JOURNAL TRANSCRIPT
BMC Musculoskeletal Disorders
BioMed Central
Open Access
Research article
Alterations in the vimentin cytoskeleton in response to single impact load in an in vitro model of cartilage damage in the rat Frances MD Henson and Thea A Vincent* Address: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK Email: Frances MD Henson -
[email protected]; Thea A Vincent* -
[email protected] * Corresponding author
Published: 24 June 2008 BMC Musculoskeletal Disorders 2008, 9:94
doi:10.1186/1471-2474-9-94
Received: 5 June 2007 Accepted: 24 June 2008
This article is available from: http://www.biomedcentral.com/1471-2474/9/94 © 2008 Henson and Vincent; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: Animal models have provided much information on molecular and cellular changes in joint disease, particularly OA. However there are limitations to in vivo work and single tissue in vitro studies can provide more specific information on individual events. The rat is a commonly used laboratory species but at the current time only in vivo models of rat OA are available to study. The purpose of this study was to investigate the damage that single impact load (SIL) of 0.16J causes in a rat cartilage in vitro model and assess whether this load alters the arrangement of vimentin. Methods: Rat cartilage was single impact loaded (200 g from 8 cm) and cultured for up to 48 hours (n = 72 joints). Histological changes were measured using a semi-quantitative modified Mankin score. Immunolocalisation was used to identify changes in vimentin distribution. Results: SIL caused damage in 32/36 cartilage samples. Damage included surface fibrillation, fissures, fragmentation, changes in cellularity and loss of proteoglycan. SIL caused a statistically significant increase in modified Mankin score and chondrocyte clusters over time. SIL caused vimentin disassembly (as evidenced by collapse of vimentin around the nucleus). Conclusion: This study describes a model of SIL damage to rat cartilage. SIL causes changes in histological/chemical parameters which have been measured using a semi-quantitative modified Mankin score. Single impact load also causes changes in the pattern of vimentin immunoreactivity, indicating vimentin dissassembley. Using a semi-quantitative scoring system the disassembly was shown to be statistically significant in SIL damaged cartilage. The changes described in this paper suggest that this novel single tissue rat model of joint damage is a possible candidate model to replace in vivo models.
Background In order to study osteoarthritis (OA) in both man and animals, much use has been made of animal models of pathology to generate consistent, reproducible articular cartilage lesions within a relatively rapid period of time [1]. Whilst animal models of pathology have provided researchers with a wealth of knowledge about events that
occur in joint disease, there are drawbacks to their use including the difficulty of isolating different tissue responses to a given insult. This is particularly true in the joint where the synovial membrane, synovial fluid, vasculature, nervous supply, cartilage and underlying subchondral bone all contribute to the end pathology, making elucidation of specific molecular events within one tissue Page 1 of 10 (page number not for citation purposes)
BMC Musculoskeletal Disorders 2008, 9:94
type difficult to isolate and more difficult to interpret. Therefore, in order to study individual tissue responses single tissue experiments are required. Single impact load (SIL) damage in articular cartilage was first described by Jeffrey et al ([2]). Subsequently a number of papers have investigated the effect of SIL and have shown alterations in matrix loss and synthesis [3], cell volume [4], and an increase in apoptosis in loaded cartilage [5,6]. In addition, a recent paper has described the validation of an in vitro SIL model of the initiation of OA-like changes in equine articular cartilage [5]. This work showed that SIL and subsequent culture of cartilage explants caused degenerative changes similar to those observed in OA, including the development of chondrocyte clusters (multinucleate groups of chondrocytes that are considered to be a characteristic histological change of OA). These changes can be quantified and compared, making the in vitro SIL model a useful single tissue tool for the elucidation of the early molecular pathways involved in the process leading from mechanical trauma to cartilage degeneration. However, whilst experiments on equine cartilage can provide extremely useful data, equine cartilage can be hard to obtain and standardized and the use of cartilage from a more readily obtainable species, e.g. rat, may be useful in the elucidation of cellular and molecular events that occur with SIL. At the current time there is, to the authors' knowledge, no description in the literature of an in vitro model of mechanical joint damage in the rat with experimental models of rat joint disease being surgically induced in vivo [7,8]. A rat SIL model of joint damage, would, therefore provide a useful experimental tool. In order to identify whether joint damage models produce changes similar to clinical disease, quantitative scoring systems have been described [9,10], which are based on structural damage to the cartilage [9]. In addition to identifying such gross structural changes in cartilage, cytoskeletal changes have also been reported in response to mechanical load, specifically changes in vimentin [11]. The chondrocyte cytoskeleton is a three-dimensional network comprised of three types of protein networks: actin microfilaments, tubulin microtubules, and vimentin intermediate filaments. The likely roles of microfilaments are in cell-matrix interactions, cell signaling, differentiation, intracellular transport, control of secretion/endocytosis and in maintaining cell shape [12]. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus, with vimentin forming a tighter and finer mesh than microtubules, and these intermediate microfilaments may play a role in the mechanotransduction process [12]. Evidence for a role in the response to load comes from a number of
http://www.biomedcentral.com/1471-2474/9/94
directions, including the response to chondrocyte swelling [11] and chondrocyte deformation experiments [13] and vimentin microfilaments have been shown to contribute to the viscoelastic properties of the chondrocyte [14]. The vimentin knockout mouse has been reported to display no obvious phenotype [15], however, a reduction in stiffness, mechanical stability, motility and directional migration in vimentin-deficient fibroblasts has been described [16]. Of interest in the context of joint disease is the observation that vimentin has also been shown to be altered in naturally occurring OA [17,18]. The aims of this study were (i) to validate a single impact load model of joint damage in rat femoral cartilage in vitro and (ii) to identify alterations in the vimentin intermediate filament cytoskeleton in this model over 48 hours in culture.
Methods Tissue samples Rat cadavers of male Sprague-Dawley rats between 20 and 26 weeks of age were obtained following euthanasia (overdose of barbiturate). After disarticulation of the femur from the acetabulum the proximal femur was dissected free from muscle. The femoral head was then severed from the femoral shaft by sharp excision to a depth of 5 mm from the articular surface using a standardized dissection procedure. The cartilage-bone unit was then placed immediately into sterile phosphate buffered saline (PBS) solution. A total of 84 femoral heads were used (42 rats). Impact loading In order to impact load the rounded cartilage surface evenly a simple cartilage/bone unit loading system was designed. This was basically a lead plate, which had, on its surface an imprint created by the articular surface of the joint. This imprint was made by using a cartilage/bone joint unit obtained as described above from an individual from the same group of rats.
Impact loading was performed by placing the femoral head articular surface downwards on the metal plate. The load was applied to the flat cut bone surface and the cartilage received load that had been transmitted through the bone to the cartilage that was adjacent to the metal plate. Single impact load was performed using a drop tower apparatus as described previously [6]. A weight of 200 g was dropped from a height of 8 cm (corresponding to an impact of 0.16J (calculated from [2]). This impact was chosen after a number of pilot experiments showed that this was the minimum load required, using this experimental design, to induce detectable damage in the cartilage, which could be scored using a modified Mankin system.
Page 2 of 10 (page number not for citation purposes)
BMC Musculoskeletal Disorders 2008, 9:94
http://www.biomedcentral.com/1471-2474/9/94
Cartilage damage scoring system A modified Mankin semi quantitative scoring system [10] was used to quantify changes within the cartilage sections (Table 1). Using this scoring system the maximum score obtainable was 15.
performed as a basic indicator of whether or not SIL could cause phosphorylation within this experimental situation. All fluorescent-labeled sections were imaged with a Leitz Laborlux 12 fluorescence microscope using digital image acquisition.
Tissue culture and histological staining Cartilage was washed three times in sterile PBS and incubated in culture medium (DMEM Sigma-Aldrich, UK) supplemented with 200 IU/ml penicillin (Invitrogen, UK), 2.5 μg/ml streptomycin (Invitrogen, UK), 500 μg/ml ascorbic acid (Sigma Aldrich, UK) and 10% fetal calf serum (Invitrogen, UK) at 37 degrees C and 5% CO2. Femoral heads were cultured for 1, 2, 4, 8, 24 and 48 hours. T = 0 was taken to represent samples that had not been cultured, however, this is roughly equivalent to t = 1 minute as this is the approximate time taken to remove the sample from the loading chamber and snap-freeze it. Non impacted femoral heads were cultured as control samples. At each time point the femoral heads were removed, the cartilage removed from the bone and snap frozen. 7 μm sections were obtained and stained with toluidine blue and H&E. All experimental time points were cultured in groups of 6.
Vimentin scoring system The integrity of the vimentin intermediate filaments can be identified by immunofluorescent staining techniques which reveal disassembly as a collapse of the vimentin microfilaments around the nucleus [19]. In order to identify and quantify intermediate filament disassembly in the chondrocytes a scoring system was devised (Figure 1). Chondrocytes with a normal cytoskeletal appearance i.e. lattice throughout the cell were scored as 0 (Figure 1a), cells with a slight increase in immunofluorescent intensity of stain around the nucleus were scored as 1 (Figure 1b), with a moderate intensity around the nucleus 2 (Figure 1c), and with the stain entirely around the nucleus 3 (Figure 1d), In each section a total of 100 chondrocytes were counted throughout the full thickness of the section and the total score for each section derived. A high vimentin score indicates a high level of intermediate filament disassembly. The scoring was performed in a blinded fashion.
Immunohistochemistry Frozen sections were fixed in 4% paraformaldehyde for 20 minutes at room temperature and washed in PBS. Immunohistochemistry was performed on cartilage sections using a standard fluorescent secondary antibody detection method. The primary antibodies used were monoclonal mouse anti-pig vimentin (Sigma, UK) at a dilution of 1 in 200, goat polyclonal anti-mouse pERK1/2 (Santa Cruz Biotechnologies) at a dilution of 1 in 100 and goat polyclonal anti-rat ERK (Santa Cruz Biotechnologies) at a dilution of 1 in 100. The secondary antibodies used were FITC-labeled anti-mouse and FITC-labeled anti-goat (Sigma, UK). Sections were counterstained with DAPi to allow identification of the nucleus (Vectorshield + DAPi, Vector Laboratories, UK). Vimentin immunofluorescence was performed at all time points, ERK and pERK at t = 0 and t = 1 hour. ERK and pERK immunofluorescence was
Statistical analysis Kruskal-Wallis and Mann-Whitney tests were used to identify whether there were significant differences between the control and SIL modified Mankin scores. In order to detect any significant difference between control and SIL vimentin scores the Chi-squared test was used. A result was considered significant when p < 0.05.
Results Histological changes Modified Mankin Score (MMS) Following SIL, cartilage damage was seen in 38/42 impacted samples and 4/42 control samples. In nonimpacted cartilage no structural damage was noted in 38/ 42 sections (Figure 2a). In impacted cartilage structural damage included surface damage and delamination of the cartilage surface (Figure 2b), cartilage fragmentation (Fig-
Table 1: The components of the modified Mankin scoring system
Structure
Cellularity
Matrix staining
Tidemark integrity Score
Smooth surface/normal Roughened surface/single crack or area of deamination Multiple cracks/moderate delamination Fragmentation in cartilage or severe delamination Loss of fragments Complete erosion to tidemark Erosion beyond tidemark
Normal arrangement Clustering in superficial layer or loss of cells up to 10% Disorganisation or loss up to 25% Cell rows absent or loss to 50%
Normal staining Slight loss of stain
Normal and intact Disrupted
Moderate loss of stain X Severe loss of stain X
2 3
Very few cells present x X
No stain present X X
4 5 6
X X X
0 1
Page 3 of 10 (page number not for citation purposes)
BMC Musculoskeletal Disorders 2008, 9:94
categories Photomicrographs the Figure vimentin 1 cytoskeleton to showwithin the representative each of the four appearance scoring of Photomicrographs to show the representative appearance of the vimentin cytoskeleton within each of the four scoring categories. Chondrocytes have been stained with monoclonal anti-vimentin antibody and visualized with an immunofluorescent secondary antibody (green). Counter stained with DAPi nuclear stain (blue). Chondrocytes with a normal cytoskeletal appearance i.e. lattice throughout the cell were scored as 0 (A), cells with a slight increase in immunofluorescent intensity of stain around the nucleus were scored as 1 (B), with a moderate intensity around the nucleus 2 (C) and with the stain entirely around the nucleus 3 (D).
ure 2c), fissures (Figure 2d) and loss of proteoglycan content of the cartilage indicated by loss of toluidine blue staining compared to non impacted cartilage (Figure 2d). Using the combined modified MMS a number of differences were shown. Firstly it was shown that, in control sections, the MMS increased with time in culture (Figure 3). This was statistically different from t = 0 at 2, 4, 8, 24 and 48 hours (p < 0.05). Secondly it was shown that, in SIL sections, a similar trend was noted i.e. the MMS increased with time in culture (Figure 3). This was statistically different from t = 0 at 1, 2, 4, 8, 24 and 48 hours. Thirdly it was shown that the MMS was statistically significantly different between control and SIL sections at t = 8, 24 and 48 hours in culture (marked as significant in Figure 3). Proteoglycan loss In both impacted and control cartilage there was loss of proteoglycan at t = 0 and a subsequent increase over time in culture. There was no statistical difference between control and SIL cartilage (results not shown).
http://www.biomedcentral.com/1471-2474/9/94
Figure 2a femur Histological of2a rat atsection 23 weeks of cartilage of age obtained from the distal 2a Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This is a control section i.e. has not received single impact load and it is has not been cultured i.e. represents a control section, T = 0. Stained with toluidine blue. There is no damage to the cartilage; the articular surface is smooth and flat. There are no micro-fractures or fragments and there is no loss of proteoglycan. Scale bar = 10 μm. 2b Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. T = 0. Stained with toluidine blue. There is marked surface damage to the cartilage including lamination. Scale bar = 10 μm. 2c Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. T = 0. Stained with toluidine blue. There is marked surface damage to the cartilage including the formation of a fragment discreet from the parent cartilage. Scale bar = 10 μm. 2d Histological section of cartilage obtained from the distal femur of a rat at 23 weeks of age. This cartilage has been impacted with a single impact load of 200 g from 8 cm. This cartilage has been cultured for 48 hours i.e. T = 48 h. Stained with toluidine blue. There is a fissure at the articular surface and marked proteoglycan loss. Scale bar = 10 μm
Structural and cellular damage Having noted that in both control and SIL cartilage there was marked proteoglycan loss during time in culture the MMS minus the proteoglycan score was quantified (Figure 4). This value clearly shows that at t = 1, 2, 4, 8, 24 and 48 hours the structural/cellular changes in the impacted cartilage are statistically increased compared to the control cartilage. Chondrocyte clusters As part of the MMS the cellularity of the cartilage was scored. This score includes a number of different parameters including loss of cells, disorganisation of the cellular arrangement and the presence of chondrocyte clusters.
Page 4 of 10 (page number not for citation purposes)
BMC Musculoskeletal Disorders 2008, 9:94
http://www.biomedcentral.com/1471-2474/9/94
period to3 show the modified Mankin score in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time Graph Figure Graph to show the modified Mankin score in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. There is a significant difference between control and single impact loaded cartilage at t = 8, 24 and 48 hours in culture (*). Chondrocyte clusters were detected in SIL cartilage at all time points. At t = 4, 8, 24, and 48 hours the numbers of chondrocyte clusters in SIL cartilage were statistically significantly increased compared both to control sections and also to SIL cartilage at both t = 0 and t = 1 hours (Fig-
ure 5). The histological appearance of the control and SIL cartilage at t = 48 hours is shown in Figures 6 and 7. In 4b there are significantly increased numbers of chondrocyte clusters.
Figure gGraph from to 84cm) show cartilage the modified over a Mankin 48 hourscore time minus periodthe proteoglycan component score in control and single impact loaded (200 Graph to show the modified Mankin score minus the proteoglycan component score in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. The SIL cartilage is significantly increased relative to the control in all sections.
Page 5 of 10 (page number not for citation purposes)
BMC Musculoskeletal Disorders 2008, 9:94
http://www.biomedcentral.com/1471-2474/9/94
Figure Graph hour time to5 show period the numbers of chondrocyte clusters in control and single impact loaded (200 g from 8 cm) cartilage over a 48 Graph to show the numbers of chondrocyte clusters in control and single impact loaded (200 g from 8 cm) cartilage over a 48 hour time period. There is a significant difference between control and single impact loaded cartilage at t= 4, 8, 24 and 48 hours in culture (*).
Vimentin Immunohistochemistry In all sections studied immunofluorescence clearly revealed the presence of vimentin intermediate filaments. The staining within each cell was assigned to one of 4 scoring groups, reflecting the degree of disassembly of the cytoskeleton. In control sections no score of 3 was assigned to any chondrocyte, i.e. no cell was seen to have vimentin staining entirely around the nucleus. In contrast, chondrocytes with a score of 3 were seen in single impact loaded sections throughout the culture period, including at time = 0.
In order to compare vimentin disassembly in control and SIL sections the total score for vimentin staining was calculated at each time point. The vimentin score was significantly increased in single impact loaded sections at all time points studied (Figure 8) compared to the control sections i.e. there was statistically significant disassembly in SIL cartilage compared to controls identified by the semi-quantitative scoring system described. The vimentin score peaked at 2 hours and then declined, this cannot be considered statistically significant evidence of re-assembly but does show a trend towards it. ERK/pERK Immunohistochemistry Immunolocalisation showed that ERK was detectable in the cytoplasm of all chondrocytes at t = 0 and t = 1 hour in control and SIL cartilage. At time = 0 there was no pERK within control sections, but in SIL cartilage strong PERK immunoreactivity was detected in the cytoplasm of all of
the chondrocytes within the section (Figure 9). By t = 1 hour pERK was detected in both impacted and control sections.
Discussion This study demonstrates that SIL and subsequent culture over 48 hours causes a response in rat femoral head articular cartilage. This response includes significant structural and cellular changes quantified by a MMS system and similar to those reported in in vivo OA animal models. SIL also causes disassembly of the vimentin intermediate filament cytoskeleton as detected by immunofluorescence. Impact load is of interest in joint pathology as it is widely reported that secondary OA is a common sequel to an impact load being applied to a joint [20]. In the experiments described here an impact energy of 0.16J was used to load a cartilage/bone unit with a time to peak load of