ARTICLE |
Correspondence to: Robert D. Young, Connective Tissue Biology Laboratories School of Biosciences, Cardiff University, PO Box 911, Museum Avenue, Cardiff, UK CF1 3US.
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Summary |
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Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmiumacetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several noncollagenous components of the extracellular matrix. (J Histochem Cytochem 48:423432, 2000)
Key Words: collagen, type II, type III, heteropolymeric fibril, immunoelectron microscopy, human cartilage, high pressure cryofixation
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Introduction |
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INDIVIDUAL COLLAGEN FIBRILS, which provide the structural framework for a wide diversity of connective tissue matrices, are themselves now recognized to be highly heterogeneous assemblies. The association of more than one collagen type to form single fibrils was first conclusively demonstrated for Types I and V collagen in avian corneal stroma (
Electron microscopy employing labeled antibodies has played a major role in studies of the distribution of different collagen types and in demonstrating conclusively by multiple immunolocalization their association within single fibrils comprising connective tissue matrices. However, collagen epitopes for specific antibodies appear intolerant to many of the methods in routine use for microscopy, particularly chemical fixation with aldehydes. Consequently, many previous investigations of collagen interactions have resorted to immunoelectron microscopy of chemically disrupted fibrils or fibrils extracted from disrupted matrix (
To overcome the difficulties with collagen sensitivity to chemical fixation and to avoid the extractive effects now known to be associated with conventional processing techniques involving aqueous fixation and dehydration (
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Materials and Methods |
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Articular cartilage was obtained from the femoral heads of patients undergoing hip replacement surgery for late-stage osteoarthritis. Samples were selected from areas of unfibrillated intact tissue and were processed for electron microscopy.
CryofixationHigh-pressure Freezing (HPF)
Full-thickness slices of cartilage were taken with a scalpel and stored for up to 16 hr, either in Eagle's MEM + 5% fetal calf serum at 37C or wrapped tightly in plastic film within a sterile vial at 4C to reduce changes in tissue hydration before cryofixation. Cylindrical full-thickness plugs of cartilage 1.7 mm in diameter were first prepared using a microtrephine. Slices approximately 0.10.2 mm thick and corresponding to the upper radial zone of the cartilage were then cut from the plugs with a scalpel and immersed in 1-hexadecene. These tissue discs, bathed in 1-hexadecene and sandwiched between two aluminum planchettes, were rapidly cryofixed at 2100 bar pressure in a Baltec 010 HPM machine. Frozen samples were maintained in liquid nitrogen until subsequent processing by freeze-substitution at -90C in a Reichert AFS system (Leica; Milton Keynes, UK), using one of two different methods.
Freeze-substitution with Chemical Fixation Followed by Conventional Epoxy Resin Embedding
Samples were freeze-substituted at -90C for 48 hr in acetone (99.9%, HPLC grade: SigmaAldrich, Gillingham, UK), containing 2% w/v osmium tetroxide (Agar Scientific; Stansted, UK) over a 4Å molecular sieve. After programmed warming to 20C at 10C/hr, the tissue blocks were infiltrated with 1:3, 1:1, 3:1 mixtures of Araldite resin:acetone, several changes of 100% resin, and then polymerized in fresh resin at 60C.
Freeze-substitution Without Fixatives Followed by Resin Embedding at Low Temperature
After freeze-substitution at -90C in pure methanol for 24 hr (99.8% anhydrous: Aldrich), the temperature was raised at 10C/hr to -50C. Samples were then infiltrated with Lowicryl HM20 resin (Agar Scientific), through 1:3, 1:1, and 3:1 resin:methanol mixtures for 1 hr each, several changes in 100% resin, and polymerized by UV light. Polymerization was carried out at -50C for 2 days and at room temperature for an additional 2 days.
Antibody Labeling
Ultrathin sections were cut from resin blocks on a Reichert Ultramicrotome equipped with a diamond knife and collected on collodion-coated nickel grids. Before immunostaining, sections were etched by exposure to enzyme solutions: bovine testicular hyaluronidase (Sigma Chemicals, Poole, UK; H-3506, 700 U/ml) and Pronase (Sigma Chemicals; Protease XIV, P5147, 2 U/ml), both for 1 hr at 37C.
The sections were then incubated on droplets of primary antibodies diluted in PBS/1% bovine serum albumin (BSA): (a) a mouse monoclonal antibody to Type II collagen (CII/CI). This antibody was developed by R. Holmdahl and K. Rubin and is available from the Developmental Studies Hybridoma Bank maintained by the University of Iowa; (b) a polyclonal antibody raised in goat to the pepsin-extracted triple-helical domain of human Type III collagen; (c) a rabbit polyclonal antibody to pN (propeptide) collagen Type III. These antibodies are the same as those used previously (
After 23-hr incubations and washing with several changes of PBS/1% BSA, the sections were transferred to secondary antibodies labeled with colloidal gold particles, either goat anti-mouse, rabbit anti-goat, or goat anti-rabbit IgG, as appropriate (Amersham International, Poole, UK, or Biocell, Cardiff, UK). Secondary antibodies were preabsorbed to exclude any cross-species reactivity and were used at a dilution of 1:20 in PBS/1% BSA.
In double-labeling experiments to localize collagen Types II and III simultaneously, the first primary and appropriate secondary antibody, followed by the second primary and secondary antibodies, were applied in sequence, with copious washings between incubations. In these experiments, secondary antibodies were employed conjugated to gold particles of different sizes with the 5-nm smaller probe applied before the 10-nm conjugate.
After immunostaining and washing steps, sections were fixed briefly by floating grids on droplets of 2% glutaraldehyde in PBS, followed by final washes in boiled Millipore-filtered distilled water. The sections were then contrasted in aqueous uranyl acetate and lead citrate for 10 and 5 min, respectively, before examination in a Philips 400 or 208 transmission electron microscope at an accelerating voltage of 80 Kv.
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Results |
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HPFAcetoneOsmium Freeze-substitutionAraldite Embedding
Collagen fibrils in the upper radial zone of articular cartilage exhibited a wide range of diameters and orientations (Fig 1), occasionally undergoing abrupt bends or twists along their longitudinal axes. Finer filamentous elements were also identified scattered among the major interstitial fibrils. In transverse section, fibrils often had an electron-lucent center, a more intensely contrasted perimeter, and were surrounded by an electron-lucent halo concentric to the fibril border. Fibril centers sometimes appeared to be less well embedded than their peripheries, although this was not evidenced in fibrils sectioned longitudinally. Some fibrils with irregular transverse section profile nevertheless were surrounded by a halo with regular smooth contours (Fig 1). This lucent perifibrillar feature was also evident in fibrils viewed in longitudinal section. The associated interfibrillar matrix in these sections displayed a uniform coarse granularity, which terminated abruptly at its interface with the perifibrillar halo.
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Attempts to immunolocalize individual collagen types with specific antibodies on sections of HPF tissue freeze-substituted in acetoneosmium were unsuccessful. However, immunogold detection of the proteoglycans keratan and chondroitin sulfate in the interfibrillar cartilage matrix of these same preparations was readily achieved (not shown).
HPFMethanol Freeze-substitution Without FixativesLow-temperature Embedding
As expected, electron contrast was reduced in Lowicryl resin sections of cartilage prepared without exposure to osmium tetroxide, even after prolonged section staining with uranyl acetate and lead citrate. The collagenous cartilage matrix was well preserved, although little nonfibrillar matrix structure could be observed and some tissue shrinkage was evident. Ultrastructural features, such as the electron-lucent perifibrillar halos and regions of reduced contrast at the centers of transverse-sectioned collagen fibrils, were absent from tissue embedded at low temperature.
In all cases in which sections were exposed to buffer solutions without anti-collagen antibodies or in which antibodies to vimentin or tropomyosin were used, immunogold labeling either was completely absent or was located in very small numbers after prolonged searching (Fig 2). However, unlike in the osmium-treated tissue, localization of collagens was possible (Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8).
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Collagen Type III could be detected both with an antibody against the N-propeptide and with one reacting with the triple-helical domain of the molecule (Fig 3 and Fig 4). Immunogold labeling was more abundant with the first antibody, which appeared densely scattered over regions containing finer fibrils but which also labeled associated fibrils of larger diameter (Fig 3). The N-propeptide antibody labeled some sites apparently unattached to fibrillar material, particularly where aggregations of fine fibrils were located. The antibody against the helical domain of collagen Type III did not appear to label fine fibrils as densely as the larger collagen fibrils. Both antibodies labeled fibrils sectioned transversely to a lesser extent than those displayed in longitudinal section. Collagen Type II immunolocalization was also evident on fibrils of widely different diameters. Immunogold labeling was best observed in larger-diameter fibrils viewed in longitudinal section (Fig 5 and Fig 6).
Sections exposed sequentially to antibodies to Type II and Type III collagens, followed, after each, by the appropriate gold-conjugated secondary antibody, clearly showed the presence of both collagen types within individual collagen fibrils (Fig 7 and Fig 8). Co-localization of Type II and Type III collagens was most evident on large-diameter fibrils sectioned longitudinally and was not detected on the finer fibrils in the matrix. Experiments applying the two anti-collagen primary antibodies in different sequence yielded similar staining patterns. In addition, Type II:Type III collagen co-localization was demonstrated with either of the anti-Type III collagen antibodies, with similar results.
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Discussion |
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The collagenous fibrillar matrix of mammalian articular cartilage is composed primarily of Type II collagen, with at least ten additional collagens, including Types VI, IX, X, and XI, present as minor components (
This study is in accord with earlier reports of Type III collagen present as a minor component of articular cartilage (
Collagen Type III, like collagens I and II, conforms to the classical interstitial molecular structure with 300-nm rod-like tropocollagen molecules produced from procollagen by enzyme-induced cleavage of terminal N- and C-propeptides before their assembly into fibrils in the extracellular matrix. Collagen Type III retains the N-propeptide for some time after fibrillogenesis (
A major question remains concerning the significance of Type III collagen in the sequence of fibril assembly in cartilage and its relationship to the pathogenesis of osteoarthritis. At present, it is unknown whether cartilage fibrils incorporate Type III during synthesis of new fibrils or whether this collagen is assembled onto the surface of existing Type II/Type IX/Type XI polymers as part of age- or disease-related degradation or repair. Our previous study showed Type III collagen to be present on fibrils in the matrix of normal as well as osteoarthritic human articular cartilage (
Cryofixation, by high-pressure freezing, and freeze-substitution followed by ambient or low-temperature embedding, has now been used by many investigators as a means to prepare cartilage for ultrastructural examination (
There have been relatively few reports of immunolocalization of collagens in high-pressure frozen tissues. Although immunolocalization of invertebrate tissue antigens, including cuticular collagen in Arenicola, was improved by the inclusion of osmium in acetone substitution media (
High-pressure freezing is capable of vitrifying cartilage specimens of up to 150-µm thickness (
Although it appears that the efficacy of high-pressure freezing for vitrification of bulk tissue samples may well have been overestimated previously, its potential for immunodetection remains underexploited. It therefore continues to offer an unrivaled opportunity for investigations of molecular interactions in connective tissues and of the changes that occur in disease.
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Acknowledgments |
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Supported by a grant from the Arthritis Research Campaign, UK.
Received for publication June 15, 1999; accepted October 28, 1999.
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