the elastic network of articular cartilage: an immunohistochemical study of elastin fibres and...

The elastic network of articular cartilage: animmunohistochemical study of elastin fibres andmicrofibrilsJing Yu and Jill P. G. Urban

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

Abstract

The elastic network of articular cartilage was investigated by immunohistochemistry using specific antibodies to

elastin and fibrillin-1. Articular cartilage was dissected from defined regions of bovine metacarpophalangeal

joints. Elastin fibres and microfibrils were dual-immunostained by labelling with distinct fluorescent dyes. A

conventional fluorescence microscope combined with a polarized light filter was used to study the organization

and degree of colocalization of elastin fibres, microfibrils and of the collagen network. We observed an elabo-

rately organized elastic network. In the uppermost superficial zone, where few cells were present, elastin fibres

and microfibrils formed a dense three dimensional network showing some degree of colocalization. The thick-

ness and organization of this elastic network varied dramatically from region to region and was most extensive

in the metacarpal palmar region. In the middle and deep zones, very few elastin fibres were observed but

microfibrils formed a network in the inter-territorial matrix and dense network around the cells. Our finding of

a three dimensional network of dense, well organized elastin fibres and microfibrils in the surface zone of the

articular cartilage matrix, and a dense network of microfibrils around the cells deeper into the tissue suggests

the elastic network could play both a mechanical and a biological role in articular cartilage.

Key words articular cartilage; elastic fibres; elastic network; elastin; fibrillin-1; microfibrils; oxytalan fibrils.

Introduction

Articular cartilage covers the articular ends of subchondral

bones and acts to provide a low-friction gliding surface and

to minimize contact stresses (Gore et al. 1983; Bhosale &

Richardson, 2008). Its matrix is composed mainly of water,

collagens and proteoglycans together with numerous non-

collagenous proteins, which, though forming only a small

fraction of the total matrix, are vital for cartilage health.

Collagens, mainly composed of type II collagen together

with type IX and XI collagens, form a fibrillar network

which provides tensile stiffness and strength to the tissue

while collagen III and collagen VI are mainly pericellular in

healthy cartilage (Eyre, 2004). The large aggregating pro-

teoglycan, aggrecan, is a major component, stabilizing

water and providing the tissue with stiffness to resist com-

pressive loads. Small proteoglycans, including decorin,

biglycan and fibromodulin, are also present and may help

to stabilize the matrix as well play a biological role in bind-

ing growth factors (Yamaguchi et al. 1990; Svensson et al.

2001; Martel-Pelletier et al. 2008).

The structure and organization of collagens and proteo-

glycans in articular cartilage have been studied extensively,

but the existence of an elastic network has been ignored

until very recently, mainly because a small number of earlier

histochemical studies reported that little elastin was present

in articular cartilage (Cotta-Pereira et al. 1984; Ortega et al.

2001; Naumann et al. 2002). Recently, however, well orga-

nized elastin fibres have been found in the superficial zone

of articular cartilage using multiphoton microscopy together

with histochemistry (Yeh et al. 2005) or immunohisto-

chemistry (Mansfield et al. 2009). Elastin fibres, however, do

not usually exist in isolation but are the core components of

an elastic network. Elastic networks have been considered to

include three types of fibres, i.e. elastic fibres, elaunin fibres

and oxytalan fibres (Montes, 1996). Both elastic fibres and

elaunin fibres consist of a central core elastin surrounded by

microfibrils, i.e. fibrils of ca. 10–12 nm in diameter under an

electron microscope (Greenlee et al. 1966; Montes, 1996)

Correspondence

Jing Yu, Department of Physiology, Anatomy and Genetics, Univer-

sity of Oxford, Parks Road, Oxford OX1 3PT, UK. T: + 44 1865

272479; F: + 44 1865 272469; E: [email protected]

Accepted for publication 22 December 2009

Article published online 10 February 2010

ªª 2010 The AuthorsJournal compilation ªª 2010 Anatomical Society of Great Britain and Ireland

J. Anat. (2010) 216, pp533–541 doi: 10.1111/j.1469-7580.2009.01207.x

Journal of Anatomy

with fibrillin-1 as the major component (Sakai et al. 1986;

Handford et al. 2000; Ramirez et al. 2004). Oxytalan fibrils

are composed only of microfibrils and contain no elastin and

have been identified in the extracellular matrix surrounding

the chondrocytes (Cotta-Pereira et al. 1984; Ortega et al.

2001); their organization in this region is reported to change

with age (Keene et al. 1997). However, the organization of

microfibrils throughout different depths in the articular carti-

lage tissue has not been well studied and so little is known

of the association of microfibrils with elastin fibres in articu-

lar cartilage.

The objectives of this study were thus to use immunohis-

tochemical methods to investigate the overall elastic net-

work. The ultimate aim of this work is to provide insight

into the role of the elastic network in articular cartilage.

Materials and methods

Bovine metacarpophalangeal joints from 18–24 month old steers

were obtained from a local abattoir. Seven joints from different

animals were used and in total nine different regions of the car-

tilage were investigated in this study. Figure 1 illustrates the

preparation of the specimens. Cartilage tissues slices were dis-

sected from the joint with a scalpel blade within 4 h of slaugh-

ter. Tissue slices were snap-frozen and stored at )80 �C until

use.

Immunohistology

Fresh specimens were used throughout this study as fixation

with formalin was found to reduce the activity of the antibodies

significantly. Tissue sections with a thickness of 20 lm were

cut with a cryomicrotome, mounted on polylysine-coated

microscope slides (VWR International Ltd) and immediately

immunostained using the method reported previously (Yu et al.

2007).

Briefly, the procedures were carried out at room tempera-

ture, and PBS (phosphate-buffered saline) was used for wash-

ing (10 min, three times) between each incubation, unless

otherwise indicated. After washing, specimen sections were

blocked with 6% normal donkey serum (Stratech Scientific Ltd,

diluted with PBS) for 30 min at room temperature. Then the

specimen was incubated with a polyclonal rabbit anti-human

alpha elastin (cross-reacted with bovine, MorphoSys UK Ltd,

Cat. no: 4060-1060, 1 : 50 dilution with PBS) for 2 h at room

temperature. After washing, specimens were incubated with

donkey anti-rabbit IgG conjugated with a fluorescent Cy3 dye

(Stratech Scientific Ltd, Cat. no: 711-165-152, 1 : 100 dilution

with PBS) for 30 min at room temperature. The specimens

were then washed with Tris buffer (50 mM Tris–HCl containing

10 mM calcium acetate) and blocked with 6% normal donkey

serum diluted with the Tris buffer for 30 min. The specimens

were then incubated with monoclonal mouse anti-bovine fibril-

lin-1 antibody (GenWay Biotech, Inc. San Diego, CA, USA, Cat.

no: 20-787-275143, 1 : 50 dilution with the Tris buffer) over-

night at 4 �C. After washing, specimens were then incubated

with donkey anti-mouse IgG conjugated with fluorescein FITC

dye (Stratech Scientific Ltd, Cat. no: 715-095-150) for 30 min.

After washing, specimens were mounted with hardset mount-

ing medium containing DAPI (Vector Laboratory, Cat. no:

H-1500), which is able to stain cell nuclei.

Negative controls were incubated with normal rabbit serum

instead of the elastin antibody and normal mouse IgG instead

of the fibrillin-1 antibody (all at the same dilution factor); no

cross-reactivity was found.

A B

C D

Fig. 1 Schematic view of the cartilage specimen showing the different regions referred to in the text. (A,B) Opened bovine metacarpophalangeal

joint. R, region, which is numbered to indicate the region studied. (C) Diagram of a histological sample in three dimensions, dashed line indicates

that half of the sample was used for transverse and half for cross-section cutting. (D,E) Transverse (sections cut parallel to the surface) and radial

cross-sections (surface-to-bone sections) respectively.


The elastic network of articular cartilage, J. Yu and J. P. G. Urban534

Microscopy

The fluorescent signal was detected using a conventional fluo-

rescence microscope (Leica DMRB) with three different filters

for the three different dyes: single fluorochrome filters for DAPI

(blue), and two filter cubes from Chroma Technology Corp.

(Cat. nos 41001 HQ and 31014 WB) for FITC and Cy3, respec-

tively. A Nikon DXM 1200 digital camera were used to take the

images. Different images of three dyes can be taken sequen-

tially at the exactly same site of the tissue; gentle switching

between the filters manually, ensures no movement of the spec-

imen. A light micrograph under a polarized filter can be taken

on the exactly same site by manually switching off the fluores-

cent light and turning on the conventional light.

Image processing and analysis

ADOBE PHOTOSHOP software was used to process the images. DAPI

and FITC are blue and green fluorescent dyes, respectively. Cy3

is a yellow-orange-red fluorescent dye in nature, but was pro-

cessed to be red to improve image contrast when merging

images from the same site of the tissue. All merged individual

images were taken from exactly the same sites.

Results

Throughout this study elastin fibres were defined as fibres

which were immunostained by the elastin antibody,

whereas microfibrils were defined as the fibres which were

immunostained with the fibrillin-1 antibody. Based on the

classical definition of elastic networks (Montes, 1996), the

elastic network here includes elastin fibres and microfibrils.

The elastin fibre network

Microscopically, articular cartilage can be divided into four

different zones (Khan et al. 2008), viz. a superficial zone

(tangential zone), a middle zone (transitional zone), a deep

zone (radial zone) and a calcified zone. Figure 2, a radial

cross-sectional view of cartilage from surface to bone, illus-

trates these different zonal regions.

Generally, elastin fibres were found in the superficial

zone of all the regions examined (Fig. 1). Interestingly, elas-

tin fibres were found in the middle and deep zones of the

metacarpal palmar region but were very sparse in these

zones in the metacarpal cortical ridge, proximal phalanx

and sesamoids.

Elastin fibres in articular cartilage viewed in radial

cross-section

Figure 3 shows the typical organization of the elastin

fibres viewed in radial cross-section and also the relation-

ship between the elastin and collagen networks. At the

metacarpal cortical ridge (Fig. 3A–F), proximal phalanx

(Fig. 3G–I) and sesamoids (data not shown), elastin fibres

were mostly found in the uppermost surface zone where

few cells were present, with zone thickness varying from

10 to 35 lm depending on the region and joint. No pre-

ferred orientation of these fibres is obvious in the superfi-

cial zone. Little elastin was observed in the middle and

deep zones (Fig. 3A,D,G). In contrast, in the metacarpal

palmar region (Fig. 3J–O), elastin fibres were distributed

not only in the uppermost surface zone but also pene-

trated deeper into the middle zone, up to 200 lm from

the cartilage surface (Fig. 3J). Such an extensive organiza-

tion of elastin fibres has not been reported previously. It

can be noted that here elastin also seems strongly associ-

ated with cells (white arrowheads in Fig. 3M). As our nega-

tive control for elastin using normal rabbit serum showed

no positive signal in this region, we suggest elastin may be

present in the cytoplasm of chondrocytes in the middle

and deep zones, noting our image collects signals from

total tissue thickness on the slide, viz. 20 lm. Obviously a

more detailed study is needed to confirm this observation,

but is beyond the scope of this paper. Therefore the discus-

sion of elastin expression in the cell is not included further

in this manuscript.

Elastin fibres in articular cartilage viewed in

transverse section

In transverse view, the elastin fibres generally appeared

much better organized than in cross-sectional view. Fig-

ure 4 shows the typical results. The elastin fibres form a

dense and well organized network in all the regions shown.

The dominant elastin fibres (white arrowheads in Fig. 4)

appear parallel to each other. However, a large number of

elastin fibres also can be found lying at different angles to

the main elastin fibre arrays (yellow arrowheads in Fig. 4).

Furthermore, many elastin fibres appear to lie vertically

toward the cutting planes (dots indicated with white

arrows in Fig. 4); such vertically organized elastin fibres

have not been reported previously, possibly because of the

Fig. 2 A light micrograph of a radial cross-section through articular

cartilage to illustrate the different zonal regions from the surface to

the bone.


The elastic network of articular cartilage, J. Yu and J. P. G. Urban 535

25 µm

25 µm 25 µm 25 µm

25 µm

50 µm 50 µm 50 µm

25 µm 25 µm

25 µm 25 µm 25 µm

25 µm 25 µm

A BElastin Collagen Merged

C

D E F

G H I

J K L

M N O

Fig. 3 Typical radial cross section view of elastin fibre organization (A,D,G,J,M) in different regions of cartilage. The network varies from region to

region. Collagen structure (B,E,H,K,N) was demonstrated using a polarized filter. The micrographs are orientated so that the cartilage surface is

uppermost and is indicated with white arrows. Merged images (C,F,I,L,O) indicate that elastin fibres are seen mainly in the uppermost superficial

zone of the cortical ridge (A–C: R-1 in Fig. 1; D–F: R-2 in Fig. 1) and phalanx (G–I: R-5 in Fig. 1), but in the palmar region (J–L: R-2 in Fig. 1; M–O:

R-4 in Fig. 1) are found deeper into the tissue and are seen also in the middle zone. White arrowheads in (M) indicate elastin associated with cells.

R, region of the joint as shown in Fig. 1.



resolution of the MPM was more limited. The elastin fibres

thus appear to form an oriented 3-D network.

Microfibril network and its colocalization with

elastin fibres

Generally, the network of microfibrils was denser, more

extensive and more elaborate than that of the elastin fibre

network. Microfibrils were seen in all the regions examined

not only in the superficial zone but also throughout the

middle and deep zones. In addition, the fibril organization

and localization varied with depth and with region of the

joint. Interestingly microfibrils did not always colocalize

with elastin fibres.

Microfibril organization in articular cartilage viewed

in radial cross-section

Figure 5 shows the typical organization of microfibrils and

their degree of colocalization with elastin fibres in cross-

sectional view.

At the metacarpal cortical ridge (Fig. 5A), proximal pha-

lanx and sesamoids (data not shown), thicker microfibrils

(white arrow in Fig. 5A) generally appeared only in the

uppermost superficial layer, where the orientation of micro-

fibrils was perpendicular to the cartilage surface (Fig. 5A,

white arrow); in general, there was no dominant fibre

direction (data not shown) but network organization varied

between regions and from joint to joint. Microfibrils gener-

ally appeared in the same orientation as the elastin fibres in

the surface zone (Fig. 5B, white arrow). In the middle and

deep zones, microfibrils appeared finer than at the surface

(Fig. 5A) and were distributed throughout areas of the tis-

sue where little elastin was observed (Fig. 5B). Microfibrils

were oriented mainly perpendicular to the cutting plane

(dots in Fig. 5A) through the whole matrix (yellow arrow in

Fig. 5D), which indicates that most of microfibrils were

organized parallel to the cartilage surface at the middle

and deep zones. The microfibril network was clearly denser

around the cells (white arrow in Fig. 5D) than in the inter-

territorial matrix.

In the metacarpal palmar region, by contrast, thicker

microfibrils were found in the middle zone (Fig. 5G,I),

where elastin fibres were also observed (Fig. 5H).

In the sagittal ridge (palmar), the uppermost cartilage

surface is indicated by white arrowheads in Fig. 5G,H. Both

microfibrils and elastin fibres appeared finer and more fila-

mentous in the uppermost superficial zone than in the mid-

dle zone (Fig. 5I). In terms of colocalization, microfibrils did

not appear always to colocalize with elastin fibres (white

arrows in Fig. 5G,H), although the elastin network in

Fig. 5H was very similar in organization to that of the

microfibrils (Fig. 5G).

Microfibril organization in articular cartilage viewed

in transverse section

Microfibril organization and its colocalization with elastin

fibre organization viewed in cartilage transverse section

was investigated using specimens from the cortical ridge of

A B C

D E F

R-2 R-4

25 µm25 µm 25 µm

R-8

Fig. 4 Typical micrographs of transverse sections of articular cartilage, showing the elastin fibre organization in different regions of the joint.

White arrowheads indicate the main elastin fibre array. Yellow arrowheads highlight elastin fibres forming an angle to the main elastin fibre

arrays. Elastin fibres are also seen vertical to the cutting plane (dots indicated with white arrows). Enlarged figures (D–F) are taken from the

highlighted areas (yellow squares) in (A–C), respectively. R, region of the joint as shown in Fig. 1.



regions 2 and 4 (sagittal ridge in Fig. 1). Figure 6 shows typ-

ical results. Generally, microfibrils appeared dense, filamen-

tous, long (more than 100 lm in length, indicated by white

arrows in Fig. 6A,D,G), and also oriented parallel to each

other and to the cartilage surface. However, the colocaliza-

tion of microfibrils with elastin fibres appeared strikingly

different between the two regions studied. The two net-

works colocalized highly in region 2 (Fig. 6A–C), but mostly

were not colocalized in region 4 (Fig. 6D–I). Microfibril

organization appeared to be depth-dependent in region 4;

the fibrils appeared finer and more filamentous in the

uppermost cutting plane (Fig. 6D) from superficial zone

than in lower cutting plane from the middle zone of the

tissue (Fig. 6G). This corresponds to the observations in

Fig. 5G, where microfibrils were finer and more filamentous

in the superficial zone than in the middle zone in transverse

view. In Fig. 6G, arrays of shorter microfibrils (yellow

arrows) appear almost vertically oriented (sometime also

obliquely, data not shown) to the long main stream of mi-

crofibrils and appear to connect a colocalized network of

long elastin fibres and microfibrils (Fig. 6I).

Discussion

Here we investigated the organization of the elastic net-

work of bovine articular cartilage by using specific dual

immunostaining of elastin and fibrillin-1. We observed net-

works of elastin fibres and microfibrils whose distribution

varied with depth from the surface and from region to

region. In the uppermost superficial zone where few cells

are present, elastin fibres and microfibrils formed a dense

three-dimensional extracellular network (Fig. 3–6) which

co-localized to some extent (Figs 5 and 6). This layer was

most extensive in the palmar regions (Fig. 5I). In the middle

and deeper zones, elastin fibres were sparse, whereas

microfibrils formed a fine filamentous network in the extra-

25 µm 25 µm

Microfibrils Elastin fibres Merged

25 µm

25 µm

10 µm 10 µm 10 µm

25 µm 25 µm

A B C

D E F

G H I

Fig. 5 Typical cross-sectional views of cartilage showing microfibril organization (A,D,G) and its colocalization (C,F,I) with elastin fibres (B,E,H) in

different regions of the joint. (A–C) Tissue from cortical ridge (R-1 in Fig. 1) with white arrows indicating the cartilage surface. (D–F) Higher

magnification view of microfibrils and elastin at the middle and deeper zones, with white arrow indicating the pericellular matrix and yellow arrow

the interterritorial matrix. (G–I) Tissue from palmer region at sagittal ridge (R-4 in Fig. 1) with white arrowheads indicating the topmost cartilage

surface at the sagittal ridge and white arrows the non-colocalized microfibrils and elastin fibres.



cellular matrix and denser network around the cells

(Fig. 5A–F). It thus appears microfibrils fulfil different

functions in different regions of cartilage.

The diameter of the fibrils varied, with some thick

straight fibrils of both elastin and fibrillin-1 visible. Microfi-

brils are defined traditionally as fibrils of 10 nm in diameter

as seen by electron microscopy (Greenlee et al. 1966; Robin-

son et al. 2006). The considerably thicker fibrils staining

positive for fibrillin-1 observed here (Figs 5A,G and 6G)

could thus be bundles of parallel arrays of microfibrils simi-

lar to those observed in articular cartilage (Hesse, 1987;

Keene et al. 1997) by electron microscopy. Elastin fibres also

appeared filamentous in some regions, particularly in the

uppermost surface layer (Figs 3A,G, 5H and 6B); however,

thicker fibres were also visible in this region (Figs 5 and 6).

In Montes’ review (Montes, 1996), elaunin fibres were

classed as fibres containing less elastin in the central core

than elastic fibres. The variation in the diameter of elastin

fibre found in the surface layer could be regarded as a mix-

ture of elaunin fibres (filamentous elastin fibres) and elastic

fibres (thicker elastin fibres). The elastic network of skin has

been reported to be a continuous network formed from all

25 µm

Microfibrils Elastin Merged

25 µm 25 µm

25 µm 25 µm 25 µm

10 µm 10 µm 10 µm

A B C

D E F

G H I

J K L

Fig. 6 Typical micrographs of the transverse sections of cartilage from the cortical ridge showing the organization of microfibrils (A,D,G) and their

colocalization (C,F,I) with elastin fibres (B,E,H); micrographs show sections from region 2 (A–C) and region 4 (D–I). Images (D–I) are from same

tissue specimen, but at different cutting planes; images (D–F) from the uppermost cutting plane of the superficial zone, and images (G–I) from the

lower cutting plane (the second 20-lm section, i.e. about 20–40 lm depth from the cartilage surface). White arrows indicate long fibres

(> 100 lm in length). Yellow arrows in (G) indicate a microfibrillar network almost vertical to the main fibre array, enlarged in (J) from highlighted

area (yellow square). White arrowheads in (E) and (H) indicate fibres which are vertical or oblique to the cutting plane (dots in the images),

enlarged in (K) and (L) from highlighted areas (yellow squares) in (E) and (H), respectively.



oxytalan fibres (microfibrils), elaunin fibres and elastic

fibres. Such a network connects the epidermis with the

dermis (Montes, 1996). The elastic network of the super-

ficial zone of articular cartilage could play a similar role

in connecting the tangential zone with the transitional

zone.

In the uppermost superficial zone, elastin fibres (Fig. 4)

and microfibrils (Fig. 6) were dense and oriented mostly

parallel to each other and to the cartilage surface, but some

were oriented obliquely, and large fibres perpendicular to

the cutting plane were also visible (dots in Figs 4 and 5).

The elastic network at the cartilage surface thus appeared

to form a dense three-dimensional network varying in

thickness from about 10 lm (Fig. 3) up to 200 lm (Figs 3J

and 5) depending on the region of cartilage examined; it

was generally thickest in the palmar regions. The observed

variation in the organization of the elastic network across

the cartilage surface could result from differences in the

pattern of mechanical loading across different regions of

the joint (Brama et al. 2001) and be important functionally.

In other tissues, expression of microfibrils is partially regu-

lated by mechanical stress (Lorena et al. 2004); thus the

organization of the network could result from remodelling

in response to customary load. Examination of changes in

the elastic network in articular cartilage during the first few

months of life as mechanical stresses change across the joint

would useful to clarify this point, but is beyond the scope

of this study.

The superficial zone of the articular cartilage has

attracted the attention of many researchers recently, as pro-

genitor cells (Dowthwaite et al. 2004) were identified in this

layer and also because growth factors including TGF-bs

(transforming growth factor beta) (Hayes et al. 2001), FGF-2

(fibroblast growth factor) (Vincent et al. 2007), IGF (insulin-

like growth factor) and IGF-binding proteins (Archer et al.

1994; Hayes et al. 2001; Vincent et al. 2007) as well as perl-

ecan and versican (Hayes et al. 2008) are reported to be

strongly expressed in this zone.

Microfibrils are now known to play an important role in

controlling the sequestration of the growth factor TGF-b in

the matrix (Neptune et al. 2003; Ramirez et al. 2004; Cohn

et al. 2007). Microfibrils could thus, as well as playing a

mechanical role, act as the repository for TGF-bs in the

superficial zone of articular cartilage and also throughout

cartilage because of their pericellular distribution in the

deeper zones.

Although the functions of microfibrillar and elastic net-

works in cartilage have yet to be investigated, the detailed

organization of these networks indicates that their role can

no longer be ignored.

Acknowledgements

Authors would like to thank Arthritis Research Campaign (grant

no. 16480) and the Fondation Yves Cotrel for financial support.

Author contributions

Jing Yu: concept ⁄ design, acquisition of data, data analysis ⁄ inter-

pretation, drafting of the manuscript. Jill Urban: con-

cept ⁄ design, data analysis ⁄ interpretation, critical revision of the

manuscript and approval of the article.

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