Original Article
Investigations of wear particles and selected cytokines in human osteoarthritic knee joints
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(11) 1176–1182 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914559570 pih.sagepub.com
Meiling Wang1, Natkunam Ketheesan2 and Zhongxiao Peng1
Abstract Inflammation of the synovial membrane (synovitis) is considered to drive the process that leads to osteoarthritis. However, the relationships between the mediators of inflammation and the properties of wear particles are not fully understood. In this study, the levels of IL-6 and IL-8 were assessed in different grades of osteoarthritis to determine whether their concentrations in the synovial fluid correlate with specific characteristics of wear particles. This study has found that the size, adhesion and nano-surface roughness of wear particles have medium strong to strong correlations with IL-6 and IL-8. This study provided evidence that the characteristics of wear particles contain valuable information for grading the disease process and the need for further evaluation of the association of properties of wear particles and the inflammatory process.
Keywords Wear particles, inflammatory cytokines, knee osteoarthritis, IL-6 and IL-8
Date received: 5 August 2014; accepted: 21 October 2014
Introduction Osteoarthritis (OA) of the human knee is one of the most common forms of arthritis and affects the elderly worldwide. It is also being increasingly recognised among young people. More than 83% of males and 87% of females had radiological evidence of OA in the age range of 55–64 years.1 The OA process is often characterised by the damage to the articular cartilage, the subchondral bone and the synovium associated with activating the inflammatory response.2,3 The process involves mechanical, biological, biochemical, molecular and enzymatic feedback loops.4,5 The final pathway of the disease is the disruption of the homeostatic balance between the matrix synthesis and degradation.4 Patients suffer pain, stiffness, increasing disability, reduced movement and swelling in the diseased knee joint. Mechanical factors play an important role in OA progression.6 Mechanical wear is a pivotal factor in causing cartilage degeneration during the disease process.6,7 In the articulation process of the knee, wear particles are produced from the articular cartilage. Therefore, similar to the composition of articular cartilage, they were mainly composed of collagen and proteoglycans (PGs). Wear particles may play an important role in the pathogenesis of degenerative
arthritis.8 Wear particles contain useful information on the knee joint conditions. For example, larger and irregular wear particles often indicate severe OA.9,10 The surface topographies and mechanical properties of wear particles contain information on particle formation and the wear mechanism of the knee joint. However, their surface topographical and mechanical properties have not been well investigated in relation to OA progression. In our previous study,11 an immobilisation procedure to prepare human knee wear particles was established to study the nano-mechanical properties and surface topographies of human wear particles in a near to their physiological environment using atomic force microscopy (AFM). In this study, the surface morphology, Young’s moduli and adhesion of wear particles will be investigated to understand the 1
School of Mechanical and Manufacturing Engineering, The University of New South Wales, Sydney, NSW, Australia 2 Infectious Diseases and Immunopathogenesis Research Group, Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia Corresponding author: Zhongxiao Peng, School of Mechanical and Manufacturing Engineering, The University of New South Wales, Sydney, NSW 2052, Australia. Email: [email protected]
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Table 1. Patient information and cytokine levels (mean) in osteoarthritic synovial fluid. OA grade
OA classification
Number of subjects Sex (M:F)
Mean age (range)
IL-6 (pg/mL)
IL-8 (pg/mL)
1 2
Softening and swelling Fragmentation and fissuring in an area 1/2 in or less in diameter Fragmentation and fissuring in an area 1/2 in or more in diameter Erosion of cartilage is down to bone
3 (1:2) 6 (2:4)
45 (17–65) 56 (45–65)
5257.8 139.0
646.5 2404.7
21 (6:15)
61 (47–81)
90.4
277.8
23 (12:11)
64 (44–89)
343.5
790.4
3 4
OA: osteoarthritis; M: male; F: female.
relation between wear particles and inflammatory changes occurring in OA of the human knee. Cytokines released from the inflamed synovium contribute to degradation in OA.2 For example, interleukin-1b (IL-1b) suppresses the anabolic mechanism in the cartilage by inhibiting the expression of type II collagen and PGs in chondrocytes.2 IL-6 from the synovial lining12 induces proteolytic cleavage.13 IL-6 was detected in the synovial fluid of OA patients2 and its concentrations correlated with radiographic changes in OA of the knee.14 IL-8 promotes the homing of inflammatory leukocytes into the synovium. The activation of these cells induces synthesis of matrix metalloproteinases (MMP)-13 in articular chondrocytes, chondrocyte apoptosis and loss of PGs.15 MMPs are involved in the progression of OA.16 IL-8 is considered to play a role in chondrocyte metabolism and OA development.2 Although wear particles can cause synovial inflammation,6 the relationships between the features of wear particles such as size, shape, surface topography and stiffness have not been investigated with the severity of the inflammation in the OA progression. As OA is a disease involving metabolic, biochemical and biomechanical factors,2 investigations of the relationships between the features of wear particles and the levels of cytokines in human knee synovial fluid could provide useful information for further understanding the OA process. The objectives of this study were to analyse the levels of IL-6 and IL-8 in osteoarthritic human knee synovial fluid and investigate the relations between the characteristics of wear particles.
Materials and methods Patients’ information and human samples’ collection The synovial fluid samples were collected from 53 patients (mean age: 61 years; 21 males and 32 females as shown in Table 1) undergoing knee arthroscopy or total knee replacement surgery in Queensland, Australia, under human ethic approval (EC00412). Together with the clinical synovial fluid samples, the patients’ arthroscopic images, MRI, X-ray reports and clinical data on OA symptoms were collected and analysed. The OA affected subjects were assessed using the Outerbridge grade system,17 which is clarified in Table 1.
Samples’ preparation Following collection of the fresh synovial fluid, the samples were centrifuged with a relative centrifugal force of 2500 g and at 4 °C for 15 min. The supernatant of the synovial fluid was taken out using a pipette and then stored at 220 °C for further analysis. Since wear particles were mainly precipitated at the bottom of the tube, the remaining synovial fluid was mixed using a vortex to obtain an evenly suspended wear particle solution. The wear particles were isolated and collected using the filtergram method and a 3 mm filter membrane.18 The collected wear particles were then deposited onto an aldehyde functional plasma polymer surface. These prepared samples were stored in a 4 °C fridge overnight so that particles could bind to the substrate before being examined. Preparation procedures have been previously described.11
Experimental measurements Synovial fluid samples were stored at 220 °C and thawed prior to use for the cytokine assays. Markers of inflammation in the synovial fluid were analysed using human cytokine multiplex kit (Bender Medsystems; Jomar Diagnostics Pty Ltd, Australia). Previous studies revealed that the concentrations of IL-6 were related to the radiographic changes in OA14 and IL-8 played a role in OA development.2 Therefore, IL-6 and IL-8 were assessed using this kit. The protocol recommended by the manufacturer was followed. Briefly, bead mixture, biotin-conjugate mixture, standard mixture and dilutions were prepared. Blank control, standard dilutions and synovial fluid samples in duplicate (25 mL, post centrifugation at 18,000 g for 5 min) were incubated with the bead mixture and biotin-conjugate mixture at room temperature for 2 h in the dark. The fluid samples were then washed using 1 mL of assay buffer twice. The supernatant was discarded following centrifugation (200 g for 5 min) and the remaining pellet resuspended. Streptavidin-phycoerythrin (PE) solution (50 mL/sample) was then added and samples were incubated for 1 h at room temperature in the dark. The samples were then washed twice as previously described and 350 mL of assay buffer was added to the samples prior to being subjected to flow cytometer (FACSCalibur; BD Biosciences, Australia). Calibration was carried out using calibration beads provided by the
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manufacturer and analysed by the software provided (FlowCytomix 3.0; Bender MedSystems, Austria). An optical microscope (Olympus U-LH100HG, Japan) was used to study the sizes and size distributions of the collected wear particles. An objective lens of 503 and a high quality charge-coupled device (CCD) camera were chosen to image hundreds of wear particles in each OA grade. The lengths in the major dimensions of wear particles were measured to evaluate their sizes. Wear particles smaller than 10 mm were classified as small particles. Particles between 10 and 20 mm were defined as medium sized wear particles. Particles greater than 20 mm were grouped as large particles. A MultiMode 8 (Bruker, Singapore) atomic force microscope was operated in a fluid mode. The PeakForce QNM (Quantitative NanoMechanics) tapping mode was used to quantitatively measure the surface topographies, Young’s moduli and adhesion of wear particles in an area of 5 mm 3 5 mm. DNP tips (tip height: 2.5–8.0 mm; front angle: 15 6 2.5°; nominal tip radius: 20 nm; nominal spring constant: 0.35 N/m) were used in the measurements. In this process, the deformation was controlled at ;10 nm.11 The scan rate was 0.1–0.5 Hz and the scan lines were 256 3 256.11Figure 1 shows the AFM images of wear particles collected from OA grades 1–4 patients. Young’s modulus was calculated using the Derjaguin Muller Toporov (DMT) model.19
Numerical and statistical analyses Based on the three-dimensional surface data acquired using the AFM, quantitative characterisations of wear particle surfaces were performed using numerical parameters defined in ISO 25178-220 and measured using the SPIP image analysis software. During the measurements, the software identified the mean plane before calculating the surface morphology values. For the nano-scaled roughness measurement, the standard cut-off lengths at a micrometre or millimetre scale cannot be applied. The definitions of key numerical parameters are provided as follows: The roughness average, Sa, is defined as Sa =
1 X N1 X 1 M j z ð xk , y l Þ j MN k = 0 l = 0
ð1Þ
where z(x, y) is height of the scale limited surface at position (x, y). M is the number of points of per profile and N is the number of profiles. The mean half wavelength, Shw, is defined as Shw =
dxðM 1Þ rmax 0:5
ð2Þ
The surface area ratio, Sdr, is defined as Sdr =
ðSurface texture areaÞ ðCross sectional areaÞ Cross sectional area ð3Þ
The mean summit curvature, Ssc, is defined as Ssc =
1 N
ðð
2 ∂2 zðx, yÞ ∂ zðx, yÞ + dxdy ∂x2 ∂y2
ð4Þ
Summitarea
Correlation analyses between the levels of IL-6 and IL8 and the features of wear particles were carried out in a 95% confidence level. Correlation coefficients indicate the strength and the direction of the linear regression model between two variables. The negative values of the correlation coefficients reveal that as one variable increases, the other decreases. The positive values denote where the two variables react in the same way, increasing or decreasing together. A large value implies a strong correlation and vice verse. Values close to or equal to 0 suggest that there is no linear relationship between the two variables. Values greater than 0.8 indicate strong correlations in this study.
Results The levels of IL-6 and IL-8 in the OA grades 1–4 synovial fluid samples are presented in Table 1. The level of IL-6 was highest at the OA grade 1 samples, continually decreased until OA grade 3 and then increased in the OA grade 4 samples. The level of IL-6 revealed a correlation with increasing OA grade and associated with a correlation coefficient of -0.753 and determination of coefficient of 0.567, which indicated that an inverse linear relation between the level of IL-6 and the increasing OA grade existed, and this linear relation could account for 56.7% of the total variation. The level of IL-8 increased substantially in OA grades 1–2 samples, decreased until the OA grade 3 samples, and then increased when OA grade 4 was reached. Correlation analysis was carried out between wear particles and detected mean levels of IL-6 and IL-8 in OA grades 1–4. Correlation coefficients between sizes of wear particles and the detected mean levels are presented in Table 2. The correlation coefficients varied with the particle sizes, which may indicate that large, medium and small wear particles interact differently with IL-6 and IL-8. IL-6 shows a negative correlation with the sizes of wear particles in OA degradation, while IL-8 reveals a positive correlation. Specifically, IL-6 had a strong correlation with medium sized particles with a correlation coefficient of 20.96 in OA degradation of grades 1–4. The results indicated that the sizes of wear particles might influence types and amounts of cytokines produced. The numerical parameters of the surface topography of wear particles were correlated with IL-6 and IL-8. The correlation coefficients between IL-6 and the numerical descriptions of wear particles were higher than those between IL-8 and wear particles. The surface roughness, Sa, had a medium strong correlation with IL-6. Half of the radial wavelength, Shw, revealed medium strong correlations with IL-6 and IL-8.
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Figure 1. AFM images for typical morphologies (2D (left) and 3D (right)) of particles collected from (a) OA grade 1, (b) OA grade 2, (c) OA grade 3 and (d) OA grade 4 knee joints.
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Table 2. Correlation coefficients of sizes, numerical descriptions, Young’s moduli and adhesion of particles and detected mean levels of IL-6 and IL-8 in synovial fluid in OA progression (OA grades 1–4). Particle Size
IL-6 IL-8
Numerical descriptions of wear particles
Large . 20 mm
Medium 10–20 mm
Small \ 10 mm
Sa (nm)
Shw (nm)
Sdr (%)
Ssc (1/nm)
Young’s moduli (MPa)
Adhesion (nN)
20.73 0.59
20.96 0.53
20.55 0.75
20.62 20.32
0.7 20.67
20.67 20.48
20.88 20.14
20.24 20.17
20.61 0.61
The surface area ratio, Sdr, presented a medium strong correlation with IL-6. Ssc showed a strong correlation with IL-6, suggested by the correlation coefficient of 20.88 in the OA progression of grades 1–4. The correlation analysis results between the nanomechanical properties (Young’s moduli and adhesion) of wear particles and IL-6 and IL-8 are presented in Table 2. Young’s moduli of wear particles had a correlation coefficient of 20.24 with IL-6 and 20.17 with IL-8. The correlation coefficients between the adhesion of wear particles and IL-6 and IL-8 were 20.61 and 0.61, separately. The results indicate that Young’s moduli of wear particles may have a weak correlation with IL-6 and IL-8. The adhesion of wear particles may have a medium to strong correlation with IL-6 and IL-8 in the degradation process of human knee. OA progression can be assessed in terms of the sizes, surface roughness, the Young’s moduli of wear particles, the detectable levels of IL-6 and IL-8 in the synovial fluid as well as by the clinical symptoms (Tables 1 and 3). Multiple OA features can be described as follows:
Young’s moduli and adhesion
OA grade 1: wear particles + detectable level of IL-6 + detectable level of IL-8 + pain, constrained physical activities (e.g. bending down); some patients felt discomfort or pain only with movement; OA grade 2: (increasing rough + softening) wear particles + decreasing level of IL-6 + increasing level of IL-8 + pain like throbbing and sharp burning, occasional swelling and constrained physical activities; OA grade 3: (increasing rough + softening) wear particles + decreasing level of IL-6 + decreasing level of IL-8 + swelling, pain and difficulty in walking; OA grade 4: (increasing rough + stiffening) wear particles + increasing level of IL-6 + increasing level of IL-8 + stiffness and pain in movement, low mobility and knee lock.
Discussion OA progression involves depletion of PGs and then deterioration of collagen II, which causes mechanical failure and complete erosion of the articular cartilage.21 This project has examined the relationships between
Table 3. Means of size, surface roughness (Sa) and Young’s moduli (E) of wear particles in different OA grades. OA grade
Size (mm)
Sa (nm)
E (MPa)
1 2 3 4
12.73 14.80 13.57 11.99
47 49 55 62
10.08 9.34 9.09 25.23
OA: osteoarthritis.
the sizes, mechanical and surface topographical properties of wear particles and the levels of IL-6 and IL-8 in the human osteoarthritic knee synovial fluid with OA progression. To the authors’ knowledge, this study has been the first investigation to the relations between wear particles and inflammation cytokines (IL-6 and IL-8) in OA process. The findings in this study indicate that selected properties of wear particles in human osteoarthritic knee joints may influence the disease process. The correlation analyses (Table 2) suggest that changes of the wear particle morphological and nanomechanical properties are associated with the concentrations of IL-6 and IL-8 observed in OA. IL-6 is involved in inducing joint and cartilage destruction.22 IL-6 can inhibit the expression of type II collagen.2 IL-8 promotes chondrocyte apoptosis and loss of PGs.12 Changes in concentrations of IL-6 and IL-8 indicate that inflammation contributes to the OA progression. The level of IL-8 increased substantially from OA grade 1 (646.5 pg/mL) to OA grade 2 (2404 pg/mL), which implies that IL-8 may impact strongly on the depletion of PGs from OA grades 1 to 2. It was observed that in OA process, accompanied with the general size increment of particles, the level of IL-6 generally decreased while that of IL-8 increased from OA grades 1 to 2. Their correlation results confirmed that above observations. OA is a complex process and involves mechanical, biochemical and biological factors.4 IL-6 can cause knee joint and cartilage destruction,22 while IL-8 could promote loss of PGs12 and recruitment of inflammatory cells to the site of inflammation. Therefore, both IL-6 and IL-8 may contribute to the generation of wear particles. Particles might cause further damage to the knee and facilitate further production of pro-inflammatory cytokines. It is known that human knee has a limited capability of
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repair itself.6 The interactions between wear particles and cytokines could be complex, and furthermore, in vitro animal studies are warranted to determine their role in OA progression. In this study, we have demonstrated that size of particles played an important role in the level of IL-6 and IL-8 observed. The size of the wear particle could also act as a possible indicator of the condition of the human knee. Collagen makes up 20% of cartilage in wet weight and PG constitutes of 7% of that.6 As the wear products of articular cartilage, wear particles probably contain more content of collagen than PGs. Previous studies have demonstrated that IL-6 can inhibit the expression of type II collagen2 and IL-8 promotes loss of PGs.12 However, future analysis is required to determine whether the composition of wear particles has an effect on OA progression. As IL-6 and IL-8 are directly involved in the disease process and associated with the different characteristics of the wear particles, it is observable that the properties of wear particles may also influence the disease process and drive the deterioration process causing damage to the knee. IL-6 is a pro-inflammatory cytokine secreted by macrophages and osteoblasts that increases inflammatory changes in the synovium and caused tissue damage. IL-8 is a major chemotactic factor that also attracts more inflammatory cells to sites of inflammation. Therefore, the larger, rougher and stiffer the particles are, the more damage and higher production of cytokines there would be in the early stages of OA. The causes for the above results are not entirely clear. However, it is believed that wear particles may be phagocytosed by magrophages.8 Human macrophages are approximately 21 mm.23 Production of IL-6 may induce proteolytic cleavage of the knee joint tissue. Macrophages are usually the first cells to release IL-8 to attract other cells.24 In the process, the majority of wear particles which are smaller than macrophages might be treated as cellular debris or an aggregate by these cells. These wear particles thus might be engulfed by the macrophages and may stimulate the inflammatory response. There is also a possibility that wear particles become involved in initiating a secondary inflammatory process.8 The correlation between the features of wear particles (e.g. size, Sa and adhesion) and levels of IL-6 and IL-8 support the above model. In this study, techniques to investigate the relation between wear particles and inflammation in the human knee OA have been developed. This is the first time that the progression of OA features has been characterised using combined information from the surface roughness and stiffness of wear particles, and detectable levels of cytokines in synovial fluid as well as the clinical symptoms. In addition to existing OA features (e.g. depletion of PGs and disruption of collagen meshwork), this project has revealed new features using the properties of wear particles and detectable levels of cytokines, which can be combined with clinical symptoms. To have a further insight into the disease process, a more comprehensive
study with more samples in the different OA grades with age-matched participants will enable a better understanding of the association between the properties of wear particles and the inflammatory changes they may participate in OA. Furthermore, properties of wear particle may be useful markers to not only grade OA but also have prognostic values.
Conclusion This study has investigated the relationships between two well-characterised inflammatory mediators involved in the disease process of OA and wear particles. It was observed that the larger, rougher and stiffer the particles were, the more damage and higher production of cytokines there would be in the early stages of OA. The results revealed that wear particle sizes may influence the concentrations of these cytokines. Surface roughness, mean half wavelength, mean summit curvature and adhesion of wear particles showed strong correlations with IL-6 and IL-8. This study demonstrates the need to study a variety of wear particle properties including size, surface roughness, stiffness and adhesion to determine the dynamic process that is involved in OA. The findings reported here assist a further understanding of the OA process. Acknowledgements The authors would like to thank Dr James Price for providing the clinical samples and associated data for the project. The authors also acknowledge Laurice Baxter, Emma Horrocks, Peter Bui, Tyler Chin and Yuan Tian for their assistance in the sample collection process. Associate Professor K. Vasilev at University of South Australia, Australia, is thanked for proving aldehyde surfaces. Dr Venkat Vangaveti is acknowledged for carrying out the cytokines analysis. Thanks go to the Mark Wainwright Analytical Centre (Biomedical Imaging facility) at the University of New South Wales (UNSW Australia) for their support in providing the AFM facility for the study. China Scholarship Council (CSC) is acknowledged for providing Meiling Wang’s PhD scholarship during her PhD candidature at UNSW Australia. Faculty of Engineering at UNSW is thanked for awarding a postdoctoral writing fellowship. Declaration of conflicting interests The authors declare that there is no conflict of interest. Funding This study was funded by the Australian Research Council (ARC) (DP1093975). References 1. Kean W, Kean R and Buchanan W. Osteoarthritis: symptoms, signs and source of pain. Inflammopharmacology 2004; 12: 3–31. 2. Haseeb A and Haqqi TM. Immunopathogenesis of osteoarthritis. Clin Immunol 2013; 146: 185–196.
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3. Hunter D. Osteoarthritis. Best Pract Res Clin Rheumatol 2011; 25: 801–814. 4. Martel-Pelletier J, Boileau C, Pelletier J, et al. Cartilage in normal and osteoarthritis conditions. Best Pract Res Clin Rheumatol 2008; 22: 351–384. 5. Felson D, Lawrence R, Dieppe P, et al. Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med 2000; 133: 635–646. 6. Ewing J. Articular cartilage and knee joint function: basic science and arthroscopy. New York: Raven Press, 1990. 7. Aigner T and McKenna L. Molecular pathology and pathobiology of osteoarthritic cartilage. Cell Mol Life Sci 2002; 59: 5–18. 8. Mears DC, Hanley EN, Rutkowski R, et al. Ferrography: its application to the study of human joint wear. Wear 1978; 50: 115–125. 9. Kuster M, Podsiadlo P and Stachowiak G. Shape of wear particles found in human knee joints and their relationship to osteoarthritis. Br J Rheumatol 1998; 37: 978–984. 10. Zha ZG, Yao C-C, Tu M, et al. Correlation of the size and shape of the solid particles in synovial fluid and knee joint disease. Curr Appl Phys 2007; 7: e112–e115. 11. Wang M, Peng Z, Vasilev K, et al. Investigation of wear particles generated in human knee joints using atomic force microscopy. Tribol Lett 2013; 51: 161–170. 12. Doss F, Menard J, Hauschild M, et al. Elevated IL-6 levels in the synovial fluid of osteoarthritis patients stem from plasma cells. Scand J Rheumatol 2007; 36: 136–139. 13. Smith KA. Interleukin-2: inception, impact, and implications. Science 1988; 240: 1169–1176. 14. Tsuchida AI, Beekhuizen M, Rutgers M, et al. Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model. Arthritis Res Ther 2012; 14(6): 1–12.
15. Chauffier K, Laiguillon MC, Bougault C, et al. Induction of the chemokine IL-8/Kc by the articular cartilage: possible influence on osteoarthritis. Joint Bone Spine 2012; 79: 604–609. 16. Punzi L, Oliviero F, Ramonda R, et al. Laboratory findings in osteoarthritis. Semin Arthritis Rheum 2004; 34: 58–61. 17. Outerbridge R and Dunlop J. The problem of chondromalacia patellae. Clin Orthop Relat Res 1975; 110: 177–196. 18. Peng Z. Osteoarthritis diagnosis using wear particle analysis technique: investigation of correlation between particle and cartilage surface in walking process. Wear 2007; 262: 630–640. 19. Derjaguin BV, Muller VM and Toporov YP. Effect of contact deformations on the adhesion of particles. J Colloid Interface Sci 1975; 53: 314–326. 20. ISO 25178-2: 2006. Geometrical product specifications (GPS) – surface texture: areal – part 2: terms, definitions and surface texture parameters. 21. Stolz M, Gottardi R, Raiteri R, et al. Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy. Nat Nanotechnol 2009; 4: 186–192. 22. Drake R, Vogl W, Mitchell A, et al. Gray’s anatomy for students. Philadelphia, PA: Elsevier/Churchill Livingstone, 2005. 23. Krombach F, Mu¨nzing S, Allmeling, et al. Cell size of alveolar macrophages: an interspecies comparison. Environ Health Perspect 1997; 105(Suppl. 5): 1261–1263. 24. Jones SA, Horiuchi S, Topley N, et al. The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J 2001; 15: 43–58.
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Investigations of wear particles and selected cytokines in human osteoarthritic knee joints
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(11) 1176–1182 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914559570 pih.sagepub.com
Meiling Wang1, Natkunam Ketheesan2 and Zhongxiao Peng1
Abstract Inflammation of the synovial membrane (synovitis) is considered to drive the process that leads to osteoarthritis. However, the relationships between the mediators of inflammation and the properties of wear particles are not fully understood. In this study, the levels of IL-6 and IL-8 were assessed in different grades of osteoarthritis to determine whether their concentrations in the synovial fluid correlate with specific characteristics of wear particles. This study has found that the size, adhesion and nano-surface roughness of wear particles have medium strong to strong correlations with IL-6 and IL-8. This study provided evidence that the characteristics of wear particles contain valuable information for grading the disease process and the need for further evaluation of the association of properties of wear particles and the inflammatory process.
Keywords Wear particles, inflammatory cytokines, knee osteoarthritis, IL-6 and IL-8
Date received: 5 August 2014; accepted: 21 October 2014
Introduction Osteoarthritis (OA) of the human knee is one of the most common forms of arthritis and affects the elderly worldwide. It is also being increasingly recognised among young people. More than 83% of males and 87% of females had radiological evidence of OA in the age range of 55–64 years.1 The OA process is often characterised by the damage to the articular cartilage, the subchondral bone and the synovium associated with activating the inflammatory response.2,3 The process involves mechanical, biological, biochemical, molecular and enzymatic feedback loops.4,5 The final pathway of the disease is the disruption of the homeostatic balance between the matrix synthesis and degradation.4 Patients suffer pain, stiffness, increasing disability, reduced movement and swelling in the diseased knee joint. Mechanical factors play an important role in OA progression.6 Mechanical wear is a pivotal factor in causing cartilage degeneration during the disease process.6,7 In the articulation process of the knee, wear particles are produced from the articular cartilage. Therefore, similar to the composition of articular cartilage, they were mainly composed of collagen and proteoglycans (PGs). Wear particles may play an important role in the pathogenesis of degenerative
arthritis.8 Wear particles contain useful information on the knee joint conditions. For example, larger and irregular wear particles often indicate severe OA.9,10 The surface topographies and mechanical properties of wear particles contain information on particle formation and the wear mechanism of the knee joint. However, their surface topographical and mechanical properties have not been well investigated in relation to OA progression. In our previous study,11 an immobilisation procedure to prepare human knee wear particles was established to study the nano-mechanical properties and surface topographies of human wear particles in a near to their physiological environment using atomic force microscopy (AFM). In this study, the surface morphology, Young’s moduli and adhesion of wear particles will be investigated to understand the 1
School of Mechanical and Manufacturing Engineering, The University of New South Wales, Sydney, NSW, Australia 2 Infectious Diseases and Immunopathogenesis Research Group, Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australia Corresponding author: Zhongxiao Peng, School of Mechanical and Manufacturing Engineering, The University of New South Wales, Sydney, NSW 2052, Australia. Email: [email protected]
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Table 1. Patient information and cytokine levels (mean) in osteoarthritic synovial fluid. OA grade
OA classification
Number of subjects Sex (M:F)
Mean age (range)
IL-6 (pg/mL)
IL-8 (pg/mL)
1 2
Softening and swelling Fragmentation and fissuring in an area 1/2 in or less in diameter Fragmentation and fissuring in an area 1/2 in or more in diameter Erosion of cartilage is down to bone
3 (1:2) 6 (2:4)
45 (17–65) 56 (45–65)
5257.8 139.0
646.5 2404.7
21 (6:15)
61 (47–81)
90.4
277.8
23 (12:11)
64 (44–89)
343.5
790.4
3 4
OA: osteoarthritis; M: male; F: female.
relation between wear particles and inflammatory changes occurring in OA of the human knee. Cytokines released from the inflamed synovium contribute to degradation in OA.2 For example, interleukin-1b (IL-1b) suppresses the anabolic mechanism in the cartilage by inhibiting the expression of type II collagen and PGs in chondrocytes.2 IL-6 from the synovial lining12 induces proteolytic cleavage.13 IL-6 was detected in the synovial fluid of OA patients2 and its concentrations correlated with radiographic changes in OA of the knee.14 IL-8 promotes the homing of inflammatory leukocytes into the synovium. The activation of these cells induces synthesis of matrix metalloproteinases (MMP)-13 in articular chondrocytes, chondrocyte apoptosis and loss of PGs.15 MMPs are involved in the progression of OA.16 IL-8 is considered to play a role in chondrocyte metabolism and OA development.2 Although wear particles can cause synovial inflammation,6 the relationships between the features of wear particles such as size, shape, surface topography and stiffness have not been investigated with the severity of the inflammation in the OA progression. As OA is a disease involving metabolic, biochemical and biomechanical factors,2 investigations of the relationships between the features of wear particles and the levels of cytokines in human knee synovial fluid could provide useful information for further understanding the OA process. The objectives of this study were to analyse the levels of IL-6 and IL-8 in osteoarthritic human knee synovial fluid and investigate the relations between the characteristics of wear particles.
Materials and methods Patients’ information and human samples’ collection The synovial fluid samples were collected from 53 patients (mean age: 61 years; 21 males and 32 females as shown in Table 1) undergoing knee arthroscopy or total knee replacement surgery in Queensland, Australia, under human ethic approval (EC00412). Together with the clinical synovial fluid samples, the patients’ arthroscopic images, MRI, X-ray reports and clinical data on OA symptoms were collected and analysed. The OA affected subjects were assessed using the Outerbridge grade system,17 which is clarified in Table 1.
Samples’ preparation Following collection of the fresh synovial fluid, the samples were centrifuged with a relative centrifugal force of 2500 g and at 4 °C for 15 min. The supernatant of the synovial fluid was taken out using a pipette and then stored at 220 °C for further analysis. Since wear particles were mainly precipitated at the bottom of the tube, the remaining synovial fluid was mixed using a vortex to obtain an evenly suspended wear particle solution. The wear particles were isolated and collected using the filtergram method and a 3 mm filter membrane.18 The collected wear particles were then deposited onto an aldehyde functional plasma polymer surface. These prepared samples were stored in a 4 °C fridge overnight so that particles could bind to the substrate before being examined. Preparation procedures have been previously described.11
Experimental measurements Synovial fluid samples were stored at 220 °C and thawed prior to use for the cytokine assays. Markers of inflammation in the synovial fluid were analysed using human cytokine multiplex kit (Bender Medsystems; Jomar Diagnostics Pty Ltd, Australia). Previous studies revealed that the concentrations of IL-6 were related to the radiographic changes in OA14 and IL-8 played a role in OA development.2 Therefore, IL-6 and IL-8 were assessed using this kit. The protocol recommended by the manufacturer was followed. Briefly, bead mixture, biotin-conjugate mixture, standard mixture and dilutions were prepared. Blank control, standard dilutions and synovial fluid samples in duplicate (25 mL, post centrifugation at 18,000 g for 5 min) were incubated with the bead mixture and biotin-conjugate mixture at room temperature for 2 h in the dark. The fluid samples were then washed using 1 mL of assay buffer twice. The supernatant was discarded following centrifugation (200 g for 5 min) and the remaining pellet resuspended. Streptavidin-phycoerythrin (PE) solution (50 mL/sample) was then added and samples were incubated for 1 h at room temperature in the dark. The samples were then washed twice as previously described and 350 mL of assay buffer was added to the samples prior to being subjected to flow cytometer (FACSCalibur; BD Biosciences, Australia). Calibration was carried out using calibration beads provided by the
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manufacturer and analysed by the software provided (FlowCytomix 3.0; Bender MedSystems, Austria). An optical microscope (Olympus U-LH100HG, Japan) was used to study the sizes and size distributions of the collected wear particles. An objective lens of 503 and a high quality charge-coupled device (CCD) camera were chosen to image hundreds of wear particles in each OA grade. The lengths in the major dimensions of wear particles were measured to evaluate their sizes. Wear particles smaller than 10 mm were classified as small particles. Particles between 10 and 20 mm were defined as medium sized wear particles. Particles greater than 20 mm were grouped as large particles. A MultiMode 8 (Bruker, Singapore) atomic force microscope was operated in a fluid mode. The PeakForce QNM (Quantitative NanoMechanics) tapping mode was used to quantitatively measure the surface topographies, Young’s moduli and adhesion of wear particles in an area of 5 mm 3 5 mm. DNP tips (tip height: 2.5–8.0 mm; front angle: 15 6 2.5°; nominal tip radius: 20 nm; nominal spring constant: 0.35 N/m) were used in the measurements. In this process, the deformation was controlled at ;10 nm.11 The scan rate was 0.1–0.5 Hz and the scan lines were 256 3 256.11Figure 1 shows the AFM images of wear particles collected from OA grades 1–4 patients. Young’s modulus was calculated using the Derjaguin Muller Toporov (DMT) model.19
Numerical and statistical analyses Based on the three-dimensional surface data acquired using the AFM, quantitative characterisations of wear particle surfaces were performed using numerical parameters defined in ISO 25178-220 and measured using the SPIP image analysis software. During the measurements, the software identified the mean plane before calculating the surface morphology values. For the nano-scaled roughness measurement, the standard cut-off lengths at a micrometre or millimetre scale cannot be applied. The definitions of key numerical parameters are provided as follows: The roughness average, Sa, is defined as Sa =
1 X N1 X 1 M j z ð xk , y l Þ j MN k = 0 l = 0
ð1Þ
where z(x, y) is height of the scale limited surface at position (x, y). M is the number of points of per profile and N is the number of profiles. The mean half wavelength, Shw, is defined as Shw =
dxðM 1Þ rmax 0:5
ð2Þ
The surface area ratio, Sdr, is defined as Sdr =
ðSurface texture areaÞ ðCross sectional areaÞ Cross sectional area ð3Þ
The mean summit curvature, Ssc, is defined as Ssc =
1 N
ðð
2 ∂2 zðx, yÞ ∂ zðx, yÞ + dxdy ∂x2 ∂y2
ð4Þ
Summitarea
Correlation analyses between the levels of IL-6 and IL8 and the features of wear particles were carried out in a 95% confidence level. Correlation coefficients indicate the strength and the direction of the linear regression model between two variables. The negative values of the correlation coefficients reveal that as one variable increases, the other decreases. The positive values denote where the two variables react in the same way, increasing or decreasing together. A large value implies a strong correlation and vice verse. Values close to or equal to 0 suggest that there is no linear relationship between the two variables. Values greater than 0.8 indicate strong correlations in this study.
Results The levels of IL-6 and IL-8 in the OA grades 1–4 synovial fluid samples are presented in Table 1. The level of IL-6 was highest at the OA grade 1 samples, continually decreased until OA grade 3 and then increased in the OA grade 4 samples. The level of IL-6 revealed a correlation with increasing OA grade and associated with a correlation coefficient of -0.753 and determination of coefficient of 0.567, which indicated that an inverse linear relation between the level of IL-6 and the increasing OA grade existed, and this linear relation could account for 56.7% of the total variation. The level of IL-8 increased substantially in OA grades 1–2 samples, decreased until the OA grade 3 samples, and then increased when OA grade 4 was reached. Correlation analysis was carried out between wear particles and detected mean levels of IL-6 and IL-8 in OA grades 1–4. Correlation coefficients between sizes of wear particles and the detected mean levels are presented in Table 2. The correlation coefficients varied with the particle sizes, which may indicate that large, medium and small wear particles interact differently with IL-6 and IL-8. IL-6 shows a negative correlation with the sizes of wear particles in OA degradation, while IL-8 reveals a positive correlation. Specifically, IL-6 had a strong correlation with medium sized particles with a correlation coefficient of 20.96 in OA degradation of grades 1–4. The results indicated that the sizes of wear particles might influence types and amounts of cytokines produced. The numerical parameters of the surface topography of wear particles were correlated with IL-6 and IL-8. The correlation coefficients between IL-6 and the numerical descriptions of wear particles were higher than those between IL-8 and wear particles. The surface roughness, Sa, had a medium strong correlation with IL-6. Half of the radial wavelength, Shw, revealed medium strong correlations with IL-6 and IL-8.
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Figure 1. AFM images for typical morphologies (2D (left) and 3D (right)) of particles collected from (a) OA grade 1, (b) OA grade 2, (c) OA grade 3 and (d) OA grade 4 knee joints.
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Table 2. Correlation coefficients of sizes, numerical descriptions, Young’s moduli and adhesion of particles and detected mean levels of IL-6 and IL-8 in synovial fluid in OA progression (OA grades 1–4). Particle Size
IL-6 IL-8
Numerical descriptions of wear particles
Large . 20 mm
Medium 10–20 mm
Small \ 10 mm
Sa (nm)
Shw (nm)
Sdr (%)
Ssc (1/nm)
Young’s moduli (MPa)
Adhesion (nN)
20.73 0.59
20.96 0.53
20.55 0.75
20.62 20.32
0.7 20.67
20.67 20.48
20.88 20.14
20.24 20.17
20.61 0.61
The surface area ratio, Sdr, presented a medium strong correlation with IL-6. Ssc showed a strong correlation with IL-6, suggested by the correlation coefficient of 20.88 in the OA progression of grades 1–4. The correlation analysis results between the nanomechanical properties (Young’s moduli and adhesion) of wear particles and IL-6 and IL-8 are presented in Table 2. Young’s moduli of wear particles had a correlation coefficient of 20.24 with IL-6 and 20.17 with IL-8. The correlation coefficients between the adhesion of wear particles and IL-6 and IL-8 were 20.61 and 0.61, separately. The results indicate that Young’s moduli of wear particles may have a weak correlation with IL-6 and IL-8. The adhesion of wear particles may have a medium to strong correlation with IL-6 and IL-8 in the degradation process of human knee. OA progression can be assessed in terms of the sizes, surface roughness, the Young’s moduli of wear particles, the detectable levels of IL-6 and IL-8 in the synovial fluid as well as by the clinical symptoms (Tables 1 and 3). Multiple OA features can be described as follows:
Young’s moduli and adhesion
OA grade 1: wear particles + detectable level of IL-6 + detectable level of IL-8 + pain, constrained physical activities (e.g. bending down); some patients felt discomfort or pain only with movement; OA grade 2: (increasing rough + softening) wear particles + decreasing level of IL-6 + increasing level of IL-8 + pain like throbbing and sharp burning, occasional swelling and constrained physical activities; OA grade 3: (increasing rough + softening) wear particles + decreasing level of IL-6 + decreasing level of IL-8 + swelling, pain and difficulty in walking; OA grade 4: (increasing rough + stiffening) wear particles + increasing level of IL-6 + increasing level of IL-8 + stiffness and pain in movement, low mobility and knee lock.
Discussion OA progression involves depletion of PGs and then deterioration of collagen II, which causes mechanical failure and complete erosion of the articular cartilage.21 This project has examined the relationships between
Table 3. Means of size, surface roughness (Sa) and Young’s moduli (E) of wear particles in different OA grades. OA grade
Size (mm)
Sa (nm)
E (MPa)
1 2 3 4
12.73 14.80 13.57 11.99
47 49 55 62
10.08 9.34 9.09 25.23
OA: osteoarthritis.
the sizes, mechanical and surface topographical properties of wear particles and the levels of IL-6 and IL-8 in the human osteoarthritic knee synovial fluid with OA progression. To the authors’ knowledge, this study has been the first investigation to the relations between wear particles and inflammation cytokines (IL-6 and IL-8) in OA process. The findings in this study indicate that selected properties of wear particles in human osteoarthritic knee joints may influence the disease process. The correlation analyses (Table 2) suggest that changes of the wear particle morphological and nanomechanical properties are associated with the concentrations of IL-6 and IL-8 observed in OA. IL-6 is involved in inducing joint and cartilage destruction.22 IL-6 can inhibit the expression of type II collagen.2 IL-8 promotes chondrocyte apoptosis and loss of PGs.12 Changes in concentrations of IL-6 and IL-8 indicate that inflammation contributes to the OA progression. The level of IL-8 increased substantially from OA grade 1 (646.5 pg/mL) to OA grade 2 (2404 pg/mL), which implies that IL-8 may impact strongly on the depletion of PGs from OA grades 1 to 2. It was observed that in OA process, accompanied with the general size increment of particles, the level of IL-6 generally decreased while that of IL-8 increased from OA grades 1 to 2. Their correlation results confirmed that above observations. OA is a complex process and involves mechanical, biochemical and biological factors.4 IL-6 can cause knee joint and cartilage destruction,22 while IL-8 could promote loss of PGs12 and recruitment of inflammatory cells to the site of inflammation. Therefore, both IL-6 and IL-8 may contribute to the generation of wear particles. Particles might cause further damage to the knee and facilitate further production of pro-inflammatory cytokines. It is known that human knee has a limited capability of
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repair itself.6 The interactions between wear particles and cytokines could be complex, and furthermore, in vitro animal studies are warranted to determine their role in OA progression. In this study, we have demonstrated that size of particles played an important role in the level of IL-6 and IL-8 observed. The size of the wear particle could also act as a possible indicator of the condition of the human knee. Collagen makes up 20% of cartilage in wet weight and PG constitutes of 7% of that.6 As the wear products of articular cartilage, wear particles probably contain more content of collagen than PGs. Previous studies have demonstrated that IL-6 can inhibit the expression of type II collagen2 and IL-8 promotes loss of PGs.12 However, future analysis is required to determine whether the composition of wear particles has an effect on OA progression. As IL-6 and IL-8 are directly involved in the disease process and associated with the different characteristics of the wear particles, it is observable that the properties of wear particles may also influence the disease process and drive the deterioration process causing damage to the knee. IL-6 is a pro-inflammatory cytokine secreted by macrophages and osteoblasts that increases inflammatory changes in the synovium and caused tissue damage. IL-8 is a major chemotactic factor that also attracts more inflammatory cells to sites of inflammation. Therefore, the larger, rougher and stiffer the particles are, the more damage and higher production of cytokines there would be in the early stages of OA. The causes for the above results are not entirely clear. However, it is believed that wear particles may be phagocytosed by magrophages.8 Human macrophages are approximately 21 mm.23 Production of IL-6 may induce proteolytic cleavage of the knee joint tissue. Macrophages are usually the first cells to release IL-8 to attract other cells.24 In the process, the majority of wear particles which are smaller than macrophages might be treated as cellular debris or an aggregate by these cells. These wear particles thus might be engulfed by the macrophages and may stimulate the inflammatory response. There is also a possibility that wear particles become involved in initiating a secondary inflammatory process.8 The correlation between the features of wear particles (e.g. size, Sa and adhesion) and levels of IL-6 and IL-8 support the above model. In this study, techniques to investigate the relation between wear particles and inflammation in the human knee OA have been developed. This is the first time that the progression of OA features has been characterised using combined information from the surface roughness and stiffness of wear particles, and detectable levels of cytokines in synovial fluid as well as the clinical symptoms. In addition to existing OA features (e.g. depletion of PGs and disruption of collagen meshwork), this project has revealed new features using the properties of wear particles and detectable levels of cytokines, which can be combined with clinical symptoms. To have a further insight into the disease process, a more comprehensive
study with more samples in the different OA grades with age-matched participants will enable a better understanding of the association between the properties of wear particles and the inflammatory changes they may participate in OA. Furthermore, properties of wear particle may be useful markers to not only grade OA but also have prognostic values.
Conclusion This study has investigated the relationships between two well-characterised inflammatory mediators involved in the disease process of OA and wear particles. It was observed that the larger, rougher and stiffer the particles were, the more damage and higher production of cytokines there would be in the early stages of OA. The results revealed that wear particle sizes may influence the concentrations of these cytokines. Surface roughness, mean half wavelength, mean summit curvature and adhesion of wear particles showed strong correlations with IL-6 and IL-8. This study demonstrates the need to study a variety of wear particle properties including size, surface roughness, stiffness and adhesion to determine the dynamic process that is involved in OA. The findings reported here assist a further understanding of the OA process. Acknowledgements The authors would like to thank Dr James Price for providing the clinical samples and associated data for the project. The authors also acknowledge Laurice Baxter, Emma Horrocks, Peter Bui, Tyler Chin and Yuan Tian for their assistance in the sample collection process. Associate Professor K. Vasilev at University of South Australia, Australia, is thanked for proving aldehyde surfaces. Dr Venkat Vangaveti is acknowledged for carrying out the cytokines analysis. Thanks go to the Mark Wainwright Analytical Centre (Biomedical Imaging facility) at the University of New South Wales (UNSW Australia) for their support in providing the AFM facility for the study. China Scholarship Council (CSC) is acknowledged for providing Meiling Wang’s PhD scholarship during her PhD candidature at UNSW Australia. Faculty of Engineering at UNSW is thanked for awarding a postdoctoral writing fellowship. Declaration of conflicting interests The authors declare that there is no conflict of interest. Funding This study was funded by the Australian Research Council (ARC) (DP1093975). References 1. Kean W, Kean R and Buchanan W. Osteoarthritis: symptoms, signs and source of pain. Inflammopharmacology 2004; 12: 3–31. 2. Haseeb A and Haqqi TM. Immunopathogenesis of osteoarthritis. Clin Immunol 2013; 146: 185–196.
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