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Rheumatology Advance Access published online on September 16, 2008
Rheumatology, doi:10.1093/rheumatology/ken341
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Morphology of the bovine chondrocyte and of its cytoskeleton in isolation and in situ: are chondrocytes ubiquitously paired through the entire layer of articular cartilage?

Y. Sasazaki1, B. B. Seedhom1 and R. Shore2

1Division of Bioengineering, Academic Unit of Musculoskeletal Disease and 2Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, UK

Correspondence to: Y. Sasazaki, Clinical Research Centre, National Hospital Organization Murayama Medical Centre, 2-37-1 Gakuen, Musasimurayamasi, Tokyo 208-0011, Japan. E-mail: medys{at}leeds.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. We compared the morphology and cytoskeleton of chondrocytes seeded in monolayer or in agarose gels with those retained in situ i.e. within their extracellular matrix—the chondrocyte's natural habitat.

Methods. Cartilage specimens were harvested from adult bovine femora. Chondrocytes were either enzymatically isolated to seed in both monolayer and agarose gel culture conditions or retained in situ. Full thickness cartilage on bone was sliced both parallel and perpendicular to the articular surface. After immunostaining, the morphology of chondrocytes and of their cytoskeletal organization, i.e. distribution of actin and vimentin, in chondrocytes seeded both in monolayer and 3D agarose and those retained in situ, were assessed using confocal laser scanning microscopy.

Results. The general cytoskeletal disposition of chondrocytes in situ was similar to that in agarose gel. Actin was seen to form stress fibres only in 2D culture, but not in 3D culture and in situ. In these latter conditions, actin showed a punctate staining pattern. The vimentin meshwork spanned the cytoplasm from the plasma membrane to the nuclear membrane in all culture conditions. However, the organization of the vimentin had a radiate organization in chondrocytes in monolayer and a more circumferential arrangement both in agarose gel and in situ. We further observed: (i) the prevalence of a bichondral configuration of chondrocytes in situ and (ii) the existence of a vimentin link joining some of the sister cells in situ.

Conclusions. Bichondral configuration linked with cytoskeletal elements may potentially be significant for the normal function of the chondrocytes, and therefore have implications for approaches to tissue engineering of cartilage.

KEY WORDS: Articular cartilage, Chondrocyte, Cytoskeleton


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It is of note that the various studies on the morphology and on the responses of the chondrocyte, and of its cytoskeleton, to mechanical strain have been primarily conducted on chondrocytes isolated from their extra cellular matrix (ECM)—their natural habitat, and cultured either in monolayer or in agarose gels [1–5]. Chondrocytes seeded in monolayer may be the least relevant in this regard as they experience significant changes in their morphology and phenotype (dedifferentiation), a process that increases with passage numbers. Chondrocytes isolated and then cultured in agarose gels would, on the face of it, appear to be retained within a much more in situ-like environment, and therefore might possibly be expected to respond in a manner similar to chondrocytes retained within their natural matrix and thus provide a suitable cell stock for further study and autologous chondrocyte implantation (ACI) for cartilage repair [6]. However, by isolating chondrocytes from their natural ECM in this way, the mechanism of transfer of load and, in turn, the conclusions about mechanotransduction may be only partially correct. One study on the effect of mechanical strain on chondrocytes in situ by Durrant et al. [7] suggested that the chondrocytes maintained their actin cytoskeleton and modified their vimentin in response to changing mechanical conditions, in this case a compressive load. Cartilage does also experience tensile stresses as a result of compressive loading [8]. Deformation and rupture of the articular surface under dynamic and static compression and the response of both the chondrocyte and its cytoskeleton need to be investigated. However, it was appropriate to study the morphology of the chondrocyte and of its cytoskeleton in situ prior to a serious study of their responses to different modalities of loading.

Using confocal laser scanning microscopy, we aimed in this study to compare directly the morphology and distribution of the same major cytoskeletal elements investigated by Durrant et al. [7] (namely actin and vimentin) of chondrocytes seeded in monolayer and in agarose gels and those retained in situ, i.e. in their natural habitat—the ECM. By choosing to investigate actin and vimentin, this allowed direct comparison with this earlier work on in situ chondrocyte cytoskeletal morphology. The implicit hypothesis behind this study is that the morphology of chondrocytes and of their cytoskeleton differs when observed within their natural habitat i.e. in situ and when observed in isolation in different culture conditions.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Materials
Three knees from skeletally mature bovines (aged 24, 32 and 33 months) were obtained from a local abattoir within 2 h after slaughter. The knee joints were aseptically exposed and observed by the naked eye to ensure that no degenerative changes were present on the articular surfaces. Articular cartilage specimens from the femoral trochlea were then immediately processed as per the following protocols for subsequent observations.

Methods
Cell isolation and labelling for culture in monolayer and in agarose gel
Slices of cartilage were aseptically removed from the articular surfaces using disposable scalpels and finely chopped and digested with 0.25% (w/v) trypsin (Sigma, Poole, UK) in Dulbecco's phosphate-buffered saline (DPBS) at 37°C for 20 min with magnetic stirring at 60 r.p.m., then with 0.25% (w/v) collagenase type 1A (Sigma) for 3 h in DMEM (Sigma) containing 10% (v/v) fetal bovine serum, 1% (v/v) antibiotic and anti-mycotic solution (final concentration: 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin) and 10 mg/ml N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES). The digest was then centrifuged and the cell pellet suspended in serum-free DMEM before passing through a 70 µm nylon filter (Falcon 2350 Cell Strainer, Becton Dickinson Labware, Franklin Lakes, NJ, USA) to remove undigested residue. The chondrocytes were again isolated by centrifugation and re-suspended in DMEM containing 10% FBS. The cell density was adjusted to 4 x 106 cells/ml. Trypan blue dye exclusion revealed that 95.3% of the cells remained viable.

For the 2D culture, the cells adjusted to 4 x 106 cells/ml were directly seeded into the Lab-Tek II chamber slide system (LAB-TEK®, Naperville, IL, USA) (1.6 x 106 cells/well) and maintained with DMEM containing 10% FBS. For 3D culture 8% (w/v) suspension of agarose (type VII, low gelling temperature, Sigma) in PBS was prepared by autoclaving at 126°C for 11 min and subsequently cooled to 37°C. The 8% agarose was then mixed with an equal volume of concentrated DMEM to give a concentration of 4% agarose. The cell suspension was added to an equal volume of 4% agarose to give a final concentration of 2 x 106 cells/ml in 2% agarose. The chondrocyte-seeded agarose was gently applied into wells (diameter: 6 mm; depth: 15.6 mm; volume: 0.44 ml) (0.88 x 106 cells/well) in a custom-made nylon disk and gelled at 4°C for 20 min. Finally, the agarose gel cylinders containing chondrocytes were expelled from the nylon disk and maintained in DMEM containing 10% FBS at 37°C in a humidified incubator under 5% CO2 in air. Before staining, the agarose-gel cylinders were cut into 2-mm thick slices.

Immunostaining was performed 5 days after cell isolation, as studies by Lee et al. [5] had demonstrated that the chondrocyte cytoskeleton becomes well organized between days 3 and 6 after chondrocytes are isolated. Both groups of chondrocytes in vitro were fixed with 3.7% paraformaldehyde in 0.1 M PBS (pH 7.4) for 15 min at ambient temperature, followed by permeabilization with 0.5% Triton X-100 (BDH, Poole, UK) in 0.1 M PBS for 10 min and washing with 0.1 M PBS. They were then pre-incubated with 1% BSA (Sigma) in 0.1 M PBS for 5 min to block the non-specific staining. After shielding from the light, filamentous actin (F-actin) was stained with 1 U/ml concentration of the Alexa-Fluor 488 conjugated phalloidin (Molecular Probes, Eugene, OR, USA) in 1% BSA in 0.1 M PBS for 15 min, followed by washing with 0.1 M PBS for 5 min. The vimentin was then directly labelled with Cy3-conjugated anti-vimentin antibody (Sigma) diluted 1 : 100 with 1% BSA in 0.1 M PBS for 30 min at ambient temperature, followed by washing with 0.1 M PBS for 5 min. Finally, nuclei were counterstained with TO-PRO-3 (Molecular probes, Eugene, OR, USA). Chondrocytes in both monolayer and in agarose gel were mounted with a cover slip using an anti-fade reagent (Vector Laboratories, Burlingame, CA, USA).

Cartilage specimen preparation for confocal microscopy observations
Eight osteochondral plugs were harvested from each femoral trochlea using a cylindrical reamer of a 12 mm inside diameter. After fixation with 3.7% paraformaldehyde and decalcification with EDTA for 2 days, each plug was embedded in OCT compound (Tissue Tek®, Tokyo, Japan) and the different plugs were sliced either parallel or perpendicular to the articular surface using a cryo-microtome (Bright, Huntingdon, UK). The slices parallel to the articular surface were used to demonstrate the en face view of chondrocytes within the articular surface and the superficial layer. Those perpendicular to the articular surface were made through the full thickness of the cartilage layer and were used to demonstrate the chondrocytes organization throughout the same full thickness. In order to visualize, in 3D detail, the organization of individual chondrons, 30 µm sections were obtained from slices in both orientations mentioned above.

Immunostaining was performed using a protocol similar to that described earlier. Each section was mounted on a glass slide and a cover slip was applied with the anti-fade reagent.

Confocal laser scanning microscopy
Beam path setting
Confocal laser scanning microscopy was performed using a Leica TCS-SP2 AOBS (Leica, Heidelberg, Germany) equipped with air lenses (HC PL FLUOTAR, Leica, Germany) and an oil immersion lens (100x/1.35 N.A., Uplan Apo, Nikon, Japan). The 488 nm argon, 543 nm argon and 633 nm He–Ne laser lines were set at 5%, 20–30% and 20–40% of full power, respectively, for the excitation of the Alexa Fluor 488 (for F-actin), the Cy3 (for vimentin) and TO-PRO-3 (for nuclei). Emission spectra were acquired between 500 and 535 nm for the Alexa Fluor 488 and between 560 and 625 nm for the Cy3 and 650–700 for the TO-PRO-3. The fluorescence images of actin, vimentin and nuclei and phase-contrast transmitted light images were acquired simultaneously using two emission detectors (with sequential frame scanning) and a transmission detector, respectively. Fluorochromes with different excitation wavelengths frequently have emission spectra that show a wide overlap. This ‘cross-talk’ can be avoided by the application of sequential frame scanning. As the main subject of the confocal laser scanning microscopy (CLSM) observations in the present study was to reconstruct the 3D organization of the chondrocyte cytoskeleton, a z-series of optical sections with a z-spacing of 0.2 µm was acquired using this scanning mode at a slow scan speed (200 Hz) with four times linear averaging prior to image capture.

Image processing
The acquired images were processed using channel dye finder for further avoidance of the ‘cross-talk’ between the three fluorochromes. The 3D projection images of fluorescence derived from actin, vimentin and nuclei were overlaid using the Leica confocal software. The lower magnification images were acquired to investigate the overall orientation of the chondrocytes, whilst the higher magnification images were acquired to investigate the detail of the cytoskeleton. Further 3D rendering of image stacks was performed using ‘Volocity’ visualization software (Improvision Ltd, Coventry, UK).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Configuration and morphology of the chondrocyte and cytoskeleton in monolayer and 3D cultures, and in situ
In monolayer culture, the chondrocytes were amoeboid in shape and their diameter was greater than that of chondrocytes either in 3D culture or in situ. Actin formed stress fibres predominantly located just beneath the cell membrane where the cells attached to the culture well substrate (Fig. 1A1), but not in their upper portion where the actin demonstrated a punctate staining pattern. A vimentin meshwork spanned the cytoplasm in a radial arrangement from the plasma membrane to the (presumed) nuclear membrane (Fig. 1A1–A3).


Figure 1
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  		FIG. 1. Confocal 3D projection images demonstrating the cytoskeleton of a chondrocyte seeded in monolayer (A) and in agarose gel (B). Distribution of actin (A1, B1), vimentin (A2, B2) and their superimposition (A3, B3) are demonstrated. In monolayer condition, the chondrocytes are amoeboid in shape. Actin (green) forms stress fibres predominantly located just beneath the cell membrane. A vimentin (red) meshwork spans the cytoplasm from the cell membrane to the nuclear membrane. Bar: 20 µm. In 3D culture condition (in agarose gel), the chondrocytes are spheroid in shape. Instead of stress fibres, actin demonstrates a punctate staining pattern throughout and distributed evenly beneath the cell membrane. The vimentin meshwork spans the cytoplasm from the plasma membrane to the nuclear membrane. Bar: 5 µm.

 
In 3D culture, the chondrocytes were spheroid in shape and instead of stress fibres, actin adopted a punctate staining pattern throughout and was distributed evenly beneath the cell membrane. The vimentin meshwork again spanned the cytoplasm from the plasma membrane to the nuclear membrane but in a concentric arrangement (Fig. 1B1–B3).

In intact cartilage, the distribution of actin and vimentin within the chondrocytes was largely symmetrical and similar to that observed in chondrocytes cultured in agarose gel. However, there appeared to be a prevalence of a bi-cellular configuration of the chondrocytes observed throughout the layers of intact cartilage from the surface to the subchondral bone. This arrangement, both in terms of chondrocyte morphology and bi-cellularity, appeared to be a consistent feature of all the cartilage plugs examined. Figure 2A and B shows low magnification images of a section through the entire thickness of a cartilage specimen, demonstrating a ubiquitous presence of this bi-cellular (bichondral) configuration throughout the cartilage layer. This is particularly evident in Fig. 2B, (a less oblique, but slightly less complete view of the same section shown in Fig. 2A), where almost all cells appear paired, with the long axes of the cell pair pointing in the same direction. Counting revealed that in this field some 336 cells (~81% of a total population of 416 cells) occurred as a member of a pair, the remaining ones being singles. This can be readily explained when it is remembered that Fig. 2B is a confocal image, of all chondrocytes in the cartilage slice defined by the two optical slicing planes. The chondrocytes shown in that figure must therefore include (i) paired cells, (ii) single cells where the sister cell of a pair falls outside the slice defined by a sectioning plane and (iii) sections through single or paired cells. Thus, the appearance of a single cell in the field is most likely due to the sister cells of a pair falling either side of the slicing plane. Figure 3A shows a higher magnification image of the chondrocytes observed in the surface layer when viewed en-face, again demonstrating the prevalence of this bichondral configuration. Figure 3B1–B3 are high magnification images of one such pair in the surface layer. The two chondrocytes are discoid and symmetrical in shape, being 10–15 µm in diameter.


Figure 2
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  		FIG. 2. (A) A low magnification image of a section through the entire thickness of a cartilage specimen, demonstrating the ubiquity of this bi-cellular configuration throughout the cartilage layer. This is particularly evident in (B), [a less oblique, but incomplete view of the same section in (A)], where almost all cells appear paired. Actin was stained with the Alexa-Fluor 488 conjugated phalloidin and shown in green.

 

Figure 3
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  		FIG. 3. (A) A low magnification image of the chondrocytes observed in the surface layer when viewed en-face, again demonstrating the prevalence of this bi-cellular configuration. The cells are stained with the Alexa-Fluor 488 conjugated phalloidin and shown in green (B1–B3) are high magnification images of one such pair in the surface layer. The two chondrocytes are discoid and symmetrical in shape, being 10–15 µm in diameter. The distributions of actin (in B1, green) and vimentin (in B2, red) within each pair of chondrocytes was largely symmetrical and similar to that observed in chondrocytes cultured in agarose gel. B3 shows the composite projection of B1 and B2. Bar: 5 µm.

 
Another interesting and significant observation was the presence of a link/projection of vimentin connecting some of the sister cells within a pair. Figure 4 demonstrates examples of this phenomenon.


Figure 4
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  		FIG. 4. Confocal 3D projection images of the pairs of chondrocytes observed in the surface layer when viewed en-face demonstrating the presence of a link/projection of vimentin (red) connecting some of the sister cells. Vimentin was labelled with Cy3-conjugated anti-vimentin antibody. Actin was labelled with the Alexa-Fluor 488 conjugated phalloidin and shown in green. Nuclei was counterstained with TO-PRO-3 and shown in blue.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Cytoskeleton of chondrocytes in monolayer and 3D cultures, and in situ
Chondrocyte cytoskeleton morphology/organization has been mainly investigated in isolated chondrocytes in monolayer and 3D cultures [1–5], few studies having investigated those aspects of chondrocytes in situ [9, 10]. This present study demonstrated the differences (or similarities) in the cytoskeletal morphology/organization of chondrocytes in different modes of culture (i.e. 2D or 3D) and that of chondrocytes in their natural habitat, the ECM. Actin was seen to form stress fibres only in 2D culture conditions, but not in 3D culture and in situ. In these two latter conditions, actin showed a punctate staining pattern. The vimentin meshwork spanned the cytoplasm from the plasma membrane to the nuclear membrane of chondrocytes in both cultures and in situ. However, the organization of the vimentin had a radiate organization in chondrocytes in monolayer and a more concentric arrangement where either an artificial or natural ECM was present.

There are factors, which have been shown to affect the morphology of chondrocytes in monolayer culture. It is widely recognized that if the chondrocytes are not seeded at a sufficiently high density they will start to de-differentiate within a short period. This was evidenced in the Blain et al. [11] study where chondrocytes seeded at a high density (1 x 106 cells/well of a 24-well plate) and maintained for 7 days retained the same characteristic actin, vimentin and tubulin cytoskeletal morphology as those in the 3D agarose constructs. The cell seeding density in the present study exceeded this value (i.e. 1.6 x 106 cells/well). Additionally, the use of 10% FCS may encourage chondrocytes to differentiate into a fibroblastic morphology, and there is evidence in the literature suggesting that the use of a supplement containing insulin–transferrin–selenium (ITS) instead of FCS will sustain the chondrocytic phenotype [12]. The current data would suggest that the substitution of FCS by ITS is necessary for the retention of the chondrocyte phenotype only when a matrix is absent.

Is the bi-chondral configuration the ubiquitous micro anatomical unit of articular cartilage?
The most significant finding of this study is perhaps the prevalence of a bi-chondral configuration throughout the cartilage layers. The authors propose that this might indeed be the predominant morphology of the main anatomical and functional unit in articular cartilage—the chondron.

The concept of the chondron was proposed by Benninghoff [13]. Morphologically, each chondron was thought to consist of a single chondrocyte linked at its surface to a transparent pericellular glycocalyx that is confined and enclosed by a fibrillar pericellular capsule. That the chondron might contain one, two or multiple chondrocytes has been inferred from images of the superficial zone of canine cartilage [14, 15]. The study undertaken by Chi et al. [16] who, using CLSM and a fluorescent recovery after photo-bleaching technique (FRAP), has suggested that at least some chondrocytes in the superficial zone appeared to be paired. Another relevant study is that by Schumacher et al. [17], which was undertaken on the chondrocytes in the superficial zones of the normal ankle (talocrural) and the normal knee (tibio-femoral) joints. When the chondrocytes from both joints were compared in serial horizontal sections, the chondrocytes in the superficial zone of the knee cartilage were described either as isolated single cells or as doublets. It is appropriate to point out that scanning electron micrographs of pairs of chondrocytes in the articular surface have appeared in much earlier studies by Meachim and Stockwell [18] and Stockwell and Meachim [19], but these were described as having the appearance of a ‘figure of eight’.

The prevalence of this bi-cellular configuration demonstrated in our study was more readily evident because of the visualization modality that we have adopted in this investigation. In particular, it is the low magnification 3D projection images, obtained by CLSM from relatively thick sections of intact articular cartilage that has highlighted the apparent ubiquity of the bi-cellular configuration throughout the cartilage depth. This ubiquity would be much more difficult to demonstrate with other microscopy modalities, whether light or transmission electron microscopy (TEM) used in many previous studies. The reason for this is that, the thicknesses of sections that are normally used for light microscopy (5–10 µm) and TEM (50–70 nm) are too thin in comparison with the overall diameter of a bi-cellular unit (20–30 µm). In particular, TEM sections are almost two orders of magnitude smaller than the dimension of the bi-cellular unit. Thus, while some such sections might occasionally include a pair of cells, they are too thin to include and visualize consistently and simultaneously many bi-cellular units. Therefore, TEM cannot demonstrate the ubiquity of chondrocyte pairing in cartilage that CLSM has demonstrated in this study.

Significance of a ubiquitous bi-cellular configuration
The functional significance of this bi-cellular configuration in cartilage has been discussed by Chi, Rattner and Matyas [16], who suggested that rapid cellular communication is possible between these paired cells. Their electron micrographs of the apposing surfaces of a chondrocyte pair showed small cytoplasmic projections extending from all surfaces of the chondrocytes and the close proximity of the processes in the thin plate of matrix separating the apposing surfaces.

The present study has shown the presence of cytoskeletal projections (containing vimentin) that appear to connect the sister cells, the importance of vimentin in the correct functioning of the chondrocyte having been highlighted in previous work [11]. This might add weight to the presumption that these cells are functionally linked, although the aforementioned projections do not appear in all units. The prevalence of paired cells over any other reported configuration might also strengthen the hypothesis that paired cells are functionally linked. Were this to be the case it would have serious consequences for surgical treatments available for cartilage repair including autologous chondrocyte implantation (ACI). In ACI, two variants of treatment exist. In the first, the enzymatically isolated chondrocytes are expanded in monolayer (2D) cultures, which are then implanted in the osteochondral lesion. In the second, the chondrocytes expanded in monolayer (2D) cultures are seeded onto 3D scaffolds, which are then implanted in the osteochondral lesion. A common difference between both variants and chondrocytes in situ would seem to be the bichondral arrangement in the latter. This prevalence of a bichondral configuration within normal articular cartilage might be significant for its normal function. Therefore, it might be appropriate to aim to retain this cellular configuration in ACI. The two adjacent chondrocytes might have an important symbiotic function, which may cease to exist once the pairs of chondrocytes are separated. It may be the absence of this bichondrality in the various cell-based modalities of tissue engineering of articular cartilage that explains the subsequent development of repair tissue that is dissimilar to natural cartilage tissue both in histology and mechanical properties. In the development of tissue engineering techniques for cartilage regeneration, it is clear that a detailed knowledge of cartilage organization is required and should be the subject of further investigations.

However, it is important to emphasize that the data presented here have been obtained from relatively few specimens, and more importantly, from just one species i.e. bovines. Furthermore, the data have been obtained from one site only—the femoral trochlea. The following questions will therefore be of particular relevance: (i) is the bichondral configuration species and/or site and/or age-specific and (ii) what is the prevalent configuration of chondrocytes in osteoarthritic joints. Thus, before embarking on other major investigations it will be essential to investigate this issue of ubiquitous pairing of chondrocytes at different sites in joints at different stages of maturity in other species including normal and arthritic humans. The pairing has also to be investigated in joints at different sites.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors would like to acknowledge Dr Fujikawa, former professor of National Defence Medical College, and Prof. Toyama, Associate Prof. Matsumoto and Prof. Otani in the Department of Orthopaedic Surgery of Keio University for their support. They are grateful to Mrs Hudson for her technical support.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Knight MM, Lee DA, Bader DL. The influence of elaborated pericellular matrix on the deformation of isolated articular chondrocytes cultured in agarose. Biochim Biophys Acta (1998) 1405:67–77.[Medline]
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Submitted 11 July 2007; revised version accepted 16 July 2008.
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