Osteoarthritis and Cartilage (2002) 10, 333–343 © 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved. 1063–4584/02/$35.00/0 doi:10.1053/joca.2002.0519, available online at http://www.idealibrary.com on Histochemical studies of the extracellular matrix of human articular cartilage—a review J. L. Hyllested, K. Veje and K. Ostergaard Osteoarthritis Research Unit, Institute for Inflammation Research (IIR), 7521 Finsencentre, National University Hospital, Rigshospitalet, Copenhagen, Denmark Summary Objective: This paper reviews the histochemistry of the extracellular matrix of human articular cartilage. No systematic review of histochemical knowledge and techniques in the study of articular cartilage has been published previously. Methods and results: Literature was searched in the Winspirs Medline database from 1960 to 2000. Only techniques applicable for bright field or polarization microscopy were considered. Unless otherwise noted, all applies to hyaline cartilage. The most widely used fixatives are adequate for routine staining of proteins, but proteoglycan fixation is problematic, and no one fixative can be recommended. Proteoglycan can be stained reliably but it is problematic that, at low substrate concentrations, these methods are not stoichiometric. Collagen can be stained efficiently, although attempts to differentiate collagen types have not been successful. Conclusions: Detailed studies of fixation and staining procedures should be carried out and standards for cartilage sampling, handling and evaluation agreed upon if results from different laboratories are to be compared. © 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved. Key words: Human cartilage, Histochemistry, Fixation, Staining. Introduction avascular matrix composed of water (70% wet w/w)7, proteoglycans8, collagens (50–90% dry w/w)9 and non- Human articular cartilage is the site of osteoarthritis (OA), collagenous proteins10. The non-collagenous proteins the most common joint disease and a cause of pain and can be subdivided into proteins present only in articular disability in a very large number of people. cartilage11, and proteins present in other tissues as well. As cartilage research expands, so does the need for However, the non-collagenous proteins are too minor standardization of techniques. It is essential that labora- constituents to be differentially detected by available histo- tories across the world are able to compare results. Histo- 1,2 chemical methods, and are best visualized by more specific logical grading systems for OA for example, depend in methods such as immunohistochemistry. Many of these part upon the stoichiometric staining characteristics of proteins help to determine the configuration of collagens proteoglycan stains for the grading of the severity of the and glycosaminoglycans. This may influence the staining osteoarthritic lesion. pattern of these components, but this has not been studied. Tissue sampling, fixation, staining and assessment are Leaching of the small structural and non-structural proteins cornerstones of histochemistry and plenty of histological during tissue processing has not been studied, and the and histochemical textbooks are available, encompassing 3–6 significance of this with regards to histochemistry is almost any known technique in use . However, no unknown. account or review of histochemical knowledge and tech- nique in the particular study of the extracellular matrix articular of cartilage has been published. Sampling, preparation and handling of tissue Articular cartilage Adequate fixation and staining of tissue samples require reliable and standardized sampling methods. Articular cartilage comprises only one cell type, i.e. Factors such as the source individual, joint type, top- the chondrocyte. The chondrocyte is embedded in an ography of sampling site within a joint, depth of sampling and the presence of joint (cartilage) disease should Received 25 August 2001; accepted 7 January 2002. be considered and standardized12. Ideally, though not Financial support was granted by the Danish Rheumatism practically feasible, a normal as well as an osteo- Association, the Danish Biotechnology Programme and EU—OA- arthritic cartilage section could be included as control contract QLRT1999-02072. samples in every staining and fixation run. The included Address correspondence to: Jacob L. Hyllested, Osteoarthritis sections should be known with regards to staining Research Unit, Institute for Inflammation Research (IIR), 7521, Finsencentre, National University Hospital (Rigshospitalet), pattern. Blegdamsvej 9, DK-2100 Copenhagen East, Denmark. Tel: All the steps in tissue processing should be standardized +45 3545 7517; Fax: +45 3545 7554; E-mail: hyllested@oa- to avoid variation, within the laboratory as well as between research.dk; Website: www.oa-research.dk laboratories. 333 334 J. L. Hyllested et al.: Cartilage matrix histochemistry Fixation and fixatives been shown not to prevent glycosaminoglycans (GAG) and proteoglycan leaching from tissue24, a finding contrary to AIMS OF FIXATION that of Kiviranta and co-workers (1984)20, who found that neutral buffered formalin was adequate for proteoglycan Fixation means ‘to make stable and stationary’ and fixation, and that almost no proteoglycans or GAG were lost hence implies that this process (1) prevents tissue autolysis following fixation by neutral buffered formalin. Alcoholic and bacterial attack; (2) prevents the tissue from rearrange- formalin was found to improve fixation of GAG in deminer- ment and changing morphological appearance during alized cartilage25, but the cellular appearance was poorly subsequent processing; (3) prevents loss of tissue compo- preserved. Addition of Safranin O to the fixative solution nents due to ‘washing out’; (4) does not produce artefacts, 13 may also prevent GAG loss, but this approach is impracti- (5) allows for clear and good staining of tissue . cal, since it may interfere with future use of the tissue, e.g. Different tissue components are retained in the tissue to for immunohistochemistry26. differing degrees by various fixatives and that retention, For better aldehyde fixation of cartilage proteins, proteo- although a prerequisite for staining, is not in itself enough, glycan or GAG, a mixture of formalin and cetyl pyridinium preservation of the tissue component is also important. chloride27, cetrimide24, cetyltrimethylammonium28 could be used. Good results have also been obtained with a mixture TYPES OF FIXATION of formalin and alcohol for GAG fixation20,29. These com- Fixatives may conveniently be classified as (1) cross- binations may work by precipitating carbohydrates and thus linking (aldehydes i.e. formaldehyde and glutaraldehyde), decrease diffusion of protein, proteoglycans and GAG (2) protein-denaturing, coagulating agents (i.e. methanol, from cartilage. A review of formalin fixation for immuno- histochemistry can be found in the American Journal of ethanol and acetone), (3) oxidizing agents (i.e. osmium 30 tetroxide and potassium permanganate), (4) other cross- Surgical Pathology, 2000 . linking agents (i.e. carbodiimides), (5) physical (i.e., heat and microwaves) and (6) miscellaneous chemical Glutaraldehyde reagents. Time of fixation, pH, temperature, osmolarity14, fixative Glutaraldehyde–protein cross-linking is increased with penetration (diffusion) of tissue15 as well as volume, buff- temperature, pH and glutaraldehyde concentration31. The ering and freshness of liquid solutions should always be cross-links are rapidly formed and irreversible; proteins considered, since these factors are important for effi- may suffer loss of up to 30% of -helix structure13,18, and cacy16,17. The time interval between death of tissue (ces- consequently the ultrastructure of collagen is not well sation of circulation) and proper fixation should clearly be preserved32. Glutaraldehyde fixation reduces the activity of kept at an absolute minimum (although rarely practically most enzymes and may introduce Schiff-positive aldehyde possible; preferably less than 6 h), although avascular by groups33. Note that articular cartilage is normally PAS- nature, articular cartilage does not undergo autolysis as negative. quickly as most other vascular tissues. It has been shown that up to 40% of proteoglycans and Most aqueous fixatives are used at temperatures GAG are lost from cartilage during aqueous fixation with between 4°C and 40°C, the higher temperatures translating glutaraldehyde19, which is deleterious both when assess- into shorter fixation times. ing morphology and when assessing proteoglycan content Fixative to tissue ratio and fixative to tissue penetration by staining. as a function of time, concentration, tissue block size and fixative volume may influence the fixative’s overall ability to retain a certain tissue component. Penetration of fixative Protein-denaturing agents into tissue has been shown to depend upon a fixative Alcohol-containing fixatives have traditionally been used specific constant and, interestingly, that alcohol is superior as fixative for proteoglycans, GAG and carbohydrates. It is to both formaldehyde and glutaraldehyde14,18 . believed that alcohols denature the protein component and Except for two studies assessing proteoglycan depletion precipitate the carbohydrate onto the protein meshwork by fixation19,20 , none of the aforementioned variables has thus formed34. been thoroughly and objectively assessed with regard to