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Dysplastic Histogenesis of Cartilage Growth Plate by Alteration of Sulphation
Pathway: A Transgenic Model
Antonia Icaro Cornaglia a; Andrea Casasco a; Marco Casasco a; Federica Riva a; Vittorio Necchi b
a
Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Pavia, Italy b
Department of Human and Hereditary Pathology, University of Pavia, Pavia, Italy
Online Publication Date: 01 August 2009
To cite this Article Cornaglia, Antonia Icaro, Casasco, Andrea, Casasco, Marco, Riva, Federica and Necchi, Vittorio(2009)'Dysplastic
Histogenesis of Cartilage Growth Plate by Alteration of Sulphation Pathway: A Transgenic Model',Connective Tissue
Research,50:4,232 — 242
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Connective Tissue Research, 50:232–242, 2009
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DOI: 10.1080/03008200802684623
Dysplastic Histogenesis of Cartilage Growth Plate by
Alteration of Sulphation Pathway: A Transgenic Model
Antonia Icaro Cornaglia, Andrea Casasco, Marco Casasco, and Federica Riva
Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Pavia, Italy
Vittorio Necchi
Downloaded By: [Cornaglia, Antonia Icaro] At: 08:16 4 September 2009
Department of Human and Hereditary Pathology, University of Pavia, Pavia, Italy
Mutations in the diastrophic dysplasia sulphate transporter
(dtdst) gene causes different forms of chondrodysplasia in the
human. The generation of a knock-in mouse strain with a mutation
in dtdst gene provides the basis to study developmental dynamics in
the epiphyseal growth plate and long bone growth after impairment
of the sulphate pathway. Our microscopical and histochemical
data demonstrate that dtdst gene impairment deeply affects tissue
organization, matrix structure, and cell differentiation in the
epiphyseal growth plate. In mutant animals, the height of the
growth plate was significantly reduced, according to a concomitant
decrease in cell density and proliferation. Although the pathway
of chondrocyte differentiation seemed complete, alteration in cell
morphology compared to normal counterparts was detected. In
the extracellular matrix, it we observed a dramatic decrease in
sulphated proteoglycans, alterations in the organization of type II
and type X collagen fibers, and premature onset of mineralization.
These data confirm the crucial role of sulphate pathway in
proteoglycan biochemistry and suggest that a disarrangement of
the extracellular matrix may be responsible for the development of
dtdts cartilage dysplasia. Moreover, we corroborated the concept
that proteoglycans not only are structural components of the
cartilage architecture, but also play a dynamic role in the regulation
of chondrocyte growth and differentiation.
Keywords
Cartilage Histogenesis, Chondrodysplasia, Sulphate
Transporter, Epiphyseal Growth Plate, Extracellular
Matrix
INTRODUCTION
The epiphyseal growth plate (EGP) is a structure of hyaline
cartilage, a highly specialized connective tissue, which is
responsible for the longitudinal growth of long bones during
Received 3 September 2008; Revised 11 December 2008; Accepted
11 December 2008.
Address correspondence to Dr. Antonia Icaro Cornaglia, researcher
of Histology, Histology and Embryology Unit, Department of Experimental Medicine, University of Pavia, Via Forlanini 10, 27100
Pavia, Italy. E-mail: [email protected]
development. From the histological point of view, EGP can be
divided in four zones: the proliferation zone, the prehypertrophy
or maturation zone, the hypertrophy zone, and the mineralizing
zone [1–4]. In the proliferation zone, the cells undergo
repeated mitotic divisions, thereby forming regular cell columns
perpendicular to the growth plate. Proliferation is followed
by cellular hypertrophy during which the cytoplasm increases
in volume and appears vacuolated. Moreover, the distance
between cells increases due to extracellular matrix production.
The intercolumnar septa of the hypertrophic zone then start
to calcify, forming the border between the hypertrophic and
mineralizing zones. Finally, soon after the surrounding matrix
starts to mineralize, the chondrocytes degenerate and disappear.
Mineralized cartilage is believed to serve as a backbone for
metaphyseal bone formation that subsequently is formed by
metaphyseal bone cells.
Therefore, since a continuous supply of mineralized cartilage
is required for endochondral bone synthesis, the longitudinal
growth and expansion of cartilage is a rate-determining factor
for growth of the long bones of the skeleton. Although several
molecules involved in growth cartilage have been identified,
to date the genetic and molecular mechanisms by which
chondrocyte proliferation, differentiation and maturation are
regulated remain largely unknown.
The chemical structure of matrix components gives the
cartilage its special biomechanical and functional properties
[2, 5]. The three main components are collagen (type I and
II), glycoproteins (such as matrilin-1), and proteoglycans [6,
7]. Different proteoglycans are present in cartilage matrix,
the largest and most abundant being aggrecan, which binds
hyaluronan, chondroitinsulfate, and keratan sulphate. Other
groups of proteoglycans include dermatan sulphate proteoglycans (such as biglycan, decorin, and fibromodulin) and the
heparan sulphate containing-syndecans. The large number of
sulphates and carboxyl groups in these glycosaminoglycans
creates an exceptional charge density and a high osmotic
pressure.
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DYSPLASTIC HISTOGENESIS OF EGP
Any disruption to the balance among proliferation, hypertrophy, and death in the relevant zones of EGP can lead to skeletal
defects and, in particular, chondrodysplasias. Human chondrodysplasias are a clinically and genetically heterogeneous
group of diseases, including over 200 different phenotypes, that
affect the skeleton development [8–12]. The severity of these
diseases ranges from mild to severe and lethal forms, and as a
group chondrodysplasia have an overall incidence of 1 per 4000
in the population.
According to the importance of sulfation processes in cartilage and other connective tissues, mutations in the diastrophic
dysplasia sulphate transporter (dtdst, also known as SLC26A2)
gene cause different forms of chondrodysplasia in the human
[13–17]. Recently, a knock-in mouse strain with a mutation in
dtdst gene was generated [18]. This mutation has been shown
to cause a partial loss of function of the sulphate transporter,
thus providing a useful model to study the physiopathology and
pharmacological treatment of chondrodysplasias.
A major problem in the study of EGP is that its anatomical
dimension, which spans from the distal epiphyseal ossification
centre to the diaphyseal ossification center, is rather small, being
less than 1 mm in the mouse [4]. Therefore, selective isolation
of the plate from the surrounding tissues (including bone, bone
marrow, blood vessels, and dense connective tissue) cannot be
obtained precisely, and reliable biochemical analyses cannot
be performed ex vivo. In line with other investigations on the
growth plate [1, 4, 19, 20, 21], we decided to investigate the
histogenesis of EGP in dtdst-mutant mouse using microscopical
methods, including histochemical and electron microscopical
analyses.
MATERIALS AND METHODS
Generation of the dtdst -Mutant Mice and Tissue
Preparation
Generation of the dtdst-mutant mice has been described
previously [18]. Transgenic mice harboring an A386V substitution in the eight transmembrane domain of the dtdts protein
were generated by homologous recombination in embryonic
stem cells. To knock-in the A386V substitution, a C1184T
transition was introduced by site-directed mutagenesis in a
cloned fragment of exon 3. This mutation was observed in
the homozygous state in a patient with a nonlethal form of
diastrophic dysplasia characterized by short stature, cleft palate,
deformity of the external ear, and “hitchhiker” thumb deformity
[16].
Wild-type (n = 12) and mutant mice (n = 12) (kindly
provided by Prof. Antonio Rossi, dept. of biochemistry,
University of Pavia) were sacrificed by cervical dislocation at 28
days of age. To detect S phase-traversing, i.e., proliferating cells,
mutant and wild-type mice were injected intraperitoneally with
0.1 mg of 5 -brome-2 -deoxyuridine (BrdUrd)/g body weight,
24 hr before sacrifice [22, 23]. Proximal epiphyses of the tibia,
containing epiphyseal growth cartilage, were dissected free from
233
corresponding diaphyses and immediately fixed for light and
electron microscopical analyses, as described below.
Light Microscopy
Samples were fixed with 4% paraformaldehyde (w/v) in
0.1 M phosphate buffer, pH 7.4, for 6 h. Decalcification was
performed in 15% EDTA in TBS for 7–10 days. Samples were
dehydrated through graded alcohols and routinely embedded in
paraffin. Sections were obtained at 5–10 µm, rehydrated, and
stained with hematoxylin and eosin or Alcian blue, or processed
for immunohistochemical stainings.
For Alcian blue staining, known to be highly specific for the
detection of acid mucopolysaccharides in tissues [24], dewaxed
slides were placed in acetic acid solution for 3 min, rinsed in
tap water, and immersed in Alcian blue solution for 30 min at
room temperature (staining kit from Biocare Medical, Walnut
Creek, CA, USA). Stained sections were observed with a Zeiss
Orthoplan microscope equipped with a Nomarski differential
interference contrast device.
Transmission Electron Microscopy
Samples were immersed in a solution containing 2.5%
glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium
cacodylate buffer, pH 7.4, for 6 hr at 4◦ C. They were then
postfixed for 1 hr in 1.33% osmium tetroxide in 0.1 M collidine
buffer. The tissues were dehydrated through graded alcohols
and embedded in Epon 812. Semithin sections were stained
with Toluidine blue on a hot plate at 50 to 60◦ C for 3 min. Thin
sections were stained with Uranile acetate and lead citrate and
observed in a Zeiss EM10 electron microscope.
Light Microscopy Immunohistochemistry
Dewaxed sections were processed, according to the indirect
Streptavidin-Biotin immunoperoxidase technique. Briefly, the
sections were incubated serially with the following solutions:
0.3% hydrogen peroxide for 30 min to remove endogenous
peroxidase activity; normal goat serum, diluted 1:20, for 30 min
to reduce nonspecific background staining; primary monoclonal
antibodies to BrdUrd, type II collagen, and type X collagen
diluted 1:100, 1:200, and 1:250 respectively, overnight at
4◦ C; biotinylated goat antimouse IgG (Super Sensitive kit,
BioGenex, San Ramon, CA, USA) for 1 hr at room temperature; streptavidin-biotinylated peroxidase complexes (Super
Sensitive kit, BioGenex) for 1 hr at room temperature; 0.03%,
3,3 diaminobenzidine tetrahydrochloride, to which hydrogen
peroxide (0.02%) was added just before use, for 5 min at room
temperature.
Each solution was prepared in 0.05 M Tris buffer, pH 7.4,
containing 0.1 mol/L NaCl (0.15M Tris-buffered saline) and
between each step of the immunostaining procedure the sections
were washed in the same buffer. Some sections were lightly
counterstained with hematoxylin. The sections were finally
dehydrated, mounted, and examined. Before immunostaining
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for type II collagen and type X collagen, the sections were
pretreated with protease (pepsin 1 mg/ml in Tris- HCl pH 2 for
10 min at 37◦ C).
Histological Identification
The epiphyseal growth plate can be localized between
the distal epiphyseal ossification front and the diaphyseal
ossification front, i.e., in the transition region between epiphysis
and diaphysis of long bones including tibias. It is easily
distinguishable from the birth throughout the period of skeleton
growth.
The growth plate can be divided in four principal zones
[2–4]: the proliferation zone, the prehypertrophic or maturation
zone, the hypertrophic zone, and the mineralizing zone. Under
normal conditions, cells withdraw from the cell cycle and
form regular cell columns parallel to the long bone axis but
have not undergone hypertrophy yet: this area is referred to
as prehypertrophic or maturation zone. Cells of the column
then become hypertrophic, thus forming the hypertrophic zone.
Finally, the intercolumnar septa of extracellular matrix start to
calcify, thus defining the border between the hypertrophic zone
and mineralizing zones; in this zone cartilage cells become more
vacuolated and start degenerating or transforming.
Cell Counts, Measurements of Different EGP Zones of
and Immunohistochemical Reactions
The analyses were performed on dewaxed sections stained
with hematoxylin-eosin or Epon-embedded semithin sections
stained with Toluidine blue. Cell counts, measurements of
histological zones, and evaluation of staining intensity and
pattern were performed by three independent observers in
three different sections of each samples (12 mutant versus 12
wild-type mice). Data were expressed as means +/− standard
error (SE) and two sample t-test analysis was made between
data obtained from normal wild-type mice and mutant dtdts
–defective mice.
The staining intensity for Alcian blue and type II collagen
was assessed according to a semiquantitative scale (no reaction,
faint, moderate, or strong reaction).
Antibodies, Controls of Immunohistochemical Reaction,
and Reagents
Primary monoclonal antibodies to BrdUrd (clone BU-1, class
IgG2a, Amersham International, Amersham, England), type II
collagen (clone 2B1.5, class IgG2a, Neomarkers, Fremont, CA,
USA), and type X collagen (clone COL-10, Sigma, St. Louis,
MO, USA) have been previously characterized [23–27]. Specificity controls included the omission of the primary antibodies
and the substitution of the primary antibodies with nonimmune
sera or monoclonal antibodies from the same immunoglobulin
subclasses [28]. No immunostaining was observed after control
procedures.
Secondary biotinylated antibodies and streptavidinbiotynilated peroxidase complexes were purchased from
BioGenex; all other reagents were purchased from Sigma
Chemical Company.
RESULTS
Microscopical observations were performed using normal
wild-type mice as control and analyzing the differences between
control and mutant dtdts defective mice.
General Architecture
The height of the total growth plate (mean value +/− SE) in
the mutant mice was greatly reduced (µm 220 +/− 5.2) compared to normal mice (300 µm +/− 10.3; Figure 1) (p < 0.05).
In both normal and mutant mice, different histogenetic zones,
namely proliferation, maturation, hypertrophic, and mineralizing zones, could be observed (Figure 1). Although the organization in cell columns was maintained, cell alignment was less
preserved in mutant mice. Moreover, in mutants chondrocyte
size and shape were more variable in each stage of differentiation
compared to controls. In particular, in the proliferation zone
cells were irregularly disposed and we observed wedge-shape
cells which were not present in normal growth plate (Figures 2B,
3B). On the proximal and distal fronts of the growth plate, which
includes most immature and mature chondrocytes, respectively,
the growth plate appeared irregularly delineated and trabecular
structures are less delineated in the border between bone and
mineralizing cartilage (Figure 1B). The extracellular matrix
in mutant animals appeared less acidic compared to controls
(Figure 1).
In mutants, the number (+/− SE) of cartilage cells per
each column was reduced (21.2 +/− 1.2) compared to normal
plates (25.3 +/− 1.3) (p < 0.05). Each zone in mutant animals
contained fewer cells compared to corresponding zone of normal
animals; the difference was striking in the proliferation zone
where cells in mutants were about 50% less compared to normals
(Figure 1, P).
Histochemical Stainings
After staining with Alcian blue and Toluidine blue, known to
have high affinity for acidic glycosaminoglycans, the cartilage
matrix in mutant animals appeared less intensely stained compared with wild-type littermates (Figures 2A, 2B). Accordingly,
metachromasia was also less intense. Such aspects were more
evident in the proliferation and hypertrophic zones of the
growth plate. Immunostaining for type II collagen was observed
in all zones of the growth plate, both in normal as well as
mutant animals. However, in dtdts-defective mice the intensity
of immunoreaction was greatly reduced (Figures 2C, 2D).
Moreover, in mutants we could not reveal any immunostaining
for collagen type X, which could be readily identified in the
hypertrophic zone of normal growth plates (Figures 2E, 2F).
BrdUrd immunostaining was detectable within the nuclei of S
phase traversing cells. As expected, labelled cells were observed
exclusively in the proliferation zone and early maturation zone
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DYSPLASTIC HISTOGENESIS OF EGP
235
FIG. 1. Epiphyseal growth plate from a normal, wild-type mouse (wt, A) and from a mutant mouse in the diastrophic dysplasia sulphate transporter gene (dtdst,
B) of the same age (28 days). In both the normal and mutant mice, different histogenetic zones, namely proliferation (P), maturation (M), hypertrophic (H),
and mineralizing (MZ) zones, are recognizable. However, the growth plate height (i.e., the distance from solid line to solid line in each growth plate) is greatly
reduced in the mutant mouse, the number of cartilage cells per column being reduced compared to normal plate. Trabecular structures are less delineated in the
border between bone and mineralizing cartilage (MZ in B). The extracellular matrix in mutant animal (B) appears less acidic compared to normal counterpart (A)
(Hematoxylin-eosin staining).
of the plate (Figures 2G, 2H). BrdUrd labelling index (+/− SE)
was 17.8 +/− 0.5 in wild-type animals and 13.7 +/− 0.4 in
mutants. Moreover, the tissue distribution of BrdUrd staining
confirmed that the proliferation zone in mutant animals was
reduced in the cell number compared to controls (p < 0.05).
Electron Microscopical Analyses
Ultrastructural features of cartilage cells in different zones
were similar in wild-type and mutant animals; in particular
the distribution of intracytoplasmic organelles appeared mostly
identical (Figures 3, 4). However, in the proliferation zone of
mutants we observed “wedge-shaped” and ovoid cells which
could not be identified in control animals (Figure 3B). Within the
hypertrophic zone of mutant animals, cells were more irregular
in size and shape. Moreover, in this zone we observed occasional
cells with an ovoid shape, instead of a prismatic shape as in
wild-type (Figure 3D).
The most striking aspect that could be observed in the
extracellular matrix of mutants was that collagen fibers were
readily visible, disposed according to a parallel array and
concentrated close to the cells (Figure 4B). Conversely, in
the matrix of wild-type animals, collagen fibers formed a
fine meshwork which appeared regularly disposed in the
extracellular space (Figure 4A).
Finally, in the matrix around the cells of late maturation
and hypertrophic zone of dtdst mutants, we detected initial
deposition of calcified matrix (Figures 3D, 4D), suggesting
that the onset of mineralization is anticipated with respect to
normal animals where initial mineralization was observed in the
“mineralizing zone” proper. Main differences between normal
and mutant mice are reported in Table 1.
DISCUSSION
Long-bone growth is the result of an endochondral ossification process which involves a cartilage intermediate called
epiphyseal growth plate (EGP). During endochondral ossification, chondrocytes deposit a cartilage-specific extracellular
matrix (ECM) and undergo terminal differentiation. At the same
time differentiating osteoblasts, together with blood vessels and
osteoclasts, invade the zone of hypertrophic chondrocytes where
ECM has started mineralizing. While osteoclasts degrade the
hypertrophic cartilage matrix, osteoblasts produce bone-specific
matrix using the degraded cartilage matrix as a scaffold [2,
3, 29, 30]. Therefore, in long bone epiphyses the pathways
of chondrocyte and osteoblast differentiation as well as ECM
synthesis are interconnected and coordinated.
The disruption of endochondral ossification in the EGP leads
to delayed and irregular bone formation and can result in a
A. I. CORNAGLIA ET AL.
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236
FIG. 2. Epiphyseal growth plate from a normal, wild-type mouse (wt, A, C, E, G) and from a mutant mouse in the diastrophic dysplasia sulphate transporter gene
(dtdst, B, D, F, H) of the same age (28 days). The intensity of staining with Alcian blue or Toluidine blue, as well as the corresponding metachromasia, in mutated
animal (B) is faint compared to normal (A), suggesting that content in acidic glycosaminoglycans is reduced. Also the intensity of immunostaining for type II
collagen is reduced in dtdts mutant animals (D vs. C), whereas immunostaining for type X collagen is completely absent in mutant (F vs. E). Bromodeoxyuridine
immunostaining shows that proliferating cells are localized within the proliferation zone of the growth plate in both normal (G) and mutant (H) animals; however,
the labelling index is reduced in mutants (H vs. G). Alcian blue (A, B) and indirect immunoperoxidase (C–H) stainings.
heterogeneous group of genetic disorders referred to as “chondrodysplasias.” The dtdst gene encodes for a sulphate/chloride
antiporter of the cell membrane which is crucial for the uptake
of inorganic sulphate by the cells [17, 31]. Although dtdst gene
is expressed in many organs and tissues, the main phenotypic
consequences of its functional impairment are restricted to bone
and joints [12, 13]. Indeed, mutations in this gene cause a
family of recessively inherited chondrodysplasias in the human
including, in order of decreasing severity, achondrogenesis type
1B, atelosteogenesis 2, diastrophic dysplasia, and recessive
multiple epiphyseal dysplasia [12].
To determine the mechanisms that underpin the pathophysiology of dtdst chondrodysplasias, a knock-in mouse
model recently has been generated [18]. We have studied the
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DYSPLASTIC HISTOGENESIS OF EGP
FIG. 3. Ultrastructural aspects of epiphyseal growth plate from a wild type (A, C, E) and from a mutant mouse in the diastrophic dysplasia sulphate transporter
gene (dtdst, B, D, F). Corresponding histogenetic zones (proliferation, A vs. B; hypertrophic, C vs. D; and mineralizing, E vs. F) are shown. Although the
organization in columns is maintained, cell alignment is less preserved in mutant mice (B vs. A); moreover, in the mutant, chondrocyte size and shape are
variable in each stage of cell differentiation compared to controls (B vs. A; D vs. C). In mutant, wedge-shape cells (solid arrows in B) within the proliferation
zone and ovoid cells (dotted arrows in B and D) within proliferation and hypertrophic zones, which are not present in corresponding normal zones, can be seen.
Within hypertrophic zone matrix of dtdst mutants, it is possible to detect initial deposition of calcified matrix (white arrows in D), suggesting that the onset of
mineralization is anticipated with respect to normal animals where initial mineralization was observed in the mineralizing zone proper (E).
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A. I. CORNAGLIA ET AL.
FIG. 4. Ultrastructural aspects of epiphyseal growth plate from a wild-type (A, C) and from a mutant mouse in the diastrophic dysplasia sulphate transporter
gene (dtdst, B, D). Intracytoplasmic features of cartilage cells in different zones are comparable between wild-type and mutant animals, including the distribution
of cytoplasmic organelles (A vs. B; C vs. D). Conversely, significant differences are detectable in the extracellular matrix of proliferation (A vs. B) and hypertrophic
(C vs. D) zones. In the matrix of wild-type animals, collagen fibers form a fine meshwork which appears regularly disposed in the extracellular space (A); in the
matrix of mutants, collagen fibers are readily visible, disposed according to a parallel array and concentrated close to the cells (B). Early deposition of calcified
matrix is anticipated in the hypertrophic zone of dtdst mutants (asterisks in D) compared to normal histogenesis. RER-rough endoplasmic reticulum; N-nucleus.
histogenesis of EGP in this model using different microscopical
techniques, as it done previously to study other forms of
chondrodysplasia in the animal [19–21]. In particular, using
ultrastructural and histochemical methods, it was possible to
reveal several differences in the growth plate of dtdst knock-in
mouse compared to normal mouse; such differences included
reduction in chondrocyte growth, modifications in chondrocyte differentiation, and alterations in ECM organization and
composition. Histochemical staining provided evidence that
both mucopolysaccharides and collagen contents are altered in
dtdst-mutant animals.
In dtdts-defective animals, although classical zones of
the EGP can be identified (namely proliferation, maturation,
hypertrophic, and calcifying) and organization in cell columns
is somehow maintained, the EGP undergoes a dysplastic
development. Cell alignment appears disturbed, cell shape much
variable and, as a whole, the EGP is shorter (about 25% less)
compared to controls. The mean cell number per column is
reduced, the number of cells per each zone being defective.
Although the tissue distribution of proliferating cells as revealed
by BrdUrd incorporation was similar to controls, BrdUrd
labelling index was lower in dtdst mutants, thus suggesting that
cell proliferation is reduced. This may account for the reduced
cell number per column as well as for shorter length of the
mutant EGP.
Morphological analysis reveals that the onset of mineralization is developed early in mutants with respect to normal
animals. This aspect may be related to alterations in the
ECM composition, which finely regulates calcium deposition
and crystal formation in calcifying tissues. This hypothesis is
supported by the findings that undersulphated proteoglycans
in cartilage are less effective inhibitors of hydroxyapatite
formation than normally sulphated proteoglycans [32].
It is generally believed that the rate of endochondral bone
growth is the result of three processes which occur within
the EGP: cell proliferation in the proliferation zone, cell
hypertrophy in the maturation and hypertrophic zones, and
turnover of the ECM in the calcifying zone where cartilage is
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DYSPLASTIC HISTOGENESIS OF EGP
TABLE 1
Comparison between microscopical aspects of epiphyseal growth plates of normal versus dtdts-mutant animals
Normal (wild-type) animal
300 +/− 10.3
25.3 +/− 1.3
Yes
Regular
Yes
Strong (+++)
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17.8 +/− 0.5
Mineralization zone
Diffuse and strong
Positive in the hypertrophic zone
Forming a fine meshwork and regularly
disposed in the extracellular space
Histological or cytological feature
Height of growth plate (µm; mean value
+/− SE)
Number of cells/column (mean value +/−
SE)
Presence of all differentiation zones
Cell alignment within the column
Homogeneity in cell size and shape within
each zone
Alcian blue/Toluidine blue stain and
related metachromasia
Bromodeoxyuridine labelling index (mean
value +/− standard error)
Zone of initial mineralization
Type II collagen immunoreactivity
Type X collagen immunoreractivity
Ultrastructural aspect of collagen fibers
substituted by new bone tissue [33, 3, 4]. As to the first process,
cell proliferation in the EGP of dysplastic animals is reduced
compared to normals, as suggested by BrdUrd immunohistochemistry. The process of cell hypertrophy seems normal in
mutants, although cells of different size and shape are visible
among hypertrophic ones. As to the ECM, profound alteration
can be observed in each zone of dysplastic EGP. According to
previous biochemical data [18], histochemical stainings show
that proteoglycans are undersulphated. Ultrastructural analysis
of the ECM reveals that the organization of collagen fibrils
is altered in dtdts mutant. This aspect is not surprising since
deposition and assembly of collagen fibers in cartilage are
regulated by proteoglycans [34, 35]. In particular, evidence
exists that undersulphation may impair the binding properties
of proteoglycans to type II collagen [10].
Interestingly, in the electron microscopical pictures of the
ECM, bundles of fibrils, presumably made by type II collagen,
are more evident in dtdts animals compared to normals.
Conversely, immunoreactivity for type II collagen is faint in
mutants and evident in normals. Although these findings appear
conflicting, there are at least two possible explanations: either
the amount of type II collagen is lower in dysplastic growth plate
or the epitope of type II collagen is less accessible by 2B1.5
monoclonal antibody since the fibril conformation is altered in
mutants, as suggested by electron microscopical analysis. This
epitope has been mapped at the carboxyl-terminus of type II
collagen [26].
Negative immunostaining for collagen type X is consistent
with the hypothesis that the overall composition and organization of the ECM are altered in dtdst chondroplastic animals.
dtdts-mutant animal
220 +/− 5.2
21.2 +/− 1.2
Yes
Irregular
No
Faint (+)
13.7 +/− 0.4
Late maturation/hypertrophic zone
Diffuse and faint
Negative
Evident, with parallel array pattern and
concentrated close to cell surface
Interestingly, the expression of collagen type X also was not
recognizable in the growth plate of type 2 collagen–defective
animals [20].
These considerations raise some questions as to molecular
mechanisms which determine chondrodysplasia in dtdst-mutant
animals. It is known that much of the sulphate is incorporated in
glycosaminoglycans, the oligosaccharide part of proteoglycans
that are synthesized by many cells and are used as cellular
or ECM components [5]. However, besides proteoglycans,
sulphation involves many substances including proteins, lipids,
hormones, and drugs. The sulphation pathway involves several
steps: sulphate transport through the cell membrane, activation
of sulphate to 3 -phosphoadenosine 5 -phosphosulphate (PAPs),
transport of PAPs to the Golgi apparatus, and sulphation of
macromolecules by a range of specific sulphotransferases [36,
37]. Regulation of sulphation pathway may occur at each step
and the degree of proteoglycan sulphation depends on the
availability of PAPs which in turn depends on the intracellular
level of sulphate [38]. Accordingly, in case the intracellular
sulphate pool is decreased by reducing the availability of
extracellular sulphate or by inhibiting transmembrane sulphate
transport, the proteoglycans are undersulphated [39].
Alteration of sulphation pathway, which results in undersulphated proteoglycans in patients and dtdst animals [18], is
presumed to be responsible for the disarrangement of the ECM
network in dysplastic growth plate. Potentially, modifications of
the ECM may produce profound effects on tissue development
and homeostasis. In fact, it is well known that cell behaviors and
activities required for developmental control, including growth,
differentiation, polarity, adhesion, motility, and programed cell
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240
A. I. CORNAGLIA ET AL.
death, are influenced by ECM [40, 41]. An interesting molecular
biophysical model, called “tensegrity,” has been proposed
[42]. According to this theory, cell behavior emerges from
biophysical interactions among different filament networks,
so that forces applied via ECM induce the activation of
integrin mechanotransduction pathways; these pathways affects
gene and protein signalling networks to produce characteristic
phenotypes.
Since ECM and cognate integrins modulate chondrocyte
activities including proliferation, shaping, and survival [43–46],
it can be reasonably presumed that defective ECM interferes
with proper interstitial growth and with normal sequence of
chondrocyte maturation in dysplastic EGP. In this view, it is also
important to consider that proteoglycans function as modulators
of growth factor binding to cell membrane receptors [37, 47,
48, 49]. According to this scenario, it is not surprising that
chondrocyte differentiation in dtdst mutants is abnormal under
several aspects, including cell proliferation, matrix secretion,
and cell shape, size, and alignment.
To gain new insight in the pathogenesis of different forms
of human chondrodysplasias, different mouse strains bearing
natural or artificially induced mutations have been studied,
including brachymorphic mice with natural alterations in
sulphation pathway and transgenic mice carrying mutations in
ECM protein genes [19–21]. Interestingly, our data concerning
cell proliferation in dtdts mutant animals are consistent with
those observed in brachymorphic mice [19]. Consistent with
our hypotheses, although with different ultrastructural aspects,
impaired alterations in the expression of type II collagen—and
matrilin 3—genes not only determines perturbation in the
cartilage ECM, but also exert profound effects on chondrocyte
differentiation [20, 21].
In this study we have not focused our observations on terminal stages of chondrocyte differentiation since the interpretation
of these stages is still a matter of debate even in normal EGP.
In fact, to date no conclusive consensus exists about the fate of
terminally differentiated chondrocytes of the calcifying zone [3,
50]. Although most chondrocytes undergo classical apoptosis
[51, 52], it has been suggested that they may also undergo
alternative programmed cell death (e.g., chondroptosis [53])
or transdifferentiation into osteoblasts [54, 55]. It is conceivable
that alterations in terminal stages of chondrocytes, including
self-destruction by apoptosis, may occur in dtdts mutants.
Moreover, we hypothesized that modifications in cartilage
ECM have significant implications on osteoblasts differentiation and bone formation at the boundary between the EGP
and the forming bone. Although not demonstrated, the latter
implications may have the ultimate responsibility for the
incomplete lengthening of long bones which is observed in
chondrodiastrophic patients and dtdst mutant animals.
Future studies will be addressed to understand molecular
mechanisms linking chondrocyte differentiation and ECM. Also
studies will attempt to identify therapeutical solutions efficient
in balancing sulphate deficiency that occurs in dtdst dysplastic
growth plate.
ACKNOWLEDGMENTS
We thank Prof. Antonio Rossi, dept. of biochemistry,
University of Pavia, for providing wild-type and dtdst mice for
the experiments and we are grateful to Aurora Farina, Dr. Paola
Giannella, and Dr. Luca Piccoli (department of experimental
medicine, University of Pavia) for valuable technical assistance.
This research was supported by grants from the University of
Pavia (F.A.R.), Banca del Monte di Lombardia Foundation (AC,
2004–2006) and COFIN (AC, 2003) from the Italian Ministry
of Education, University and Research.
Declaration of Interest: The authors report no conflicts of
interest. The authors alone are responsible for the content and
writing of this article.
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