% (w/v) dextran sulphate in prehy-
bridization solution, was carried out at 50°C overnight. Aliquots of 50 /A were
applied to each slide and covered with siliconized coverslips, they were immersed
in 2XSSC and 10 mM DTT to remove the coverslips. After hybridization the
tissue sections were immersed in 2 X SSC and washed at room temperature for 1
h. The sections were then washed for 4 h at 50°C in wash buffer, consisting of
50% formamide, 0.6 mM NaCl, 0.02 mM Tris-HCl (pH 8), 1 mM EDTA (pH
8), 10 mM DTT and 1 X denharts. Sections were subsequently washed in 10 mM
Tris-HCl (pH 8) containing 0.5 M NaCl, and 1 mM EDTA (NTE buffer).
Digestion with RNAse A (20 mg/ml) and RNAse Tl (100 units) was then carried
out at 37°C for 30 min, after which the slides were washed in wash buffer
overnight at 50°C. The sections were then washed in 2 X SSC at room tempera-
ture for 30 min, followed by washing in 0.1 X SSC for 30 min at room tempera-
ture. Finally, the slides were dehydrated and air dried for autoradiology.
Hybridization of poly d(T) probe
Prehybridization treatment was similar to that quoted for cDNA with the excep-
tion that after the fixation step, the sections were treated with prehybridization
buffer; 0.6 M NaCl 1 X phosphoethanolamine. 0.01% (w/v) salmon sperm DNA
and 10% (v/v) polyethylene glycol 6000, at 37°C for 1 h. Following this, the
hybridization mixture (prehybridization buffer with the addition of the digoxi-
genin-labelled probe) was added to the sections and incubated overnight at 37°C.
The sections were washed three times in 2 X SSC at 37°C for 10 min and then
washed in 2 X SSC for 10 min at room temperature. Sections were blocked using
Boehringer Blocking reagent for 30 min. Following this step, the buffer was
removed and the anti-digoxigenin-AP applied (diluted 1 in 250 in blocking
solution) on to sections for 2 h. The sections were then washed twice with buffer
1; 100 mM Tris (pH 7.4), 150 mM NaCl, for 15 min, followed by a 3-min wash
in veronal acetate buffer; 30 mM NaAc, 30 mM sodium barbitone, 100 mM
NaCl and 50 mM MgCl2 6H2O, (pH 9.2). Detection solution, (dissolved initially
in dimethyl formamide) made up in veronal acetate buffer, was then applied to
the sections, incubated for 1 h at room temperature. Sectionswere counterstained
with haematoxylin, mounted and examined.
Immunoperoxidase localization
Paraffin-embedded, decalcified tissue sections of 7 ¿an were mounted on to
310 C. TSEUEPIS ETAL.
silanated slides and baked for 1 h at 60°C. After dewaxing in xylene, sections were
washed in 99% alcohol and immersed in methanolic peroxidase; 3% (v/v) hydro-
gen peroxide in 99% methylated spirit, rinsed in distilled water and sections
incubated at 37°C in 50 mM Tris-HCl pH 7.4 containing 0.14 M NaCl (TBS).
Sections were then treated with either 0.01% (w/v) chymotrypsin in 0.1% (w/v)
CaCl2 in TBS, or bovine testicular hyaluronidase (300 units/ml) in 50 mM
Tris-HCl (pH 7.4), 0.15 M NaCl and 0.1% (w/v) CaCl2. For the immunolocal-
izations of chondroitin-4 and 6-sulphate stubs using monoclonal antibodies 2B6
and 3B3, sections were pretreated with chondroitinase ABC (0.25 units/ml at
37°C in TBS containing 0.1% (w/v) BSA, 0.1% (v/v) Tween 20 and 0.05% (w/v)
sodium azide) to expose chóndroitin-6-sulphate and chondroitin-4-sulphate/der-
matan sulphate, respectively. Sections were rinsed twice in TBS for 5 min at room
temperature, and then blocked in 20% (v/v) swine serum in TBS for 20 min,
monoclonal antibodies—2B6, 3B3 and 5D4 (gifts from Professor C. Caterson,
University of Wales, Cardiff), or rabbit anti-bovine decorin antiserum (kindly
donated by Professor D. Heingard, University of Lund, Sweden), diluted in TBS,
were applied and incubated for 1 h at room temperature. The sections were
rinsed twice for 5 min in TBS, after which the secondary anti-rabbit or anti-
mouse antibody was applied for 30 min at room temperature. Sections were
washed twice in TBS for 5 min and then an avidin-biotin complex was applied for
30 min at room temperature. Following a 10 min wash in TBS, sections were
immersed in 0.02 mg/ml DAB, 0.05% (v/v) H2O2 in TBS for 20 min. Sections
were then washed in water and stained with haematoxylin, dehydrated in a series
of alcohols and mounted.
Immunofluorescence
For the immunolocalizations of collagens type II and X, the proximal tibial
epiphyses were removed from the freshly killed 3-week-old chickens and frozen
immediately in liquid N2 and stored at — 70°C. Sections (9 mm thick) were cut
using a PMV 450 MP cryomicrotome (Palmistiermas Instrument AB) at — 25°C
from fresh frozen blocks mounted in 1.6% carboxymethyl cellulose (Sigma
Chemical Co.) and pressed on to a microscope slide precoated with
polyvinylpyrrolidine (Sigma Chemical Co.). Freshly-cut sections were fixed in 4%
(v/v) buffered formalin for 5 min, and then equilibrated for 10 to 15 min in
phosphate buffered saline (PBS). Sections to be digested with enzyme were
incubated with 50 jul of bovine testicular hyaluronidase (300 units/ml) in PBS-
Tween (0.05% (v/v)) for 15 min in a moist chamber at room temperature, while
undigested controls were incubated with PBS-Tween only for the same length of
time. Sections were washed twice in PBS-Tween BSA [0.1% (w/v)], incubated
for 1 h at room temperature with anti-type X collagen monoclonal antibody
(MAI diluted 1:100) (Kwan et al, 1989) or goat anti-type II polyclonal antibody
1320-01 (1:10 or 1:5) (Southern Biotechnology Associates, Birmingham, Ala-
bama). For the immunolocalization of biglycan, paraffin-embedded sections were
used. Sections were dewaxed and treated as described above. The rabbit anti-
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 1 1
mouse biglycan antiserum LF106 (donated by Dr Larry Fischer, NIH) was used
at a dilution of 1 in 200. Sections were washed in PBS and incubated at 30°C for
1 h with the appropriate FITC-conjugated secondary antibody (Dakopatts, High
Wycombe, Bucks.j England) and applied at a dilution of 1:50. The slides were
washed thoroughly in PBS, then mounted in PBS containing 90% (v/v) glycerol
and 0.25% (w/v) 1, 4-diazabicyclo [2.2.2.] octane (Sigma Chemical Co.) cor-
rected to pH 8.6 with 1 M HC1, to minimize photobleaching. Control sections
were incubated with either non-immune mouse or goat serum instead of the
respective antibodies at the appropriate protein concentrations.
RESULTS
Histology of TD growth plates
Approximately 10% of birds fed on the control diet had dyschondroplastic
lesions, whereas the unbalanced diet resulted in an incidence of TD of approxi-
mately 40%. The normal epiphyseal plate consisted of flattened proliferating
cells, a narrow band of rounded transitional chondrocytes and a wide zone of
large, rounded hypertrophie chondrocytes (Figure 1A, B). The hypertrophie zone
contained metaphyseal vessels. In the dyschondroplastic physes there was an
accumulation of transitional chondrocyte-like cells (Figure 1C), in the centre of
the accumulation some of the chondrocytes were strongly eosinophilic and some
nuclei were pyknotic (Figure ID). An accumulation of disorganized proliferative
chondrocytes, is seen in hypocalcaemic rickets and has been reported as a
consequence in some birds of feeding the unbalanced diet (Rennie et al., 1993).
Accumulations of proliferative chondrocytes were not seen in any of the physes
selected for further study.
In situ hybridization studies
The expression of aggrecan, biglycan, decorin and type II collagen were investi-
gated by in situ hybridization on tissue sections from 10 normal and 10 dyschon-
droplastic growth plates. Figure 2Á shows the in situ hybridization of aggrecan
mRNA in the normal epiphyseal plate. As expected, expression of aggrecan core
protein was found throughout the different zones of the normal chicken growth
plate except the resting zone. The concentration of the probes for aggrecan
mRNA appeared to decrease gradually in the calcified cartilage. Areas of normal
hypertrophie cartilage (as shown in Figure IB) and TD cartilage (as shown in
Figure ID) were examined after in situ hybridizations. Relatively less aggrecan
expression was observed in the dyschondroplastic lesion when compared to the
normal hypertrophie chondrocytes (Figure 2B & C). In normal growth cartilage,
expression of biglycan is observed throughout the plate except the resting zone.
High expression of biglycan is detected within the hypertrophie zone (Figure 2E).
In the chondrodysplastic growth plate, the over-riding feature was the reduction
in the level of biglycan mRNA in the dyschondroplastic chondrocytes when
312 C. TSELEPIS ETAL.
Figure 1. Photomicrographs of histológica! sections from the proximal tibial growth plates
of 3-week-old normal and chondrodysplastic chickens. (A) Longitudinal section of normal
growth plate showing flattened proliferating chondrocytes [P] at the top and large rounded
fully hypertrophied chondrocytes [H] between the metaphyseal vessel at the bottom of the growth
plate. An arrow indicates the narrow band containing rounded transitional chondrocytes, bar = 100
p.m. (B) Normal hypertrophie chondrocytes at higher magnification showing the morphology
of hypertrophie cells and their regular columnar arrangement, bar= 100 ¡an. (C) Section of
the dyschondroplastic growth plate showing thickening of the physis due to apparent accumulation
of transitional chondrocyte-like cells [between arrows], bar= 600 \xm. (D) Higher magnification
photomicrograph of the affected physis, cells are separated by a larger volume of interterritorial
matrix. Strong eosinophilic cells are found and some nuclei werepyknotic (arrows), bar = 100 \an.
Haematoxylin and eosin stained.
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 1 3
compared to the normal hypertrophie chondrocytes (Figure 2F). Figures 2G and
2H show low concentrations of decorin mRNA in both the normal and dyschon-
droplastic chondrocytes when compared to that of aggrecan and biglycan. Figure
2D shows a typical control hybridization experiment with the corresponding
radiolabelled sense riboprobe, in that, no significant amount of specific signal was
detected on sections with the corresponding sense-riboprobes. No significant
comparative changes in the concentration of mKNA of type II were observed
(Figure 3 A, B). Furthermore,in situ hybridization of total poly-A RNAs suggested
no quantitative differences between normal and TD chondrocytes (Fig. 4A,B).
These results indicated that chondrocytes within the TD lesion are not degener-
ating cells as previously reported and the reduction in the expressions of the
proteoglycan core proteins are specific for certain extracellular matrix compo-
nents, but not a general failure of protein synthesis in TD.
Immunohistochemistry
The distribution of aggrecan, biglycan, decorin, and collagen types II and X was
investigated either by indirect immunofluorescence or immunoperoxidase label-
ling of longitudinal sections through the proximal tibia of normal and dyschon-
droplastic chickens. Monoclonal antibodies 2B6, 3B3 (recognize the
chondroitin-4-sulphate and chondroitin-6-sulphate stubs of proteoglycans after
chondroitinase ABC digestion), and 5D4 (recognizes keratan sulphate epitopes)
were used to immunolocalize proteoglycans containing these glycosaminoglycan
(GAG) epitopes in normal and tibial dyschondroplastic growth plates. Figures 5A
to D show the localization of keratan sulphate epitope in normal and dyschondro-
plastic growth plate cartilage using monoclonal antibody 5D4. In the resting zone
of the normal growth plate there was little detectable labelling though the matrix
surrounding the proliferative and hypertrophie chondrocytes labelled intensely,
with little cellular labelling. Within the calcifying cartilage region of the metaphy-
sis including residual cartilage within bone trabeculae, intense matrix labelling for
keratan sulphate containing proteoglycans was also observed. (Figure 5A B). The
labelling pattern for keratan sulphate in the TD lesion was markedly different
from that of normals. An apparent intracellular accumulation of the immunoper-
oxidase product was observed within the TD lesion and very weak labelling was
found in the matrix of the TD cartilage (Figure 5C, D). Similar immunoperoxi-
dase labelling patterns were observed with monoclonal antibodies 2B6 and 3B3
(results not shown). According to the specificities of the three antibodies, co-lo-
calization of these epitopes is highly indicative of the distribution of the large
aggregating proteoglycan, aggrecan. This observation is consistent with the result
from the in situ hybridization studies and indicates a reduction in the synthesis
and deposition of aggrecan within the TD lesion.
Immunofluorescent localization of biglycan in the normal chick growth plate
showed that in the resting zone there was little labelling though there was a sharp
transition with progression into the prehypertrophic chondrocytes where strong
intracellular labelling for biglycan was observed (Figure 5E). Such an observation
314 C. TSEIJEPIS ETAL.
Figure 2. In situ hybridization of aggrecan, biglycan anddecorin in the proximal growth plates of
3-week-old normal and dyschondroplastic chickens. Slides were exposed in emulsion for 14 days. (A)
Dark-field photomicrograph of in situ hybridisation of aggrecan core protein mRNA using a
32P-labelled-antisenseprobe in normal chick growthplate cartilage, [r] the resting zone, [p]proliferative
zone and [h] the hypertrophie zone and [m] calcified cartilage, bar= 100 pirn. (B) Photomicrograph
of normal hypertrophie cartilage, hybridized with a 32P-labelled-antisense aggrecan probe, showing
relatively high expression of aggrecan in this zone, bar= 100 ¡an. (C) Apparent low expression of
aggrecan mRNA isjound in the dyschondroplastic cartilage/dyschondroplastic lesion. Sections
hybridized with a 32P-labelled-antisense aggrecan probe, bar= 100 um. (D) Control in situ
hybridization of aggrecan mRNA on TD cartilage using a 32P-labelled-sense probe, bar = 50 \im. (E)
Dark-field photomicrograph of normal chick hypertrophie cartilage hybridized with a 32P-labelled-an-
tisense probe for biglycan core protein mRNA in growth plate cartilage high level ofbiglycan expression
is detected in the normal hypertrophie chondrocytes, bar= 100 um. (F) Low level ofbiglycan expression
in dyschondroplastic chondrocytes, sections hybridized with a 32P-labeUed-antisense probe, bar= 50
p.m. (G) In situ hybridization ofdecorin core protein mRNA using a 32P-labelled-antisense probe in
normal hypertrophie cartilage, bar = 50 urn, and (H) dyschondroplastic cartilage, bar= 100 ¡im.
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 315
Figure 3. In situ hybridization of type II collagen using a 3SS-dCTP-labeBed cDNA probes. (A)
Photomicrograph shovñng the expression of type II collagen mRNA in normal hypertrophie cartilage
and (B) in dyschondroplastic cartilage. Bar= 25 urn.
Figure 4. In situ hybridization of total mRNA using a digoxigenin-labetted oligo dTprobe. (A)
Section from normal hypertrophie cartilage and (B) from dyschondroplastic cartilage. Bar = 50 um.
316
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CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 1 7
was in contrast to the hypertrophie cartilage where intense labelling within the
extracellular matrix was observed, but relatively weak intracellular labelling (Fig-
ure 5F). Biglycan, as expected, was also in abundance in the osteoid with little
labelling inside the osteocytes. This observation is consistent with evidence which
suggests that biglycan is predominantly found in bone extracts (Fisher et al.,
1987). In the dyschondrôplastic growth plate, no detectable signal for biglycan
was observed within the resting zone. In contrast, within the prehypertrophic
zone, and in the lesion, strong intracellular labelling for biglycan was apparent,
with little or no labelling within the surrounding matrix (Figure 5G). This
observation indicates that there was no deposition of biglycan from the dyschron-
droplastic chondrocytes.
Decorin was found to be localized mainly to the newly-formed osteoid and no
apparent difference in the distribution of this proteoglycan was found between
normal and T D cartilage (Figure 5H & I). Consistent with immunohistological
observations previously reported by us and by others (Schmid & linsenmayer,
1985; Kwan et al,. 1986), type X collagen labelling was confined to the hyper-
trophic and mineralizing cartilage in the normal growth plate (Figure 6A).
Although the early hypertrophie cells appeared to contain some intracellular
collagen X, the highest labelling was found in the matrix of the late hypertrophie
and calcifying cartilage. Cells in these regions were surrounded by a strongly
labelled pericellular band but contained little or no intracellular type X collagen
labelling. Distribution of collagen X in the TD lesion was very different from that
observed in normal chickens. Type X collagen labelling was mostly intracellular
with a greatly reduced amount of collagen X in the matrix and little pericellular
fluorescence (Figure 6B).
Type II collagen was found throughout the epiphyseal and growth plate
cartilage in both normal and TD sections (Figure 6C & D). Type II collagen was
localized to the matrix within the resting, proliferative, hypertrophie and calcify-
ing regions of the normal growth plate.
DISCUSSION
Studies performed in the past decade have now identified numerous members of
(B) labelling is abundant within the calcified cartilage zone. (C) Within the dyschondrôplastic
matrix, no immurtoperoxidase product was detected. (D) Higher magnification photomicrograph
showing apparent intracellular accumulation of labelling. Bar = 100 ¡un. (E) Im-
munofluorescence localization of biglycan, with rabbit anti-mouse biglycan antiserum LF106, in
normal growth plate [r], [p] and Pi] = resting, proliferative and hypertrophie zone, respectively.
Bar•= 100 um. (F) Higher magnification photomicrograph showing matrix localization of
biglycan in normal hypertrophie cartilage. Bar = 50 fim. (G) Immunofluorescence localization of
biglycan in dyschondrôplastic cartilage. Bar= SO ¡im. (H and I) Immunoperoxidase localization
of decorin, with rabbit anti-bovine decorin antiserum, in normal and dyschondrôplastic growthplate respectively. Bar= 100 fim.
318 C. TSELEPIS ETAL.
Figure 6. Immunqfluorescence localization of types II and X collagen in normal and
dyschondroplastic cartilage. (A) Localization of type X collagen in normal hypertrophie cartilage.
Bar = 50 fim. (B) Intracellular accumulation of type X collagen in dyschondroplastic cartilage.
Bar — 50 ¡an. (C and D) Sections shozoing distribution of type II collagen in normal hypertrophie
and dyschondroplastic cartilage, respectively. Bar= 100 fim.
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 1 9
different collagen and proteoglycan families that are differentially expressed
in cartilage during normal growth and development, and in pathology.
Such findings indicate that the cartilage ECM has a complex, but dynamic
ultrastructural organization. Alterations to the intricate matrical organization
of cartilage are implicated in the mechanisms of pathological skeletal
development.
Avian TD is a disease which is a consequence of impaired chondrocyte
hypertrophy leading to impaired ossification and bone morphogenesis (Hargest et
al, 1985; Farquharson et al, 1992; Loveridge et al, 1993; Thorp et al, 1993).
This skeletal disorder is not restricted to broiler chickens, but is also found in
other species, and is hence a convenient and economical model for studying
aberrant endochondral ossification. A number of studies have suggested an
altered extracellular matrix composition within the cartilage lesion, which may
have implications in the initiation and the progression of the disease (Lowther et
al, 1974; Thorp et al, 1993). In this study, we examined the expression and
deposition of aggrecan, biglycan, decorin, and types II and X collagen in normal
and TD avian growth plate cartilage by in situ hybridization and immunohisto-
chemistry. From our observations, it appears that within the dyschondroplastic
lesion the expression and deposition of the major aggregating proteoglycan,
aggrecan, is greatly reduced, when compared to that of normal hypertrophie
chondrocytes. The reduced levels of aggrecan in TD lesion is reflected by the
reduced staining with monoclonal antibodies 5D4, 2B6 and 3B3 which recognize
potential GAG epitopes of aggrecan. The use of these antibodies showed loss of
GAGs in the TD cartilage which contrast observations reported by Farquharson
et al. (1994). These investigators demonstrated that in TD cartilage, the concen-
tration of sulphated glycosaminoglycans, determined by alcian blue staining, is
higher than that of normal growth plate cartilage. However, reduction in ex-
pression and deposition of aggrecan in TD cartilage corroborates our earlier
biochemical studies showing a 3-fold reduction in extractable aggrecan core
protein in TD cartilage and intracellular accumulation of aggrecan core protein
in the rough endoplasmic reticulum of TD chondrocytes (C. Tselepis and A. P.
L. Kwan, unpublished data). Further biochemical analysis of cartilage extracts
would be required to clarify the discrepancies in glycosaminoglycan concentration
in TD cartilage. In the light of our findings, i.e. reduced expression and the
intracellular accumulation of newly synthesized aggrecan, avian TD shares some
common features with avian nanomelia—a lethal genetic mutation of chickens
characterized by shortened and malformed limbs. It has been shown that
nanomelic chondrocytes fail to translocate normal aggrecan precursor but pro-
duce a truncated form of aggrecan (O'Donnell et al, 1988; Vertel et al, 1993).
In both skeletal disorders the lack of aggrecan within the cartilage matrix appears
to have severe implications in their pathologies. As aggrecan is the major struc-
tural proteoglycan in the cartilage matrix it is therefore reasonable to speculate
that the loss of aggrecan in the growth cartilage may alter the mechanical
properties of the interterritorial matrix which mediates mechanical influences/load
on the constituent chondrocytes. The change in the mechanical properties of
320 C. TSELEPIS ETAL.
cartilage may also be important in the development and progression of the disease
in which deformation of the long bone is apparent.
The functional roles of biglycan in cartilage are not well defined although
it has been suggested that biglycan may have a role in promoting cell differen-
tiation. Biglycan has been shown to bind Transforming Growth Factor-/?
(TGF-jß; Hildebrand et al., 1994). A marked decrease in TGF-03 in TD
cartilage has been demonstrated recently (Thorp et al., 1995). Because this
growth factor has been implicated in the cascade of events associated with
chondrocyte differentiation during endochondral ossification, the reduced deposi-
tion of biglycan in the cartilage matrix may contribute to the reduction of the
levels of matrix TGF-03.
A recent study by Chen et al. (1993) has shown no difference in the concen-
tration of type X collagen mRNA between normal and dyschondroplastic chon-
drocytes. Although similar observation was obtained in our previous Northern
hybridization studies (R. E. Barber and A. P. L. Kwan, unpublished data) this is
contrary to work by Bashey et al. (1989) suggesting collagen X synthesis is
reduced within the TD lesion. However, in our immunohistochemical analysis of
collagen X in TD cartilage, type X collagen was observed to be accumulated
intracellularly in TD chondrocytes. The function of this short-chain collagen in
the growth plate has been linked to mineralization, vascularization and other
processes in endochondral ossification (Schmid & linsenmayer, 1987). Type X
collagen has been demonstrated to form a regular hexagonal lattice (Kwan et al.,
1991). This novel macromolecular structure may take part in the modification of
the cartilage extracellular matrix for the subsequent steps in endochondral bone
formation. The consequences of the lack of collagen X in the ECM is still not
fully understood but the deposition of collagen X in die hypertrophie cartilage
may allow full cell hypertrophy to occur.
In this series of studies, we have been able to identify some of the key matrix
components which appear to be essential for the processes of endochondral
ossification. The apparent lower amount of aggrecan, biglycan and type X
collagen in the TD lesion may be caused by either: (i) failure for these secretory
proteins to be exported into the extracellular matrix possibly from a defect in
post-translational modification or (ii) failure of deposition of these molecules in
the extracellular matrix from disruptions in extracellular matrix assembly.
Deficiency in the incorporation of some extracellular proteins in TD cartilage is
not a consequence of a failure in protein synthesis in general, because the
depositions of decorin and type II collagen are not affected in the TD growth
plate. Furthermore, we have shown by the use of a poly-d(T) oligonucleotide
digoxygenin-labelled probe, and with a Type II collagen cDNA probe, that
protein synthesis remains normal in the dyschondroplastic chondrocytes which
synthesize normal amounts of mRNA, and that the chondrocytes are not under-
going necrosis. It is conceivable that changes in the molecular composition of the
matrix will alter the patterns of intermolecular interactions or cell-matrix interac-
tions, and eventually lead to the break down in tissue function and integrity. The
present series of studies, therefore, provides a basis for further investigations of
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 3 2 1
the structure-function relationship of the extracellular cellular matrix in the
development of the growth plate in health and disease.
ACKNOWLEDGEMENTS
The financial support of the BBSRC and the Medical Research Council is
gratefully acknowledged. BHT is funded by a commission from the Ministry of
Agriculture, Fisheries and Food. Thanks to S. Rennie and H. McCormack for
technical assistance.
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RESUME
Expression et distribution des macromolécules de la matrice carti-
lagineuse dans la dyschondroplasie tibiale aviaire
La dyschondroplasie tibiale (TD) est un trouble de l'ossification endochondrale caractérisée
par la persistance d'un cartilage avaseulaire non minéralisé s'étendant de la plaque de
croissance vers la métaphyse. Les cellules du cartilage dyschondroplasique ne parviennent plus
à atteindre le stade de l'hypertrophie complète et, à la place, s'arrêtent au stade "pré-hyper-
trophique ou à un stade de "transition". La synthèse et la distribution de protéoglycanes telles
que l'aggrécane, le biglycane, la décorine et des collagènes type II et X dans les plaques de
croissance des poulets normaux et dyschondroplasiques ont été étudiées par hybridation
CARTILAGE MATRIX SYNTHESIS IN DYSCHONDROPLASIA 323
in situ et inmuno localisation. Des réductions importantes dans les quantités des ARNm des
noyaux protéiques d'aggrecane et de biglycane, ont été observées dans les lésions de dyschon-
droplasie tibiale par hybridation in situ. La réduction de synthèse d'ARNm semble être
spécifique des composants de la matrice extracellulaire puisque l'expressiontotale des ARNm
ne montre pas de différence significative entre le cartilage normal et dyschondroplasique. De
plus, l'expression du collagène type II et decorine ne diffère pas significativement entre le
cartilage normal et dyschondroplasique. La distribution des aggrecane, biglycane, decorine,
collagènes type II et X a été examinée par immunohistochimie. Le cartilage hypertrophique
normal a montré un fort marquage de la matrice pour l'aggrecane et le biglycane. Dans le
cartilage hypertrophique normal, le collagène type X est fortement marqué au niveau de la
matrice et en zone péri cellulaire, tandis que dans le cas de cartilage dyschondroplasique, le
marquage des aggrecane, biglycane et collagène X est très faible et observé uniquement en
localisation intracellulaire. Au contraire, le collagène de type II a été localisé dans toute la
matrice extracellulaire aussi bien dans la plaque de croissance normale que dyschondro-
plasique, suggérant que les différences observées dans la distribution des aggrecane, biglycane
et collagène type X sont spécifiques de ces protéines et non dues à un trouble général du
métabolisme macromoléculaire de la matrice. La réduction du dépôt de ces macromolécules
dans la matrice doit avoir des implications dans le développement de l'os normal et
pathologique.
Z U S A M M E N F A S S U N G
Expression und Verteilung von Knorpelmatrix-Makromolekülen bei der
aviären Tibia-Dyschondroplasie
Die Tibia-Dyschondroplasie (TD) ist eine Störung der Knorpelossifikation und ist durch das
Vorhandensein einer gefäßlosen, nicht mineralisierten Knoxpelläsion charakterisiert, die sich
von der Wachstumsplatte bis in die Metaphyse erstreckt. Die Zellen innerhalb der TD-Wach-
stumsplatte verfehlen ihre Differenzierung zur vollen Hypertrophy und scheinen stattdessen
einen "prähypertrophen" Zustand oder "Übergangszustand" beizubehalten. Die Synthese und
Verteilung von Aggrecan, Biglykan, Dekorin und den Kollagen-Typen II und X in den
Wachstumsplatten von normalen Küken und Küken mit TD wurde mit Hilfe von in situ-
Hybridisierung und Immunolokalisation untersucht. Ausgeprägte Mengenreduzierungen der
Aggrecan- und Biglykan-Kernprotein-mRNAs wurden in der TD-Läsion mittels in
situ-Hybridisierung festgestellt. Die Verminderng der mRNA-Produktion schien für die ex-
trazellulären Matrixkomponenten spezifisch zu sein, da es bei der Expression der Gesamt-
mRNA keinen signifikanten Unterschied zwischen normalem und dyschondroplastischem
Knorpel gab. Außerdem gab es hinsichtlich der Expression von Kollagen des Typs II und
Dekorin keine signifikanten Unterschiede zwischen normalem und TD-Knorpel. Die
Verteilung von Aggrecan, Biglykan, Dekorin und den Kollagen-Typen II und X wurde
immunhistochemisch untersucht. Normaler hypertrophischer Knorpel wies eine starke Aggre-
can- und Biklykan-spezifische Matrixmarkierung auf. Kollagen vom Typ X zeigte im normalen
hypertrophischen Knorpel eine starke perizelluläre und Matrix-Verteilung, während sich die
Markierung zum Nachweis von Aggrecan, Biglykan und Kollagen X intrazellulär befand, mit
einem sehr schwachen Signal in der Matrix. Im Gegensatz dazu ließ sich Kollagen vom Typ
II überall in der extrazellulären Matrix sowohl der normalen Wachstumsplatte als auch der
TD-Läsion nachweisen, was darauf schließen läßt, daß die bei der Verteilung von Aggrecan,
Biglykan und dem Kollagen-Typ X gefundenen Unterschiede spezifisch für diese Proteine sind
und nicht für eine allgemeine Störung des makromolekularen Metabolismus in der Matrix. Die
reduzierte Ablagerung dieser Makromoleküle könnte Auswirkungen auf die normale und
pathologische Knochenentwicklung haben.
324 C. TSELEPIS ET AL.
RESUMEN
Expresión y distribución de macromoléculas de la matriz cartilaginosa en
la discondroplasia tibial aviar
La discondroplasia tibial (TD) es una alteración de la osificación endocondral caracterizada
por la presencia de una lesión avascular y un cartílago no mineralizado que se extiende desde
la placa de crecimiento hasta la metáfisis. Las células cartilaginosas en la placa de crecimiento
de los animales con TD no llegaron a diferenciar al estado de hipertrofia sino que se
mantuvieron en el estado de "prehipertrofia" o "transición". Se investigó la síntesis y
distribución de agrecan, biglucan, decorina y colágeno de tipo II y X en las placas de
crecimiento en aves normales y con discondroplasia tibial mediante hibridación in situ e
inmunolocalización. La hibridación in situ detectó una reducción marcada de la cantidad de
ARN mensajeros de las proteínas de agrecan y biglucan en la discondroplasia tibial. La
reducción en la producción de ARN mensajero parecía ser específica de los componentes de
la matriz extracelular puesto que la expresión total de ARN fue diferente entre el cartílago
normal y el discondroplástico. La expresión de colágeno de tipo II y decorina no fue
significativamente distinta entre el cartílago discondroplásico y el normal. Se estudió mediante
inmunohistoquímica la distribución de agrecan, biglucan, decorina, colágenos de tipo II y X.
El cartílago hipertrófico normal mostró un mareaje intenso en la matriz para agrecan y
biglucan. El cartílago normal tenía una intensa reacción pericelular y en la matriz para colágeno
X mientras que el cartílago TD presentó una reacción intracelular para el agrecan, biglucan y
colágeno X y una reacción muy débil en la matriz. Por el contrario, se encontró colágeno II
por toda la matriz extracelular de ambos grupos de animales sugiriendo que las diferencias
observadas en agrecan, biglucan y colágeno X eran específicas para estas proteínas y no una
alteración general del metabolismo macromolecular de la matriz. El depósito reducido de estas
macromoléculas puede tener implicaciones en el desarrollo normal y patológico del tejido óseo.