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Osteoarthritic cartilage loses its ability to remain avascular
Osteoarthritis and Cartilage (1999) 7, 441–452 © 1999 OsteoArthritis Research Society International Article No. joca.1998.0238, available online at http://www.idealibrary.com on
1063–4584/99/090441+12 $12.00/0
Osteoarthritic cartilage loses its ability to remain avascular S. A. Fenwick*, P. J. Gregg† and P. Rooney Department of Orthopaedic Surgery, University of Leicester, Glenfield Hospital NHS Trust, Groby Road, Leicester, LE3 9QP, *Rheumatology Research Unit, Box 194, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, †Department of Surgery, The Medical School, University of Newcastle-Upon-Tyne, Newcastle-Upon-Tyne, NE2 4HH Summary Objective: To determine whether human osteoarthritic (OA) cartilage loses its ability to remain avascular when placed into the in-vivo model of angiogenesis, the chick embryo chorio-allantoic membrane (CAM), and to determine specific changes that occur in the cartilage matrix when the cartilage is exposed to an active vasculature. Design: Articular cartilage from OA and non-OA joints was grafted onto the CAM for up to 5 days before fixing and processing for histological, histochemical and immunological examination for specific changes in proteoglycan and collagen. Results: OA cartilage, but not non-OA cartilage, showed invasion of its matrix by blood vessels from the CAM to various extents. Associated with these blood vessels was a loss of staining for proteoglycans and cartilage specific glycosaminoglycans (GAG). A deposition of collagen types I and X was also visualized around the invasive vessels. Conclusions: OA cartilage loses or has already lost its ability to remain avascular when placed onto the chick CAM. Changes occur in the matrix around the invasive blood vessels, specifically a loss of proteoglycan and GAG, and the deposition of new collagen types, notably I and X. © 1999 OsteoArthritis Research Society International Key words: Osteoarthritis, Cartilage, Vascularization, CAM Graft.
capillary endothelial cells in-vitro, and angiogenesis in-vivo.14 Cartilage vascularization is thought important in three steps of OA pathology, the formation and growth of osteophytes, the advancement of the underlying subchondral bone and remodeling of the joint.16 The process of cartilage vascularization is extremely complex and probably involves cartilage mineralization, changes in protease activity and changes in inhibition of proteases.15–17 It is also possible that there is release of angiogenic factors encouraging vascularization to occur, and indeed a very low molecular weight substance (600 daltons) from cartilage, termed endothelial cell stimulating angiogenic factor (ESAF), has been described to stimulate vascularization, and also to activate latent matrix metalloproteinases (MMP) in cartilage.16 At the cellular level, normal cartilage shows only a very low level of matrix biosynthesis.18 There is, however, evidence to suggest that during OA, the chondrocytes within articular cartilage respond to the disease by mounting a biosynthetic response, considered to be an attempt at repair.2,19,20 The use of radiolabelled precursors of both collagen and proteoglycan have shown a new wave of synthesis of these components by the damaged tissue.3,19–24 A number of studies have also been performed to investigate whether there is a change in the phenotype of chondrocytes and/or a change in the ECM of articular cartilage during OA.7,25–31 It has been shown that significant alterations in the collagen expression of chondrocytes does occur during OA,25,28–30 particularly for a phenotypic switch of the chondrocytes to produce
Introduction 1
Osteoarthritis (OA) is a disease of complex etiology, often considered to be a slowly progressive disorder occurring late in life.2 The result of OA is a loss of the normal function of the joint due to a breakdown of the articular cartilage.1 Macroscopically, OA appears focal.2–4 The cartilage loses its normal glistening appearance, and yellow or brown spots appear.4 The cartilage becomes frayed and/or fibrillated with penetrating fissures or clefts and there is loss of cartilage tissue.5–8 As the severity of the disease increases, there is cartilage erosion, often with eburnation, and focal exposure of the subchondral bone.4,5,7 Changes are also known to occur within the underlying subchondral bone,9,10 including bony sclerosis, cyst and osteophyte formation.2,6,11,12 One of the less recognized changes which occurs during OA, is the violation of the tidemark between the subchondral bone and articular cartilage by blood vessels.2 This concept of neovascularization in OA cartilage is relatively new.2,13 The avascular nature of articular cartilage is not clearly understood although it is generally considered to be a function of anti-angiogenic molecules within the tissue.14,15 A specific angiogenic inhibitor, cartilage derived inhibitor (CDI), has been isolated from cartilage, and has been shown to inhibit the proliferation and migration of
Received 5 June 1998; accepted 14 December 1998. Address for correspondence: Steven A. Fenwick, Rheumatology Research Unit, Box 194, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ.
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442 collagen types I, III7,25,29,32 and X.26,28,30 Type I and III collagen have been identified immunohistochemically in OA articular cartilage in the pericellular matrix of some chondrocytes,7,25 although biochemical analysis has demonstrated that most of the collagen in OA cartilage is still type II.33 Although levels of types I and III collagen in OA cartilage may not be significant relative to the amount of type II collagen,7 the minor differences are indicative of important modifications in chondrocyte metabolism and the collagen network during cartilage degeneration in OA.33 Cartilage mineralization and the formation of bone on calcified cartilage are thought to be key phases during bone remodeling in OA.34 Type X collagen is believed to function during the calcification of cartilage during endochondral ossification.35 The presence of type X collagen has been shown in normal articular cartilage immunochemically at the cartilage surface,28 and at the mRNA level in some chondrocytes adjacent to the calcified cartilage layer.26 In OA cartilage, type X collagen has been localized immunochemically around chondrocyte clusters in fibrillated, severely damaged OA cartilage in the upper and middle zones, in hypertrophic cells28,30 and within the chondrocytes of the calcified layer.26 The earliest and most prominent change in OA cartilage, at the histological level is a progressive increase in the loss of proteoglycans,20,23,24 shown by a moderate decrease in metachromatic staining.2 The release of proteoglycan from OA cartilage is believed to be greater than that from normal cartilage.36 As the disease progresses, there is a progressive loss of colour with the metachromatic stains, such as alcian blue, and the orthochromatic stains, such as safranin-O,2 which can be explained by the extensive removal of aggrecan seen in severe OA.8 The role of neovascularization of articular cartilage in OA has not been well studied, and little is known about the changes that occur within the cartilage relating to cartilage vascularization. We describe here the loss of avascularity of human osteoarthritic articular cartilage when it is placed into the in-vivo model of vascularization, the chick embryo chorio-allantoic membrane, and show a loss of proteoglycan and glycosaminoglycan, and a deposition of collagen types I and X around the invasive vasculature.
Materials and methods HUMAN TISSUE HARVESTING
Human OA cartilage was obtained direct from theatre after total knee replacement (TKR) from five male and four female patients between the ages of 58 and 79. Non-OA human cartilage was obtained from the ankle joint of a female aged 67 undergoing above knee amputation and from three femoral heads of three females aged 68, 71 and 77, undergoing hip replacement for fractured neck of femur. Cartilage was taken from theatre and harvested aseptically in a class 2 microbiological safety cabinet. The femoral condyles of the OA knees were cut into thin strips with a pair of bone cutters. The cartilage, taken from areas of relatively healthy looking cartilage of at least 3 mm in depth, was then carefully cut away from the subchondral bone at the bone/cartilage interface with a scalpel to maintain the full thickness of the cartilage. Slices of this cartilage 1-mm thick were taken for use in CAM grafting experiments. Slices of ankle and femoral head cartilage were cut off with a scalpel as close to the underlying bone as possible to maintain as full thickness of the cartilage as
S. A. Fenwick et al.: Osteoarthritic cartilage vascularization possible. Histological observation did not show any evidence of the calcified cartilage layer. Slices of 1-mm thickness were again taken for use in CAM grafting experiments.
CAM GRAFTS
Fertilized White Leghorn chicken eggs were obtained from a registered supplier and incubated in a humidified incubator at 37°C. The eggs were windowed at 3 days to check fertility, sealed with sellotape and returned to the incubator until 10 days of age. The 1-mm thick slices of cartilage were cut in half. One half was laid flat onto the CAM of 10-day-old chick embryos, three pieces per CAM in each of 12 eggs, and incubated at 37°C for 5 days. After the incubation period, the grafts were removed, the excess CAM tissue removed, and the grafts prepared for either wax histology or cryosectioning. The second, ungrafted, half was prepared directly for similar treatment.
HISTOLOGY AND HISTOCHEMISTRY
Grafted and ungrafted cartilage was fixed in 10% neutral buffered formalin for up to a week before processing for wax histology and histochemistry. The grafted cartilage was further trimmed of excess CAM tissue before processing. Sections (7 ìm) were cut on a sledge microtome and mounted on 3-aminopropyltriethoxy-silane (silane) (Sigma, Poole) coated slides. Every fourth section was stained with haemotoxylin and eosin for general morphology. A number of other sections were stained with safranin-O (BDH) (0.5% in 0.1M acetate buffer) to detect proteoglycans or with alcian blue at critical electrolyte concentrations (CEC) of 0.7 M MgCl2 to detect cartilage specific GAG, according to the method of Scott and Dorling.37
CRYOSECTIONING
Grafted cartilage was excised and trimmed of excess CAM tissue before embedding in Cryo-M-Bed (Bright) on a piece of cork and snap frozen in liquid nitrogen. Nongrafted cartilage was snap frozen in an identical manner. Samples were stored at −80°C until ready for sectioning. Sections were cut on a Bright cryostat model OTF with a blade specifically designed for skeletal tissue. Sections (7 ìm) were cut and mounted on clean silane (Sigma) coated slides, before air-drying at room temperature for 60 min. The sections were then fixed in analytical grade acetone (Fisher) for 10 min and air-dried before wrapping in foil and storage at −20°C until ready for staining.
IMMUNOSTAINING
Sections were brought to room temperature then unwrapped and hydrated in TBS. After treatment with testicular hyaluronidase (Type I-S, Sigma) 1 mg/ml in acetate buffer (50 mM NaAc+2 mM NaCl) for 1 h, the sections were rinsed in TBS and flooded with normal rabbit serum (Serotec) diluted 1:5 with TBS for 10 min. Serum was then removed but not rinsed, before addition of primary antibody. The primary antibodies used were goat antihuman type I collagen (Southern Biotechnologies), mouse anti-type II collagen (CIICI, DSHB) and mouse anti-human
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Fig. 1. (a) OA cartilage and (b) healthy cartilage grafted onto the CAM of a 10 day old chick embryo for 4 days. Blood vessels and CAM tissue can be seen growing over both pieces of cartilage (×10). (c indicates the cartilage, arrows indicate blood vessels within the overlying CAM, the blurred edges in (a) are the egg shell).
type X collagen (MB6, A kind gift from Dr A. Kwan, University of Wales, Cardiff). Dilutions were made as recommended, 1:20 for type I , 1:5 for type II and 1:100 for type X. All dilutions were made in TBS. Slides were incubated for 1 h at room temperature, then rinsed in TBS before addition of appropriate secondary antibody, either rabbit anti-goat peroxidase conjugated secondary antibody, or rabbit anti-mouse biotin conjugated antibody respectively (Dako) at 1:200 dilution in TBS for 1 h at room
temperature. Peroxidase-labeled sections were rinsed and incubated in DAB/H2O2 solution (Sigma Fast DAB Tablets, Sigma) for 3–5 min, rinsed again before counterstaining in Mayers haemotoxylin, dehydration and mounting in DPX (BDH). Biotin labeled sections were rinsed before addition of ABComplex (Dako) for 20–30 min, rinsed again then incubated with DAB (Sigma) for 3–5 min. Slides were counterstained with haemotoxylin then dehydrated before mounting in DPX mountant (BDH) and visualized on an
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(b)
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Fig. 2. Invasion of OA cartilage by CAM tissue after being grafted onto the CAM (a) a cellular infiltrate with no indication of vessel formation, (b) a line of cells and (c) an actual blood vessel (H&E ×100) (arrows indicate the invasive cells/vessel, t is the CAM tissue).
Osteoarthritis and Cartilage Vol. 7 No. 5 Olympus BH2 light microscope. Negative controls had the primary antibody omitted.
Results All OA tissue generally had lost the shiny hyaline look associated with normal articular cartilage.2,4,6 The tissue varied in thickness from approx. 1 mm to 3–4 mm. Only the thicker pieces were used for grafting. During incubation on the CAM, almost all pieces of cartilage, both OA and non-OA, became at least partially enveloped in CAM tissue containing blood vessels (Fig. 1). However, there was no leakage of blood around the grafts. This is in contrast to when chick embryonic long bone rudiment cartilage is grafted onto the CAM in an identical manner.38 HISTOLOGY AND HISTOCHEMISTRY
CAM grafted sections stained with H&E revealed that all OA cartilage had become invaded by CAM derived cells to varying extents. The invasive tissue appeared as either groups of single cells, lines of cells or occasionally as a proper lumen containing erythrocytes (Fig. 2). None of the non-OA cartilage, despite becoming enveloped in CAM tissue containing blood vessels, showed any vascular invasion [Fig. 3(a)]. Invasion of the OA cartilage occurred through the deep layers and possibly from the side of the deep layers of the sections, it was never seen to occur through the surface layers [Fig. 3(b)]. The majority of the invasive cells were morphologically fibroblastic in nature (see Fig. 1). Histochemical staining with safranin-O showed a loss of metachromatic staining around invasive vessels, indicating a loss of proteoglycans [Fig. 4(a)]. Alcian blue at CEC of 0.7 M MgCl2 for cartilage specific GAG also showed a loss of staining around invasive vessels [Fig. 4(b)]. Throughout the rest of the cartilage in these sections, the staining for both proteoglycan and GAG was extremely variable, although generally the surface and upper layers had lost a lot of their staining properties while the deep layers stained relatively strongly. Non-OA cartilage stained uniformly throughout except the very surface layer, which is known to have a low proteoglycan content.39 IMMUNOHISTOCHEMISTRY
Antibody staining was performed for collagen types I, II and X on both CAM grafted and non-CAM grafted cartilage to discern any effect that the CAM may have on the collagen content of the matrix. TYPE I COLLAGEN
Type I collagen was localized to the surface layer of OA cartilage [Fig. 5(a)], whether CAM grafted or not, although some areas of the surface did not stain, most likely due to previous erosion during the disease process. In CAM grafted cartilage, the chondrocytes were negative for type I collagen staining. When vascular invasion was clearly seen, the edges of the invasive site stained very intensely for type I collagen [Fig. 5(a) and (b)]. Non-CAM grafted OA cartilage showed a number of chondrocytes at all depths staining positively for type I collagen. Non-OA cartilage, whether grafted or not had some surface staining, but
445 generally, there was little other indication of any positive staining. TYPE II COLLAGEN
When stained for type II collagen, CAM grafted and non-CAM grafted non-OA cartilage showed identical results. Two patterns of staining were seen similar to those described previously.27,29,31 Some samples showed uniform staining throughout the cartilage while other samples showed intense pericellular staining, with a lower intensity in the interterritorial matrix, or a combination of the two [Fig. 6(a)]. The chondrocytes themselves stained negatively. The chondrocytes in grafted and non-grafted OA cartilage were also negative for type II collagen, although staining of the matrix showed much variability. There was a tendency towards pericellular staining [Fig. 6(b)]. In the grafted cartilage, where invasion was visible, there was no lack of staining intensity. TYPE X COLLAGEN
Unlike the staining for collagen types I and II, where there was very little variation in the staining properties between different samples, staining for type X collagen showed a number of patterns which has been described previously.30 All samples showed positive staining of the chondrocytes to varying extents [Fig. 7(a)]. Non-grafted non-OA cartilage generally showed a positive result in all chondrocytes throughout the sections, although the matrix showed no evidence of positive staining except in the occasional section where there was faint staining at the extreme surface. In non-OA grafted cartilage, no indication of surface staining could be seen. Non-grafted OA cartilage stained very similarly to the non-OA cartilage although occasionally, the surface showed some positive staining, and some deep cells showed some faint pericellular staining [Fig. 7(a)]. Grafted OA cartilage showed general positive staining of all chondrocytes and some indications in a number of sections of positive surface staining. The matrix showed no indication of staining except around sections of invading vasculature where a narrow rim of the matrix around the entire invasion stained positively [Fig. 7(b)]. This rim of positive staining was not as intense and did not penetrate as deeply into the matrix as the type I collagen staining.
Discussion It has been demonstrated here that human OA articular cartilage loses its ability to remain avascular when placed on to an active vasculature, that of the chick CAM. Changes in the ECM components were examined by both histochemical and immunohistochemical staining. Most notable is the observation that vascular invasion was always associated with a localized loss of proteoglycan/ GAG staining within the cartilage ECM and a deposition of collagen types I and X along the rim of invasive vessels. CAM grafting did not have any noticeable effect on the rest of the matrix. This, however, may not be such a surprise. As incubation time is relatively short and it is known that cartilage ECM components have a slow turnover time within cartilage matrix,2,21 there is unlikely to be a significant cartilage mediated change over such a short period of time.
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Fig. 3. (a) Section of a grafted piece of healthy cartilage, there is no indication of any vascular invasion in the deep layers (s indicates the surface). (b) Section of a grafted piece of OA cartilage. Two small invasions can be seen into the deep layers (arrows) but there is no indication of invasion through the surface layers (s) (×100).
The results of this study are similar to previous studies that have examined types I, II and X collagen and proteoglycan/GAG in cartilage.3,24–26,28,29,40,41 Type I collagen is often found confined to the surface layer of normal cartilage. Type II collagen showed general uniform staining throughout both control and OA cartilage sections, as would be expected, and has been shown previously.7,29 Aigner et al.27 however, also showed some irregular staining of type II collagen in OA cartilage, via enhanced
pericellular deposition and diminished inter-territorial staining. Type X collagen stained to varying degrees in both OA and non-OA cartilage at the surface layers, and in the pericellular region of a number of the chondrocytes and also intra-cellularly in the majority of chondrocytes. Healthy cartilage has previously shown little or no type X collagen staining,30,42 whilst OA cartilage has been shown to stain for type X collagen both intra-cellularly and pericelluarly,
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Fig. 4. (a) Loss of metachromatic staining for proteoglycans with safranin-O around an invasive vessel (×200). (b) Loss of cartilage specific GAG staining shown by a decrease in alcian blue CEC staining around an invasive vessel (×100) (arrows indicate the area of staining loss).
most prominently around hypertrophic cells and proliferating clusters of cells in the upper and middle zones of the cartilage.28,30 However, the most prominent feature of the staining of the cartilage was of the invasive vessels seen in CAM grafted OA cartilage. The rim of matrix around the invasion stained positively for both type I collagen and type X collagen. Type I collagen is associated with de-differentiated chondrocytes.42–44 It is also a component of blood vessels and is known to be upregulated in endothelial cells undergoing angiogenesis.45 Type X has been hypothesized to be a factor in the calcification/ mineralization and vascularization of cartilage.46–49 The source of the type I collagen could either be cartilage or vasculature derived as the anti-type I antibody is a
polyclonal shown to cross react with both human and chick type I. The anti-type X antibody is specific for human type X collagen, therefore the type X collagen laid down in the matrix must be cartilage derived. It has been shown that the underlying subchondral bone and the calcified cartilage between the subchondral bone and articular cartilage contains blood capillaries,50 this is part of the normal structure of the joint, and as such is not a symptom of the OA process. However vascularization of the articular cartilage above the original ‘tidemark’, a defined layer between the calcified cartilage and the articular cartilage, is an important step in the pathological process of OA.16 The vascularization of cartilage above this tidemark is partly responsible for the conversion of cartilage to bone, a process which requires a mineralized cartilage
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Fig. 5. (a) Surface staining for type I collagen and along the rim of an invasive vessel (s indicates the surface layer, arrow indicates the vessel) (×40). (b) Higher power micrograph of an invasive vessel showing a rim of positive staining for type I collagen (v is the vessel, arrows indicate type I positive staining) (×200).
intermediate51 (un-mineralized cartilage is generally resistant to vascularization52), there is therefore, an advancement of the tidemark as the cartilage is calcified, and subsequently eroded.4 However, in the work described above, there was no histological evidence of cartilage mineralization in tissue sections in any of the tissue samples analysed, indicating that the process on the CAM may not require the cartilage to be mineralized for vascularization. The invasive process may be more like the
vascularization of avian cartilage, which, during development, is known not to mineralize before vascular invasion.53 Alongside the cartilage erosion associated with the pathology of OA, there is also renewed growth of cartilage (and bone) at the joint margin, resulting in the formation of osteophytes.2,6,54 New vasculature is required for this process, the formation and growth of the osteophyte being reliant upon the proliferation of blood vessels into
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Fig. 6. (a) Staining of cartilage with anti-type II antibody in a section of grafted OA cartilage showing an almost blanket staining effect with some regions of only pericellular staining (indicated by arrow) (×40). (b) Intense pericellular staining for type II collagen in a section of grafted non-OA cartilage (arrows) (×200).
degenerate articular cartilage.54 The vasculature therefore, is partly responsible for the erosion of degenerate cartilage and the formation of new bone via endochondral ossification, yet at the same time aids in the growth of new cartilage within the same joint. This new cartilage appears similar to normal articular cartilage at the histological level (personal observation), which as such is not surprising as the growth of osteophytes is considered to be a mechanism of attempted repair.54 Despite its now accepted role in the OA process, cartilage vascularization has more commonly been associated
with rheumatoid arthritis, an inflammatory disease of the articular cartilage and surrounding joint. Chronic inflammation is very much associated with angiogenesis,55 however, osteoarthritis is considered to be a noninflammatory disease. If OA is non-inflammatory, then the mediators of the angiogenic response may be very different from those associated with an inflammatory response. The presence or absence of angiogenesis mediating cells, such as macrophages or mast cells, needs to be assessed, along with the presence of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic
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Fig. 7. (a) Faint staining of the pericellular matrix of non-grafted OA cartilage with the anti-type X antibody (short arrows), the chondrocytes are also intensely stained (long arrows) (×100). (b) Deposition of type X collagen along the rim of an invasive vessel (arrows indicate positive staining) (×100).
fibroblast growth factor (bFGF). The role of MMP and TIMP in the process is also of great interest, as evidence suggests that cartilage turnover and degradation is mediated by an imbalance between the activity of MMP and their inhibitors.56,57 An examination of cartilage and vascular derived MMP and TIMP may provide much information about the mechanism of cartilage destruction during vascular invasion, and the role that both the cartilage itself, and the invasive vasculature play during the process. OA cartilage has never previously been grafted onto the CAM, therefore this work provides a novel model for the study of active vascularization of human OA cartilage.
The process of vascularization of human OA cartilage on the CAM occurs rapidly. The study of this process is therefore enhanced by this model which may aid in elucidating the reasons as to why the process occurs, along with the cell biological, biochemical and molecular mechanisms behind the process. In conclusion, human OA cartilage loses its avascular state when placed onto the chick CAM. There is a loss of non-specific and cartilage-specific GAG and proteoglycans around the invasion site, and deposition of type I and type X collagen along the rim of vascular invasion, which may have a role in the process.
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Acknowledgments We would like to acknowledge the University of Leicester for funding this research. The monoclonal antibody CIICI developed by Holmdahl and Rubin was obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICHD.
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emphasis on type VI Collagen. Biochim Biophys Acta 1990;1038:222–30. Sokoloff L. Osteoarthritis as a remodelling process. J Rheumatol 1987;14 (suppl 1):7–10. Schmid TM, Linsenmayer TF. Type X collagen. In: Mayne R, Burgeson RE, Eds. Structure and Function of Collagen Types. New York: Academic Press 1987:223–59. Yamada N, Nakagawa T, Stephens RW, Nagai Y. Proteinases and their inhibitors in normal and osteoarthritic articular cartilage. Biomed Res 1987;8:289– 300. Scott JE, Dorling J. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie 1965;5:221–33. Fenwick SA, Gregg PJ, Kumar S, Ponting J, Rooney P. Intrinsic control of vascularisation in developing cartilage rudiments. Int J Exp Pathol 1997;78:187–96. Schumacher BL, Block JA, Schmid TM, Aydelotte MB, Kuettner KE. A novel proteoglycan synthesised and secreted by chondrocytes of the superficial zone of articular cartilage. Archives Biochem and Biophys 1994;311:144–52. Venn M, Maroudas A. Chemical composition and swelling of normal and osteoarthritic femoral head cartilage. I. Chemical composition. Ann Rheum Dis 1977;36:121–9. Christensen SB, Reimann I. Differential histochemical staining of glycosaminoglycans in the matrix of osteoarthritic cartilage. Acta Path Microbiol Scand Sect A 1980;88:61–8. Kirsch T, Swoboda B, von der Mark K. Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and X collagen and matrix mineralisation. Differentiation 1992;52:89–100. Mayne R, Vail MS, Mayne PM, Miller EJ. Changes in the type of collagen synthesised as clones of chick chondrocytes grow and eventually lose division capacity. Proc Natl Acad Sci USA 1976;73:1674–8. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C et al. Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res 1994;212:97–104. Rooney P, Wang M, Kumar P, Kumar S. Angiogenic oligosaccharides of hyaluronan enhance the produc-
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tion of collagen by endothelial cells. J Cell Sci 1993;105:213–8. Schmid TM, Popp RG, Linsenmayer TF. Hypertrophic cartilage matrix type X collagen: supramolecular assembly and calcification. Ann New York Acad Sci 1990;580:64–73. Iyama K, Ninomiya Y, Olsen BR, Linsenmayer TF, Trelstad RL, Hayashi M. Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 1991;229:462–72. Rooney P, Kumar S. Inverse relationship between hyaluronan and collagens in development. Differentiation 1993;54:1–9. Wallis GA. Here today, bone tomorrow. Curr Biol 1993;3:687–9. Bullough PG, Jagannath A. The morphology of the calcification front in articular cartilage. J Bone Joint Surg 1983;65B:72–8. Brown RA, Weiss JB. Neovascularisation and its role in the osteoarthritic process. Annals Rheum Dis 1988;47:881–95. Reddi AH, Kuettner KE. Vascular invasion of cartilage: correlation of morphology with lysozyme, glycosaminoglycans, protease and protease inhibitory activity. Dev Biol 1981;82:217–23. Roach HI, Shearer JR. Cartilage resorption and endochondral bone formation during the development of long bones in chick embryos. Bone and Mineral 1989;6:289–309 . Harrison MHM, Schajowicz F, Trueta J. Osteoarthritis of the hip: a study of the nature and evolution of the disease. J Bone Joint Surg 1953;35B:598–626. Jackson JR, Seed MP, Kircher CH, Willoughby DA, Winkler JD. The codependence of angiogenesis and chronic inflammation. FASEB J 1997;11:457–65. Dean DD, Martell-Pelletier J, Pelletier JP. Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J Clin Invest 1989;84:678–85. Pelletier JP, Mineau F, Faure MP, Martell-Pelletier J. Imbalance between the mechanisms of activation and inhibition (TIMP) of metalloproteases in early lesions of experimental osteoarthritis. Arth Rheum 1990;33:1466–76.
1063–4584/99/090441+12 $12.00/0
Osteoarthritic cartilage loses its ability to remain avascular S. A. Fenwick*, P. J. Gregg† and P. Rooney Department of Orthopaedic Surgery, University of Leicester, Glenfield Hospital NHS Trust, Groby Road, Leicester, LE3 9QP, *Rheumatology Research Unit, Box 194, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, †Department of Surgery, The Medical School, University of Newcastle-Upon-Tyne, Newcastle-Upon-Tyne, NE2 4HH Summary Objective: To determine whether human osteoarthritic (OA) cartilage loses its ability to remain avascular when placed into the in-vivo model of angiogenesis, the chick embryo chorio-allantoic membrane (CAM), and to determine specific changes that occur in the cartilage matrix when the cartilage is exposed to an active vasculature. Design: Articular cartilage from OA and non-OA joints was grafted onto the CAM for up to 5 days before fixing and processing for histological, histochemical and immunological examination for specific changes in proteoglycan and collagen. Results: OA cartilage, but not non-OA cartilage, showed invasion of its matrix by blood vessels from the CAM to various extents. Associated with these blood vessels was a loss of staining for proteoglycans and cartilage specific glycosaminoglycans (GAG). A deposition of collagen types I and X was also visualized around the invasive vessels. Conclusions: OA cartilage loses or has already lost its ability to remain avascular when placed onto the chick CAM. Changes occur in the matrix around the invasive blood vessels, specifically a loss of proteoglycan and GAG, and the deposition of new collagen types, notably I and X. © 1999 OsteoArthritis Research Society International Key words: Osteoarthritis, Cartilage, Vascularization, CAM Graft.
capillary endothelial cells in-vitro, and angiogenesis in-vivo.14 Cartilage vascularization is thought important in three steps of OA pathology, the formation and growth of osteophytes, the advancement of the underlying subchondral bone and remodeling of the joint.16 The process of cartilage vascularization is extremely complex and probably involves cartilage mineralization, changes in protease activity and changes in inhibition of proteases.15–17 It is also possible that there is release of angiogenic factors encouraging vascularization to occur, and indeed a very low molecular weight substance (600 daltons) from cartilage, termed endothelial cell stimulating angiogenic factor (ESAF), has been described to stimulate vascularization, and also to activate latent matrix metalloproteinases (MMP) in cartilage.16 At the cellular level, normal cartilage shows only a very low level of matrix biosynthesis.18 There is, however, evidence to suggest that during OA, the chondrocytes within articular cartilage respond to the disease by mounting a biosynthetic response, considered to be an attempt at repair.2,19,20 The use of radiolabelled precursors of both collagen and proteoglycan have shown a new wave of synthesis of these components by the damaged tissue.3,19–24 A number of studies have also been performed to investigate whether there is a change in the phenotype of chondrocytes and/or a change in the ECM of articular cartilage during OA.7,25–31 It has been shown that significant alterations in the collagen expression of chondrocytes does occur during OA,25,28–30 particularly for a phenotypic switch of the chondrocytes to produce
Introduction 1
Osteoarthritis (OA) is a disease of complex etiology, often considered to be a slowly progressive disorder occurring late in life.2 The result of OA is a loss of the normal function of the joint due to a breakdown of the articular cartilage.1 Macroscopically, OA appears focal.2–4 The cartilage loses its normal glistening appearance, and yellow or brown spots appear.4 The cartilage becomes frayed and/or fibrillated with penetrating fissures or clefts and there is loss of cartilage tissue.5–8 As the severity of the disease increases, there is cartilage erosion, often with eburnation, and focal exposure of the subchondral bone.4,5,7 Changes are also known to occur within the underlying subchondral bone,9,10 including bony sclerosis, cyst and osteophyte formation.2,6,11,12 One of the less recognized changes which occurs during OA, is the violation of the tidemark between the subchondral bone and articular cartilage by blood vessels.2 This concept of neovascularization in OA cartilage is relatively new.2,13 The avascular nature of articular cartilage is not clearly understood although it is generally considered to be a function of anti-angiogenic molecules within the tissue.14,15 A specific angiogenic inhibitor, cartilage derived inhibitor (CDI), has been isolated from cartilage, and has been shown to inhibit the proliferation and migration of
Received 5 June 1998; accepted 14 December 1998. Address for correspondence: Steven A. Fenwick, Rheumatology Research Unit, Box 194, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ.
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442 collagen types I, III7,25,29,32 and X.26,28,30 Type I and III collagen have been identified immunohistochemically in OA articular cartilage in the pericellular matrix of some chondrocytes,7,25 although biochemical analysis has demonstrated that most of the collagen in OA cartilage is still type II.33 Although levels of types I and III collagen in OA cartilage may not be significant relative to the amount of type II collagen,7 the minor differences are indicative of important modifications in chondrocyte metabolism and the collagen network during cartilage degeneration in OA.33 Cartilage mineralization and the formation of bone on calcified cartilage are thought to be key phases during bone remodeling in OA.34 Type X collagen is believed to function during the calcification of cartilage during endochondral ossification.35 The presence of type X collagen has been shown in normal articular cartilage immunochemically at the cartilage surface,28 and at the mRNA level in some chondrocytes adjacent to the calcified cartilage layer.26 In OA cartilage, type X collagen has been localized immunochemically around chondrocyte clusters in fibrillated, severely damaged OA cartilage in the upper and middle zones, in hypertrophic cells28,30 and within the chondrocytes of the calcified layer.26 The earliest and most prominent change in OA cartilage, at the histological level is a progressive increase in the loss of proteoglycans,20,23,24 shown by a moderate decrease in metachromatic staining.2 The release of proteoglycan from OA cartilage is believed to be greater than that from normal cartilage.36 As the disease progresses, there is a progressive loss of colour with the metachromatic stains, such as alcian blue, and the orthochromatic stains, such as safranin-O,2 which can be explained by the extensive removal of aggrecan seen in severe OA.8 The role of neovascularization of articular cartilage in OA has not been well studied, and little is known about the changes that occur within the cartilage relating to cartilage vascularization. We describe here the loss of avascularity of human osteoarthritic articular cartilage when it is placed into the in-vivo model of vascularization, the chick embryo chorio-allantoic membrane, and show a loss of proteoglycan and glycosaminoglycan, and a deposition of collagen types I and X around the invasive vasculature.
Materials and methods HUMAN TISSUE HARVESTING
Human OA cartilage was obtained direct from theatre after total knee replacement (TKR) from five male and four female patients between the ages of 58 and 79. Non-OA human cartilage was obtained from the ankle joint of a female aged 67 undergoing above knee amputation and from three femoral heads of three females aged 68, 71 and 77, undergoing hip replacement for fractured neck of femur. Cartilage was taken from theatre and harvested aseptically in a class 2 microbiological safety cabinet. The femoral condyles of the OA knees were cut into thin strips with a pair of bone cutters. The cartilage, taken from areas of relatively healthy looking cartilage of at least 3 mm in depth, was then carefully cut away from the subchondral bone at the bone/cartilage interface with a scalpel to maintain the full thickness of the cartilage. Slices of this cartilage 1-mm thick were taken for use in CAM grafting experiments. Slices of ankle and femoral head cartilage were cut off with a scalpel as close to the underlying bone as possible to maintain as full thickness of the cartilage as
S. A. Fenwick et al.: Osteoarthritic cartilage vascularization possible. Histological observation did not show any evidence of the calcified cartilage layer. Slices of 1-mm thickness were again taken for use in CAM grafting experiments.
CAM GRAFTS
Fertilized White Leghorn chicken eggs were obtained from a registered supplier and incubated in a humidified incubator at 37°C. The eggs were windowed at 3 days to check fertility, sealed with sellotape and returned to the incubator until 10 days of age. The 1-mm thick slices of cartilage were cut in half. One half was laid flat onto the CAM of 10-day-old chick embryos, three pieces per CAM in each of 12 eggs, and incubated at 37°C for 5 days. After the incubation period, the grafts were removed, the excess CAM tissue removed, and the grafts prepared for either wax histology or cryosectioning. The second, ungrafted, half was prepared directly for similar treatment.
HISTOLOGY AND HISTOCHEMISTRY
Grafted and ungrafted cartilage was fixed in 10% neutral buffered formalin for up to a week before processing for wax histology and histochemistry. The grafted cartilage was further trimmed of excess CAM tissue before processing. Sections (7 ìm) were cut on a sledge microtome and mounted on 3-aminopropyltriethoxy-silane (silane) (Sigma, Poole) coated slides. Every fourth section was stained with haemotoxylin and eosin for general morphology. A number of other sections were stained with safranin-O (BDH) (0.5% in 0.1M acetate buffer) to detect proteoglycans or with alcian blue at critical electrolyte concentrations (CEC) of 0.7 M MgCl2 to detect cartilage specific GAG, according to the method of Scott and Dorling.37
CRYOSECTIONING
Grafted cartilage was excised and trimmed of excess CAM tissue before embedding in Cryo-M-Bed (Bright) on a piece of cork and snap frozen in liquid nitrogen. Nongrafted cartilage was snap frozen in an identical manner. Samples were stored at −80°C until ready for sectioning. Sections were cut on a Bright cryostat model OTF with a blade specifically designed for skeletal tissue. Sections (7 ìm) were cut and mounted on clean silane (Sigma) coated slides, before air-drying at room temperature for 60 min. The sections were then fixed in analytical grade acetone (Fisher) for 10 min and air-dried before wrapping in foil and storage at −20°C until ready for staining.
IMMUNOSTAINING
Sections were brought to room temperature then unwrapped and hydrated in TBS. After treatment with testicular hyaluronidase (Type I-S, Sigma) 1 mg/ml in acetate buffer (50 mM NaAc+2 mM NaCl) for 1 h, the sections were rinsed in TBS and flooded with normal rabbit serum (Serotec) diluted 1:5 with TBS for 10 min. Serum was then removed but not rinsed, before addition of primary antibody. The primary antibodies used were goat antihuman type I collagen (Southern Biotechnologies), mouse anti-type II collagen (CIICI, DSHB) and mouse anti-human
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Fig. 1. (a) OA cartilage and (b) healthy cartilage grafted onto the CAM of a 10 day old chick embryo for 4 days. Blood vessels and CAM tissue can be seen growing over both pieces of cartilage (×10). (c indicates the cartilage, arrows indicate blood vessels within the overlying CAM, the blurred edges in (a) are the egg shell).
type X collagen (MB6, A kind gift from Dr A. Kwan, University of Wales, Cardiff). Dilutions were made as recommended, 1:20 for type I , 1:5 for type II and 1:100 for type X. All dilutions were made in TBS. Slides were incubated for 1 h at room temperature, then rinsed in TBS before addition of appropriate secondary antibody, either rabbit anti-goat peroxidase conjugated secondary antibody, or rabbit anti-mouse biotin conjugated antibody respectively (Dako) at 1:200 dilution in TBS for 1 h at room
temperature. Peroxidase-labeled sections were rinsed and incubated in DAB/H2O2 solution (Sigma Fast DAB Tablets, Sigma) for 3–5 min, rinsed again before counterstaining in Mayers haemotoxylin, dehydration and mounting in DPX (BDH). Biotin labeled sections were rinsed before addition of ABComplex (Dako) for 20–30 min, rinsed again then incubated with DAB (Sigma) for 3–5 min. Slides were counterstained with haemotoxylin then dehydrated before mounting in DPX mountant (BDH) and visualized on an
(a)
(b)
(c)
Fig. 2. Invasion of OA cartilage by CAM tissue after being grafted onto the CAM (a) a cellular infiltrate with no indication of vessel formation, (b) a line of cells and (c) an actual blood vessel (H&E ×100) (arrows indicate the invasive cells/vessel, t is the CAM tissue).
Osteoarthritis and Cartilage Vol. 7 No. 5 Olympus BH2 light microscope. Negative controls had the primary antibody omitted.
Results All OA tissue generally had lost the shiny hyaline look associated with normal articular cartilage.2,4,6 The tissue varied in thickness from approx. 1 mm to 3–4 mm. Only the thicker pieces were used for grafting. During incubation on the CAM, almost all pieces of cartilage, both OA and non-OA, became at least partially enveloped in CAM tissue containing blood vessels (Fig. 1). However, there was no leakage of blood around the grafts. This is in contrast to when chick embryonic long bone rudiment cartilage is grafted onto the CAM in an identical manner.38 HISTOLOGY AND HISTOCHEMISTRY
CAM grafted sections stained with H&E revealed that all OA cartilage had become invaded by CAM derived cells to varying extents. The invasive tissue appeared as either groups of single cells, lines of cells or occasionally as a proper lumen containing erythrocytes (Fig. 2). None of the non-OA cartilage, despite becoming enveloped in CAM tissue containing blood vessels, showed any vascular invasion [Fig. 3(a)]. Invasion of the OA cartilage occurred through the deep layers and possibly from the side of the deep layers of the sections, it was never seen to occur through the surface layers [Fig. 3(b)]. The majority of the invasive cells were morphologically fibroblastic in nature (see Fig. 1). Histochemical staining with safranin-O showed a loss of metachromatic staining around invasive vessels, indicating a loss of proteoglycans [Fig. 4(a)]. Alcian blue at CEC of 0.7 M MgCl2 for cartilage specific GAG also showed a loss of staining around invasive vessels [Fig. 4(b)]. Throughout the rest of the cartilage in these sections, the staining for both proteoglycan and GAG was extremely variable, although generally the surface and upper layers had lost a lot of their staining properties while the deep layers stained relatively strongly. Non-OA cartilage stained uniformly throughout except the very surface layer, which is known to have a low proteoglycan content.39 IMMUNOHISTOCHEMISTRY
Antibody staining was performed for collagen types I, II and X on both CAM grafted and non-CAM grafted cartilage to discern any effect that the CAM may have on the collagen content of the matrix. TYPE I COLLAGEN
Type I collagen was localized to the surface layer of OA cartilage [Fig. 5(a)], whether CAM grafted or not, although some areas of the surface did not stain, most likely due to previous erosion during the disease process. In CAM grafted cartilage, the chondrocytes were negative for type I collagen staining. When vascular invasion was clearly seen, the edges of the invasive site stained very intensely for type I collagen [Fig. 5(a) and (b)]. Non-CAM grafted OA cartilage showed a number of chondrocytes at all depths staining positively for type I collagen. Non-OA cartilage, whether grafted or not had some surface staining, but
445 generally, there was little other indication of any positive staining. TYPE II COLLAGEN
When stained for type II collagen, CAM grafted and non-CAM grafted non-OA cartilage showed identical results. Two patterns of staining were seen similar to those described previously.27,29,31 Some samples showed uniform staining throughout the cartilage while other samples showed intense pericellular staining, with a lower intensity in the interterritorial matrix, or a combination of the two [Fig. 6(a)]. The chondrocytes themselves stained negatively. The chondrocytes in grafted and non-grafted OA cartilage were also negative for type II collagen, although staining of the matrix showed much variability. There was a tendency towards pericellular staining [Fig. 6(b)]. In the grafted cartilage, where invasion was visible, there was no lack of staining intensity. TYPE X COLLAGEN
Unlike the staining for collagen types I and II, where there was very little variation in the staining properties between different samples, staining for type X collagen showed a number of patterns which has been described previously.30 All samples showed positive staining of the chondrocytes to varying extents [Fig. 7(a)]. Non-grafted non-OA cartilage generally showed a positive result in all chondrocytes throughout the sections, although the matrix showed no evidence of positive staining except in the occasional section where there was faint staining at the extreme surface. In non-OA grafted cartilage, no indication of surface staining could be seen. Non-grafted OA cartilage stained very similarly to the non-OA cartilage although occasionally, the surface showed some positive staining, and some deep cells showed some faint pericellular staining [Fig. 7(a)]. Grafted OA cartilage showed general positive staining of all chondrocytes and some indications in a number of sections of positive surface staining. The matrix showed no indication of staining except around sections of invading vasculature where a narrow rim of the matrix around the entire invasion stained positively [Fig. 7(b)]. This rim of positive staining was not as intense and did not penetrate as deeply into the matrix as the type I collagen staining.
Discussion It has been demonstrated here that human OA articular cartilage loses its ability to remain avascular when placed on to an active vasculature, that of the chick CAM. Changes in the ECM components were examined by both histochemical and immunohistochemical staining. Most notable is the observation that vascular invasion was always associated with a localized loss of proteoglycan/ GAG staining within the cartilage ECM and a deposition of collagen types I and X along the rim of invasive vessels. CAM grafting did not have any noticeable effect on the rest of the matrix. This, however, may not be such a surprise. As incubation time is relatively short and it is known that cartilage ECM components have a slow turnover time within cartilage matrix,2,21 there is unlikely to be a significant cartilage mediated change over such a short period of time.
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Fig. 3. (a) Section of a grafted piece of healthy cartilage, there is no indication of any vascular invasion in the deep layers (s indicates the surface). (b) Section of a grafted piece of OA cartilage. Two small invasions can be seen into the deep layers (arrows) but there is no indication of invasion through the surface layers (s) (×100).
The results of this study are similar to previous studies that have examined types I, II and X collagen and proteoglycan/GAG in cartilage.3,24–26,28,29,40,41 Type I collagen is often found confined to the surface layer of normal cartilage. Type II collagen showed general uniform staining throughout both control and OA cartilage sections, as would be expected, and has been shown previously.7,29 Aigner et al.27 however, also showed some irregular staining of type II collagen in OA cartilage, via enhanced
pericellular deposition and diminished inter-territorial staining. Type X collagen stained to varying degrees in both OA and non-OA cartilage at the surface layers, and in the pericellular region of a number of the chondrocytes and also intra-cellularly in the majority of chondrocytes. Healthy cartilage has previously shown little or no type X collagen staining,30,42 whilst OA cartilage has been shown to stain for type X collagen both intra-cellularly and pericelluarly,
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Fig. 4. (a) Loss of metachromatic staining for proteoglycans with safranin-O around an invasive vessel (×200). (b) Loss of cartilage specific GAG staining shown by a decrease in alcian blue CEC staining around an invasive vessel (×100) (arrows indicate the area of staining loss).
most prominently around hypertrophic cells and proliferating clusters of cells in the upper and middle zones of the cartilage.28,30 However, the most prominent feature of the staining of the cartilage was of the invasive vessels seen in CAM grafted OA cartilage. The rim of matrix around the invasion stained positively for both type I collagen and type X collagen. Type I collagen is associated with de-differentiated chondrocytes.42–44 It is also a component of blood vessels and is known to be upregulated in endothelial cells undergoing angiogenesis.45 Type X has been hypothesized to be a factor in the calcification/ mineralization and vascularization of cartilage.46–49 The source of the type I collagen could either be cartilage or vasculature derived as the anti-type I antibody is a
polyclonal shown to cross react with both human and chick type I. The anti-type X antibody is specific for human type X collagen, therefore the type X collagen laid down in the matrix must be cartilage derived. It has been shown that the underlying subchondral bone and the calcified cartilage between the subchondral bone and articular cartilage contains blood capillaries,50 this is part of the normal structure of the joint, and as such is not a symptom of the OA process. However vascularization of the articular cartilage above the original ‘tidemark’, a defined layer between the calcified cartilage and the articular cartilage, is an important step in the pathological process of OA.16 The vascularization of cartilage above this tidemark is partly responsible for the conversion of cartilage to bone, a process which requires a mineralized cartilage
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Fig. 5. (a) Surface staining for type I collagen and along the rim of an invasive vessel (s indicates the surface layer, arrow indicates the vessel) (×40). (b) Higher power micrograph of an invasive vessel showing a rim of positive staining for type I collagen (v is the vessel, arrows indicate type I positive staining) (×200).
intermediate51 (un-mineralized cartilage is generally resistant to vascularization52), there is therefore, an advancement of the tidemark as the cartilage is calcified, and subsequently eroded.4 However, in the work described above, there was no histological evidence of cartilage mineralization in tissue sections in any of the tissue samples analysed, indicating that the process on the CAM may not require the cartilage to be mineralized for vascularization. The invasive process may be more like the
vascularization of avian cartilage, which, during development, is known not to mineralize before vascular invasion.53 Alongside the cartilage erosion associated with the pathology of OA, there is also renewed growth of cartilage (and bone) at the joint margin, resulting in the formation of osteophytes.2,6,54 New vasculature is required for this process, the formation and growth of the osteophyte being reliant upon the proliferation of blood vessels into
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Fig. 6. (a) Staining of cartilage with anti-type II antibody in a section of grafted OA cartilage showing an almost blanket staining effect with some regions of only pericellular staining (indicated by arrow) (×40). (b) Intense pericellular staining for type II collagen in a section of grafted non-OA cartilage (arrows) (×200).
degenerate articular cartilage.54 The vasculature therefore, is partly responsible for the erosion of degenerate cartilage and the formation of new bone via endochondral ossification, yet at the same time aids in the growth of new cartilage within the same joint. This new cartilage appears similar to normal articular cartilage at the histological level (personal observation), which as such is not surprising as the growth of osteophytes is considered to be a mechanism of attempted repair.54 Despite its now accepted role in the OA process, cartilage vascularization has more commonly been associated
with rheumatoid arthritis, an inflammatory disease of the articular cartilage and surrounding joint. Chronic inflammation is very much associated with angiogenesis,55 however, osteoarthritis is considered to be a noninflammatory disease. If OA is non-inflammatory, then the mediators of the angiogenic response may be very different from those associated with an inflammatory response. The presence or absence of angiogenesis mediating cells, such as macrophages or mast cells, needs to be assessed, along with the presence of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic
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Fig. 7. (a) Faint staining of the pericellular matrix of non-grafted OA cartilage with the anti-type X antibody (short arrows), the chondrocytes are also intensely stained (long arrows) (×100). (b) Deposition of type X collagen along the rim of an invasive vessel (arrows indicate positive staining) (×100).
fibroblast growth factor (bFGF). The role of MMP and TIMP in the process is also of great interest, as evidence suggests that cartilage turnover and degradation is mediated by an imbalance between the activity of MMP and their inhibitors.56,57 An examination of cartilage and vascular derived MMP and TIMP may provide much information about the mechanism of cartilage destruction during vascular invasion, and the role that both the cartilage itself, and the invasive vasculature play during the process. OA cartilage has never previously been grafted onto the CAM, therefore this work provides a novel model for the study of active vascularization of human OA cartilage.
The process of vascularization of human OA cartilage on the CAM occurs rapidly. The study of this process is therefore enhanced by this model which may aid in elucidating the reasons as to why the process occurs, along with the cell biological, biochemical and molecular mechanisms behind the process. In conclusion, human OA cartilage loses its avascular state when placed onto the chick CAM. There is a loss of non-specific and cartilage-specific GAG and proteoglycans around the invasion site, and deposition of type I and type X collagen along the rim of vascular invasion, which may have a role in the process.
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Acknowledgments We would like to acknowledge the University of Leicester for funding this research. The monoclonal antibody CIICI developed by Holmdahl and Rubin was obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICHD.
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