Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinβ1 and focal adhesion kinase signaling cues
© Prasadam et al.; licensee BioMed Central Ltd. 2013
Received: 7 March 2013
Accepted: 17 September 2013
Published: 9 October 2013
Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function.
De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies.
The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell’s attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression.
This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.
Bone matrix serves as an organized framework for bone as a tissue, offering mechanical support and mediating biological activities of bone cells and signals that maintain bone homeostasis and remodelling. Bone cells, like most other matrix-associated cells, cannot survive or differentiate without adhesion to their matrix[2, 3]. Consequently, bone cell morphology and functions can depend strongly on matrix quality under conditions in which biological signals are constant. In osteoarthritis (OA) it is well-known that subchondral bone matrix, structure, organisation, composition and mineralisation are abnormal when compared to normal bone.
Osteocytes are the most abundant and longest-living cells in the adult skeleton. The importance of osteocytes in regulating bone remodeling and turnover has been generally acknowledged. Our recent study demonstrated that various functional and morphological properties of osteocytes appear to be hampered in patients with OA, indicating that these cells could play an important pathological role in subchondral bone sclerosis. However, the potential molecular mechanism behind this abnormal osteocyte behaviour in OA patients is yet to be identified.
In vivo, osteocyte cells under normal conditions, contact a complex mixture of secreted ‘extracellular matrix’ (ECM) proteins called the bone matrix. The bone matrix isolates osteocytes from each other and instead osteocytes interact with other osteocytes and other bone cells by an elaborate network of osteocytes (dendritic) processes. The contact with the bone matrix is a critical mechanism providing cues via cytoplasmic processes called canalicules to form a cellular network to sense efficiently both mechanical and systemic stimuli. On the other hand, it seems that osteocytes which become transformed in diseases such as osteoporosis and OA are characterised by loose contact with ECM substrate leading to morphological and functional bony changes[6, 8]. Primarily based on our previous observations, in this study we hypothesised that altered mineralisation and the ECM quality of the subchondral bone matrix is the trigger for the osteocyte abnormalities seen in OA.
In vivo cell adhesion to the ECM is mediated by integrinβ1 receptors. Bone ECMs are composed of several macromolecules including fibronectin, laminin, collagens and proteoglycans. A number of these ECM proteins contain the three amino acid sequence Arg-Gly-Asp (RGD), which is exclusively recognised by corresponding integrinβ1 receptors[9, 10]. Attachment of integrins with the above macromolecules can activate the downstream signalling focal adhesion kinase (FAK) and vinculin that can initiate a cascade of phosphorylation events that fine-tune cell-type-specific phenotypes. Maintenance of integrin linkages is essential for cell adhesion, proper cytoskeletal organisation and function of the specific cell types. It has been demonstrated previously that disruption of these attachments, via addition of neutralising antibodies or peptides, can induce cells to detach from the ECM resulting in apoptosis, structural alterations and cellular dysfunction. The aim of this study is to test how normal and OA bone ECM differentially regulates the function of the osteocytes. The other objective of the present study is to reveal the critical role of cell-matrix adhesions governing this process, notably involving the integrinβ1-FAK signalling axis.
Subchondral bone osteoblast (SBO) isolation and characterisation
The Ethics Committee of Queensland University of Technology and the Prince Charles Hospital approved this study and the participants’ written consent was obtained according to the Declaration of Helsinki (Ethics Number: 0700000157). Knee bone specimens were taken within 5 mm of the subchondral bone plate as described previously in our studies[12–14]. OA SBOs were cultured from bone sourced from the medial compartment of the knee from patients suffering advanced OA, where the cartilage was degraded and showed prominent subchondral bone sclerosis and density (n = 5) (age: 61.5 ± 4 years). Normal SBOs were cultured from bone collected from trauma patients who were undergoing above the knee amputations with no evidence of subchondral bone sclerosis or cartilage degeneration on top of it (n = 4) (age: 60.1 ± 6 years). The criteria for the OA diagnosis were those established according to the American College of Rheumatology. None of the normal patients had any musculoskeletal disorders, such as osteoporosis. SBOs were isolated according to the method described by Beresford[16, 17]. Isolated normal and OA SBOs were characterised for their phenotype as described in our previous studies[12, 13]. Passage one SBOs were used for this study.
Subchondral bone osteoblast differentiation and preparation of de-cellularised matrices
Osteogenic differentiation of normal and OA SBOs (20,000 cells per well) was performed in (D)MEM medium containing osteogenic supplements (10 nM dexamethasone, 10 mM β-glycero-phosphate, 50 μg/mL ascorbic acid) on coverslips (NUNC, Roskilde, Denmark) (placed on 24 well plates) for five weeks. After five weeks, SBOs were rinsed two times in 1X PBS. Next, to produce the de-cellularised matrices, 0.02 M ammonium hydroxide in ddH2O was applied for 20 to 30 minutes at room temperature intermittently visualising under a light microscope for cell roundup and lysis. Then ammonium hydroxide was removed by inverting the culture surface, with plates washed several times with sterile 1X PBS before seeding osteocytes on top of them.
Characterisation of de-cellularised matrices
The removal of cells and the retention of a collagenous matrix were verified following de-cellularisation. Briefly, cells were fixed in 4% paraformaldehyde for ten minutes, washed three times with 1X PBS and permeabilised with 0.1% Triton for five minutes and stained with ProLong Gold Antifade Reagent (Invitrogen, Life Technologies Australia Pty Ltd, Victoria, Australia) with 4',6-diamidino-2-phenylindole (DAPI) on a glass slide. The absence of nuclei was confirmed using fluorescence microscopy (Zeiss, using AxioVision Image analysis software). The presence of collagen I and various ECM proteins in the de-cellularised matrices was confirmed by immunostaining and western blotting following the protocol described in our previous studies[12, 13]. The presence of mineral content was confirmed by staining matrices with 1% alizarin red. Scanning electron microscopy (SEM) with energy dispersive X-ray analysis (SEM/EDX) (FEI Quanta 200 Environmental SEM equipped with an Evarhart Thomley secondary electron detector) was used to examine the morphology of the calcium phosphate deposit and to obtain the elemental composition in normal and OA matrices.
MLOY4 osteocyte cell line culture
The well-characterised osteocytic line, MLOY4 (provided by Dr. Lynda Bonewald) was cultured on T75 tissue culture flasks coated with type I collagen (0.30 mg/ml; Sigma, St. Louis, MO, USA) in osteocyte culture medium containing alpha modified essential medium (αMEM; GIBCO BRL, Grand Island, NY, USA), 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 5% calf serum (CS), and 1% penicillin and streptomycin (GIBCO BRL).
Seeding MLOY4 cells on normal and osteoarthritis matrices
MLOY4 cells (10,000 cells/cover slip) were seeded directly on the top of the normal and OA matrices prepared above and incubated in osteocyte medium at 37°C containing 5% CO2/95% atmospheric air at different time points.
Integrin and focal adhesion kinase signalling studies
To confirm the integrinβ1 mediated molecular mechanism, MLOY4 cells were cultured on normal and OA matrices in the presence or absence of blocking-integrinβ1 (10 μm) antibody (Clone P5D2, Chemicon, Millipore, MERCK Pvt Ltd, Victoria, Australia) or with an irrelevant antibody. P5D2 react with the human β1 integrin subunit and block adhesion of cells to ECM proteins. FAK activation and de-activation was analysed by anti-phospho- FAK and anti-total FAK antibodies (Cell Signaling Technology, Gene search Pty Ltd, Queensland, Australia).
Scanning electron microscopy
The morphology of osteocytes grown on normal and OA osteoblast matrices were assessed by SEM as described previously. (+/- MLOY4 cells) were fixed with a solution containing 3% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer solution (pH 7.3) for one hour at 4°C and postfixed in 1% osmium tetroxide for one hour. The samples were dehydrated in increasing concentrations of ethanol (from 50%, 70%, 90% to 100%) and were critical-point-dried. Cover slips were mounted on aluminium stubs before being sputter coated with a thin layer of gold in a SC500, Bio-Rad sputter coater (Bio-Rad, BioRad Laboratories Pty Ltd, New south wales, Australia) before examination using a FEI Quanta 200 scanning electron microscope (FEI, Hillsboro, OR, USA). Backscatter imaging on in vivo samples was performed as described previously.
To examine focal adhesion formation and cytoskeletal organisation, osteocytes cultured on normal and OA ECM substrates were fixed in 4% paraformaldehyde (pH 7.4 in PBS) for 10 minutes, and then permeabilised with 0.2% Triton X-100 for 10 minutes. After washing and blocking with 1% BSA, cells were incubated with primary antibody vinculin 5 μg/ml (Sigma Aldrich, New south wales, Australia), SOST 5 μg/ml (R&D Systems, Sapphire Biosciences Pty Ltd, New south wales, Australia), DMP1 5 μg/ml (gift from Professor Jian Feng) and E11 5 μg/ml at 4°C overnight followed by secondary antibody (Alexa Fluor 488-labeled goat anti mouse immunoglobulin G (IgG), Invitrogen) incubation. F-actin distribution was visualised using Alexa Fluor 568 nm-labeled phalloidin (Invitrogen) staining. The samples were washed with 1X PBS three times, blown dry with air and mounted with Prolong Gold antifade solution containing DAPI for cellular nuclei staining and fluorescence preservation. Samples were then visualised using a DeltaVision PDV microscopy system equipped with an Olympus IX70 inverted microscope (Olympus). After acquisition of the Z-series images, they were deconvolved using the software attached in the system (Softworx; Applied Precision). The shapes of the fluorescent signals in osteocytes cultured on the normal and OA matrix were analysed using the integrated morphometry shape factor analysis of Metamorph software version 7.1.2 (Universal Imaging). This algorithm assigns a value from 0 to 1, describing the shape of the fluorescence signal where a perfect circle attains a value of 1 and a line is assigned a 0.
Cell attachment assays
MLOY4 cells were seeded directly on normal and OA matrices with a seeding density of 10,000 viable cells per well. Cells were allowed to attach in the humidified incubator for two different time periods, 3 and 24 hours. At the end of each culture period cells were stained for 10 minutes with 0.2% crystal violet (Sigma) in 20% methanol. After washing with H2O, plates were dried overnight at room temperature. Cells were dissolved in 1% SDS (150 μl), and the absorbance at 570 nm was measured.
Cell proliferation assays
Cell proliferation was determined using the CyQUANT NF Cell Proliferation Assay Kit (Molecular Probes, Invitrogen) at 48 hours of MLOY4 cell culture on normal and OA matrices according to the manufacturer’s instructions. Plates were then analysed by using a microplate reader (excitation: 485 nm, emission: 520 nm).
The viability of MLOY4 cells was quantified by flow cytometry with the AnnexinV-FLOUOS apoptosis staining kit (Roche Applied Science, Germany, Roche Australia PVT LTD, New south wales, Australia) following the manufacturer’s protocol.
Quantitative real time PCR (qRT-PCR), zymography, and western blotting
Comparisons between groups were carried out by Student’s t-test and for multiple comparisons analysis of variance (ANOVA) was used where P ≤0.05 was considered significant. Results are presented as a mean ± standard deviation (SD).
Confirmation of de-celluarisation
Characterisation of ECM secreted by normal and OA osteoblasts
Differential cell-matrix adhesions regulate morphology of osteocytic cells scattered within the normal and osteoarthritis osteoblast derived extracellular matrix
These differences in morphology suggest underlying differences in the organisation of the respective cytoskeletons. Osteocytic cells that were grown on normal matrix had straight actin fibres, which were visualised by phalloidin. When the cells were grown on the OA matrix, actin filament fibres were observed randomly and visually no fibres were present at the center of the osteocytic cells (Figure 3C).
The formation of focal adhesion complexes is a prerequisite for cell-ECM adhesion. Therefore, it is possible that the altered cell morphology capabilities of osteocytic cells on the OA matrix may result from alterations in focal adhesion functions. We thus used immunofluorescence staining to examine the expression of focal adhesions. As shown in Figure 3D, osteocytic cells grown on normal matrices formed multiple focal adhesion structures at plasma membranes and cytoplasm, as visualised by staining for vinculin, an abundant cytoskeleton protein localised at focal adhesions. In marked contrast, osteocytic cells on an OA matrix showed fewer focal adhesion contact points. Confocal images of osteocytic cells shown in Figure 3C were quantitatively analysed for shape factor. Cell shape factor changed from 0.1 to 0.4, indicating a transformation from a non-circular spreading pattern as the cells developed spindle shaped morphology to a rounded/circle pattern when grown on an OA matrix (Figure 3E).
Altered functional and gene expression characteristics of osteocytic cells grown on osteoarthritis osteoblast-derived matrices
Expression of integrinβ1-FAK signalling in osteocytic cells cultured in normal and osteoarthritis bone matrices
Integrinβ1 blocking diminished phenotypic and genotypic capacity of osteocytic cells attached on the normal matrices
It is now well accepted that joint cartilage degeneration is associated with intensified remodelling of the subchondral bone and increased bone stiffness. Understanding which cells/molecules are involved in bone sclerosis could help us find ways to manipulate such molecules to slow down the progression of OA.
Earlier studies have shown that the de-cellularised matrix produced by osteoblasts is able to induce attachment and spreading of many adherent cell types, including mesenchymal stem cells[27–29]. In principle, the use of NH4OH leads to hypoxia and subsequent cell lysis leaving the matrix intact. It is thus regarded as an in vitro model to study the molecular basis of cell attachment, spreading and signalling from the ECM. In the present study, we confirmed that de-cellularised osteoblast matrices represent the in vivo bone matrix characterised by accumulation of the mineral content and expression of various collagenous and non-collagenous proteins, thus providing the ideal environment to study cell-matrix interactions. First, we observed significant differences in the mineral content of matrices secreted by normal and OA osteoblasts. These results are in agreement with previous reports suggesting the abnormally low mineralisation of OA osteoblasts. The present study also showed that human osteoblast ECM derived from primary bone cell cultures has the capacity to produce a wide range of matrix proteins. Surprisingly, we observed that the levels of fibronectin, laminin and versican were decreased in OA matrices compared to normal. In general, fibronectin is presumed to contribute more to general structure and load bearing, whereas versican and laminin interact with other matrix components as well as with cell surface adhesion receptors via well-defined domains. Low expression of these molecules is suggestive of abnormal ECM composition in OA matrices. Previous studies demonstrated an increase of COL1 in OA osteoblasts at the cellular level. In this study, the matrix deposited by OA osteoblasts showed a dispersed COL1 staining compared to normal matrix. These results corroborate the results of studies showing disorganised collagen fibres in OA bone compared to normal. Furthermore, although not statistically significant, we observed that expression of OCN was higher in OA patients. However, many other studies showed a significant increase of OCN in OA patients[30, 35]. This disparity in the results could be attributed to biological variation, process variation and system variation.
Based on the above results, it is apparent that the ECM secreted by normal and OA osteoblasts is different. We next tested whether these matrix differences can contribute to the poor morphological and phenotypic properties of osteocytes seen in OA bone. Our results suggest that osteocyte cell lines, such as MLOY4, could be altered in response to the ECM of OA osteoblasts compared to the ECM generated from normal osteoblasts. At the gene expression level, when osteocytic cells were grown on OA matrices a decrease in DMP1 and SOST expression and an increase in E11 and MEPE expression were found. Although E11, MEPE, DMP1 and SOST are known markers of the osteocyte, each gene has a distinct function and their expression levels change according to the specific stage of differentiation. Osteocyte expression of SOST and DMP is a delayed event and it is produced only by osteocytes after they become embedded deeply in matrix that has been fully mineralised. Our results showed that OA osteoblasts failed to lay a proper mineral or matrix. Therefore, these matrix properties might have influenced the behaviour of osteocytes, stopping them from expressing those mature markers; whereas E11 and MEPE are early markers and they start to express when osteocytes are in the process of embedding into the matrix that is not fully mineralised. Failure of osteocytic cells to produce these proteins when cultured on the OA matrix also suggests a state of immature osteocytes. These results together suggest that in OA matrices osteocytic cells remained in an immature phenotype; however, when they were cultured on the normal matrices, the osteocytes differentiated from an immature to mature phenotype. Likewise, on the OA matrices an increase in osteocytic cell death was observed which can possibly be related to increased skeletal fragility, linked to the loss of ability to sense microdamage and/or signal repair of in vivo bone.
The observation that the morphological and phenotypic characteristics of osteocytic cells attached to the normal and the OA matrices differ from each other, suggests that specific signalling pathways must arise or alter between matrix and cells. Of note, we observed that the expression of integrinβ1 was much less in osteocytic cells that were cultured on the OA matrices compared to normal matrices indicating that the decrease of integrinβ1 expression could be responsible for the observed phenotypic changes of osteocytes in OA.
How and to what extent integrinβ1 is involved in the transduction of a matrix signal to modulate osteocyte function was investigated by a series of further experiments. We found that the blocking integrinβ1 activity promoted the elongated cell morphology to a rounded phenotype even when osteocytic cells are cultured on normal osteoblast matrices. This series of events occurred via the down regulation of FAK phosphorylation levels. The effect of integrinβ1 blocking on cell morphology was possibly due to FAK signals leading to actin contractility events and the dynamic regulation of viniculin. Normally, integrinβ1-FAK signalling, apart from playing a role as a hook to attach the cell to matrix, generates a signal to enforce the cell towards functions such as attachment, apoptosis and differentiation. In this study, we observed that osteocytic cells on OA matrices and integrinβ1 blocking showed an increased apoptosis and altered osteocyte gene expression, indicating that the expression of integrinβ1-FAK signalling is important to maintain the normal osteocyte-matrix interactions. In vitro data presented here are consistent with results from an integrinβ1 negative mouse model in which mice exhibited less mineralised bone, reduced tibial curvature and decreased femoral strength. Thus, this study provides evidence that integrinβ1 may initiate intracellular signals either by organisation of the cytoskeleton and alteration of cell shape or through mechanisms akin to osteocyte signalling. In this study, we observed that the expression of Vβ3 and Vβ5 were not changed. Although different integrin receptors perform common functions and can share identical ligands, each member seems to be highly specific, since mice carrying gene deletions of the different integrin chains often show non-overlapping phenotypes. We are confident that there might be other integrins participating in the interaction and further studies are warranted to determine their role.
In summary, this study demonstrated that ECM from OA osteoblasts can induce significant alteration of osteocytic cells via a focal adhesion mediated integrinβ1-FAK cell signalling pathway, a possible mechanism of OA subchondral bone sclerosis and OA progression.
Bovine serum albumin
Back scattered electron microscopy
Bone sialo protein
Type I collagen
Type III collagen
Dentin matrix protein
Dentin matrix protein 1
(Dulbecco’s)_modified Eagle’s medium
Energy dispersive X-ray
Focal adhesion kinase
Matrix extracellular phosphoglycoprotein
Phosphate-regulating neutral endopeptidase
Quantitative polymerase chain reaction
Subchondral bone osteoblasts
Scanning electron microscopy
This study is supported by the Prince Charles Hospital Foundation (MS2010-02), NHMRC project fund (APP1032738), and the Australian Orthopaedics Association Research Foundation. The authors would like to thank, Dr Anna Taubenberger for her kind inputs in the de-cellularisation protocols and Dr Abhishek Kashyap for provision of integrin antibodies.
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