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Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage
OsteoArthritis and Cartilage (2004) 12, 17–24 © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2003.09.001
International Cartilage Repair Society
Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage I. Holloway†,‡, M. Kayser†, D. A. Lee§, D. L. Bader§, G. Bentley† and M. M. Knight§* †Institute of Orthopaedics, University College London Medical School, Brockley Hill, Stanmore, U.K. ‡Orthopaedic Department, Northwick Park Hospital, Watford Road, Harrow, U.K. §IRC in Biomedical Materials and Medical Engineering Division, Department of Engineering, Queen Mary University of London, Mile End Road, London, U.K. Summary Objective: This study examined the morphology of chondrocytes in articular cartilage from osteoarthritic (OA) and non-OA human femoral heads and in particular the appearance of a sub-population of cells with multiple elongated processes radiating up to 30 µm into the extracellular matrix. Methods: Cartilage explants were removed from 8 anatomical sites over the surface of OA (n=6) and non-OA (n=5) femoral heads. Cells were labeled for vimentin intermediate filaments and visualized using epi-fluorescence and confocal microscopy. The percentage of cells with elongated processes was correlated with macroscopic and histological indicators of osteoarthritis. Results: Cells with processes accounted for less than 10% of the total cell population in non-OA cartilage. By contrast, in the peripheral regions of the OA femoral head these cells accounted for 20–45% of the total cell population, the differences being statistically significant. These peripheral areas are habitually non-load bearing and were also the most likely to show gross fibrillation and pannus formation. A statistically significant correlation was demonstrated between the percentage of cells with processes and the histological extent of the OA degradation, quantified in terms of the Mankin score. Conclusions: The extension of cell processes, which may be associated with localized breakdown of the pericellular matrix, will undoubtedly alter numerous aspects of cell function including phenotypic expression and mechanotransduction. Hence these significant changes in chondrocyte morphology are likely to have important implications for the aetiology of osteoarthritis and the development of potential treatment strategies. © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Osteoarthritis, Cartilage, Cytoskeleton, Vimentin, Confocal, Chondrocyte.
TNF-5–8. Cell metabolism is known to be influenced by cell morphology and associated cytoskeletal organization9. For example, a rounded cell morphology and the absence of actin stress fibres is necessary for the maintenance of chondrocytic phenotype as demonstrated by the synthesis of type II collagen and aggrecan10–12. However, it remains unclear the extent to which changes in chondrocyte metabolism in osteoarthritic cartilage are associated with alterations in cell morphology. This study utilized epifluorescence and confocal microscopy to test the hypothesis that morphological differences exist between chondrocytes within cartilage from non-osteoarthritic (nonOA) and osteoarthritic (OA) femoral heads. In particular, the study focuses on the appearance of a sub-population of cells with multiple elongated processes, identified by immunofluorescent labelling of the cytoskeletal protein, vimentin. Although previous studies on articular cartilage have described the appearance of chondrocytes with limited cell processes13,14, cells with elongated processes extending up to 30 µm have only, very recently been reported in one study of OA cartilage from human tibia plateaus15. In this study, the presence of cells with elongated processes in non-OA and OA femoral head cartilage is examined in relation to anatomical position in
Introduction Osteoarthritis is a widespread clinical problem affecting many joints of the body, particularly the hip and the knee, resulting in swelling, stiffness and pain. Pathological changes in osteoarthritis are characterized by softening, fibrillation, abrasion and ultimate loss of the articular cartilage exposing the underlying bone1,2. Cartilage breakdown is associated with changes in extracellular matrix composition with reduced keratan sulphate content and increased water content3. These mechanical and biochemical changes are partly attributed to damage to the collagen network such that it is unable to restrain the swelling pressure produced by the glycosaminoglycan (GAG). Osteoarthritis is also associated with differences in chondrocyte gene expression4 and metabolism characterized by increased cell proliferation, synthesis of fibrocartilage matrix and secretion of matrix metalloproteases (MMPs) and inflammatory cytokines such as IL1- and *Address correspondence to: Dr Martin Knight, IRC in Biomedical Materials, Queen Mary University of London, Mile End Road, London E1 4NS. Tel.: +44-(0)-207-882-5512; E-mail: [email protected] Received 28 April 2003; revision accepted 6 September 2003.
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I. Holloway et al.: Cell morphology in OA cartilage
Fig. 1. Schematic diagram showing the location of the anatomical sites across the femoral head from which cartilage specimens were removed.
the joint and histological and macroscopic indicators of osteoarthritis.
Materials and methods PREPARATION OF CARTILAGE SPECIMENS
This study examined the articular cartilage from the femoral heads of patients in two groups. An osteoarthritic (OA) group, who received total hip replacement as a treatment for osteoarthritis, and an age-matched non-OA group who required hemiarthroplasty for fractured neck of femur. The number of femoral heads used in this study was 5 in the non-OA group and 6 in the OA group. The mean ages of patients in the non-OA and OA groups was 81.6 and 67.3 respectively. Patients were seen pre-operatively and consented according to regulations laid down by the local Research Ethics Committee. In both groups, the presence of osteoarthritis in the operated hip was assessed based on the diagnostic criteria established by the American Rheumatism Association2. All patients in the osteoarthritic group had clinical evidence of osteoarthritis where as no patients in the non-OA group showed any evidence of osteoarthritis. Femoral heads were collected during surgery and maintained briefly in Dulbecco’s Minimal Essential Medium supplemented with 10% (v/v) foetal calf serum (DMEM+10% FCS, Gibco, Poole, U.K.). Explants of full thickness cartilage with underlying bone, approximately 1 cm3, were excised from eight sites on each femoral head: four parafoveal sites (superior, inferior, anterior, posterior) and four corresponding sites in the peripheral cartilage as indicated in Fig. 1. The cartilage at each site was assessed for macroscopic signs of surface fibrillation. Explants were cut in two halves such that one half was processed for histology while the other half was used for analysis of cell morphology. HISTOCHEMICAL STAINING
For histological investigation specimens were examined with Haematoxylin and Eosin staining and Safranin-O
staining following standard protocols. Briefly, cartilagebone explants were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, washed in Phosphate Buffered Saline (PBS) and then slowly decalcified at 4°C in 0.1 M sodium cacodylate buffer containing 0.1 M ethylenediamine-N,N,N′,N′-tetracetic acid (EGTA) and 4% glutaraldehyde. Full decalcification was confirmed using X-ray analysis. Explants were embedded in paraffin and 5 µm sections prepared. The sections were deparaffinized with xylene, alcohol and water and then stained with Harris’s acid alum haematoxylin (HS355, HD Supplies, Aylesbury, U.K.) for 9 min. Specimens were further washed in water, differentiated in acid alcohol for 3–5 s and washed again. All specimens were counterstained for 5 min in either 1% Eosin (colour index 45380, HD1320, HD Supplies, Aylesbury, U.K.) or 0.1% Safranin O (colour index 50240, HD1850, HsD Supplies, Aylesbury, U.K.). Specimens were then washed, dehydrated through a series of alcohols and xylene and finally mounted in DePeX (BDH Merk). Light microscopy was used to visualize the specimens to enable histological grading of the cartilage according to the Mankin score1. Statistical comparison of Mankin scores at the 8 anatomical sites in non-OA and OA femoral heads was conducted using the Student’s t test with a value of P<0.05 indicating a significant difference.
ANALYSIS OF CELL MORPHOLOGY
Cartilage explants were fixed for 20 min at 37°C in 4% formaldehyde prepared in PBS and then washed with PBS. A microtome was used to section the explants perpendicular to the articular surface yielding individual specimens 150 µm in thickness. To aid the penetration of the antibodies, the extracellular matrix components underwent partial enzymatic digestion. Specimens were incubated on rollers for 2 h at 37°C in 0.125 units/ml chondroitinase ABC (Sigma, Poole, U.K.) and 1500 units/ml bovine testicular hyaluronidase (Sigma, Poole, U.K.) prepared in modified Hanks Balanced Salt Solution (mHBSS). Modified Hanks Balanced Salt Solution at pH 6.5 consisted of HBSS (10X, Gibco, Paisley, U.K.)+2 mM MgCl2+4 mM NaHCO3+2 mM EGTA (Sigma, Poole, U.K.)+0.11% w/v MES (Sigma,
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Fig. 2. Photograph of (a) non-OA and (b) OA human femoral heads showing the gross appearance of the articular cartilage. Arrow indicates the fovea.
Poole, U.K.) as described by the Langelier et al.16. Following enzymatic digestion, samples were washed 3 times for 5 min in mHBSS. The staining protocol for vimentin intermediate filament cytoskeleton followed that previously optimized for isolated bovine articular chondrocytes seeded in agarose17–19. Specimens were maintained for 5 min at 4°C in cell permeabilizing buffer consisting of 10.3 g sucrose, 0.292 g sodium chloride, 0.006 g magnesium chloride, 0.476 g HEPES (Sigma, Poole, U.K.) and 0.5 ml Triton X dissolved in 100 ml distilled water. Specimens were then washed with 1% bovine serum albumin (BSA, Sigma, Poole, U.K.) in mHBSS and incubated on rollers for 2 h at 37°C in primary mouse anti-vimentin clone 9 (Sigma, Poole, U.K.) prepared at 1:100 in mHBSS. To remove unbound primary antibody the specimens were washed 3 times for 10 min in PBS+0.5% (v/v) Tween 20 (BDH Laboratory Supplies, Poole, U.K.). Specimens were then incubated on rollers for 1 h at 37°C in rabbit anti-mouse FITC-conjugated secondary antibody (Sigma, Poole, U.K.) prepared at 1:20 in mHBSS and finally washed 3 times for 10 min in PBS+0.5% (v/v) Tween 20. All specimens were stored at 4°C in mHBSS prior to epi-fluorescence microscopy. Each specimen was mounted on a coverslip, hydrated in PBS and placed on the stage of an inverted microscope (Eclipse, Nikon, U.K.) attached to a confocal laser scanning microscope system (Ultra View, Perkin Elmer, Cambridge, U.K.). Standard epi-fluorescence and confocal microscopy
with a 40x/0.95 NA Plan Apo objective lens (Nikon, U.K.) was used to assess the morphology of fluorescently labeled cells. For each specimen, a total of nine separate fields of view, approximately 500 µm in diameter, were analysed at 3 different depths below the articular surface, approximately corresponding to the surface, middle and deep zones where possible. The percentage of cells exhibiting elongated processes over 10 µm in length, was recorded within each field of view and the mean percentage value calculated at each depth. The procedure was repeated for specimens from the 8 anatomical sites from each femoral head in the two groups. Non-parametric statistical analysis was performed using the Mann–Whitney U-test with P<0.05 indicating a statistically significant difference.
Results All the femoral heads in the OA group exhibited areas of macroscopic fibrillation which varied across the joint surface (Fig. 2, Table I). In the non-OA group the cartilage appeared normal with no sign of fibrillation except at the inferior parafoveal site (PFI) where mild fibrillation and surface unevenness was present in all the femoral heads. A summary of the areas demonstrating fibrillation for both groups is presented in Table I.
Table I Numerical data describing the cartilage at the 8 anatomical sites for non-OA (n=5) and OA (n=6) femoral heads. Anatomical Site
% of femoral head with fibrillation % femoral heads showing pannus Mankin Score (mean ± SE) % cells with processes in non-OA cartilage (median)
Non-OA OA Non-OA OA Non-OA OA surface middle deep
PFI
PFS
PFA
PFP
PI
PS
PA
PP
100% 83% 0% 50% 1.8±0.5 5.3±1.3 38.9 5.2 2.5
0% 17% 0% 17% 1.3±0.5 7.6±1.5 2.9 4.6 0.0
0% 33% 0% 17% 0.8±0.6 5.0±1.2 13.1 4.3 0.0
0% 83% 0% 33% 0.0±0.0 4.6±1.2 5.9 5.9 0.0
0% 50% 0% 50% 1.4±0.5 5.3±0.7 6.9 2.9 2.0
0% 83% 0% 83% 0.3±0.3 8.0±0.7 4.3 6.8 2.2
0% 67% 0% 50% 1.0±0.8 6.7±1.3 2.1 3.9 0.0
0% 50% 0% 50% 1.3±1.3 5.5±1.0 3.1 3.0 2.1
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I. Holloway et al.: Cell morphology in OA cartilage
Fig. 3. Histological appearance of representative articular cartilage specimens stained with H&E (a–d) or Safranin O (e–h). Images indicate the characteristic appearance of cartilage from a non-OA femoral head with a Mankin score of 0 (a, e) and from OA femoral heads with Mankin scores of 3 (b–f), 7 (c, g) and 11 (d, h). Arrows indicate the presence of a pannus layer at the articular surface (c, g). Scale bars represent 10 µm.
HISTOLOGICAL APPEARANCE OF THE TISSUE
Representative histological images of non-OA and OA cartilage stained with Haematoxylin and Eosin or Safranin-O are shown in Fig. 3. The average Mankin scores for cartilage at the 8 anatomical sites in non-OA and OA femoral heads are presented in Table I. At all locations over the femoral head, the Mankin score for OA cartilage was higher than that for non-OA cartilage, the differences being statistically significant. The articular surfaces of all 6 femoral heads from the OA group were, in part, covered by a fibrous avascular pannuslike tissue which showed negative Safranin-O staining (Fig. 3c,g) as previously described20. This tissue occurred most frequently at the peripheral anatomical sites and inferior to the fovea (Table I). Using identical histological staining, pannus was not observed in any of the femoral heads from the non-OA group. ANALYSIS OF CELL MORPHOLOGY
Cells in non-OA cartilage exhibited characteristic zonal variation in morphology and arrangement, such that cells in the surface zone appeared flattened and disc-shaped where as cells in the middle and deep zones were more spherical and arranged in columns running perpendicular to the articular surface. This distinction between the different zones was less obvious in OA cartilage. In OA specimens where pannus was identified by histology, the pannus
layer was distinguishable from the underlying cartilage by the high cell density and the fibroblastic appearance of the cells (Fig. 4). Cells within the cartilage tissue were catagorized into two types, with and without elongated cell processes. Typical examples of these cell types are shown in Fig. 5. In both cell types the vimentin intermediate filament cytoskeleton appeared as a complex fibrous structure extending throughout the cytoplasm. The percentage of cells exhibiting elongated processes in the surface, middle and deep zones of non-OA cartilage is presented in Table I. Within non-OA cartilage, less than 10% of cells exhibited processes with the exception of the surface zone at the PFI site where the value was 38.9% (Table I). At all anatomical sites there was a greater percentage of cells with processes in the surface and middle zones than in the deep zone. Data obtained at the 3 different depths was pooled to give a mean value for each specimen. The median values at each anatomical site for OA and non-OA femoral heads are plotted in Fig. 6. At the 4 peripheral sites (PS, PI, PA and PP) there was a greater percentage of cells with processes in the OA cartilage compared to the non-OA, the differences being statistically significant.
Discussion This study describes a sub-population of chondrocytes with multiple elongated processes in human femoral head
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Fig. 4. Confocal 3D reconstruction showing cells within OA cartilage labeled for vimentin intermediate filaments and visualized close to the surface of the tissue. Scale bar indicates 10 µm.
cartilage. Differences between cells in cartilage from non-OA and OA joints were examined at 8 anatomical sites over the surface of the femoral head. Immunofluorescent labeling of the cytoskeletal protein, vimentin, was used as a marker of cell morphology. This technique has the advantage over more general cytoplas-
mic stains which are less suitable for labeling thin cell processes and show poor stability following fixation. Vimentin intermediate filament cytoskeleton in both OA and non-OA cartilage exhibited a characteristic intracellular fibrous structure as previously reported16,18. In addition, vimentin staining also revealed the presence of multiple
Fig. 5. Representative confocal 3D reconstruction images of individual cells in (a) non-OA and (b) OA cartilage. The latter shows the appearance of elongated cell processes. Cells were immunolabelled for vimentin intermediate filaments. Scale bars indicate 5 µm.
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I. Holloway et al.: Cell morphology in OA cartilage
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Fig. 6. Percentage of cells with elongated cell processes in cartilage from the eight anatomical sites in non-OA and OA femoral heads. Values represent medians with error bars indicating the inter-quartile range. Statistically significant differences have been indicated for P<0.05 (*).
vimentin-rich cell processes which extended up to 30 µm into the surrounding matrix (Figs. 4 and 5b). Epifluorescence and 3D confocal microscopy were used to visualise these cell processes which radiate from the cell body in all directions. With the exception of a recent confocal study by Bush and Hall15, such extensive cell processes have not previously been reported in intact cartilage, possibly due to the widespread use of light and transmisson electron microscopy techniques which require specimens to be cut into sections less than 10 µm in thickness. Indeed, in the present study cell processes were not visible in the thin sections used for histology (Fig. 3). Cells with elongated processes were present in both non-OA and OA cartilage. However, in the non-OA tissue they accounted for less than 10% of the total cell population with the exception of the surface zone immediately inferior to the fovea (PFI site), which contained a higher percentage of cells with elongated processes than all the surrounding tissue. This region also consistently showed mild fibrillation and increased Mankin scores in otherwise healthy joints in agreement with previous studies20,21. In non-OA cartilage cells with elongated processes were most prevalent in the surface and middle zones of the tissue (Table I). However, a comprehensive analysis of the zonal distribution was not possible in OA cartilage due to the loss of clear zonal differences, tissue swelling and frequent abrasion of the surface zone. In the peripheral cartilage of OA femoral heads there was a significantly greater percentage of cells with elongated processes compared to that observed in corresponding areas of non-OA femoral heads (Fig. 6). Both the peripheral cartilage and the area inferior to the fovea universally experience cartilage degradation in osteoarthritis21. It has been suggested that these areas are more susceptible to injury as they are not normally load bearing and consequently exhibit inferior mechanical integrity21. In the present study the highest mean percentage of cells with processes in OA cartilage (42.1%) occurred at the peripheral superior (PS) site. This site also showed the greatest
histological and macroscopic signs of OA (Table I) with highest average Mankin score (8.0) and the most likely site for surface fibrillation (83% of femoral heads) and pannus formation (83% of femoral heads). This site may be susceptible to injury as age- and disease-related increased conformity between the femoral head and the acetabulum may change this area from a habitual non-load bearing area to one experiencing high loads21. In the present study, all tissue was histologically graded using the well established Mankin score to determine the extent of any osteoarthritis (Table I)1. However, it is recognized that the use of a single observer limits the reproducibility and accuracy of these gradings22,23. To further examine the relationship between changes in cell morphology and cartilage degradation, the Mankin score was plotted against the percentage of cells with processes, for both non-OA and OA specimens (Fig. 7). The data indicate a statistically significant positive correlation between the presence of cells with processes and the Mankin score, such that tissue showing histologically more advanced OA had a higher percentage of cells with processes. By contrast, the recent study by Bush and Hall found no correlation between the frequency of chondrocytes demonstrating elongated projections in OA cartilage and the degree of tissue degradation15. However this previous study used human tibeal plateaus rather than femoral heads and did not perform the thorough histological assessment based on the Mankin score, as adopted in the present study. Fig. 7 shows the data plotted separately for fibrillated and non-fibrillated specimens and those covered by pannus. Cells with processes were observed in OA specimens both with and without pannus (Fig. 7). Traditionally, pannus is most commonly associated with rheumatoid arthritis where it forms an invasive granulation tissue composed of macrophage- and fibroblast-like cells24,25. However pannus has also been widely reported in osteoarthritis where it may contribute to cartilage degradation by the release of catabolic factors including IL1-ß and MMP31,3,20,26. In the present study, pannus occurred most
Osteoarthritis and Cartilage Vol. 12, No. 1
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0DQNLQ6FRUH Fig. 7. Relationship between histological appearance and the presence of cells with elongated processes. A statistically significant linear model has been fitted to the data (P<0.001, r=0.63). Symbols indicate the data for non-OA and OA specimens with and without fibrillation and with and without pannus.
frequently at the peripheral sites on the femoral head (Table I) in agreement with previous studies20. The fact that these sites also contained the highest percentage of cells with processes supports the hypothesis that pannus induces cartilage degradation leading to the appearance of elongated processes. Indeed it has been suggested that cells from the articular cartilage are recruited into the pannus layer which may explain the similar morphological appearance20. It is also evident from Fig. 7, that cartilage with a Mankin score between 0 and 2 appeared histologically normal with few cells with processes and no fibrillation except at the PFI site. For Mankin scores of 3–6 there was a greater likelihood of fibrillation and an increased presence of cells with elongated processes. At Mankin scores of 7 or above, the majority of specimens were fibrillated with up to 56% of cells showing elongated processes. Despite the correlation with pannus and histological changes to the matrix, the reason for the increased appearance of cells with elongated processes in OA cartilage remains uncertain. Previous studies, using cytoskeletal staining and confocal microscopy, have reported cells with similar morphology in the surface zone of meniscal cartilage27. This tissue is known to be fibrocartilagenous in nature and thus the appearance of similar fibroblast-like cell morphologies in OA cartilage may indicate an extracellular matrix transition from articular cartilage to fibrocarti-
lage. In the present study, cells with elongated processes in OA cartilage exhibited a fibroblast-like ultrastructure when examined using electron microscopy (Holloway, unpublished observations). It has also been reported that isolated articular chondrocytes exhibit similar morphological features when subjected to tensile strain28 or cultured in very low modulus agarose scaffolds29. Thus the altered cell morphology in OA cartilage may reflect a localized changes in the pericellular mechanical environment allowing the extension of cell processes into the surrounding matrix. Indeed the mechanical properties of the pericellular matrix associated with chondrons isolated from OA cartilage is significantly reduced compared to the chondron matrix from non-OA cartilage30 and similarly there is a negative correlation between Mankin score and cartilage stiffness31. In conclusion, this study describes the increased presence of a sub-population of cells with multiple elongated processes in cartilage from OA femoral heads. These cells are found predominantly at the periphery of the joint and are associated with histological changes reflected by increased Mankin scores. It remains to be seen whether these cells directly contribute to localized cartilage breakdown through the release of catabolic agents from the cell processes as seen in growth-plate cartilage32 or whether the extension of cell processes is a direct response to matrix degradation. Either way, such profound changes in morphology will undoubtedly alter numerous aspects of cell
24 function including cell phenotype and mechanotransduction with implications for the aetiology of osteoarthritis and potential treatment strategies.
References 1. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971;53:523–37. 2. Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039–49. 3. Meachim G. Articular cartilage lesions in osteo-arthritis of the femoral head. J Pathol 1972;107:199–210. 4. Reginato AM, Olsen BR. The role of structural genes in the pathogenesis of osteoarthritic disorders. Arthritis Res 2002;4:337–45. 5. Goldring MB. Osteoarthritis and cartilage: the role of cytokines. Curr Rheumatol Rep 2000;2:459–65. 6. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum 2000;43:1916–26. 7. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res 2001;3:107–13. 8. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology 2002;39:237–46. 9. Ingber DE, Dike L, Hansen L, Karp S, Liley H, Maniotis A, et al. Cellular tensegrity: Exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. International Review of Cytology 1994;150:173–218. 10. von der Mark K, Gauss V, von der Mark H, Muller P. Relationship between cell shape and type of collagen synthesized-chondrocytes lose their cartilage phenotype in culture. Nature 1977;267:531–2. 11. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phentotype when cultured in agarose gels. Cell 1982;30:215–24. 12. Aydelotte M, Keuttner KE. Differences between subpopulations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect Tissue Res 1988;18:205–22. 13. Ghadially FN. Fine structure of synovial joints. London: Butterworth & Co. Ltd 1983:42–79. 14. Weiss C, Rosenberg L, Helfet A. An ultrastructural study of normal young adult human articular cartilage. J Bone Joint Surg Am 1968;50-A:663–73. 15. Bush PG, Hall AC. The volume and morphology of chondrocytes within non-degenerate and degenerate human articular cartilage. Osteoarthritis Cartilage 2003;11:242–51. 16. Langelier E, Suetterlin R, Hoemann CD, Aebi U, Buschmann MD. The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments. J Histochem Cytochem 2000;48:1307–20.
I. Holloway et al.: Cell morphology in OA cartilage 17. Idowu BD, Knight MM, Bader DL, Lee DA. Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose. Histochem J 2000;32:165–74. 18. Durrant LA, Archer CW, Benjamin M, Ralphs JR. Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture. J Anat 1999;194:343–53. 19. Knight MM, Idowu BD, Lee DA, Bader DL. Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique. Med Biol Eng Comput 2001;39:397–404. 20. Shibakawa A, Aoki H, Masuko-Hongo K, Kato T, Tanaka M, Nishioka K, et al. Presence of pannus-like tissue on osteoarthritic cartilage and its histological character. Osteoarthritis Cartilage 2003;11:133–40. 21. Bullough P, Goodfellow J, O’Conner J. The relationship between degenerative changes and loadbearing in the human hip. J Bone Joint Surg Br 1973; 55:746–58. 22. Ostergaard K, Petersen J, Andersen CB, Bendtzen K, Salter DM. Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity. Arthritis Rheum 1997;40:1766–71. 23. van der Sluijs JA, Geesink RG, van der Linden AJ, Bulstra SK, Kuyer R, Drukker J. The reliability of the Mankin score for osteoarthritis. J Orthop Res 1992; 10:58–61. 24. Nathan JZ. Etiology and pathogenesis of rheumatoid arthritis. In: William JK, Ed. Arthritis and applied conditions: A textbook of rheumatology. Lippincott: Williams & Wilkins 2001;659–73. 25. Fassbender HG, Simmling-Annefeld M. The potential aggressiveness of synovial tissue in rheumatoid arthritis. J Pathol 1983;139:399–406. 26. Meachim G, Osborne GV. Repair at the femoral articular surface in osteo-arthritis of the hip. J Pathol 1970; 102:1–8. 27. Le Graverand MP, Eggerer J, Rattner J, Barclay L, Hart DA. Identification of four major morphologically distinct cell types in rabbit menisci. Trans Orthop Res 2001;811. 28. Vanderploeg EJ, Levenston ME. Oscillatory tension modulates chondrocyte biosynthesis and morphology. Trans Orthop Res 2002;378. 29. Bush PG, Hoemann CD, Hall AC. A similarity between morphology of chondrocytes within degenerate cartilage and those cultured in a weak 3D matrix. Trans Orthop Res 2002;661. 30. Jones WR, Ting-Beall HP, Lee GM, Kelley SS, Hochmuth RM, Guilak F. Alterations in the Young’s modulus and volumetric properties of chondrocytes isolated from normal and osteoarthric human cartilage. J Biomech 1999;32:119–27. 31. Franz T, Hasler EM, Hagg R, Weiler C, Jakob RP, Mainil-Varlet P. In situ compressive stiffness, biochemical composition, and structural integrity of articular cartilage of the human knee joint. Osteoarthritis Cartilage 2001;9:582–92. 32. Stockwell RA, Meachim G. The chondrocytes. In: Freeman MAR, Ed. Adult Articular Cartilage. London: Pitman Publishing Ltd 1979;69–144.
International Cartilage Repair Society
Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage I. Holloway†,‡, M. Kayser†, D. A. Lee§, D. L. Bader§, G. Bentley† and M. M. Knight§* †Institute of Orthopaedics, University College London Medical School, Brockley Hill, Stanmore, U.K. ‡Orthopaedic Department, Northwick Park Hospital, Watford Road, Harrow, U.K. §IRC in Biomedical Materials and Medical Engineering Division, Department of Engineering, Queen Mary University of London, Mile End Road, London, U.K. Summary Objective: This study examined the morphology of chondrocytes in articular cartilage from osteoarthritic (OA) and non-OA human femoral heads and in particular the appearance of a sub-population of cells with multiple elongated processes radiating up to 30 µm into the extracellular matrix. Methods: Cartilage explants were removed from 8 anatomical sites over the surface of OA (n=6) and non-OA (n=5) femoral heads. Cells were labeled for vimentin intermediate filaments and visualized using epi-fluorescence and confocal microscopy. The percentage of cells with elongated processes was correlated with macroscopic and histological indicators of osteoarthritis. Results: Cells with processes accounted for less than 10% of the total cell population in non-OA cartilage. By contrast, in the peripheral regions of the OA femoral head these cells accounted for 20–45% of the total cell population, the differences being statistically significant. These peripheral areas are habitually non-load bearing and were also the most likely to show gross fibrillation and pannus formation. A statistically significant correlation was demonstrated between the percentage of cells with processes and the histological extent of the OA degradation, quantified in terms of the Mankin score. Conclusions: The extension of cell processes, which may be associated with localized breakdown of the pericellular matrix, will undoubtedly alter numerous aspects of cell function including phenotypic expression and mechanotransduction. Hence these significant changes in chondrocyte morphology are likely to have important implications for the aetiology of osteoarthritis and the development of potential treatment strategies. © 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Osteoarthritis, Cartilage, Cytoskeleton, Vimentin, Confocal, Chondrocyte.
TNF-5–8. Cell metabolism is known to be influenced by cell morphology and associated cytoskeletal organization9. For example, a rounded cell morphology and the absence of actin stress fibres is necessary for the maintenance of chondrocytic phenotype as demonstrated by the synthesis of type II collagen and aggrecan10–12. However, it remains unclear the extent to which changes in chondrocyte metabolism in osteoarthritic cartilage are associated with alterations in cell morphology. This study utilized epifluorescence and confocal microscopy to test the hypothesis that morphological differences exist between chondrocytes within cartilage from non-osteoarthritic (nonOA) and osteoarthritic (OA) femoral heads. In particular, the study focuses on the appearance of a sub-population of cells with multiple elongated processes, identified by immunofluorescent labelling of the cytoskeletal protein, vimentin. Although previous studies on articular cartilage have described the appearance of chondrocytes with limited cell processes13,14, cells with elongated processes extending up to 30 µm have only, very recently been reported in one study of OA cartilage from human tibia plateaus15. In this study, the presence of cells with elongated processes in non-OA and OA femoral head cartilage is examined in relation to anatomical position in
Introduction Osteoarthritis is a widespread clinical problem affecting many joints of the body, particularly the hip and the knee, resulting in swelling, stiffness and pain. Pathological changes in osteoarthritis are characterized by softening, fibrillation, abrasion and ultimate loss of the articular cartilage exposing the underlying bone1,2. Cartilage breakdown is associated with changes in extracellular matrix composition with reduced keratan sulphate content and increased water content3. These mechanical and biochemical changes are partly attributed to damage to the collagen network such that it is unable to restrain the swelling pressure produced by the glycosaminoglycan (GAG). Osteoarthritis is also associated with differences in chondrocyte gene expression4 and metabolism characterized by increased cell proliferation, synthesis of fibrocartilage matrix and secretion of matrix metalloproteases (MMPs) and inflammatory cytokines such as IL1- and *Address correspondence to: Dr Martin Knight, IRC in Biomedical Materials, Queen Mary University of London, Mile End Road, London E1 4NS. Tel.: +44-(0)-207-882-5512; E-mail: [email protected] Received 28 April 2003; revision accepted 6 September 2003.
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I. Holloway et al.: Cell morphology in OA cartilage
Fig. 1. Schematic diagram showing the location of the anatomical sites across the femoral head from which cartilage specimens were removed.
the joint and histological and macroscopic indicators of osteoarthritis.
Materials and methods PREPARATION OF CARTILAGE SPECIMENS
This study examined the articular cartilage from the femoral heads of patients in two groups. An osteoarthritic (OA) group, who received total hip replacement as a treatment for osteoarthritis, and an age-matched non-OA group who required hemiarthroplasty for fractured neck of femur. The number of femoral heads used in this study was 5 in the non-OA group and 6 in the OA group. The mean ages of patients in the non-OA and OA groups was 81.6 and 67.3 respectively. Patients were seen pre-operatively and consented according to regulations laid down by the local Research Ethics Committee. In both groups, the presence of osteoarthritis in the operated hip was assessed based on the diagnostic criteria established by the American Rheumatism Association2. All patients in the osteoarthritic group had clinical evidence of osteoarthritis where as no patients in the non-OA group showed any evidence of osteoarthritis. Femoral heads were collected during surgery and maintained briefly in Dulbecco’s Minimal Essential Medium supplemented with 10% (v/v) foetal calf serum (DMEM+10% FCS, Gibco, Poole, U.K.). Explants of full thickness cartilage with underlying bone, approximately 1 cm3, were excised from eight sites on each femoral head: four parafoveal sites (superior, inferior, anterior, posterior) and four corresponding sites in the peripheral cartilage as indicated in Fig. 1. The cartilage at each site was assessed for macroscopic signs of surface fibrillation. Explants were cut in two halves such that one half was processed for histology while the other half was used for analysis of cell morphology. HISTOCHEMICAL STAINING
For histological investigation specimens were examined with Haematoxylin and Eosin staining and Safranin-O
staining following standard protocols. Briefly, cartilagebone explants were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, washed in Phosphate Buffered Saline (PBS) and then slowly decalcified at 4°C in 0.1 M sodium cacodylate buffer containing 0.1 M ethylenediamine-N,N,N′,N′-tetracetic acid (EGTA) and 4% glutaraldehyde. Full decalcification was confirmed using X-ray analysis. Explants were embedded in paraffin and 5 µm sections prepared. The sections were deparaffinized with xylene, alcohol and water and then stained with Harris’s acid alum haematoxylin (HS355, HD Supplies, Aylesbury, U.K.) for 9 min. Specimens were further washed in water, differentiated in acid alcohol for 3–5 s and washed again. All specimens were counterstained for 5 min in either 1% Eosin (colour index 45380, HD1320, HD Supplies, Aylesbury, U.K.) or 0.1% Safranin O (colour index 50240, HD1850, HsD Supplies, Aylesbury, U.K.). Specimens were then washed, dehydrated through a series of alcohols and xylene and finally mounted in DePeX (BDH Merk). Light microscopy was used to visualize the specimens to enable histological grading of the cartilage according to the Mankin score1. Statistical comparison of Mankin scores at the 8 anatomical sites in non-OA and OA femoral heads was conducted using the Student’s t test with a value of P<0.05 indicating a significant difference.
ANALYSIS OF CELL MORPHOLOGY
Cartilage explants were fixed for 20 min at 37°C in 4% formaldehyde prepared in PBS and then washed with PBS. A microtome was used to section the explants perpendicular to the articular surface yielding individual specimens 150 µm in thickness. To aid the penetration of the antibodies, the extracellular matrix components underwent partial enzymatic digestion. Specimens were incubated on rollers for 2 h at 37°C in 0.125 units/ml chondroitinase ABC (Sigma, Poole, U.K.) and 1500 units/ml bovine testicular hyaluronidase (Sigma, Poole, U.K.) prepared in modified Hanks Balanced Salt Solution (mHBSS). Modified Hanks Balanced Salt Solution at pH 6.5 consisted of HBSS (10X, Gibco, Paisley, U.K.)+2 mM MgCl2+4 mM NaHCO3+2 mM EGTA (Sigma, Poole, U.K.)+0.11% w/v MES (Sigma,
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Fig. 2. Photograph of (a) non-OA and (b) OA human femoral heads showing the gross appearance of the articular cartilage. Arrow indicates the fovea.
Poole, U.K.) as described by the Langelier et al.16. Following enzymatic digestion, samples were washed 3 times for 5 min in mHBSS. The staining protocol for vimentin intermediate filament cytoskeleton followed that previously optimized for isolated bovine articular chondrocytes seeded in agarose17–19. Specimens were maintained for 5 min at 4°C in cell permeabilizing buffer consisting of 10.3 g sucrose, 0.292 g sodium chloride, 0.006 g magnesium chloride, 0.476 g HEPES (Sigma, Poole, U.K.) and 0.5 ml Triton X dissolved in 100 ml distilled water. Specimens were then washed with 1% bovine serum albumin (BSA, Sigma, Poole, U.K.) in mHBSS and incubated on rollers for 2 h at 37°C in primary mouse anti-vimentin clone 9 (Sigma, Poole, U.K.) prepared at 1:100 in mHBSS. To remove unbound primary antibody the specimens were washed 3 times for 10 min in PBS+0.5% (v/v) Tween 20 (BDH Laboratory Supplies, Poole, U.K.). Specimens were then incubated on rollers for 1 h at 37°C in rabbit anti-mouse FITC-conjugated secondary antibody (Sigma, Poole, U.K.) prepared at 1:20 in mHBSS and finally washed 3 times for 10 min in PBS+0.5% (v/v) Tween 20. All specimens were stored at 4°C in mHBSS prior to epi-fluorescence microscopy. Each specimen was mounted on a coverslip, hydrated in PBS and placed on the stage of an inverted microscope (Eclipse, Nikon, U.K.) attached to a confocal laser scanning microscope system (Ultra View, Perkin Elmer, Cambridge, U.K.). Standard epi-fluorescence and confocal microscopy
with a 40x/0.95 NA Plan Apo objective lens (Nikon, U.K.) was used to assess the morphology of fluorescently labeled cells. For each specimen, a total of nine separate fields of view, approximately 500 µm in diameter, were analysed at 3 different depths below the articular surface, approximately corresponding to the surface, middle and deep zones where possible. The percentage of cells exhibiting elongated processes over 10 µm in length, was recorded within each field of view and the mean percentage value calculated at each depth. The procedure was repeated for specimens from the 8 anatomical sites from each femoral head in the two groups. Non-parametric statistical analysis was performed using the Mann–Whitney U-test with P<0.05 indicating a statistically significant difference.
Results All the femoral heads in the OA group exhibited areas of macroscopic fibrillation which varied across the joint surface (Fig. 2, Table I). In the non-OA group the cartilage appeared normal with no sign of fibrillation except at the inferior parafoveal site (PFI) where mild fibrillation and surface unevenness was present in all the femoral heads. A summary of the areas demonstrating fibrillation for both groups is presented in Table I.
Table I Numerical data describing the cartilage at the 8 anatomical sites for non-OA (n=5) and OA (n=6) femoral heads. Anatomical Site
% of femoral head with fibrillation % femoral heads showing pannus Mankin Score (mean ± SE) % cells with processes in non-OA cartilage (median)
Non-OA OA Non-OA OA Non-OA OA surface middle deep
PFI
PFS
PFA
PFP
PI
PS
PA
PP
100% 83% 0% 50% 1.8±0.5 5.3±1.3 38.9 5.2 2.5
0% 17% 0% 17% 1.3±0.5 7.6±1.5 2.9 4.6 0.0
0% 33% 0% 17% 0.8±0.6 5.0±1.2 13.1 4.3 0.0
0% 83% 0% 33% 0.0±0.0 4.6±1.2 5.9 5.9 0.0
0% 50% 0% 50% 1.4±0.5 5.3±0.7 6.9 2.9 2.0
0% 83% 0% 83% 0.3±0.3 8.0±0.7 4.3 6.8 2.2
0% 67% 0% 50% 1.0±0.8 6.7±1.3 2.1 3.9 0.0
0% 50% 0% 50% 1.3±1.3 5.5±1.0 3.1 3.0 2.1
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I. Holloway et al.: Cell morphology in OA cartilage
Fig. 3. Histological appearance of representative articular cartilage specimens stained with H&E (a–d) or Safranin O (e–h). Images indicate the characteristic appearance of cartilage from a non-OA femoral head with a Mankin score of 0 (a, e) and from OA femoral heads with Mankin scores of 3 (b–f), 7 (c, g) and 11 (d, h). Arrows indicate the presence of a pannus layer at the articular surface (c, g). Scale bars represent 10 µm.
HISTOLOGICAL APPEARANCE OF THE TISSUE
Representative histological images of non-OA and OA cartilage stained with Haematoxylin and Eosin or Safranin-O are shown in Fig. 3. The average Mankin scores for cartilage at the 8 anatomical sites in non-OA and OA femoral heads are presented in Table I. At all locations over the femoral head, the Mankin score for OA cartilage was higher than that for non-OA cartilage, the differences being statistically significant. The articular surfaces of all 6 femoral heads from the OA group were, in part, covered by a fibrous avascular pannuslike tissue which showed negative Safranin-O staining (Fig. 3c,g) as previously described20. This tissue occurred most frequently at the peripheral anatomical sites and inferior to the fovea (Table I). Using identical histological staining, pannus was not observed in any of the femoral heads from the non-OA group. ANALYSIS OF CELL MORPHOLOGY
Cells in non-OA cartilage exhibited characteristic zonal variation in morphology and arrangement, such that cells in the surface zone appeared flattened and disc-shaped where as cells in the middle and deep zones were more spherical and arranged in columns running perpendicular to the articular surface. This distinction between the different zones was less obvious in OA cartilage. In OA specimens where pannus was identified by histology, the pannus
layer was distinguishable from the underlying cartilage by the high cell density and the fibroblastic appearance of the cells (Fig. 4). Cells within the cartilage tissue were catagorized into two types, with and without elongated cell processes. Typical examples of these cell types are shown in Fig. 5. In both cell types the vimentin intermediate filament cytoskeleton appeared as a complex fibrous structure extending throughout the cytoplasm. The percentage of cells exhibiting elongated processes in the surface, middle and deep zones of non-OA cartilage is presented in Table I. Within non-OA cartilage, less than 10% of cells exhibited processes with the exception of the surface zone at the PFI site where the value was 38.9% (Table I). At all anatomical sites there was a greater percentage of cells with processes in the surface and middle zones than in the deep zone. Data obtained at the 3 different depths was pooled to give a mean value for each specimen. The median values at each anatomical site for OA and non-OA femoral heads are plotted in Fig. 6. At the 4 peripheral sites (PS, PI, PA and PP) there was a greater percentage of cells with processes in the OA cartilage compared to the non-OA, the differences being statistically significant.
Discussion This study describes a sub-population of chondrocytes with multiple elongated processes in human femoral head
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Fig. 4. Confocal 3D reconstruction showing cells within OA cartilage labeled for vimentin intermediate filaments and visualized close to the surface of the tissue. Scale bar indicates 10 µm.
cartilage. Differences between cells in cartilage from non-OA and OA joints were examined at 8 anatomical sites over the surface of the femoral head. Immunofluorescent labeling of the cytoskeletal protein, vimentin, was used as a marker of cell morphology. This technique has the advantage over more general cytoplas-
mic stains which are less suitable for labeling thin cell processes and show poor stability following fixation. Vimentin intermediate filament cytoskeleton in both OA and non-OA cartilage exhibited a characteristic intracellular fibrous structure as previously reported16,18. In addition, vimentin staining also revealed the presence of multiple
Fig. 5. Representative confocal 3D reconstruction images of individual cells in (a) non-OA and (b) OA cartilage. The latter shows the appearance of elongated cell processes. Cells were immunolabelled for vimentin intermediate filaments. Scale bars indicate 5 µm.
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I. Holloway et al.: Cell morphology in OA cartilage
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Fig. 6. Percentage of cells with elongated cell processes in cartilage from the eight anatomical sites in non-OA and OA femoral heads. Values represent medians with error bars indicating the inter-quartile range. Statistically significant differences have been indicated for P<0.05 (*).
vimentin-rich cell processes which extended up to 30 µm into the surrounding matrix (Figs. 4 and 5b). Epifluorescence and 3D confocal microscopy were used to visualise these cell processes which radiate from the cell body in all directions. With the exception of a recent confocal study by Bush and Hall15, such extensive cell processes have not previously been reported in intact cartilage, possibly due to the widespread use of light and transmisson electron microscopy techniques which require specimens to be cut into sections less than 10 µm in thickness. Indeed, in the present study cell processes were not visible in the thin sections used for histology (Fig. 3). Cells with elongated processes were present in both non-OA and OA cartilage. However, in the non-OA tissue they accounted for less than 10% of the total cell population with the exception of the surface zone immediately inferior to the fovea (PFI site), which contained a higher percentage of cells with elongated processes than all the surrounding tissue. This region also consistently showed mild fibrillation and increased Mankin scores in otherwise healthy joints in agreement with previous studies20,21. In non-OA cartilage cells with elongated processes were most prevalent in the surface and middle zones of the tissue (Table I). However, a comprehensive analysis of the zonal distribution was not possible in OA cartilage due to the loss of clear zonal differences, tissue swelling and frequent abrasion of the surface zone. In the peripheral cartilage of OA femoral heads there was a significantly greater percentage of cells with elongated processes compared to that observed in corresponding areas of non-OA femoral heads (Fig. 6). Both the peripheral cartilage and the area inferior to the fovea universally experience cartilage degradation in osteoarthritis21. It has been suggested that these areas are more susceptible to injury as they are not normally load bearing and consequently exhibit inferior mechanical integrity21. In the present study the highest mean percentage of cells with processes in OA cartilage (42.1%) occurred at the peripheral superior (PS) site. This site also showed the greatest
histological and macroscopic signs of OA (Table I) with highest average Mankin score (8.0) and the most likely site for surface fibrillation (83% of femoral heads) and pannus formation (83% of femoral heads). This site may be susceptible to injury as age- and disease-related increased conformity between the femoral head and the acetabulum may change this area from a habitual non-load bearing area to one experiencing high loads21. In the present study, all tissue was histologically graded using the well established Mankin score to determine the extent of any osteoarthritis (Table I)1. However, it is recognized that the use of a single observer limits the reproducibility and accuracy of these gradings22,23. To further examine the relationship between changes in cell morphology and cartilage degradation, the Mankin score was plotted against the percentage of cells with processes, for both non-OA and OA specimens (Fig. 7). The data indicate a statistically significant positive correlation between the presence of cells with processes and the Mankin score, such that tissue showing histologically more advanced OA had a higher percentage of cells with processes. By contrast, the recent study by Bush and Hall found no correlation between the frequency of chondrocytes demonstrating elongated projections in OA cartilage and the degree of tissue degradation15. However this previous study used human tibeal plateaus rather than femoral heads and did not perform the thorough histological assessment based on the Mankin score, as adopted in the present study. Fig. 7 shows the data plotted separately for fibrillated and non-fibrillated specimens and those covered by pannus. Cells with processes were observed in OA specimens both with and without pannus (Fig. 7). Traditionally, pannus is most commonly associated with rheumatoid arthritis where it forms an invasive granulation tissue composed of macrophage- and fibroblast-like cells24,25. However pannus has also been widely reported in osteoarthritis where it may contribute to cartilage degradation by the release of catabolic factors including IL1-ß and MMP31,3,20,26. In the present study, pannus occurred most
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0DQNLQ6FRUH Fig. 7. Relationship between histological appearance and the presence of cells with elongated processes. A statistically significant linear model has been fitted to the data (P<0.001, r=0.63). Symbols indicate the data for non-OA and OA specimens with and without fibrillation and with and without pannus.
frequently at the peripheral sites on the femoral head (Table I) in agreement with previous studies20. The fact that these sites also contained the highest percentage of cells with processes supports the hypothesis that pannus induces cartilage degradation leading to the appearance of elongated processes. Indeed it has been suggested that cells from the articular cartilage are recruited into the pannus layer which may explain the similar morphological appearance20. It is also evident from Fig. 7, that cartilage with a Mankin score between 0 and 2 appeared histologically normal with few cells with processes and no fibrillation except at the PFI site. For Mankin scores of 3–6 there was a greater likelihood of fibrillation and an increased presence of cells with elongated processes. At Mankin scores of 7 or above, the majority of specimens were fibrillated with up to 56% of cells showing elongated processes. Despite the correlation with pannus and histological changes to the matrix, the reason for the increased appearance of cells with elongated processes in OA cartilage remains uncertain. Previous studies, using cytoskeletal staining and confocal microscopy, have reported cells with similar morphology in the surface zone of meniscal cartilage27. This tissue is known to be fibrocartilagenous in nature and thus the appearance of similar fibroblast-like cell morphologies in OA cartilage may indicate an extracellular matrix transition from articular cartilage to fibrocarti-
lage. In the present study, cells with elongated processes in OA cartilage exhibited a fibroblast-like ultrastructure when examined using electron microscopy (Holloway, unpublished observations). It has also been reported that isolated articular chondrocytes exhibit similar morphological features when subjected to tensile strain28 or cultured in very low modulus agarose scaffolds29. Thus the altered cell morphology in OA cartilage may reflect a localized changes in the pericellular mechanical environment allowing the extension of cell processes into the surrounding matrix. Indeed the mechanical properties of the pericellular matrix associated with chondrons isolated from OA cartilage is significantly reduced compared to the chondron matrix from non-OA cartilage30 and similarly there is a negative correlation between Mankin score and cartilage stiffness31. In conclusion, this study describes the increased presence of a sub-population of cells with multiple elongated processes in cartilage from OA femoral heads. These cells are found predominantly at the periphery of the joint and are associated with histological changes reflected by increased Mankin scores. It remains to be seen whether these cells directly contribute to localized cartilage breakdown through the release of catabolic agents from the cell processes as seen in growth-plate cartilage32 or whether the extension of cell processes is a direct response to matrix degradation. Either way, such profound changes in morphology will undoubtedly alter numerous aspects of cell
24 function including cell phenotype and mechanotransduction with implications for the aetiology of osteoarthritis and potential treatment strategies.
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I. Holloway et al.: Cell morphology in OA cartilage 17. Idowu BD, Knight MM, Bader DL, Lee DA. Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose. Histochem J 2000;32:165–74. 18. Durrant LA, Archer CW, Benjamin M, Ralphs JR. Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture. J Anat 1999;194:343–53. 19. Knight MM, Idowu BD, Lee DA, Bader DL. Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique. Med Biol Eng Comput 2001;39:397–404. 20. Shibakawa A, Aoki H, Masuko-Hongo K, Kato T, Tanaka M, Nishioka K, et al. Presence of pannus-like tissue on osteoarthritic cartilage and its histological character. Osteoarthritis Cartilage 2003;11:133–40. 21. Bullough P, Goodfellow J, O’Conner J. The relationship between degenerative changes and loadbearing in the human hip. J Bone Joint Surg Br 1973; 55:746–58. 22. Ostergaard K, Petersen J, Andersen CB, Bendtzen K, Salter DM. Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity. Arthritis Rheum 1997;40:1766–71. 23. van der Sluijs JA, Geesink RG, van der Linden AJ, Bulstra SK, Kuyer R, Drukker J. The reliability of the Mankin score for osteoarthritis. J Orthop Res 1992; 10:58–61. 24. Nathan JZ. Etiology and pathogenesis of rheumatoid arthritis. In: William JK, Ed. Arthritis and applied conditions: A textbook of rheumatology. Lippincott: Williams & Wilkins 2001;659–73. 25. Fassbender HG, Simmling-Annefeld M. The potential aggressiveness of synovial tissue in rheumatoid arthritis. J Pathol 1983;139:399–406. 26. Meachim G, Osborne GV. Repair at the femoral articular surface in osteo-arthritis of the hip. J Pathol 1970; 102:1–8. 27. Le Graverand MP, Eggerer J, Rattner J, Barclay L, Hart DA. Identification of four major morphologically distinct cell types in rabbit menisci. Trans Orthop Res 2001;811. 28. Vanderploeg EJ, Levenston ME. Oscillatory tension modulates chondrocyte biosynthesis and morphology. Trans Orthop Res 2002;378. 29. Bush PG, Hoemann CD, Hall AC. A similarity between morphology of chondrocytes within degenerate cartilage and those cultured in a weak 3D matrix. Trans Orthop Res 2002;661. 30. Jones WR, Ting-Beall HP, Lee GM, Kelley SS, Hochmuth RM, Guilak F. Alterations in the Young’s modulus and volumetric properties of chondrocytes isolated from normal and osteoarthric human cartilage. J Biomech 1999;32:119–27. 31. Franz T, Hasler EM, Hagg R, Weiler C, Jakob RP, Mainil-Varlet P. In situ compressive stiffness, biochemical composition, and structural integrity of articular cartilage of the human knee joint. Osteoarthritis Cartilage 2001;9:582–92. 32. Stockwell RA, Meachim G. The chondrocytes. In: Freeman MAR, Ed. Adult Articular Cartilage. London: Pitman Publishing Ltd 1979;69–144.