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. 2014 Sep 1;225(5):548–568. doi: 10.1111/joa.12232

Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage

Maria Löfgren 1, Stina Ekman 1, Emilia Svala 1,2, Anders Lindahl 2, Cecilia Ley 1, Eva Skiöldebrand 1,2
PMCID: PMC4292756  PMID: 25175365

Abstract

Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell-like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor-like domain 7 (EGFL7), the stem cell-indicating factor Stro-1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin-1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro-1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro-1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell-like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage.

Keywords: EGFL7, extracellular matrix, horse, Notch signalling, Stro-1

Introduction

The formation of synovial joints includes a series of events regulated by different factors and signalling pathways (Archer et al. 2003). The immature skeleton comprises a cartilage model, formed via chondrogenesis, which is gradually replaced by bone during endochondral ossification (Goldring, 2012). The endochondral ossification of vascular growth cartilage follows a distinct pattern, with chondrocytes in resting, proliferative and hypertrophic stages. During this process, chondrocytes synthesise variable extracellular matrix (ECM) components that contribute to the zonal properties of the articular cartilage and in the growth plate (Behonick & Werb, 2003). The terminally differentiated hypertrophic chondrocytes in the growth plate secrete and modulate an ECM that mineralises and is used as a scaffold for bone formation (Dreier, 2010; Mackie et al. 2011).

The growth plate is surrounded by a fibrochondrosseous structure that encircles the bone and was first described by Ranvier in 1873. The zone harbours progenitor cells and is called the perichondrial groove of Ranvier (Shapiro et al. 1977). Further studies have identified a stem cell niche in this area (Karlsson et al. 2009), with transit amplifying cells present along a potential migration route towards the articular cartilage (Henriksson et al. 2013).

In contrast to the vascular growth cartilage, mature articular cartilage comprises avascular hyaline cartilage with a zone of calcified cartilage adjacent to the subchondral bone. Articular cartilage is organised in superficial, middle and deep zones (Pritzker & Aigner, 2010), and the zones contain chondrocytes with an ECM composition that varies with depth and proximity to the cells (Las Heras et al. 2012).

The expression and organisation of the ECM in the developing equine articular and growth cartilage largely remains to be elucidated. Currently, studies of equine developing cartilage have focused on the organisation and structure of the collagen network (Brama et al. 2000, 2009; Cluzel et al. 2013). The organisation of the collagen network is regulated by a number of other ECM proteins, such as cartilage oligomeric matrix protein (COMP), matrilin-1, fibromodulin and chondroadherin (Heinegård, 2009); however, whether they are present and their location in the equine joint and growth cartilage is not known. The interaction between matrix molecules and between matrix molecules and cells provides a network that defines the functional properties of the tissue (Heinegård, 2009).

The signalling pathways regulating the development of zone-specific chondrocyte phenotypes have not been fully clarified (Goldring et al. 2006). However, Notch signalling is a likely regulator of cartilage development considering its involvement in the development of various cell types including chondrocytes (Hayes et al. 2003; Watanabe et al. 2003; Dowthwaite et al. 2004; Hardingham et al. 2006; Karlsson et al. 2007; Williams et al. 2009). Active Notch signalling influences cell proliferation and inhibits cartilage differentiation (Watanabe et al. 2003; Dowthwaite et al. 2004; Karlsson et al. 2007). Activation of Notch signalling occurs when specific ligands interact with Notch receptors (Notch1-4 in mammals), leading to transcription of target genes, such as the hairy and enhancer of split (Hes) family. The largest class of Notch ligands is the Delta/Serrate/LAG2 (DSL) family, which in mammals includes Jagged1, Jagged2, Delta-like (Dll)1, Dll3 and Dll4 (Kopan & Ilagan, 2009; D'Souza et al. 2010; Wang, 2011). Notch receptors and ligands are present in the cartilage of varying species but with different distributions (Hayes et al. 2003; Watanabe et al. 2003; Dowthwaite et al. 2004; Hiraoka et al. 2006; Karlsson et al. 2007, 2008a; Ustunel et al. 2008; Williams et al. 2009; Mahjoub et al. 2012).

Progenitor cells expressing Notch1 have been isolated from bovine (Dowthwaite et al. 2004), human (Williams et al. 2010) and equine (McCarthy et al. 2012) articular cartilage. The progenitor cells isolated from human and equine cartilage additionally express Stro-1, a potential stem-cell marker (Williams et al. 2010; McCarthy et al. 2012). Stro-1 has also been demonstrated in chondrocytes of human, rabbit and murine articular cartilage (Grogan et al. 2009; Karlsson et al. 2009; Otsuki et al. 2010; Ozbey et al. 2014) and in cells of the perichondrial groove of Ranvier in rabbits (Karlsson et al. 2009).

Notch signalling may become dysregulated when other proteins, such as epidermal growth factor-like domain 7 (EGFL7), bind to the Notch receptor (Schmidt et al. 2009; Wang, 2011). EGFL7 is involved in vascular development and cell migration (Soncin et al. 2003; Parker et al. 2004; Campagnolo et al. 2005) and is associated with angiogenesis in vascular injury (Campagnolo et al. 2005) and hypoxia (Gustavsson et al. 2007; Badiwala et al. 2010). EGFL7 competes with Jagged ligands and modulates Notch signalling in neural stem cells, leading to decreased proliferation and increased differentiation (Schmidt et al. 2009). EGFL7 expression has, to our knowledge, not been delineated in the cartilage of any species.

The aim of this study was to increase the understanding of the regulatory factors controlling chondrocyte homeostasis and differentiation using immunohistochemistry to study equine chondrocytes in developing prenatal and postnatal growth plates, zones of Ranvier and articular cartilage from the distal radius during normal differentiation. The surface antigen Stro-1, the Notch signalling components Notch1, Jagged1, Dll4 and Hes1 were immunohistochemically localised to specific chondrocyte populations. Additionally, the Notch dysregulating protein EGFL7 was delineated with respect to a possible interaction between EGFL7 and Notch1. We also investigated the protein expression of the structural matrix molecules COMP, fibromodulin, matrilin-1 and chondroadherin, which are all known to be involved in collagen fibril formation and ECM assembly in the developing articular and growth cartilage. Our hypothesis was that the localisation of proteins present in the growth cartilage and in the zone of Ranvier during normal cell differentiation may serve as a template for identifying progenitor cells in the adult articular cartilage. This differentiation model may later aid in discovering progenitor cells and uncovering evidence of repair in cartilage pathology.

Materials and methods

Equine samples

Immature equine osteochondral tissues representing different developmental stages of the articular and growth cartilage of the distal radius (Fig. 1) were collected postmortem from two foetuses (6.5 and 10 months’ gestational age) following diseases of the mares (duration of approximately 1 week before euthanisation) and from two young horses (a 2-day-old and a 1-year-old) without clinical signs of orthopaedic disease or macroscopic lesions of the joints sampled. Growth-plate cartilage was sampled from all four individuals and articular cartilage was available from the 6.5-month-old foetus, the 2-day-old and the 1-year-old horses.

Fig. 1.

Fig. 1

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Schematic drawing of the equine forelimb showing the location used for collecting material at the distal radius (see arrow). Illustration by Alexandra Leijon.

Sample preparation

Osteochondral slabs from the distal radius, cut in a lateromedial direction (approximately 5 mm thick), were sampled using a band saw (KT-400, Klaukkala, Finland). The slabs were fixed in 10% neutral buffered formalin for 2 days, then decalcified in formic acid for 5–10 days, embedded routinely in paraffin, cut into 4-μm sections and stained with haematoxylin & eosin and picrosirius red.

Immunohistochemistry

The tissue sections were deparaffinised, dehydrated in a graded series of alcohols and washed in phosphate-buffered saline (PBS; Medicago AB, Uppsala, Sweden). Unless otherwise noted, PBS was used for all dilutions below. The sections were digested with 8000 U mL–1 hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) and then 0.05 U mL–1 chondroitinase ABC (Sigma-Aldrich) for 1 h at 37 °C. Sections were then rinsed in PBS, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide, followed by rinsing in PBS. Non-specific binding was blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) containing 100 mm glycine (Sigma-Aldrich) for matrix molecules and Stro-1 or with 3% BSA for Notch signalling components. All primary antibodies (Table 1) were diluted in 3% BSA and then added to the sections. Normal goat IgG (sc-2028; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit IgG (X0903, Dako, Glostrup, Denmark), mouse IgM (MGM00, Invitrogen, Frederick, MD, USA) or chicken IgY (AB-101-C, RnD Systems, Minneapolis, MN, USA) were run in parallel with the primary antibodies as isotype controls using the same concentrations as for the primary antibodies. The sections were incubated at 4 °C overnight, washed with PBS, blocked with 3% BSA containing 100 mm glycine for matrix molecules, and Stro-1 or 3% BSA for Notch signalling components. Sections were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Table 1). Rinsing with PBS-Tween™ (Medicago AB) was performed before and after visualisation of labelling with TSA-Direct Cy3 kit (Perkin Elmer, Waltham, MA, USA) according to the manufacturer's instructions. Sections were rinsed with PBS, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Sigma-Aldrich). Finally, sections were rinsed in PBS and mounted with ProLong® Gold Antifade Reagent (Life Technologies, Carlsbad, CA, USA). The sections were evaluated subjectively via observations using a Nikon Eclipse E600 fluorescence microscope and NIS Elements Basic Research software version 3.22.11 (Nikon Instruments Inc., Melville, NY, USA). The Notch signalling components and Stro-1 were graded as absent, a few stained cells, many stained cells or a majority of cells stained (Fig. 2A-C). The ECM was divided into interterritorial and pericellular compartments. Because it was not possible to differentiate the pericellular and territorial (between adjacent cells) ECM using light microscopy, both were referred to as pericellular areas (Kvist et al. 2008). Interterritorial and pericellular staining was graded for the matrix molecules as being absent, weak, moderate or intense (Fig. 2D-I).

Table 1.

Antibodies used for immunohistochemical staining.

Protein Primary antibody Catalogue number Dilution Secondary antibody Catalogue number Dilution
COMP Rabbit polyclonal COMP a 1 : 1000 Goat anti-rabbit IgG HRP sc-2004b 1 : 150
Fibromodulin Rabbit polyclonal Fibromodulin a 1 : 1000 Goat anti-rabbit IgG HRP sc-2004b 1 : 150
Matrilin-1 Rabbit polyclonal Matrilin-1 a 1 : 1000 Goat anti-rabbit IgG HRP sc-2004b 1 : 150
Chondroadherin Chicken polyclonal Chondroadherin a 1 : 1000 Goat anti-chicken IgY HRP sc-2428b 1 : 150
Notch1 Goat polyclonal Notch1 (C-20) sc-6014b 1 : 75 Donkey anti-goat IgG HRP sc-3851b 1 : 150
Hes1 Goat polyclonal Hes1 (N-17) sc-13842b 1 : 100 Donkey anti-goat IgG HRP sc-3851b 1 : 150
Delta4 Goat polyclonal Delta4 (T-20) sc-18639b 1 : 100 Donkey anti-goat IgG HRP sc-3851b 1 : 150
EGFL7 Rabbit polyclonal EGFL7 (M-135) sc-66875b 1 : 150 Goat anti-rabbit IgG HRP sc-2004b 1 : 150
Jagged1 Rabbit polyclonal Jagged1 (H-66) sc-11376b 1 : 75 Goat anti-rabbit IgG HRP sc-2004b 1 : 150
Stro-1 Mouse monoclonal h-STRO-1 MAB1038c 1 : 50 Goat anti-mouse IgM HRP sc-2064b 1 : 150
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a

Provided by Professor Heinegård (Lund University, Sweden).

b

Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

c

RnD Systems (Minneapolis, MN, USA).

Fig. 2.

Fig. 2

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Representative examples of the grading used for Notch signalling components and Stro-1 (A-C) and matrix molecules (D-I). (A) A few stained cells, (B) many stained cells, (C) the majority of cells stained, (D) weak interterritorial staining, (E) moderate interterritorial staining, (F) intense interterritorial staining, (G) weak pericellular staining, (H) moderate pericellular staining and (I) intense pericellular staining.

Proximity ligation assay

Duolink™ in situ Proximity Ligation Assay (PLA) technology (Olink Bioscience, Uppsala, Sweden) was used to detect proteins in proximity to each other. The tissue sections were treated as described above until the application of the primary antibodies. Antibodies for Notch1 and EGFL7, at the same dilutions as described above, were diluted together in 3% BSA in PBS, added to the sections and incubated at 4 °C overnight. Negative controls omitting one of the antibodies were run in parallel. The assay was performed according to the manufacturer's instructions using Duolink II PLA probe anti-Goat MINUS, Duolink II PLA probe anti-Rabbit PLUS, Duolink II Detection Reagents Orange, Duolink II Wash Buffers for Fluorescence and Duolink II Mounting Medium with DAPI. The sections were evaluated using a Nikon Eclipse E600 fluorescence microscope and NIS Elements Basic Research software version 3.22.11 (Nikon Instruments Inc.).

Results

Histology

The 6.5-month-old foetus (Fig. 3A) contained cartilaginous epiphyses of the distal radius with endochondral ossification at the future growth plate and a rim of avascular articular cartilage towards the radial-carpal joint. The distal radius of the 10-month-old foetus (Fig. 3B), 2-day-old (Fig. 3C) and 1-year-old horses (Fig. 3D) was characterised by a growth plate between the epiphyses and metaphyses. The growth plates of all four individuals contained a resting zone with small chondrocytes surrounded by hyaline ECM followed by a zone of proliferation. The proliferation zone presented with flat chondrocytes in rows or clusters in lacunae. The zone closest to the ossification front comprised hypertrophic chondrocytes with a thin rim of ECM that, adjacent to the bone, had calcified and lost the cells, leaving empty lacunae with vessels growing into the cartilage template. Cartilage canals in the growth plates were identified in all sections (Fig. 3A-D). The vessels within these canals varied in size and frequency and were more abundant in the foetuses and 2-day-old horse than in the 1-year-old horse.

Fig. 3.

Fig. 3

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Sections included in the study stained with haematoxylin & eosin. Growth plates including the hypertrophic, proliferative and resting zones, and articular cartilage comprising the lower deep, upper deep, middle and superficial zones are indicated according to the abbreviations below. The zone of Ranvier is denoted as boxes in the growth plates and cartilage canals are indicated by arrows. (A) 6.5-month-old foetus, (B) growth plate of the 10-month-old foetus, (C) growth plate of the 2-day-old horse, (D) growth plate of the 1-year-old horse, (E) articular cartilage of the 2-day-old horse and (F) articular cartilage of the 1-year-old horse. See Figs 5 and 5 for higher magnification of the cells in the specific zones. O, ossification; H, hypertrophic zone; P, proliferative zone; R, resting zone; PC, perichondrium; LD, lower deep zone; UD, upper deep zone; M, middle zone; SZ, superficial zone. Scale bars: 1000 μm.

The zone of Ranvier, comprising densely packed elongated cells, was identified in the periphery of the growth plates of all horses (Figs 3A-D and 4A). Picrosirius staining (Fig. 4B) further differentiated the area from the surrounding collagenous structures that stained red.

Fig. 4.

Fig. 4

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Zone of Ranvier comprising densely packed cells located adjacent to the perichondrium in the periphery of the growth plate in the 2-day-old horse stained with (A) haematoxylin & eosin and (B) picrosirius red. See box in Fig. 3C for location of the zone of Ranvier in the studied section. V, vessel; C, cartilage; ZR, zone of Ranvier; PC, perichondrium. Scale bars: 100 μm.

The articular cartilage present in the sections from the 6.5-month-old foetus (Fig. 3A) the 2-day-old (Fig. 3E) and the 1-year-old (Fig. 3F) horses were divided into zones based on the terminology suggested by Pritzker & Aigner (2010). The superficial zone comprising the surface zone without cells was located sub-adjacent to the joint cavity and the upper zone with elongated-to-flattened chondrocytes parallel to the joint surface. The middle zone, showing grouped rounded chondrocytes, was followed by the deep zone, which was divided into upper and lower parts including chondrocytes organised perpendicular to the joint surface. The deep zones of the foetus and the 2-day-old horse contained vascular canals; however, the cartilage of the 1-year-old foal was avascular (Fig. 3A,E,F).

Immunohistochemistry

Growth plate

The ECM of all growth plates showed staining for COMP and fibromodulin. The staining often showed a transition from interterritorial staining in the foetus to pericellular staining in the postnatal horses (Figs 5 and 6A-D). Matrilin-1 was present in the hypertrophic zone of both foetus, in the proliferative zone of the youngest foetus and in the resting zone of both pre- and postnatal growth cartilage. The staining in the postnatal horses was restricted to the pericellular matrix (Figs 5 and 6E,F). Chondroadherin was localised interterritorially in all zones of the 6.5-month-old foetus and in the proliferative and hypertrophic zones of the 10-month-old foetus. Pericellular staining was observed in the proliferative zone of the 2-day-old horse and in the hypertrophic zones of both postnatal horses (Figs 5 and 6G,H).

Fig. 5.

Fig. 5

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Overview of immunohistochemical staining for matrix molecules in the hypertrophic, proliferative and resting zones of the growth plates. Panels to the left in the figure contain representative images of the zones. Intensity of staining is indicated by continuous or dotted lines as described below the figure.

Fig. 6.

Fig. 6

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Immunohistochemical staining for matrix molecules in the hypertrophic zone of the growth plates (ossification in upper areas *) from the 6.5-month-old foetus (left) and the 2-day-old horse (right). (A,B) COMP, (C,D) fibromodulin, (E,F) matrilin-1, (G,H) chondroadherin. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

A majority of the hypertrophic chondrocytes from both foetuses and the postnatal horses stained positive for the Notch signalling components Notch1, Hes1 and Dll4. Cells in the cartilage bone interface were also positive for EGFL7 (Figs 7 and 8A-H). Notch1, Hes1 and Dll4 were present in proliferative cells of the 6.5-month-old foetus but not in proliferative cells of the other horses, except for a few cells positive for Notch1 in the 10-month-old foetus (Fig. 7). Notch1, Hes1 and Dll4 were present in resting cells of the growth plates in the two foetuses, whereas the postnatal horses showed cells positive for Dll4 and the 1-year-old horse additionally showed a few cells that stained for Hes1 (Fig. 7). Jagged1 was not detected in the growth plate cartilage of any individual. Stro-1 was present in a few cells of the resting zone in the foetuses and in the proliferative zone of the 2-day-old horse (Fig. 7).

Fig. 7.

Fig. 7

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Overview of immunohistochemical staining for Notch signalling components and Stro-1 in hypertrophic, proliferative and resting zones of the growth plates. Panels to the left in the figure contain representative images of the zones. Grading of stained cells is indicated by continuous or dotted lines as described below the figure.

Fig. 8.

Fig. 8

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Immunohistochemical staining for Notch signalling components in the hypertrophic zone of the growth plates (ossification in upper areas *) of the 6.5-month-old foetus (left) and the 2-day-old horse (right). (A,B) Notch1, (C,D) Hes1, (E,F) Dll4, (G,H) EGFL7. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

Zone of Ranvier and cartilage close to the perichondrium

An absence of COMP distinguished the zone of Ranvier from the surrounding cartilage (Figs 9 and 10A). Fibromodulin was detected only in the 2-day-old horse, and pericellular matrilin-1 was detected in the postnatal horses (Fig. 9). Chondroadherin was present in the zone of Ranvier in all horses (Figs 9 and 10B). The Notch signalling components Notch1, Dll4 and Hes1 were not detected in the zone of Ranvier, whereas EGFL7 and Stro-1 were observed in all horses (Figs 10C,D, 11 and 12A). In addition to cellular staining for EGFL7, there was staining in the matrix of the zone of Ranvier in the 10-month-old foetus and the 2-day-old horse (Fig. 10C).

Fig. 9.

Fig. 9

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Overview of immunohistochemical staining for matrix molecules in the zone of Ranvier, in cartilage close to perichondrium and the perivascular area. Panels to the left in the figure contain representative images of the areas where the locations of the zone of Ranvier and cartilage close to the perichondrium are indicated by dotted lines in the images. Intensity of staining is indicated by continuous or dotted lines as described below the figure.

Fig. 10.

Fig. 10

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Immunohistochemical staining in the zone of Ranvier from the 2-day-old horse. The area is characterised by absence of (A) COMP and presence of (B) chondroadherin, (C) EGFL7 and (D) Stro-1. V, vessel; C, cartilage; ZR, zone of Ranvier; PC, perichondrium. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

Fig. 11.

Fig. 11

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Overview of immunohistochemical staining for Notch signalling components and Stro-1 in the zone of Ranvier, in cells close to perichondrium and in perivascular cells. Panels to the left in the figure contain representative images of the areas where the locations of the zone of Ranvier and cartilage close to the perichondrium are indicated by dotted lines in the images. Grading of stained cells is indicated by continuous or dotted lines as described below the figure.

Fig. 12.

Fig. 12

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Schematic drawing of the distal radius (2-day-old horse) showing the location of Stro-1-positive cells, denoted as grey cells. Representative Stro-1 immunohistochemical staining is shown and the location of the areas is denoted as boxes in the schematic drawing in the (A) zone of Ranvier, (B) chondrocytes close to the perichondrium and (C) superficial and middle zones of the articular cartilage. C, cartilage; ZR, zone of Ranvier; PC, perichondrium; *, joint cavity. Positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm. Illustration by Pontus Andersson.

In all horses, Stro-1-positive cells were also localised to the cartilage close to the perichondrium, which runs from the zone of Ranvier to the articular cartilage (Figs 11, 12B and 13A,B). In the foetuses, this area was discriminated from the surrounding cartilage based on the presence of EGFL7 in the cells and matrix (Figs 11 and 13C,D). Chondroadherin was present in all horses but was most intense in the 2-day-old horse (Figs 9 and 13E,F). Moreover, Matrilin-1 was more intensely stained in this area compared with the surrounding cartilage (Fig. 9). Immunostaining for COMP (Figs 9 and 10G,H), fibromodulin, Notch1, Dll4 and Hes1 was present in the area (Figs 9 and 11).

Fig. 13.

Fig. 13

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Immunohistochemical staining in the area close to the perichondrium (*) in the 10-month-old foetus (left) and 2-day-old horse (right). (A,B) Stro-1, (C,D) EGFL7, (E,F) chondroadherin, (G,H) COMP. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

Vascular areas in the growth cartilages

The ECM of the perivascular chondrocytes stained positive for all studied matrix molecules in all four specimens (Fig. 9). Staining for Notch1 was more intense in perivascular chondrocytes than in chondrocytes in the surrounding resting zone. Similar to the surrounding resting cells, the majority of the perivascular chondrocytes stained for Dll4, and Hes1 showed some staining in perivascular chondrocytes (Fig. 11). EGFL7- and Stro-1-positive cells were more abundant in perivascular areas than in the surrounding resting zones. EGFL7 was detected in perivascular cells of all horses and Stro-1 was identified in all individuals except the 1-year-old horse (Figs 11 and 14A,B).

Fig. 14.

Fig. 14

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Immunohistochemical staining of perivascular cells surrounding a vessel with the vessel lumina indicated by *. (A) EGFL7 in the 2-day-old horse, (B) Stro-1 in the 10-month-old foetus. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

Articular cartilage

All studied matrix molecules were present in all zones of the articular cartilage (Fig. 15). A transition from interterritorial to pericellular staining was observed in the postnatal horses in the deep zones. Prominent pericellular staining of matrix molecules was observed in the middle and superficial zones in the 1-year-old horse (Figs 15 and 16A–H).

Fig. 15.

Fig. 15

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Overview of immunohistochemical staining for matrix molecules in the lower deep, upper deep, middle and superficial (SZ) zones of the articular cartilage. Panels to the left in the figure contain representative images of the zones. Intensity of staining indicated by continuous or dotted lines as described below the figure.

Fig. 16.

Fig. 16

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Immunohistochemical staining for matrix molecules in the superficial and middle zones of the articular cartilage (joint cavity *) from the 6.5-month-old foetus (left) and 1-year-old horse (right). (A,B) COMP, (C,D) fibromodulin, (E,F) matrilin-1, (G,H) chondroadherin. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

Notch1, Dll4 and Hes1 were detected in chondrocytes of all zones of the articular cartilage. The postnatal cartilage showed the most prominent staining, localised to chondrocytes in the middle and superficial zones and in the lower deep zones (Figs 17 and 18A–F). Jagged1 was detected only in a few cells of the superficial and middle zones in the 1-year-old horse (Fig. 17). EGFL7 was present in a few cells in the superficial zone of the 6.5-month-old foetus and in the superficial and middle zones of the 2-day-old horse. In the 1-year-old horse, only a non-specific edge effect was visible with no positive cells detected in the articular zones (Figs 17 and 18G,H). Stro-1 was present in a few cells in all zones of the fetal articular cartilage, whereas staining was restricted to the superficial and middle zones in the postnatal horses (Figs 12C and 17).

Fig. 17.

Fig. 17

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Overview of immunohistochemical staining for Notch signalling components and Stro-1 in lower deep, upper deep, middle and superficial (SZ) zones of the articular cartilage. Panels to the left in the figure contain representative images of the zones. Grading of stained cells is indicated by continuous or dotted lines as described below the figure.

Fig. 18.

Fig. 18

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Immunohistochemical staining for Notch signalling components in the superficial and middle zones of the articular cartilage (joint cavity *) from the 6.5-month-old foetus (left) and 1-year-old horse (right). (A,B) Notch1, (C,D) Hes1, (E,F) Dll4, (G,H) EGFL7. Photos taken at 10x magnification with positive labelling shown in yellow and nuclei shown in blue. Scale bars: 100 μm.

In situ PLA

The proximity of Notch1 to EGFL7 was studied using the Duolink™ in situ PLA technology. Positive signals were detected in hypertrophic chondrocytes in the cartilage bone interface of all four specimens (Fig. 7). Positive signals were also detected in cells of the perivascular cartilage from the 10-month-old foetus and the postnatal horses (Fig. 11). A few cells were positive in the zone of Ranvier in the 1-day-old horse, in cells close to the perichondrium in the 10-month-old foetus, and in the 2-day-old horse (Fig. 11). There were no positive signals in the articular cartilage except in a few cells of the superficial zone of the 2-day-old horse (Fig. 17).

Discussion

The results of this study show that the localisation of Notch signalling components and matrix molecules change in a distinct manner during joint development, with an apparent difference in the expression and localisation when prenatal and postnatal cartilage are compared.

A structure similar to the zone of Ranvier has not been previously described in equine cartilage and was identified as an area comprising densely packed cells in haematoxylin & eosin- and picrosirius red-stained sections. Furthermore, it was well distinguished from the surrounding tissues due to an absence of COMP and the presence of Stro-1, EGFL7 and chondroadherin. Interestingly, Stro-1 was also present in areas extending from the zone of Ranvier along the perichondrium towards the articular cartilage (Fig. 12), which corresponds to the suggested migration route in rabbit cartilage (Henriksson et al. 2013). However, this area, close to the perichondrium, did differ in protein expression from the zone of Ranvier because COMP and fibromodulin were present together with Notch signalling components, thus suggesting a different regulating signal for the potentially migrating cells and a more differentiated chondrocyte. The area close to the perichondrium was distinguished from surrounding cartilage based on the presence of cellular and matrix EGFL7 staining in the prenatal horses and prominent pericellular staining of chondroadherin in the 2-day-old horse. Matrilin-1 was present in the zone of Ranvier and close to the perichondrium with intense pericellular presence in the postnatal horses, which may be related to the influence of postnatal mechanics; the synthesis of matrilin-1 has been observed to be up-regulated by mechanical stress (Wu & Chen, 2000; Kanbe et al. 2007). The zone of Ranvier is a potential source of migrating cells via the described migration route (Henriksson et al. 2013). However, other sources of the Stro-1-positive cells, such as the perichondrium, should be considered because the periosteum has been described as a source of mesenchymal stem cells in humans (Frey et al. 2013) and horses (Radtke et al. 2013).

Progenitor cells in articular cartilage serve as a potential cellular source for the repair of damaged cartilage. The presence of Stro-1 in equine articular cartilage in this study is consistent with a study showing the isolation of Stro-1-positive progenitor cells from equine cartilage (McCarthy et al. 2012). Stro-1 has been detected previously in the zone of Ranvier and the superficial zone of the articular cartilage within the rabbit knee joint (Karlsson et al. 2009). In our study, Stro-1 was observed in chondrocytes of all zones in prenatal articular cartilage but was restricted to the superficial and middle zones of the postnatal articular cartilages. A transition of Stro-1-positive cells from all zones to a more abundant presence in the upper zones is observed in many species. However, the time-point for the transition appears to be species-specific. Stro-1 was restricted to the superficial and middle zones of mature murine cartilage. However, positive cells were observed in all zones of immature cartilage (2-week-old mice) (Otsuki et al. 2010). Studies of normal adult human cartilage show the presence of Stro-1 in all zones of the articular cartilage; however, the highest percentage of positive cells was found in the superficial zone in one study (Grogan et al. 2009) and in the deep zone in another study (Ozbey et al. 2014).

The receptor Notch1 has previously been identified in progenitor cells isolated from bovine (Dowthwaite et al. 2004), human (Ustunel et al. 2008) and equine (McCarthy et al. 2012) articular cartilage. However, Notch1 is not an optimal marker for progenitor cells because Notch1 is not expressed exclusively by progenitor cells (Karlsson et al. 2008b; Grogan et al. 2009). Notch1 was present in chondrocytes in the surface of articular cartilage in murine embryos but was restricted to the deeper zones postnatally (Hayes et al. 2003). In adult human cartilage, Notch1 was found in chondrocytes of the surface layer (Karlsson et al. 2008a) or absent from all layers (Mahjoub et al. 2012). Others have reported chondrocytes with positive immunostaining for Notch 1 in the surface and in deeper zones of normal adult human articular cartilage (Hiraoka et al. 2006; Ustunel et al. 2008; Grogan et al. 2009), which is consistent with our results. Notch signalling is induced by cell-to-cell interactions (D'Souza et al. 2010; Wang, 2011), which might explain why there are less positive cells in the upper deep zone, where the cells are separated by more ECM. No specific niche of chondrocytes can be identified in the articular cartilage using the Notch1 receptor.

This study showed the presence of Notch signalling components in the developing growth cartilage, indicating its involvement in the formation of equine synovial joints. Previous studies have shown that active Notch signalling inhibits the differentiation of chondrocytes (Watanabe et al. 2003; Karlsson et al. 2007). We observed active Notch signalling, defined as the presence of the Notch1 receptor, the Dll4 ligand and the Hes1 downstream molecule, in proliferative cells of the 6.5-month-old foetus and in resting cells of both foetuses, which indicates that differentiation is inhibited in these cells. The absence of Hes1, suggesting inactive Notch signalling, in the resting chondrocytes of the postnatal cartilage may indicate on-going cell differentiation in those cells. Active Notch signalling, observed as the presence of Hes1, in the terminally differentiated hypertrophic cells of all four individuals is consistent with a suppression of further differentiation. The Notch1 receptor has also been localised to hypertrophic cells in murine (Hayes et al. 2003) and avian (Williams et al. 2009) developing cartilage. The presence of Hes1 in hypertrophic chondrocytes may also affect endochondral ossification via the induction of matrix metalloproteinase (MMP)13 because Hes1 has been observed previously to induce MMP13 in late-stage chondrocyte differentiation (Hosaka et al. 2013). MMP13 expression by hypertrophic chondrocytes during endochondral ossification is essential for normal growth plate development because a lack of MMP13 diminishes ECM remodelling and delays vascular recruitment (Stickens et al. 2004).

The Notch ligand Jagged1 was not detected in the growth plates. This ligand was only detected in the superficial and middle zones of the articular cartilage in the 1-year-old horse, suggesting that this ligand is not involved in Notch signalling during the endochondral ossification of equine cartilage. The ligand may, however, be expressed at specific developmental stages not included in our study.

The potential Notch1 dysregulating protein EGFL7 has been suggested to affect vascular development, modulation of ECM and stem cell behaviour (Nichol & Stuhlmann, 2012). We observed EGFL7-positive endothelial cells in blood vessels of cartilage canals and at the ossification front, which is consistent with previous reports showing that EGFL7 is a marker of endothelial cells (Soncin et al. 2003; Parker et al. 2004). EGFL7 has, to our knowledge, not been identified previously in cartilage, and our findings of EGFL7-positive chondrocytes in perivascular areas and in the cartilage bone interface may suggest a role for it in the vascular infiltration of growth cartilage. Presence of EGFL7 in hypertrophic chondrocytes in the cartilage bone interface may promote the invasion of vessels, both by affecting the behaviour of endothelial cells and by modulating the ECM to favour vessel invasion. EGFL7 has been shown previously to affect the invasion of vessels by modulating Notch signalling (Nichol et al. 2010). Furthermore, Jagged1-mediated Notch signalling was inhibited by EGFL7; however, the effect was suggested to be the opposite for Dll4-mediated signalling, which indicates ligand-specific effects of EGFL7 (Schmidt et al. 2009).This finding may explain why no clear association between EGFL7 and the target gene Hes1 was observed in our study, because ligands other than those included in our study may be affected. Chondrocytes in perivascular areas of the growth cartilage stained positive for EGFL7, which may indicate on-going vessel infiltration where the positive cells may function as modulators of ECM or attractants for vessels or endothelial cell precursors. The presence of fewer EGFL7-stained chondrocytes in perivascular areas of the growth plate of the 1-year-old horse is consistent with the normal closure of cartilage canals associated with the loss of growth cartilage (Ytrehus et al. 2004).

The proximity and possible interactions between Notch1 and EGFL7 in perivascular areas was indicated by positive signals detected using in situ PLA technology and were observed primarily in the 10-month-old foetus and the 2-day-old horse. These interactions, in combination with a decline in Hes1 staining, indicate a modulation of Notch signalling, which may affect vessel infiltration. However, there were also positive signals in the hypertrophic zone without a reduction in Hes1 staining, which may be attributed to ligand-specific effects of EGFL7 (Schmidt et al. 2009). Recently, EGFL7 was discovered to be regulated by the transcription factor CASTOR (CASZ1) (Charpentier et al. 2013), which may also influence the effects of EGFL7.

All studied matrix molecules were present in the growth plates with age-related differences in expression and localisation. The most prominent change was a shift from prenatal interterritorial staining to postnatal pericellular staining of COMP, fibromodulin and chondroadherin in the hypertrophic zone. A change from interterritorial to pericellular localisation was also observed in the other zones of the growth plate and in the articular cartilage; however, the time-point of the shift varied between proteins. Increasing thickness of the pericellular matrix over time has been observed in cultured chondrocytes and is associated with increased protection against mechanical deformation (de Vries et al. 2014). The spatial change with increased pericellular staining of the more developed cartilage in our study may indicate an increase in production of the specific molecule. An altered metabolism with increased production of ECM molecules may potentially be a response to mechanical load after birth, with a structural change in the collagen network when the foetus (prenatal) is born and begins to put weight on the joint tissues (postnatal) (Cluzel et al. 2013). The pericellular matrix has been found to be important in the response to load, and increased load has been accompanied by increased organisation of the collagen fibril network (Giannoni et al. 2003). COMP, fibromodulin, chondroadherin and matrilin-1, all of which were studied in our study and have known roles in collagen fibril formation and ECM assembly (Heinegård, 2009). Age- and load-related changes in the collagen content and zonal organisation of equine cartilage has previously been reported (Brama et al. 2000, 2009; Cluzel et al. 2013). An increase of total collagen content with age was observed in articular cartilage of neonatal, 5-month-old and 1-year-old horses, and in the postnatal horses, a higher collagen content was present in sites subjected to intermittent loading compared with sites subjected to constant loading (Brama et al. 2000). Further studies showed that exercise affected the collagen parallelism and orientation in foals subjected to normal or forced exercise (Brama et al. 2009). Collagen has been suggested to show zonal development in equine fetal cartilage: a dense tangential zone in the superficial articular cartilage, a progressive underlying the transitional zone and, at 6 months of gestation, a radial layer in the deeper zones (Cluzel et al. 2013). This development proceeded until 1 year postnatally, when adult collagen organisation was present (Cluzel et al. 2013).

COMP has been identified as a catalyst in collagen fibrillogenesis (Halász et al. 2007) and mechanical load increased the amount of COMP in bovine cartilage explants at protein and RNA levels (Wong et al. 1999; Giannoni et al. 2003). The distribution of COMP in developing porcine and human cartilage varies with zones and stage of maturation. In 6-week-old pigs, the highest COMP levels were observed in the territorial ECM of the proliferative zone (Ekman et al. 1997), and a study of COMP in developing human embryonic limbs showed a transition from a presence throughout the matrix to one in the pericellular matrix of hypertrophic cells (Koelling et al. 2006). The postnatal shift to pericellular/territorial staining observed in the equine cartilage in this study is thereby consistent with previous studies. The equine postnatal articular cartilage stained similar to human adult cartilage, with pericellular staining in the deep zones and immunolabelling throughout the matrix in the superficial and middle zones (Koelling et al. 2006) and intense pericellular staining in the middle zone of the oldest horse.

Fibromodulin distribution has previously been studied in several species; however, to our knowledge, it has not been studied in equine cartilage. In articular cartilage from 2 to 2.5-year-old bovines, the strongest ultrastructural labelling of fibromodulin was observed in the superficial zones, and staining decreased gradually towards the deep zones of the articular cartilage, with the most intense labelling in the interterritorial matrix (Hedlund et al. 1994). This pattern differs from our results, where staining was present in all zones, with increased pericellular staining in the postnatal horses. This discrepancy may be related to species and/or age variations. The change in fibromodulin location from interterritorial to pericellular in articular and growth cartilage in this study may relate to a zonal change in the collagen network (Cluzel et al. 2013) because fibromodulin is involved in the formation of thick and mechanically strong collagen fibres (Svensson et al. 1999; Ezura et al. 2000; Kalamajski & Oldberg, 2010) and in the protection against cleavage of collagen by MMPs (Geng et al. 2006).

Matrilin-1 has been identified as a modulator of collagen fibrillogenesis that affects fibril diameter and organisation of the collagen network (Nicolae et al. 2007). Mechanical stress up-regulates the synthesis of matrilin-1 (Wu & Chen, 2000; Kanbe et al. 2007), and matrilins in the pericellular matrix have been suggested to regulate mechanical activation of chondrocytes (Kanbe et al. 2007). Excessive and reduced levels of matrilin-1 reduced the mechanical responsiveness of murine chondrocytes, suggesting an adaption of the chondrocytes and the prevention of further activation (Kanbe et al. 2007). The decreased staining of matrilin-1 in hypertrophic and proliferative zones of the postnatal growth plates may be explained potentially as an adaptation or response to the increased mechanical load on joint tissues following birth. The transition from interterritorial to pericellular localisation of matrilin-1 in the articular cartilage and the resting zone of growth plate cartilage may also represent an adaptation to increased postnatal mechanical load. In contrast to our results, the protein has been detected previously in the prehypertrophic and hypertrophic zones of the growth plate in chickens (Chen et al. 1995), and in the developing mouse cartilage, matrilin-1 was located in the matrix of resting, proliferative and hypertrophic chondrocytes but not in the articular cartilage (Aszódi et al. 1996). These differences in matrilin-1 localisation may be species-dependent.

The expression of chondroadherin has been delineated previously in the developing rat femoral head, where it is observed in the territorial matrix of the late proliferative zone of the growth plate and the deep zones of the articular cartilage (Shen et al. 1998). In this current study, chondroadherin was most prominent in the hypertrophic zone of the growth plate and in all zones of the articular cartilage. The differences in localisation of chondroadherin between the developing rat femoral head and the equine distal radius may be attributed to maturation stages, the different anatomical sites sampled or species differences. Chondroadherin binds to chondrocytes via the α2β1 integrin (Camper et al. 1997; Haglund et al. 2011), and the shift to pericellular staining in postnatal cartilage indicates an increased binding to the receptor that potentially affects chondrocyte phenotype and matrix synthesis. Notch signalling did not adhere strictly to the spatial change in ECM molecules; hence, the suggested increase in synthesis of ECM molecules does not appear to be influenced by Notch activity.

Conclusion

The zone of Ranvier has not been described previously in equine cartilage. The structure was specifically localised based on the presence of Stro-1, EGFL7 and chondroadherin and the absence of COMP and Notch signalling. The presented results showing Stro-1-positive cells in the fetal resting zone, the zone of Ranvier, the area close to the perichondrium and in the superficial and middle zones of the articular cartilage may strengthen Stro-1 as a marker of progenitor cells in cartilage. These resident Stro-1-positive cells may have the capacity to repair cartilage pathology.

Identification of EGFL7 and Notch signalling components in perivascular chondrocytes is a novel finding that may indicate a role in vessel infiltration of growth cartilage. The presence of EGFL7 in hypertrophic chondrocytes also reveals the possibility that vascular infiltration may be modulated by this protein during the ossification of cartilage.

This study also delineates spatial changes in the distribution and expression of matrix molecules and Notch signalling components and Stro-1 during joint development in an attempt to classify the phenotype of the chondrocytes during normal chondrogenesis. The changes in localisation of the matrix molecules in developing equine cartilage indicates that several molecular events occur during joint development, with the increased pericellular localisation of these matrix components suggesting new synthesis. However, it was not possible to characterise the phenotype of the cells based on surrounding ECM during normal chondrogenesis.

Acknowledgments

This study was supported by funding grants from the Swedish Norwegian Foundation for Equine Research (H0847237) and from The Swedish Research Council Formas (dnr, 221-2013-317). The authors would like to acknowledge Beate Hillman for technical assistance in preparing the sections for immunostaining and Alexandra Leijon for illustration.

Conflict of interests

The authors declare that they have no competing interests.

Author contributions

M.L. contributed to the design of the study, performed the experimental work, analysed and interpreted the results and drafted the manuscript. S.E. contributed to the conception and design of the study, analysed and interpreted the results and edited the manuscript. E.Sv. contributed to the experimental setup and critically revised the manuscript. A.L. provided intellectual input and edited the manuscript. C.L. collected the material for the study and critically revised the manuscript. E.Sk. contributed to the conception and design of the study, the interpretation of the results, provided intellectual input and edited the manuscript. All authors read and approved the final version of the manuscript. D.H. provided antibodies and contributed with intellectual input on the design of the study and interpretation of results.

References

  1. Archer CW, Dowthwaite GP, Francis-West P. Development of synovial joints. Birth Defects Res C Embryo Today. 2003;69:144–155. doi: 10.1002/bdrc.10015. [DOI] [PubMed] [Google Scholar]
  2. Aszódi A, Hauser N, Studer D, et al. Cloning, sequencing and expression analysis of mouse cartilage matrix protein cDNA. Eur J Biochem. 1996;236:970–977. doi: 10.1111/j.1432-1033.1996.00970.x. [DOI] [PubMed] [Google Scholar]
  3. Badiwala MV, Tumiati LC, Joseph JM, et al. Epidermal growth factor-like Domain 7 suppresses intercellular adhesion molecule 1 expression in response to hypoxia/reoxygenation injury in human coronary artery endothelial cells. Circulation. 2010;122:S156–S161. doi: 10.1161/CIRCULATIONAHA.109.927715. [DOI] [PubMed] [Google Scholar]
  4. Behonick DJ, Werb Z. A bit of give and take: the relationship between the extracellular matrix and the developing chondrocyte. Mech Dev. 2003;120:1327–1336. doi: 10.1016/j.mod.2003.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brama PAJ, Tekoppele JM, Bank RA, et al. Functional adaptation of equine articular cartilage: the formation of regional biochemical characteristics up to age one year. Equine Vet J. 2000;32:217–221. doi: 10.2746/042516400776563626. [DOI] [PubMed] [Google Scholar]
  6. Brama PAJ, Holopainen J, van Weeren PR, et al. Effect of loading on the organization of the collagen fibril network in juvenile equine articular cartilage. J Orthop Res. 2009;27:1226–1234. doi: 10.1002/jor.20866. [DOI] [PubMed] [Google Scholar]
  7. Campagnolo L, Leahy A, Chitnis S, et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol. 2005;167:275–284. doi: 10.1016/S0002-9440(10)62972-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Camper L, Heinegård D, Lundgren-Åkerlund E. Integrin α2β1 is a receptor for the cartilage matrix protein chondroadherin. J Cell Biol. 1997;138:1159–1167. doi: 10.1083/jcb.138.5.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charpentier MS, Christine KS, Amin NM, et al. CASZ1 promotes vascular assembly and morphogenesis through the direct regulation of an EGFL7/RhoA-mediated pathway. Dev Cell. 2013;25:132–143. doi: 10.1016/j.devcel.2013.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen Q, Johnson DM, Haudenschild DR, et al. Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation. Dev Biol. 1995;172:293–306. doi: 10.1006/dbio.1995.0024. [DOI] [PubMed] [Google Scholar]
  11. Cluzel C, Blond L, Fontaine P, et al. Foetal and postnatal equine articular cartilage development: magnetic resonance imaging and polarised light microscopy. Eur Cell Mater. 2013;26:33–48. doi: 10.22203/ecm.v026a03. [DOI] [PubMed] [Google Scholar]
  12. Dowthwaite GP, Bishop JC, Redman SN, et al. The surface of articular cartilage contains a progenitor cell population. J Cell Sci. 2004;117:889–897. doi: 10.1242/jcs.00912. [DOI] [PubMed] [Google Scholar]
  13. Dreier R. Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther. 2010;12:216. doi: 10.1186/ar3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. D'Souza B, Meloty-Kapella L, Weinmaster G. Chapter Three – Canonical and non-canonical Notch ligands. Curr Top Dev Biol. 2010;92:73–129. doi: 10.1016/S0070-2153(10)92003-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ekman S, Reinholt FP, Hultenby K, et al. Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in porcine growth cartilage. Calcif Tissue Int. 1997;60:547–553. doi: 10.1007/s002239900278. [DOI] [PubMed] [Google Scholar]
  16. Ezura Y, Chakravarti S, Oldberg Å, et al. Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons. J Cell Biol. 2000;151:779–788. doi: 10.1083/jcb.151.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frey SP, Jansen H, Doht S, et al. Immunohistochemical and molecular characterization of the human periosteum. ScientificWorldJournal. 2013;2013:341078. doi: 10.1155/2013/341078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geng Y, McQuillan D, Roughley PJ. SLRP interaction can protect collagen fibrils from cleavage by collagenases. Matrix Biol. 2006;25:484–491. doi: 10.1016/j.matbio.2006.08.259. [DOI] [PubMed] [Google Scholar]
  19. Giannoni P, Siegrist M, Hunziker EB, et al. The mechanosensitivity of cartilage oligomeric matrix protein (COMP) Biorheology. 2003;40:101–109. [PubMed] [Google Scholar]
  20. Goldring MB. Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis. Ther Adv Musculoskelet Dis. 2012;4:269–285. doi: 10.1177/1759720X12448454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem. 2006;97:33–44. doi: 10.1002/jcb.20652. [DOI] [PubMed] [Google Scholar]
  22. Grogan S, Miyaki S, Asahara H, et al. Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis. Arthritis Res Ther. 2009;11:R85. doi: 10.1186/ar2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gustavsson M, Mallard C, Vannucci SJ, et al. Vascular response to hypoxic preconditioning in the immature brain. J Cereb Blood Flow Metab. 2007;27:928–938. doi: 10.1038/sj.jcbfm.9600408. [DOI] [PubMed] [Google Scholar]
  24. Haglund L, Tillgren V, Addis L, et al. Identification and characterization of the integrin α2β1 binding motif in chondroadherin mediating cell attachment. J Biol Chem. 2011;286:3925–3934. doi: 10.1074/jbc.M110.161141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Halász K, Kassner A, Mörgelin M, et al. COMP acts as a catalyst in collagen fibrillogenesis. J Biol Chem. 2007;282:31166–31173. doi: 10.1074/jbc.M705735200. [DOI] [PubMed] [Google Scholar]
  26. Hardingham TE, Oldershaw RA, Tew SR. Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat. 2006;209:469–480. doi: 10.1111/j.1469-7580.2006.00630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hayes AJ, Dowthwaite GP, Webster SV, et al. The distribution of Notch receptors and their ligands during articular cartilage development. J Anat. 2003;202:495–502. doi: 10.1046/j.1469-7580.2003.00185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hedlund H, Mengarelli-Widholm S, Heinegård D, et al. Fibromodulin distribution and association with collagen. Matrix Biol. 1994;14:227–232. doi: 10.1016/0945-053x(94)90186-4. [DOI] [PubMed] [Google Scholar]
  29. Heinegård D. Proteoglycans and more – from molecules to biology. Int J Exp Pathol. 2009;90:575–586. doi: 10.1111/j.1365-2613.2009.00695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Henriksson H, Lindahl A, Skioldebrand E, et al. Similar cellular migration patterns from niches in intervertebral disc and in knee-joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit. Stem Cell Res Ther. 2013;4:104. doi: 10.1186/scrt315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hiraoka K, Grogan S, Olee T, et al. Mesenchymal progenitor cells in adult human articular cartilage. Biorheology. 2006;43:447–454. [PubMed] [Google Scholar]
  32. Hosaka Y, Saito T, Sugita S, et al. Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development. Proc Natl Acad Sci U S A. 2013;110:1875–1880. doi: 10.1073/pnas.1207458110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kalamajski S, Oldberg Å. The role of small leucine-rich proteoglycans in collagen fibrillogenesis. Matrix Biol. 2010;29:248–253. doi: 10.1016/j.matbio.2010.01.001. [DOI] [PubMed] [Google Scholar]
  34. Kanbe K, Yang X, Wei L, et al. Pericellular matrilins regulate activation of chondrocytes by cyclic load-induced matrix deformation. J Bone Miner Res. 2007;22:318–328. doi: 10.1359/jbmr.061104. [DOI] [PubMed] [Google Scholar]
  35. Karlsson C, Jonsson M, Asp J, et al. Notch and HES5 are regulated during human cartilage differentiation. Cell Tissue Res. 2007;327:539–551. doi: 10.1007/s00441-006-0307-0. [DOI] [PubMed] [Google Scholar]
  36. Karlsson C, Brantsing C, Egell S, et al. Notch1, Jagged1, and HES5 are abundantly expressed in osteoarthritis. Cells Tissues Organs. 2008a;188:287–298. doi: 10.1159/000121610. [DOI] [PubMed] [Google Scholar]
  37. Karlsson C, Stenhamre H, Sandstedt J, et al. Neither Notch1 expression nor cellular size correlate with mesenchymal stem cell properties of adult articular chondrocytes. Cells Tissues Organs. 2008b;187:275–285. doi: 10.1159/000113409. [DOI] [PubMed] [Google Scholar]
  38. Karlsson C, Thornemo M, Henriksson HB, et al. Identification of a stem cell niche in the zone of Ranvier within the knee joint. J Anat. 2009;215:355–363. doi: 10.1111/j.1469-7580.2009.01115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Koelling S, Clauditz T, Kaste M, et al. Cartilage oligomeric matrix protein is involved in human limb development and in the pathogenesis of osteoarthritis. Arthritis Res Ther. 2006;8:R56. doi: 10.1186/ar1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kopan R, Ilagan MXG. The Canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–233. doi: 10.1016/j.cell.2009.03.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kvist AJ, Nyström A, Hultenby K, et al. The major basement membrane components localize to the chondrocyte pericellular matrix — A cartilage basement membrane equivalent? Matrix Biol. 2008;27:22–33. doi: 10.1016/j.matbio.2007.07.007. [DOI] [PubMed] [Google Scholar]
  42. Las Heras F, Gahunia HK, Pritzker KPH. Articular cartilage development: a molecular perspective. Orthop Clin North Am. 2012;43:155–171. doi: 10.1016/j.ocl.2012.01.003. [DOI] [PubMed] [Google Scholar]
  43. Mackie EJ, Tatarczuch L, Mirams M. The skeleton: a multi-functional complex organ. The growth plate chondrocyte and endochondral ossification. J Endocrinol. 2011;211:109–121. doi: 10.1530/JOE-11-0048. [DOI] [PubMed] [Google Scholar]
  44. Mahjoub M, Sassi N, Driss M, et al. Expression patterns of Notch receptors and their ligands in human osteoarthritic and healthy articular cartilage. Tissue Cell. 2012;44:182–194. doi: 10.1016/j.tice.2012.03.001. [DOI] [PubMed] [Google Scholar]
  45. McCarthy HE, Bara JJ, Brakspear K, et al. The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse. Vet J. 2012;192:345–351. doi: 10.1016/j.tvjl.2011.08.036. [DOI] [PubMed] [Google Scholar]
  46. Nichol D, Stuhlmann H. EGFL7: a unique angiogenic signaling factor in vascular development and disease. Blood. 2012;119:1345–1352. doi: 10.1182/blood-2011-10-322446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nichol D, Shawber C, Fitch MJ, et al. Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7. Blood. 2010;116:6133–6143. doi: 10.1182/blood-2010-03-274860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nicolae C, Ko Y-P, Miosge N, et al. Abnormal collagen fibrils in cartilage of Matrilin-1/Matrilin-3-deficient mice. J Biol Chem. 2007;282:22163–22175. doi: 10.1074/jbc.M610994200. [DOI] [PubMed] [Google Scholar]
  49. Otsuki S, Grogan SP, Miyaki S, et al. Tissue neogenesis and STRO-1 expression in immature and mature articular cartilage. J Orthop Res. 2010;28:96–102. doi: 10.1002/jor.20944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ozbey O, Sahin Z, Acar N, et al. Characterization of colony-forming cells in adult human articular cartilage. Acta Histochem. 2014;116:763–770. doi: 10.1016/j.acthis.2014.01.004. [DOI] [PubMed] [Google Scholar]
  51. Parker LH, Schmidt M, Jin S-W, et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature. 2004;428:754–758. doi: 10.1038/nature02416. [DOI] [PubMed] [Google Scholar]
  52. Pritzker KPH, Aigner T. Terminology of osteoarthritis cartilage and bone histopathology – a proposal for a consensus. Osteoarthritis Cartilage. 2010;18(Suppl 3):S7–S9. doi: 10.1016/j.joca.2010.05.028. [DOI] [PubMed] [Google Scholar]
  53. Radtke CL, Nino-Fong R, Esparza Gonzalez BP, et al. Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells. Am J Vet Res. 2013;74:790–800. doi: 10.2460/ajvr.74.5.790. [DOI] [PubMed] [Google Scholar]
  54. Schmidt MHH, Bicker F, Nikolic I, et al. Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal. Nat Cell Biol. 2009;11:873–880. doi: 10.1038/ncb1896. [DOI] [PubMed] [Google Scholar]
  55. Shapiro F, Holtrop ME, Glimcher MJ. Organization and cellular biology of the perichondrial ossification groove of Ranvier: a morphological study in rabbits. J Bone Joint Surg Am. 1977;59:703–723. [PubMed] [Google Scholar]
  56. Shen Z, Gantcheva S, Månsson B, et al. Chondroadherin expression changes in skeletal development. Biochem J. 1998;330:549–557. doi: 10.1042/bj3300549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Soncin F, Mattot V, Lionneton F, et al. VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J. 2003;22:5700–5711. doi: 10.1093/emboj/cdg549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stickens D, Behonick DJ, Ortega N, et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development. 2004;131:5883–5895. doi: 10.1242/dev.01461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Svensson L, Aszódi A, Reinholt FP, et al. Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon. J Biol Chem. 1999;274:9636–9647. doi: 10.1074/jbc.274.14.9636. [DOI] [PubMed] [Google Scholar]
  60. Ustunel I, Ozenci AM, Sahin Z, et al. The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells. Acta Histochem. 2008;110:397–407. doi: 10.1016/j.acthis.2007.12.005. [DOI] [PubMed] [Google Scholar]
  61. de Vries S, van Turnhout M, Oomens C, et al. Deformation thresholds for chondrocyte death and the protective effect of the pericellular matrix. Tissue Eng Part A. 2014;20:1870–1876. doi: 10.1089/ten.tea.2013.0436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang MM. Notch signaling and Notch signaling modifiers. Int J Biochem Cell Biol. 2011;43:1550–1562. doi: 10.1016/j.biocel.2011.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Watanabe N, Tezuka Y, Matsuno K, et al. Suppression of differentiation and proliferation of early chondrogenic cells by Notch. J Bone Miner Metab. 2003;21:344–352. doi: 10.1007/s00774-003-0428-4. [DOI] [PubMed] [Google Scholar]
  64. Williams R, Nelson L, Dowthwaite GP, et al. Notch receptor and Notch ligand expression in developing avian cartilage. J Anat. 2009;215:159–169. doi: 10.1111/j.1469-7580.2009.01089.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Williams R, Khan IM, Richardson K, et al. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage. PLoS ONE. 2010;5:e13246. doi: 10.1371/journal.pone.0013246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wong M, Siegrist M, Cao X. Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins. Matrix Biol. 1999;18:391–399. doi: 10.1016/s0945-053x(99)00029-3. [DOI] [PubMed] [Google Scholar]
  67. Wu Q-q, Chen Q. Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals. Exp Cell Res. 2000;256:383–391. doi: 10.1006/excr.2000.4847. [DOI] [PubMed] [Google Scholar]
  68. Ytrehus B, Ekman S, Carlson CS, et al. Focal changes in blood supply during normal epiphyseal growth are central in the pathogenesis of osteochondrosis in pigs. Bone. 2004;35:1294–1306. doi: 10.1016/j.bone.2004.08.016. [DOI] [PubMed] [Google Scholar]

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