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Osteoarthritic synovial fluid rheology and correlation with protein concentration Madkhali, Anwar Ali 2013

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Osteoarthritic Synovial Fluid Rheology and Correlation with Protein Concentration  by Anwar Ali Madkhali B.Sc., King Saud University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Master of Applied Science in THE FACULTY OF GRADUATE STUDIES (Biomedical Engineering)  The University Of British Columbia (Vancouver) January 2013 © Anwar Ali Madkhali, 2013  Abstract Synovial fluid (SF) is a lubricant for articulating joints. The study of SF rheological properties has gained significance due to SF viscoelastic properties, and SF’s ability to sustain a considerable load. The rheological performance of SF is linked to the joint’s condition. A joint disease such as osteoarthritis (OA) reduces SF rheological properties. This study is aimed at investigating the shear and extensional rheological properties of osteoarthritic synovial fluid (OA SF). Additionally, this study is aimed at correlating SF rheological properties with its protein concentration. Shear rheological properties of 35 OA SF samples were investigated at a physiological temperature (37 °C) using cone-and-plate shear rheometer. Furthermore, the effects of the temperature, the centrifugation, and the storage at -20 °C for two weeks were also studied on some samples. Additionally, the time-dependent rheological properties were investigated by rotation and oscillation tests. Extensional rheological properties were studied using a capillary breakup extensional rheometer (CaBER). First, the effects of different CaBER configurations on the extensional rheological measurements were investigated in order to determine the optimal configuration. Then, the extensional rheological properties of 21 OA SF samples were studied. The protein concentrations of SF were determined using a bicinchoninic acid (BCA) protein assay kit. I also investigate the correlations between rheological properties and protein concentration. The understanding of SF rheological properties will lead to a better understanding of its lubrication properties, and to the development of a rheological analogue to SF or to a periprosthetic fluid.  ii  Preface The authors of Chapter 2 are Anwar Madkhali and Dr. Dana Grecov. Dr. Grecov recognized the need to further understand the rheological properties of synovial fluid, and the need to study the correlations between synovial fluid rheological properties and total protein concentration. I did the rheological and compositional testing. The data was analysed by Dr. Grecov and me. A version of Chapter 2 will be submitted for publication: Madkhali, Anwar and Grecov, Dana, ”Osteoarthritic synovial fluid shear rheology and correlations with protein concentration,” 2013. The authors of Chapter 3 are Anwar Madkhali, Dr. Dana Grecov and Samin Fakhraei. Dr. Grecov recognized the need to further investigate SF rheological properties under an extensional flow, and the need to correlate the synovial fluid extensional rheological properties with the synovial fluid protein concentration. I carried out the experimental testing. Samin assisted me post processing the data from the capillary breakup extensional rheometer. The data was analysed by Dr. Grecov and me. A version of Chapter 3 will be submitted for publication: Madkhali, Anwar; Fakhraei, Samin and Grecov, Dana, ”Osteoarthritic synovial fluid and viscosupplement extensional rheology,” 2013. The collection of synovial fluid from human subjects in Chapter 2 and Chapter 3 were approved by the University of British Columbia Behavioural Research Ethics Board (H08-02272).  iii  Table of Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ii  Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  iii  Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  iv  List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  vii  List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ix  Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xi  Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xv  Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xvi  Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1  Synovial Joint . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1.1  Articular Cartilage . . . . . . . . . . . . . . . . . . . . .  1  1.1.2  Synovium . . . . . . . . . . . . . . . . . . . . . . . . . .  3  1.1.3  Synovial Fluid . . . . . . . . . . . . . . . . . . . . . . .  3  1.2  Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4  1.3  Viscosupplementation . . . . . . . . . . . . . . . . . . . . . . . .  4  1.4  Lubrication of Synovial Joints . . . . . . . . . . . . . . . . . . .  5  iv  1.5  2  Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6  1.5.1  Shear Rheology . . . . . . . . . . . . . . . . . . . . . . .  7  1.5.2  Capillary Breakup Extensional Rheometer . . . . . . . . .  9  1.6  Rheology of Synovial Fluid . . . . . . . . . . . . . . . . . . . . .  12  1.7  Purpose of Research . . . . . . . . . . . . . . . . . . . . . . . .  14  1.8  Research Objectives . . . . . . . . . . . . . . . . . . . . . . . . .  15  1.9  Thesis Organization . . . . . . . . . . . . . . . . . . . . . . . . .  15  Osteoarthritic Synovial Fluid Shear Rheology and Correlations with Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . . .  17  2.1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  17  2.2  Materials and Methods . . . . . . . . . . . . . . . . . . . . . . .  19  2.2.1  Materials . . . . . . . . . . . . . . . . . . . . . . . . . .  19  2.2.2  Methods  . . . . . . . . . . . . . . . . . . . . . . . . . .  20  Results and Discussion . . . . . . . . . . . . . . . . . . . . . . .  23  2.3.1  Rheological Properties at 37 °C . . . . . . . . . . . . . .  23  2.3.2  Temperature Effect . . . . . . . . . . . . . . . . . . . . .  30  2.3.3  Centrifugation Effect . . . . . . . . . . . . . . . . . . . .  33  2.3.4  Storage Effect . . . . . . . . . . . . . . . . . . . . . . . .  36  2.3.5  Total Protein Concentration . . . . . . . . . . . . . . . .  38  2.3.6  Correlations between Total Protein Concentration and Rhe-  2.3  2.4 3  ological Properties . . . . . . . . . . . . . . . . . . . . .  39  Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  40  Osteoarthritic Synovial Fluid and Viscosupplement Extensional Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  42  3.1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  42  3.2  Materials and Methods . . . . . . . . . . . . . . . . . . . . . . .  43  3.2.1  Materials . . . . . . . . . . . . . . . . . . . . . . . . . .  43  3.2.2  Methods  . . . . . . . . . . . . . . . . . . . . . . . . . .  43  Results and Discussion . . . . . . . . . . . . . . . . . . . . . . .  46  3.3.1  Surface Tension . . . . . . . . . . . . . . . . . . . . . . .  46  3.3.2  Influence of Endplates Diameter and Final Aspect Ratio .  46  3.3  v  Influence of Step-stretch Parameters . . . . . . . . . . . .  49  3.3.4  Extensional Rheological Properties . . . . . . . . . . . .  55  3.3.5  Correlation between Total Protein Concentration and Extensional Rheology . . . . . . . . . . . . . . . . . . . . .  57  Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  61  Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  62  4.1  Shear Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . .  62  4.2  Extensional Rheology . . . . . . . . . . . . . . . . . . . . . . . .  63  4.3  Correlations between Rheological Properties and Protein Concen-  3.4 4  3.3.3  tration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  64  4.4  Contributions to the Research . . . . . . . . . . . . . . . . . . . .  64  4.5  Limitations to Study . . . . . . . . . . . . . . . . . . . . . . . .  64  4.6  Recommendations for Future Work . . . . . . . . . . . . . . . . .  65  Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  66  A Experimental Data . . . . . . . . . . . . . . . . . . . . . . . . . . . .  77  vi  List of Tables Table 1.1  The rheological properties of healthy and OA SF reported in the literature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  Table 2.1  13  Comparison of OA SF and VS rheological properties between the present study and different literature studies of OA and healthy SF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  Table 2.2  25  Percentage changes in the steady state rheological properties for OA SF samples (Average ± SD). . . . . . . . . . . . . . . . .  32  Table 2.3  Flow activation energy for six OA SF samples. . . . . . . . . .  32  Table 2.4  Percentage changes in the dynamic rheological properties for OA SF samples (Average ± SD). . . . . . . . . . . . . . . . .  Table 3.1  Extensional rheological properties for four OA SF samples using 4 mm endplates at different final aspect ratios . . . . . . .  Table 3.2  34  48  Extensional rheological properties for four OA SF samples using 6 mm endplates at different final aspect ratios . . . . . . .  50  Table 3.3  Results from repeated CaBER experiments for SF#30 . . . . .  51  Table 3.4  Extensional rheological properties for five OA SF samples and VS using three different final heights h f  Table 3.5  . . . . . . . . . . . .  51  Extensional rheological properties for six OA SF samples and VS using three different extension rates . . . . . . . . . . . . .  53  Table 3.6  Results from repeated CaBER experiments for VS . . . . . . .  56  Table 3.7  Extensional rheological properties for 21 OA SF samples . . .  58  Table A.1  Demographic, and synovial fluid appearance. . . . . . . . . . .  78  vii  Table A.2  Steady state rheological properties for OA SF samples at 37 °C.  79  Table A.3  Dynamic rheological properties for OA SF samples at 37 °C. .  80  Table A.4  Relative viscosity change for OA SF when applying constant 0.05 s−1 , for 1800 s. . . . . . . . . . . . . . . . . . . . . . . .  Table A.5  82  Relative change in storage modulus G , loss modulus G , and viscosity η when applying the oscillation/rotation/oscillation profile presented in Figure 2.1. . . . . . . . . . . . . . . . . .  83  Table A.6  Steady state rheological properties for OA SF samples at 25 °C.  83  Table A.7  Dynamic rheological properties for OA SF samples at 25 °C. .  84  Table A.8  Steady state rheological properties for OA SF samples after centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . .  Table A.9  84  Dynamic rheological properties for OA SF samples after centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . .  84  Table A.10 Steady state rheological properties for OA SF samples after storage for 2 weeks at -20 °C. . . . . . . . . . . . . . . . . . .  85  Table A.11 Dynamic rheological properties for OA SF samples after storage for 2 weeks at -20 °C. . . . . . . . . . . . . . . . . . . . .  85  Table A.12 Total protein concentration in OA SF samples. . . . . . . . . .  86  viii  List of Figures Figure 1.1  Synovial joint . . . . . . . . . . . . . . . . . . . . . . . . . .  2  Figure 1.2  Cone and plate geometry . . . . . . . . . . . . . . . . . . . .  7  Figure 1.3  Polymer molecule response to shear, extension and compression 10  Figure 1.4  Schematic diagram of CaBER geometry before and after performing a stretch experiment . . . . . . . . . . . . . . . . . .  Figure 1.5  11  Evolution of the mid-filament diameter for Newtonian, viscoelastic and adhesive materials . . . . . . . . . . . . . . . .  12  Figure 2.1  Three stages step test profile. . . . . . . . . . . . . . . . . . .  21  Figure 2.2  Shear viscosity as a function of shear rate for six OA SF samples and VS. . . . . . . . . . . . . . . . . . . . . . . . . . .  Figure 2.3  The steady state viscosity as a function of shear rate for three OA SF samples and VS (experimental and calculated). . . . .  Figure 2.4  26  The dynamic rheological properties as a function of frequency for three OA SF samples and VS. . . . . . . . . . . . . . . .  Figure 2.5  24  27  Shear stress of an OA SF sample as a function of time at fixed shear rate 0.05 s−1 . . . . . . . . . . . . . . . . . . . . . . . .  29  Figure 2.6  Structure recovery of an OA SF sample. . . . . . . . . . . . .  30  Figure 2.7  Shear viscosity as a function of shear rate for three OA SF samples and VS at 25 and 37 °C. . . . . . . . . . . . . . . . .  31  Figure 2.8  The Arrhenius plot for six OA SF samples. . . . . . . . . . .  33  Figure 2.9  The dynamic rheological properties as a function of frequency for two OA SF samples at 25 and 37 °C. . . . . . . . . . . . .  ix  34  Figure 2.10 Shear viscosity as a function of shear rate for three OA SF samples before and after centrifugation. . . . . . . . . . . . .  36  Figure 2.11 The dynamic rheological properties as a function of frequency for two OA SF samples before and after centrifugation. . . . .  37  Figure 2.12 Shear viscosity as a function of shear rate for two OA SF samples before and after centrifugation before and after storage for two weeks. . . . . . . . . . . . . . . . . . . . . . . . . . . .  38  Figure 2.13 The dynamic rheological properties as a function of frequency for two OA SF samples before and after storage for two weeks. Figure 3.1  Temporal evolution in filament profiles for SF#32 using 6 mm endplate, hi = 2 mm, h f = 10 mm, and separation time = 50 ms  Figure 3.2  53  Filament diameter thinning versus time for VS using three different final plate separation . . . . . . . . . . . . . . . . . . .  Figure 3.7  52  Filament diameter thinning over time for SF#33 using three different extension times . . . . . . . . . . . . . . . . . . . .  Figure 3.6  50  Filament diameter thinning over time for SF#33 using three different final plate separation . . . . . . . . . . . . . . . . .  Figure 3.5  48  Filament diameter thinning over time for SF#2 using 6 mm endplates and different final aspect ratios . . . . . . . . . . .  Figure 3.4  47  Filament diameter thinning over time for SF#2 using 4 mm endplates and different final aspect ratios . . . . . . . . . . .  Figure 3.3  39  54  Filament diameter thinning versus time for VS using three different extension times . . . . . . . . . . . . . . . . . . . . .  55  Figure 3.8  Filament diameter thinning over time for five OA SF samples .  57  Figure 3.9  Filament diameter thinning over time for VS . . . . . . . . .  59  Figure 3.10 The terminal extensional viscosity vs. the relaxation time . . .  60  x  Nomenclature Latin a  Carreau-Yasuda model parameter  B  Width of bearing surface  Bo  Bond number  D  Endplate diameter  D(t)  Filament diameter  D0  Initial filament diameter  E  Modulus  E  Reduced modulus of elasticity  E1  Modulus for surface 1  E2  Modulus for surface 2  Ea  Flow activation energy  fc  Cross-over frequency  g  Gravity  G  Storage (elastic) modulus  G  Loss (viscous) modulus xi  G0.5Hz  Loss (viscous) modulus at a frequency of 0.5 Hz  G2.5Hz  Loss (viscous) modulus at a frequency of 2.5 Hz  G0.5Hz  Storage (elastic) modulus at a frequency of 0.5 Hz  G2.5Hz  Storage (elastic) modulus at a frequency of 2.5 Hz  Gc  Dynamic moduli at cross-over  Gd  Amplitude ratio in oscillatory shear  h0  Minimum lubricant film thickness  h1  Initial film thickness  h2  Final film thickness  hf  Final separation  hi  Initial separation  L  Length of bearing surface  lcap  Capillary length  n  The power-law exponent for Carreau-Yasuda model  R  Radius of the cone  R1  Radius of curvature at surface 1  R2  Radius of curvature at surface 2  Rc  Reduced radius of curvature  Ru  Universal gas constant  T  Torque on the cone  t  Time  T0  Torque amplitude xii  TK  Absolute temperature  TK,0  Reference temperature  U  Average velocity  U1  Velocity at surface 1  U2  Velocity at surface 2  v  Poissons ratio  W  Applied normal load per unit width of disk  Greek β  Constant determined by B/L  ∆t  Time over which a gap between bearing surfaces is maintained  δ  Phase shift angle  γ˙  Shear rate  η0  The zero-shear rate viscosity  η∞  The infinite-shear rate plateau viscosity  ηE,t  Steady terminal extensional viscosity  η  Apparent steady-shear viscosity  η(TK )  Viscosity at TK  η(TK,0 )  Viscosity at TK,0  ηs  Solvent viscosity  η300  The viscosity at a shear rate of 300 s−1  ηt=0s  Viscosity at t = 0 s xiii  ηt=1800s  Viscosity at t = 1800 s  γ  Shear strain  γ0  Strain amplitude  λE  Relaxation time in extensional flow  Λf  Final aspect ratio  Λi  Initial aspect ratio  λ  Carreau-Yasuda model characteristic time  Ω  Angular velocity  ω  Frequency  φ0  Angular amplitude  ρ  Density  σ  Surface tension  τ  Shear stress  τ0  Stress amplitude  τzz − τrr  Normal stress difference  Θ0  Cone angle  xiv  Glossary BCA  Bicinchoninic acid  CaBER  Capillary breakup extensional rheometer  GAGs  Glycosaminoglycans  HA  Hyaluronic acid  OA  Osteoarthritis  PBS  Phosphate buffered saline  SF  Synovial fluid  VS  Viscosupplement  xv  Acknowledgments First and foremost, I would like to thank Allah for giving me the power to believe in myself and achieve my dreams. My sincere gratitude goes to my supervisors Dr. Dana Grecov and Dr. Ezra Kwok for giving me the chance and confidence to explore my research interest. I can’t thank Dr. Grecov enough for her continued support, guidance and for her patience, advice, motivation, and insightful comments. I am also thankful for Dr. Kwok for his encouragement, questions and helpful comments. This thesis would not have been possible without their guidance and encouragement. I would like to express my appreciation to my thesis committee members Dr. Peter Cripton and Dr. Savvas Hatzikiriakos for their time and advice. I cannot thank Dr. Louise Creagh enough. Her guidance and recommendation were crucial to do the protein concentration tests. I extend my thanks to Kristy Wiens for her assistance with the high speed camera setup and to Samin Fakhraei for her assistance in the post processing capillary breakup extensional rheometer. I owe my deepest gratitude to my wife, my parents, my brothers and sisters for their never-ending encouragement and support. This thesis would not have been possible without the financial support and the scholarship from the Saudi Food and Drug Authority and the Saudi Arabian Cultural Bureau in Canada.  xvi  To my family with love.  xvii  Chapter 1  Introduction This chapter first presents background information about synovial joints. Consequently, I also provide background information on osteoarthritis. Then, a particular treatment for osteoarthritis is discussed. The lubrication of the synovial joint is then discussed. Different concepts of rheological measurements are described briefly. After that, a literature review of previous research on the rheology of synovial fluid is presented. Finally, the research objectives and thesis organization are outlined.  1.1  Synovial Joint  Synovial joints (Figure 1.1) provide movement for the articulating bones at the point of contact. The main components of synovial joints are articular cartilage, synovium, and synovial fluid.  1.1.1  Articular Cartilage  Articular cartilage also called hyaline cartilage is a thin layer of fibrous connective tissue that covers the articular surface of bones. It consists of 70% water, 25% solid components and 5% cells. Collagen makes up 70% of its solid component. Type II is the most abundant collagen. 20% of its solid component is glycosaminoglycans (GAGs), and 5% is multiadhesive glycoproteins [57]. Articular cartilage is divided into four different zones. Each zone has different material properties and functions. Those zones are the superficial zone, the transi-  1  Figure 1.1: Synovial joint. tional zone, the radial zone and the calcified zone. The gliding surface for the articular joints is the superficial zone. Its superficial layer consists of randomly aligned collagen fibrils; whereas, its deeper layer has collagen fibrils that are aligned parallel to the cartilage surface and the direction of joint motion. The transitional zone has randomly oriented collagen fibrils that are thicker than those which are found in the superficial zone. In the radial zone, the largest diameter collagen fibrils are found, which are oriented perpendicularly to the subchondral bone and the cartilage surface. The chondrocytes in the radial zone are aligned in radial columns. The transitional and separation of cartilage tissue from the subchondral bone occur at the calcified zone. This zone adheres the cartilage to the bone [49].  2  1.1.2  Synovium  The synovium is a thin and flexible lining surrounding the joint. Synovial fluid is secreted by the synovium. The synovium also exports nutrients and removes waste products from the joint.  1.1.3  Synovial Fluid  In healthy individuals, articular cartilage and a thin film of synovial fluid (SF) are closely linked in providing a protective barrier between the ends of the bones and lubricating the joint. Synovial fluid is a plasma dialysate consisting mainly of hyaluronic acid (HA), lubricin, water and protein [109]. The main proteins in SF are Albumin and γ-Globulin; other proteins that exist in very low concentration are fibrinogen, immunoglobulin IgM, and α2-macroglobuline [41]. The volume of SF in a healthy knee joint is about 1 to 2 mL [16, 105]. Synovial fluid has a major role in joint lubrication, shock absorption and load bearing. Healthy SF is a viscoelastic fluid, highly viscous at low strain rates and highly elastic at high strain rates [98]. Inflammatory and degenerative joint diseases alter the compositions of SF. They reduce the molecular weight and concentration of HA resulting in a decrease of the rheological properties of SF [14, 41, 97, 105]. The molecular weight of HA for healthy SF is about 4 to 7 millions Da [16, 17, 33, 99, 105], and the concentration of HA is about 2 to 4 mg/mL [16, 17, 24, 33, 35, 99, 105]. The concentration of HA in OA SF is in the range of 0.2 to 2.5 mg/mL [67], and the molecular weight of HA ranges between 1 and 3 million Da [67, 85]. Osteoarthritis also changes the compositions of protein in SF. The protein concentration of healthy SF was reported to be in the range of 10.4 to 21.3 mg/mL [17, 93]. Osteoarthritis generally increases the protein concentration in SF. Studies showed that the protein concentration of OA SF were found to be in the range of 25 to 35 mg/mL [67]. The protein in SF may also affect the rheological properties of SF [79, 80]. Protein may aggregate to each other which causes rheopectic behaviour that is at a constant shear rate the viscosity increases in time.  3  1.2  Osteoarthritis  Osteoarthritis (OA) is a degenerative disease which is characterized by the erosion of articular cartilage. All synovial joints are susceptible to OA; however, knee joints are more commonly affected by OA [66]. The hallmarks of OA are pain, joint stiffness, sounds from joints during movement, stiffening of the subchondral bone, subchondral cysts and functional failure. The entire synovial joint is affected by OA. Osteoarthritis is associated with aging and weight [65]. 80% of people over the age of 60 show radiographic evidence of OA [65]. Aging and weight are not the sole risk factors. Osteoarthritis can also be caused by repeated high impact loading and traumatic loading [7]. Other factors may include gender, race and bone density [36, 100]. Cartilage degeneration is the primary characteristic of OA. A localized remodelling of the subchondral bone can result in a repetitive high impact load. This, in turn, causes an increase of stress in a certain area of the joint and, therefore, the articular cartilage gets more stress. The increased stress caused the deterioration of the articular cartilage. A breakdown of the articular matrix allows the cartilage to be more infused with water. The compositions of SF changes as a result of the increased fluid. Consequently, the rheological properties of SF deteriorate [25, 41, 47, 68, 69]. Thus, SF becomes a less effective lubricant [28, 37].  1.3  Viscosupplementation  Viscosupplementation was proposed by Balazs et al. [17] as a treatment of OA. The aim of this treatment is to restore the physiological properties of SF by injecting a high molecular weight and a high concentration of HA into the joint. There are different viscosupplements available at a wide range of molecular weight and concentration. The efficacy of viscosupplementation is usually determined by a decrease in pain or improved function. Viscosupplements were found to be effective by some studies [104] and ineffective by others [8]. There are also contradictory reports about the efficacy of high molecular weight HA [9, 10]. In vitro studies showed that an injection of high molecular weight and a high concentration of HA increased the rheological properties of SF [21, 22, 67]. However, the in vivo rheological 4  properties of viscosupplements are still unknown.  1.4  Lubrication of Synovial Joints  Synovial fluid lubricates the synovial joints by reducing the friction and/or the wear of the articular cartilage. The main function of SF is to keep the surfaces of the articular cartilage apart so that the interaction between the surfaces of the cartilage cannot occur; thus, friction and wear can be reduced or controlled. Different modes of lubrication in the synovial joints have been proposed [40, 45]. Among those modes are: 1. Hydrodynamic lubrication 2. Elastohydrodynamic lubrication 3. Squeeze film 4. Boundary lubrication In hydrodynamic lubrication, the generated hydrodynamic pressure in a compressed fluid film between two surfaces supports the load and keeps the sliding surfaces completely separated. For rotating disks with parallel axes, the simplified form of Reynolds equation yields: h0 ηU = 4.9 Rc W  (1.1)  where h0 is the minimum lubricant film thickness, η is the dynamic viscosity, U is the average velocity (U1 +U2 /2), U1 is the velocity at surface 1, U2 is the velocity at surface 2, W is the applied normal load per unit width of disk, and Rc is the reduced radius of curvature (1/Rc = 1/R1 + 1/R2 ). R1 is the radius of curvature at surface 1, and R2 is the radius of curvature at surface 2 [26, 40, 45]. In elastohydrodynamic lubrication, elastic deformation of the solid surfaces occurs. The Dowson-Higginson expression for minimum film thickness is: h0 ηU = 2.6 Rc W  0.7  αW Rc 5  0.54  W Rc E  0.03  (1.2)  The term E represents the reduced modulus of elasticity: 1 (1 − v1 2 ) (1 − v2 2 ) = + E E1 E2  (1.3)  where α is the pressure-viscosity coefficient, E is the modulus, v is Poissons ratio, and the subscripts 1 and 2 refer to the two solids in contact [45]. Squeeze-film lubrication can occur when two surfaces approach each other. The exerted force by the surfaces tends to squeeze out the lubricating fluid; however, the viscous forces of the lubricant are strongly resisting this action. The Reynolds equation for squeeze film gives: ∆t = β  ηB3 L 2W  1 1 − 2 2 h2 h1  (1.4)  where B and L are the width and length of each bearing surface, h1 is the initial film thickness, h2 is the final film thickness, β is a constant determined by B/L, and ∆t is the time over which a gap is maintained [101]. Boundary lubrication exists when the bearing surfaces are separated by films of molecular thickness. Boundary lubrication is independent of the bulk properties of the lubricant. It is determined by the surface properties of the bearing and the molecular natures of the lubricant. It relies on the adsorption of molecules of the lubricant to the articulating surfaces [45, 107]. There are many factors that determine the mode of lubrication such as the geometry of the cartilage, the contacts load, the sliding velocity, the physiological condition of the cartilage and SF [45]. Thus, a combination of the previous mechanisms of lubrication will come into play to lubricate synovial fluid [40, 45].  1.5  Rheology  Rheology is the science of deformation and flow of matter. Rheological studies are aimed at understanding the materials’ behaviours when they are subjected to an applied force. A rheometer is a device used to study rheological properties of materials. There are rheometers that characterize rheological properties under shear flow, and rheometers that characterize rheological properties under extensional flow.  6  Figure 1.2: Cone and plate geometry.  1.5.1  Shear Rheology  Shear rheometers can determine the rheological behaviour of the material using different flow testing techniques, such as steady shear or small amplitude oscillatory shear. There are different geometries that can be used with shear rheometers such as cone-and-plate, parallel plate, and concentric cylinder. A cone-and-plate rheometer (Figure 1.2) is commonly used to study the shear rheological properties of SF [23, 62, 97]. Steady Shear Test The viscosity is determined by the steady shear test. The viscosity is the material resistance to flow or deformation. There are three types of material viscous behaviour: Newtonian, where the viscosity is constant at any given shear rate; shear-thinning, where the viscosity decreases with increasing shear rate; shearthickening, where the viscosity increases with increasing shear rate. Non-Newtonian 7  viscosity can be expressed as: ˙ γ˙ τ = η(γ)  (1.5)  ˙ is the shear viscosity as a where τ is the shear stress, γ˙ is the shear rate and η(γ) ˙ function of γ. The measurable quantities and fixed parameters for cone-and-plate are the fixture radius R, the cone angle Θ0 , the rotational speed Ω, and the torque T . The following equations can be used to calculate quantities of rheological importance. τ=  3T 2πR3  (1.6)  Ω Θ0  (1.7)  3T Θ0 2πR3 Ω  (1.8)  γ˙ = η=  Small Amplitude Oscillatory Shear Some fluids posses both viscous and elastic behaviours. Small-amplitude oscillatory shear (SAOS) test are the most widely used experiments to determine the linear viscoelastic properties of polymers. The storage (elastic) and loss (viscous) moduli, as defined below, are used to describe the viscoelastic properties. In this test, the material is subjected to a small amplitude shear strain history, that is: γ(t) = γ0 sin(ωt)  (1.9)  where γ0 is the strain amplitude and ω is the frequency. The stress then is measured as a function of time. For very small strain amplitudes, the shear stress is sinusoidal in time and independent of strain (linear viscoelastic limit) and can be written as: τ(t) = τ0 sin(ωt + δ )  (1.10)  where τ0 is the stress amplitude and δ is a phase shift or the mechanical loss angle.  8  The amplitude ratio (Gd = τ0 /γ0 ) and the loss angle (δ ) are functions of frequency but are independent of γ0 for sufficiently small γ0 values. Equation 1.10 can be written as: τ(t) = γ0 [G (ω) sin(ωt) + G (ω) cos(ωt)]  (1.11)  where G (ω) is called the “storage (elastic) modulus” and G (ω) is called the “loss (viscous) modulus”. The two quantities can be easily calculated from: G (ω) =  τ0 cos δ γ0  (1.12)  G (ω) =  τ0 sin δ γ0  (1.13)  For a cone-and-plate rheometer, the equations for calculating the storage and loss modulus in terms of the actual test variables are as follows: G =  3Θ0 T0 cos δ 2πR3 φ0  (1.14)  G =  3Θ0 T0 sin δ 2πR3 φ0  (1.15)  where T0 is the torque amplitude, and φ0 is the angular amplitude.  1.5.2  Capillary Breakup Extensional Rheometer  High molecular weight polymer molecules can produce dramatically different responses in extensional and shear flows (Figure 1.3) [11]. Extensional strain can dissociate molecular entanglements of HA [74]. Although studying the extensional rheological properties is significant, it is a difficult task to achieve. This is due to the lack of appropriate experimental methods. Early designs of extensional rheometers required large volume of fluid and additional limitations such as the inability of producing a homogenous extensional flow. Currently, there is one method that is suitable to measure the extensional rheological properties for low viscous fluid. This method is the capillary breakup which can produce a homogenous uniaxial extensional flow.  9  Figure 1.3: Polymer molecule response to shear, extension and compression. To use CaBER, a small volume of a sample is placed between two circular endplates as shown in Figure 1.4. The upper plate is suddenly pulled apart. Once the upper plate reaches its final position, the measurements of the formed filament diameter at the middle starts. The principle of CaBER is based on the forces balance acting on the filament during extensional stretching. The capillary forces drive the thinning of the filament, whereas the viscous and elastic forces resist it. The forces balance can be approximated by: 3ηs (−  2 ∂ D(t) 2σ )= − (τzz − τrr ) D(t) ∂t D  (1.16)  where ηs is the solvent viscosity, D(t) is the filament diameter, σ is the surface tension and τzz − τrr is the first normal stress difference in axisymmetrical flows. For a viscoelastic material, the diameter decreases exponentially at the beginning, and linearly towards the end as shown in Figure 1.5. The longest relaxation time can be determined from the exponential part, and the terminal extensional viscosity can be determined from the linear part. The filament diameter in the first stage can be given by: D(t) = D0 e 10  − 3λt  E  (1.17)  Figure 1.4: Schematic diagram of CaBER geometry before and after performing a stretch experiment. where λE is the longest relaxation time. The filament diameter at the final stages of the experiment can be given by: D(t) = D0 −  σ t ηE,t  where ηE,t is the terminal extensional viscosity.  11  (1.18)  Figure 1.5: Evolution of the mid-filament diameter for Newtonian, viscoelastic and adhesive materials.  1.6  Rheology of Synovial Fluid  Healthy SF is a non-Newtonian fluid; its viscosity depends on the shear rate and it demonstrates a shear thinning effect [19, 87, 98]. Osteoarthritic synovial fluid had a lower viscosity compared to that of healthy SF [22, 38, 68, 94]. The extent , the ratio of the zero shear viscosity to of shear-thinning is measured by η0 /ηγ=300 ˙ the viscosity at a shear rate of 300 s−1 [21, 87, 98]. η0 /ηγ=300 for healthy SF is ˙ higher than that for OA SF [22, 41, 98]. The rheological properties of healthy and OA SF are summarized in Table 1.1. The viscoelastic properties of SF were also reported by numerous studies [58, 77, 81, 83, 89, 95]. Healthy SF exhibited a viscous behaviour at low frequencies of oscillation, corresponding to slow joint motion, and it exhibited an elastic-like behaviour at high frequencies of oscillation, corresponding to rapid joint motion [77]. The cross-over frequency, the frequency at which storage modulus G and loss modulus G are equal, for healthy SF can be observed at a fairly low frequency  12  Table 1.1: The rheological properties of healthy and OA SF reported in the literature.  η0 (Pa·s)  η0 /η300 λ (s)  Healthy SF  OA SF  6-12 [97] 6-175 [87] 1-100 [32] 70-250 [87] 100 [97] 40-100 [97]  0.1-1 [32, 97]  5-40 [97] 8-20 [97] 0.047-35 [68]  (∼ 0.02 Hz) [95]. In contrast, OA SF behaves generally as viscous fluids [89]. The dynamic moduli of OA SF are lower than that for healthy SF by more than one order of magnitude [14]. Some OA SF samples did not show cross-over behaviour in their investigated frequency range [14]. However, some samples exhibited crossover behaviour that occurred at a higher frequency than that of healthy SF [21, 68]. Relaxation time, which is the time that characterizes a materials stress relaxation after deformation, is the inverse of the cross over frequency. Relaxation time was shorter for OA SF compared to that of healthy SF (Table 1.1). The reduction of the rheological properties of OA SF is likely attributable to the decrease in the molecular weight and concentration of HA [113]. Hyaluronic acid exists as an extended random coil in aqueous solutions at a low concentration (<1 mg/mL) [97], whereas at higher concentrations (>1 mg/mL), a transient entanglement network was formed [34]. The increase of the concentration of HA increased the viscosity and resulted in a pronounced non-Newtonian shear thinning behaviour [62, 63]. The increase of the molecular weight of HA for solutions of the same concentration had similar effects [73]. Kobayashi et al. [60] studied the effect of the molecular weight of HA on the storage G and loss G moduli. A distinct transition from viscous to elastic behaviour was observed as the oscillation frequency increased for the higher molecular weight HA solutions. Kobayashi et al. [60] observed that HA solutions of higher molecular weights (1.2 MDa) showed a transient entanglement network. In contrast, the lower molecular weight (150 KDa) did not. A similar observation was 13  reported by Ambrosio et al. [4]. High molecular weight HA exhibited viscoelastic behaviour, and the storage and loss moduli exhibited cross-over, whereas the lower molecular weight HA behaved like a viscous fluid throughout the investigated frequencies range [4]. The time dependent rheological behaviours of SF were investigated by many researchers [22, 61, 79, 80, 82]. O’Neill and Stachowiak [82] investigated the timedependent rheological of SF at low temperatures (20 °C and below). They found that SF exhibited rheopectic behaviour that is at a constant shear rate, viscosity increased with time. Bhuanantanondh et al. [22] observed similar behaviours at 37 °C. The rheopexy of SF was observed at a low shear rate [22]. Oates et al. [79, 80] suggested that SF rheopectic behaviour is due to protein aggregation which creates weak networks around HA.  1.7  Purpose of Research  The ultimate goal of this research is to improve the condition and function level of patients suffering from OA. To achieve this goal, this study focused on the rheological characterization of OA SF. The understanding of the rheological properties of SF will lead to a better understand its lubrication properties. Furthermore, the knowledge of the rheological properties of SF will lead to the development of a rheological analogue to SF or to a periprosthetic fluid. Although there are some rheological studies that were conducted on SF both healthy and pathological, there are still unknowns about the rheological characteristics of SF. Previous studies were conducted using shear rheometry [41]. In some studies, synovial fluid samples were centrifuged [23, 69, 88]. Additionally, rheological studies were conducted at different temperatures for instance at 25 °C [48, 67, 91] or 37 °C [21, 22, 42], and/or after storing samples [23, 44, 52, 70]. Therefore, this study was aimed at investigating the effects of the centrifugation, the temperature and the storage on the rheological properties of OA SF. Synovial fluid exhibits a time dependent behaviour at low shear rates [22, 61, 79, 80, 82]. More specifically, SF exhibits rheopectic behaviour, that is the viscosity increases in time at constant shear rate. The aim of this study was to further investigate this behaviour using steady state shear and small-amplitude oscillatory  14  shear tests. Inside the joint, SF is subjected to mixed shear and extensional flows. Thus, studying the extensional rheological properties is also critical. This study aimed to perform an extensional rheological characterization of OA SF. The rheological properties of SF are mostly linked to its HA contents [113]. However, the protein content in SF may also play a role on its rheological properties [79, 80]. This study aimed to correlate the rheological properties with the protein concentration of SF. Viscosupplementation is used as treatment for OA. The aim of the treatment is to restore the rheological properties of SF to the normal characteristics. In this study, rheological characterization for one commercially available VS was performed to compare its rheological properties with the rheological properties of OA SF.  1.8  Research Objectives  The specific objectives of this study are as follows: 1. to perform a comprehensive shear rheological characterization of OA SF and VS, 2. to determine the effects of temperature, centrifugation and storage on the steady state and dynamic rheological properties of OA SF and VS, 3. to correlate the rheological properties of SF with its protein concentration, 4. to determine the optimal setup for capillary breakup extensional rheometer in order to effectively study the flow behaviour of OA SF and VS, and 5. to determine the extensional rheological properties of OA SF and VS.  1.9  Thesis Organization  In Chapter 2, the shear rheological properties of OA SF and VS are discussed. The effects of temperature, centrifugation and storage on the rheological properties of  15  OA SF and VS were studied. The rheological data were fitted to the CarreauYasuda model. Finally, I discuss the correlations between the shear rheological properties with the protein concentration in the OA SF. In Chapter 3, the extensional rheological properties of SF and VS are presented. The effect of different CaBER configurations on the extensional rheological measurements were determined. The extensional rheological properties are correlated with the protein concentration of the various samples examined. In Chapter 4, the conclusions, the contributions to knowledge, the limitations of this study, and the recommendations for future work are presented.  16  Chapter 2  Osteoarthritic Synovial Fluid Shear Rheology and Correlations with Protein Concentration 2.1  Introduction  Osteoarthritis (OA) is one of the most disabling degenerative diseases and is characterized by the breakdown of articular cartilage resulting in joint pain and stiffness [56]. In healthy individuals, articular cartilage and a thin film of synovial fluid (SF) are closely linked by providing a protective barrier between the ends of the bones and the lubricating joint. The SF volume in a healthy knee joint is about 1 to 2 mL [16, 105]. Synovial fluid plays a major role in joint lubrication, shock absorption and load bearing. Healthy SF is a viscoelastic fluid, highly viscous at low strain rates and highly elastic at high strain rates [98]. Synovial fluid is a plasma dialysate consisting mainly of hyaluronic acid (HA), protein, water and lubricin [109]. The rheological behaviour of SF is mostly determined by its HA content [46]. Hyaluronic acid plays an important role in joint lubrication, as it provides shock absorption and viscoelastic properties of SF [16, 43, 78]. The protein concentration may also affect the rheological properties of SF [79, 80]. A recent study suggested that protein may interact with HA [90]. Its interaction  17  with protein may form a physiological loose network, which plays an important role for the rheology of the SF [78, 97]. However, the interaction mechanism, and the protein’s role in SF lubrication have not been totally elucidated. Lubricin may also have a role in SF lubrication ability, especially in the boundary lubrication [54]. In an OA joint, damage to the articular cartilage causes modifications in the rheological properties of SF and its chemical composition [25, 41, 47, 68, 69]. Inflammatory and degenerative joint diseases reduce the molecular weight and concentration of HA resulting in a decrease of SF viscosity and viscoelasticity [14, 41, 97, 105]. The molecular weight of HA in healthy SF is about 4 to 7 million Da [16, 17, 33, 99, 105], and the concentration of HA is about 2 to 4 mg/mL [16, 17, 24, 33, 35, 99, 105]. The concentration of HA in OA SF is in the range of 0.2 to 2.5 mg/mL [67], and the molecular weight of HA ranges between 1 and 3 million Da [67, 85]. As a result of the degradation of HA, the SF becomes less viscous [75] and, therefore, less effective in lubricating the joint [84, 98]. In addition, the reduction in the molecular weight and concentration of HA reduces the SF viscoelasticity and sequentially its ability to protect the joint [15]. Viscosupplementation is a treatment for OA; HA is used as a viscosupplement (VS) to the diseased joint. The aim of this treatment is to restore the physiological properties of SF. In vitro studies showed that an injection of high molecular weight and concentration of HA increased the rheological properties of SF [21, 22, 67]. There are many theories about the mechanism of SF lubrication of those are boundary and fluid film lubrication [39, 50, 55, 110]. To date, there is no definite mechanism for synovial fluid lubrication. Boundary lubrication relies on the adsorption of molecules of the SF to the articulating surfaces [101]. Phospholipids [50, 96] and some proteins [86] that exist on synovial joints are potential boundary lubricants. Other possible boundary lubricants that are synthesized in synovial joints are lubricin [86, 106] and superficial zone protein [53]. Fluid film lubrication depends on the fluid rheological properties, the load, the geometry of the joint, the sliding speed, and the material properties of the joint [55]. A thorough rheological characterization of synovial fluid is required in order to better understand its role in joint lubrication. In this chapter, comprehensive shear rheological characterization of osteoarthritic synovial fluid, and the correlation be18  tween the rheological properties with protein concentrations will be demonstrated. Also, the influence of the temperature and different pre-treatments (storage, and centrifugation) on the rheological properties of synovial fluid will be illustrated. Additionally, the rheological properties of OA SF will be compared with HA by choosing a commercial viscosupplement, Orthovisc. The materials and methods will be presented first. After that, the results of shear rheological tests and the correlation with the protein concentration will be presented and discussed. Lastly, the conclusions of this part of the study will be presented.  2.2 2.2.1  Materials and Methods Materials  Synovial Fluid Synovial fluid samples were collected from OA patients undergoing total knee replacement surgery. Samples were obtained from the Orthopedic Reconstructive Service at the Vancouver Coastal Health Region in accordance with a protocol approved by the University of British Columbia’s Clinical Research Ethics Board and the Vancouver Coastal Health Research Institute. An informed consent was obtained from each patient prior to the surgery. Synovial fluid was aspirated from each patient’s knee joint into a test tube by an experienced surgeon under sterile conditions. Shear rheological studies were conducted on 35 SF samples (Appendix Table A.1). The samples were obtained from 32 subjects (16 males and 16 females) and were labelled SF#1 - SF#32 (R or L was used when two samples were collected from the same subject to indicate right or left knee respectively). The average age was 64 years. Shear rheology experiments were conducted immediately after collecting SF samples. The samples were stored at - 20 °C for two to three days before conducting protein concentration tests.  19  Viscosupplement A commercial non-crossed-linked HA (Orthovisc, Anika Therapeutics) was obtained from CanDrug Pharmacy Inc. The molecular weight of HA in Orthovisc is 1 to 2.9 million Da [59], and the concentration of HA is 15 mg/mL.  2.2.2  Methods  Rheometery The rheological properties of each SF sample were determined using a Kinexus Ultra rheometer (Malvern Instruments Ltd., Worcestershire, UK) at 25 and/or 37 °C using a stainless steel cone and plate geometry (40 mm diameter cone with a 1°cone angle). The cone and plate geometry was used in this study due to the small volume sample. The rheometer was first calibrated with Cannon Certified Viscosity Standard oil. Shear rates ranging from 10−2 to 103 s−1 were applied to each sample. It is worth noting that the shear rate in human joints can reach more than 105 s−1 [55]; however, measurements over 1000 s−1 are not possible due to an inherent limitation of the rheometer. In the small amplitude oscillatory shear test, preliminary strain sweep tests were performed on the samples in order to identify the linear viscoelastic response range of the samples. Then, frequency sweep measurements were conducted in the linear region, at 1 to 5% strain, over a frequency range of 10−2 to 10 Hz. Time dependent behaviour was investigated by applying a constant shear rate of 0.05 s−1 for 30 minutes. The change in viscosity and shear stress with time was recorded. The time dependent rheological behaviour of SF was only observed at a low shear rate. The recovery of mechanical properties after a shock was further investigated by applying a step test with three stages: oscillation/rotation/oscillation. In the first step, samples were subjected to oscillation at 1 H z and 5 % strain for 2.5 minutes; this stage was used as reference. Then, samples were subjected to a shear rate of 100 s−1 for 1 minute in order to destruct the fluid structure. In the final step, samples were subjected to oscillation at 1 H z and 5 % strain for 20 minutes. The preset profile is presented in Figure 2.1. The evolution in time of the storage modulus, loss modulus and viscosity was observed.  20  (1)  (2)  (3)  γ˙ = 100s − 1  γ = 5%  γ = 5%  f = 1H z  f = 1H z  t 0s  150 s  210 s  1410 s  Figure 2.1: Three stages step test profile. The rheometer has an accuracy of +/- 2 % of the viscosity measurements and +/- 3% of the dynamic moduli measurements. Rheological Models Experimental results were fitted using the Carreau-Yasuda model [112]. This model is used to describe shear-thinning behaviour with zero shear viscosity η0 and infinite shear viscosity η∞ . η0 is the maximum plateau value at low shear rate, and η∞ is the viscosity at infinite shear rate. The model equation is: n−1 η − η∞ ˙ a] a = [1 + (λ γ) η0 − η∞  (2.1)  where λ is the time constant and the inverse of this parameter is the critical shear rate at which viscosity begins to decrease, a determines the curvature at the top of the curve (a higher a means a sharper curvature) and n is the power-law index 21  which governs the power-law regime. The Arrhenius model was used to describe the viscosity-temperature dependence. The model equation is: η(T ) Ea = exp η(T0 ) Ru  1 1 − TK TK,0  (2.2)  where TK is the absolute temperature (K), TK,0 is a reference temperature (K), η(TK ) is the viscosity at TK , η(TK,0 ) is the viscosity at TK,0 , Ea is the flow activation energy, and Ru is the universal gas constant. Storage Eight OA SF samples were stored at -20 °C for two weeks. Rheological measurements were done before and after storage to study its effect. Centrifugation Five OA SF samples were centrifuged using a Galaxy 14D Micro-centrifuge (VWR, Batavia, IL) at 13,000 RPM for five minutes. The rheological properties of OA SF were studied before and after centrifugation to evaluate the centrifugation effect. Determination of Protein Concentration The protein concentrations of synovial fluid were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL). The preparation of standards and working reagent was done according to the manufacturer’s instruction. In order to be within the BCA working range of 20 to 2000 µg/mL, 20 µL of synovial fluid was diluted in a 1:50 ratio with phosphate buffered saline (PBS). 100 µL of standards and diluted samples were mixed with 2 mL of working reagent on cuvettes. The mixture was incubated at room temperature for two hours. The absorption was measured within 10 minutes by a spectrophotometer (Spectronic 601, Milton Roy Co., Rochester, NY) at 562 nm. Finally, a standard curve was prepared using the absorption reading of the standards, and the absorption of each sample was interpolated from the standard curve to obtain the protein concentration. The error in protein concentration determination may be up to 10% [51]. 22  Correlations between Synovial Fluid Protein Concentration and Rheological Properties The protein concentration for each OA SF samples was correlated with the following rheological properties: the zero shear viscosity (η0 ); the Carreau-Yasuda model parameters (λ , a, and n); the dynamic moduli at 0.5 Hz (G0.5Hz and G0.5Hz ), at 2.5 Hz (G2.5Hz and G2.5Hz ) and at the cross-over frequency (Gc ); and the crossover frequency ( fc ). The protein concentration was also correlated with the time dependent behaviour of OA SF.  2.3 2.3.1  Results and Discussion Rheological Properties at 37 °C  Steady State Shear Flow Osteoarthritic synovial fluid exhibited non-Newtonian shear thinning behaviour, that is the viscosity decreased with increasing shear rate as shown in Figure 2.2. The zero shear viscosity η0 , the maximum plateau value at low shear rate, varied from 0.05 to 14.40 Pa·s. At high shear rates, variations between sample viscosities were small, ranging between 0.007 and 0.025 Pa·s. The ratio of the zero shear viscosity to the viscosity at a shear rate of 300 s−1 , η0 /η300 , is commonly used in literature as a measure of shear thinning behaviour [21, 87, 98]. η0 /η300 ranged between 5 and 289. Complete results are given in Appendix Table A.2. The range of OA SF viscosity measured in this study is in agreement with previous studies [22, 41, 98]. Osteoarthritic synovial fluid samples are less viscous than healthy SF [41, 98]. It is worth noting that some of OA SF samples showed rheological properties similar to those reported for healthy SF. Healthy SF has a zero shear viscosity between 1 and 175 Pa·s [87, 98]. Many OA SF samples had a zero shear viscosity higher than 1 Pa·s. Another observation is the value of η0 /η300 for many OA SF samples that we studied were in the range of healthy SF (70 to 250) [87, 98]. These results either suggest that healthy SF can be found at higher viscosity than those reported by previous studies, or that OA does not necessarily decrease SF 23  2  10  SF #3L SF #3R SF #5 SF #11 SF #15L SF #15R VS  1  Viscosity (Pa.s)  10  0  10  −1  10  −2  10  −3  10  −2  10  −1  10  0  1  10  10  2  10  3  10  −1  Shear Rate (s )  Figure 2.2: Shear viscosity as a function of shear rate for six OA SF samples and VS. viscosity. The rheological properties of healthy SF were studied in the ’80s and early ’90s. Since then, there have been advancements in rheometers design and performance. For instance, current rheometers are able to measure shear viscosity at very low shear rates, and the accuracy and the precision have been improved. Therefore, there is a need for a more recent study for the rheological properties of healthy SF using more modern technology. A comparison between our results and the previous literature studies are presented in Table 2.1. The steady state rheological properties of VS were studied. Viscosupplement exhibited non-Newtonian shear thinning behaviour as shown in Figure 2.2. The steady state rheological properties were fitted using the Carreau-Yasuda model. Figure 2.3 shows that the Carreau-Yasuda model is a suitable model to represent the rheological behaviour of OA SF and VS. Complete results are given 24  Table 2.1: Comparison of OA SF and VS rheological properties between the present study and different literature studies of OA and healthy SF. OA SF (Current Study)  OA SF (Previous Studies)  Healthy SF  VS  η0 (Pa·s)  0.05-14.40  0.1-1 [32, 97]  49  λ (s)  0.29-18.78  8-20 [97] 0.047-35 [68]  6-12 [97] 6-175 [87] 1-100 [32] 40-100 [97]  a n η0 /η300  0.532-1.376 0.311-0.765 5 - 289  0.54±0.10 [68] 5-40 [97]  70-250 [87] 100 [97]  0.53 0.67 0.141 77  in Appendix Table A.2. The values of model parameters can be used in future OA SF simulation. Previously, Cross model [18] were used to fit OA SF results [22]; however, there was a significant lack of fit when the Cross model was used with OA SF. Fam et al. [42] were able to use a modified Cross model to fit a HA/BCS solution that had a concentration greater than 2 mg/mL; however, they were not able to use the model with HA/BCS solutions below 1 mg/mL. Dynamic Shear Flow The viscoelastic properties of OA SF were investigated by a small amplitude oscillatory flow. Figure 2.4 shows the storage (elastic) and loss (viscous) moduli for three samples at 37 °C, as well as the cross-over frequency, the frequency at which G and G cross. The cross-over frequency, fc , indicates the transition from viscous to elastic behaviour. Synovial fluid shock absorption during walking and running is related to its viscoelastic properties. Osteoarthritic synovial fluid viscoelasticity varied from sample to sample. Figure 2.4 illustrates the trends for different behaviours that were observed. Complete results are given in Appendix Table A.3. Some OA SF shows viscoelastic behaviour [76]; they have a high loss modulus at a low frequency and a high storage modulus at a high frequency. However, some low viscosity samples showed viscous behaviour [76] meaning that the loss modu-  25  2  10  1  Viscosity (Pa.s)  10  0  10  −1  10  SF #3R Data SF #3R Carreau−Yasuda Fit SF #1 Data SF #1 Carreau−Yasuda Fit SF #2 Data SF #2 Carreau−Yasuda Fit VS Data VS Carreau−Yasuda Fit  −2  10  −3  10  −2  10  −1  10  0  1  10  10  2  10  3  10  −1  Shear Rate (s )  Figure 2.3: The steady state viscosity as a function of shear rate for three OA SF samples and VS (experimental and calculated). lus G was higher than the storage modulus G at all investigated frequencies. The storage modulus at knee joint walking frequency (0.5 Hz [14]) ranged between 0.05 and 4 Pa (average ± SD =0.83±0.79), whereas the loss modulus ranged between 0.11 and 2.58 Pa (0.81±0.51). At knee joint running frequency (2.5 Hz [13]), the storage modulus ranged between 0.11 and 6.97 Pa (1.77±1.39), and the loss modulus ranged between 0.31 and 3.37 Pa (1.36±0.65). The cross-over frequency ranged between 0.06 and 5 Hz (1.26±1.32); however, some samples did not show a cross-over within the investigated range. It is worth noting that the measurements for low viscous samples were only possible between a limited frequency range due to issues of secondary flow and wall slip as shown in Figure 2.4. Our results shows that for the majority of OA SF samples both the storage and loss moduli were lower than those reported for healthy SF [14, 41, 98]. Healthy 26  3  10  Storage Modulus G′ and Loss Modulus G′′ (Pa)  SF #3R G′ SF #3R G′′ SF #9 G′ SF #9 G′′ 2  SF #22 G′  10  Running Frequency 2.5Hz→ Walking Frequency 0.5Hz→  SF #22 G′′ VS G′ VS G′′ 1  10  0  10  −1  10  −2  10  −1  0  10  10  1  10  Frequency of Oscillation (Hz)  Figure 2.4: The dynamic rheological properties as a function of frequency for three OA SF samples and VS. SF has a high loss modulus at low frequencies and a high storage modulus at high frequencies, which makes SF a suitable lubricant at a low frequency activity like walking, and a good shock absorber at a higher frequency activity like running [13, 14]. However, OA SF tends to lose its elastic properties. These results suggest that OA patients suffer more when they perform an activity at a high frequency because their SF losses its shock absorption properties. Compared to healthy SF, an increase in the cross-over frequency of OA SF samples is observed. The cross-over frequency depends on the molecular weight and concentration of HA as it reflects the mobility of the HA chain [42]. The reduction in the elastic properties and the increase of the cross-over frequency can indicate a decrease in the molecular weight and concentration of HA [42]. Viscosupplement also showed viscoelastic behaviour. The cross-over frequency 27  of VS was 1.24 Hz. Time Dependent Behaviour Previous studies found that SF exhibited a rheopectic behaviour, that is at a constant shear rate the viscosity increases with time [22, 61, 79, 80, 82]. This rheopectic behaviour was found at a low shear rate [22]. The rheopectic behaviour of OA SF was further investigated in this study. In addition to the steady and constant shear rate test, another method was used to investigate the time dependent behaviour of SF. In this method, samples were evaluated using an oscillatory flow test; the advantage of this test is that it reveals the dynamic moduli response with time. Five OA SF samples were investigated by applying a constant shear rate of 0.05 s−1 for 30 minutes and then observing the change in viscosity or shear stress with time. Synovial fluid was found to be affected by the time of the applied shear. Figure 2.5 shows that under constant shear rate, the viscosity of OA SF increased in time showing rheopectic behaviour. The relative viscosity change between the viscosity measurement at t = 1800s and the viscosity measurement at t = 0, (ηt=1800s − ηt=0s ) · 100/ηt=0s , ranged between 10.37 and 33.78 (18.88 ±9.46). Complete results are given in Appendix Table A.4. Viscosupplement exhibited thixotropic behaviour; at a constant shear rate, its viscosity decreased with time. Viscosupplements do not contain protein and only contain HA. Protein aggregation was hypothesized to cause SF rheopectic behaviour [80]. The thixotropic behaviour of VS might reinforce this hypothesis. Oates et al. [79, 80] suggested that the rheopectic behaviour of SF is due to protein aggregation which creates weak networks around HA. The rheopexy of SF indicates that SF becomes stiffer after extended inactivity. Synovial fluid is subjected to oscillatory motion in the joints; therefore, evaluating SF with oscillatory flow will give more relevant information about the time dependent rheological behaviour for SF dynamic moduli. 15 OA SF samples were investigated using three stages of (oscillation/rotation/oscillation) tests. Figure 2.6 shows that all the investigated parameters increased with time showing rheopectic behaviour, and OA SF sample was able to recover at phase 3 after being destructed at phase 2. The relative change of the storage modulus, loss modulus and viscosity  28  0.06  0.058  Shear Stress (Pa)  0.056  0.054  0.052  0.05  0.048  0.046 0  200  400  600  800  1000  1200  1400  1600  1800  Time (s)  Figure 2.5: Shear stress of an OA SF sample as a function of time at fixed shear rate 0.05 s−1 . at stage 3 are calculated as follows: Grel =  Gt=1410 − Gt=210 × 100 Gt=210  (2.3)  Grel =  Gt=1410 − Gt=210 × 100 Gt=210  (2.4)  ηrel =  ηt=1410 − ηt=210 × 100 ηt=210  (2.5)  The relative change of the storage modulus ranged between 4 to 43 % (14±13); it ranged between 2 to 13 % (6±4) for the loss modulus, and between 2 to 20 % (7±6) for the viscosity. Complete results are given in Appendix Table A.5. Viscosupplement showed thixotropic behaviour, that is at constant shear rate the  29  Viscosity (Pa.s), Storage and Loss Modulus (Pa)  7  (1)  (2)  (3)  6  Storage Modulus G′ Loss Modulus G′′ Viscosity  5  4  3  2  1  0  150 s  210 s  Time (s)  1410 s  Figure 2.6: Structure recovery of an OA SF sample. viscosity decreases with time. The relative change for the storage modulus, the loss modulus and the viscosity were -1, -2 and -1 respectively.  2.3.2  Temperature Effect  Rheological testing was done at 25 °C and 37 °C to investigate the temperature effects on the rheological properties of SF. Some previous rheological studies were conducted at either 25 °C [48, 62, 67, 79, 91, 97, 98, 108] or 37 °C [4, 21, 22, 27, 42, 70]. The SF temperature inside the joint varies depending on the surrounding environment and physical activity [20]. Therefore, neither 25 °C nor 37 °C would reflect the physiological temperature of SF; however, as human body temperature is 37 °C, it may be a more relevant reference temperature. We studied six OA SF samples at both 25 and 37 °C. Figure 2.7 shows that as  30  2  10  SF #14 at 25oC o  SF #14 at 37 C o  SF #15L at 25 C SF #15L at 37oC SF #19 at 25oC  1  10  SF #19 at 37oC o  Viscosity (Pa.s)  VS at 25 C VS at 37oC  0  10  −1  10  −2  10  −2  10  −1  10  0  1  10  10  2  10  3  10  −1  Shear Rate (s )  Figure 2.7: Shear viscosity as a function of shear rate for three OA SF samples and VS at 25 and 37 °C. the temperature increased, SF viscosity decreased as expected. Table 2.2 shows the percentage changes in the steady state rheological properties from 25 °C to 37 °C. Complete results are given in Appendix Table A.2 and Table A.6. Viscosupplement showed similar response to the test temperature when it was tested with steady state flow. The zero shear viscosity, η0 , of VS decreased by 49% when it was tested at 37 °C compared to 25 °C, and its viscosity at shear rate of 1000 s−1 decreased by 12%. The Arrhenius Equation 2.2 was used to study the temperature effect on viscosity. Figure 2.8 shows the Arrhenius curve, ln η versus 1/T , for six OA SF samples. Table 2.3 shows the flow activation energy which “characterizes the energy needed by the molecules to be set in motion against the frictional forces of the neighbouring molecules” [71]. The variation between the flow activation energy values is 31  Table 2.2: Percentage changes in the steady state rheological properties for OA SF samples (Average ± SD).  Percentage change from 25 °C to 37 °C Percentage change from uncentrifuge to centrifuge Percentage change before and after storage  η0  η1000  η0 /η300  λ  a  n  -42.9 ± 4.6  -12.2 ± 2  -37.6 ± 1.1  -43.9 ± 3  4.4 ± 4.1  4.5 ± 1.2  -23.2 ± 14  -6.1 ± 8.4  -17.5 ± 7.1  -15.6 ± 8.5  5.8 ± 9  3.5 ± 2.4  -23.7 ± 18.6  -5.8 ± 6.2  -19 ± 16.6  -18 ± 16.6  13.3 ± 21.9  4.2 ± 14.5  Table 2.3: Flow activation energy for six OA SF samples. Sample #  Activation energy (kJ/g mole)  SF#12 SF#13 SF#14 SF#15R SF#15L SF#20L  6.95 8.58 10.74 7.88 8.54 8.78  Avg ± SD  8.58 ± 1.25  small. Thus, the average value of the flow activation energy from this study can be used to predict the viscosity of SF at any arbitrary temperature if the viscosity is know at another temperature. It is worth noting that measurements were done at only two temperatures due to the limited sample volume. The dynamic moduli of OA SF are investigated by the oscillatory flow. Figure 2.9 shows the storage and loss moduli at 25 °C and 37 °C, as well as the cross-over frequency. Similar to previous steady shear rheological results, the viscoelastic properties of OA SF decreased as the temperature increased. OA SF viscoelasticity varied from sample to sample as shown in Figure 2.9. Table 2.4 shows the percentage changes in the dynamic rheological properties from 25 °C to 37 °C. Complete results are given in Appendix Table A.3 and Table A.7. The 32  2.5  2  Ln Viscosity  1.5  1  0.5 SF #12 SF #13 SF #14 SF #15R SF #15L SF #20L  0  −0.5 3.22  3.24  3.26  3.28  3.3  3.32  3.34  1/T (K−1)  3.36 −3  x 10  Figure 2.8: The Arrhenius plot for six OA SF samples. storage modulus of OA SF was more affected by the temperature, especially at a walking frequency. That suggests SF becomes a less effective lubricant in higher temperature environments or with excessive physical activity. The dynamic moduli of VS were more affected by the test temperature at both the walking and running frequencies. The storage and loss moduli of VS at the walking frequency decreased by 65% and 50%, respectively. At the running frequency, the storage and loss moduli decreased by 55% and 37%, respectively.  2.3.3  Centrifugation Effect  Centrifugation of SF samples in order to separate cells for chemical analysis was proposed by Balazs [12, 99]. Our observation of OA SF showed that it had small particles and small sediments. Those particles could change the rheological properties of OA SF. However, studying OA SF with those particles is still relevant 33  ′  ′′  Storage Modulus G and Loss Modulus G (Pa)  0  SF #13 G′ at 25 oC  10  SF #13 G′′ at 25 oC SF #13 G′ at 37 oC SF #13 G′′ at 37 oC SF #20L G′ at 25 oC SF #20L G′′ at 25 oC SF #20L G′ at 37 oC SF #20L G′′ at 37 oC  0  10  Frequency of Oscillation (Hz)  Figure 2.9: The dynamic rheological properties as a function of frequency for two OA SF samples at 25 and 37 °C.  Table 2.4: Percentage changes in the dynamic rheological properties for OA SF samples (Average ± SD). G0.5Hz (Pa)  G0.5Hz (Pa)  G2.5Hz (Pa)  G2.5Hz (Pa)  fc (Hz)  Gc (Pa)  Percentage change from 25 °C to 37 °C  -65 ± 4.8  -13 ± 3.3  -17 ± 3.2  -7 ± 2.1  86 ± 26.5  7 ± 7.7  Percentage change from uncentrifuge to centrifuge  -17.2 ± 9.1  -12 ± 8.9  -12.5 ± 9.1  -8.5 ± 8.6  8.2 ± 7.7  -10.8 ± 7.1  Percentage change before and after storage  -18.4 ± 18.2  -11.3 ± 16.5  -21.3 ± 17.7  -15.2 ± 17.9  23.3 ± 23.7  -1.4 ± 11.7  34  because it reflects the current state of SF in the joint. We were interested to see to what extent centrifugation would alter the rheological properties of SF. The steady state and dynamic rheological properties for five OA SF samples were studied before and after centrifugation. Figure 2.10 shows the results of the steady state rheological properties for three OA SF samples. Centrifugation had an effect on some samples, like sample SF#15L and SF#16; however, centrifugation did not have the same effect across all of our samples. Some of the samples were not affected by the centrifugation like sample SF#14. The average percentage changes of the steady state rheological properties for all OA SF samples from uncentrifuge measurement to centrifuge measurement are presented in Table 2.2. Complete results are given in Appendix Table A.2 and Table A.8. Viscosupplement was also tested before and after centrifugation. The rheological properties of VS were not affected by centrifugation. Centrifugation also altered the dynamic rheological properties of OA SF. Figure 2.11 shows the dynamic moduli for two OA SF samples. The dynamic moduli were reduced after centrifugation on both samples. The cross-over frequency was also shifted to a higher frequency after centrifugation. The average percentage changes of the dynamic rheological properties for all OA SF samples from uncentrifuge measurement to centrifuge measurement are presented in Table 2.4. Complete results are given in Appendix Table A.3 and Table A.9. The dynamic rheological properties of VS were not affected by centrifugation. Centrifugation possibly did not alter the composition and structure of VS, which might explain why the rheological properties of VS did not change after centrifugation. Centrifugation can alter the composition of SF [91]. In this study, centrifugation was able to reduce the viscosity of OA SF by up to 36%. The reduction of the rheological properties of OA SF after centrifugation can be attributed to the changes of the HA-protein complex structure [91], and to the elimination of small particles such as cartilage debris. Some samples might have very few small particles; thus, centrifugation might have a lesser effect on them.  35  1  10  SF #15L SF #15L Centrifuged SF #16 SF #16 Centrifuged SF #14 SF #14 Centrifuged  0  Viscosity (Pa.s)  10  −1  10  −2  10  −2  10  −1  10  0  1  10  10  2  10  3  10  −1  Shear Rate (s )  Figure 2.10: Shear viscosity as a function of shear rate for three OA SF samples before and after centrifugation.  2.3.4  Storage Effect  Storage of SF for further study was used in the literature [23, 44, 52, 70]. In the current work, the rheological properties for eight OA SF samples were studied before and after storage for two weeks at -20 °C. Some previous studies stored their samples at a similar temperature [1, 85]. Figure 2.12 presents the steady state rheological properties for two OA SF samples. Storage decreased the rheological properties of OA SF. The average percentage changes of the steady state rheological properties for all OA SF samples from initial sample measurement to stored sample measurement are presented in Table 2.2. Complete results are given in Appendix Table A.2 and Table A.10. Figure 2.13 shows the dynamic moduli for two OA SF samples. Our results  36  Storage Modulus G′ and Loss Modulus G′′(Pa)  0  10  SF #15L G′ SF #15L G′ Centrifuged SF #15L G′′ SF #15L G′′ Centrifuged SF #16 G′ SF #16 G′ Centrifuged SF #16 G′′ SF #16 G′′ Centrifuged −1  0  10  10  1  10  Frequency of Oscillation (Hz)  Figure 2.11: The dynamic rheological properties as a function of frequency for two OA SF samples before and after centrifugation. show that storage for two weeks at -20 °C reduced the dynamic rheological properties of OA SF. The storage modulus was more affected by storage. The average percentage changes of the dynamic rheological properties for all SF samples from the initial sample measurement to the stored measurement are presented in Table 2.4. Complete results are given in Appendix Table A.3 and Table A.11. The steady state and dynamic rheological properties of VS were not modified by storage. This suggests that VS maintained its molecular structure. In this study, storage was able to reduce the viscosity of OA SF by up to 48%. The reduction of the rheological properties of OA SF after storage can be attributed to the change of the HA-protein structure.  37  1  10  SF #15L SF #15L After 2 Weeks SF #10 SF #10 After 2 Weeks  0  Viscosity (Pa.s)  10  −1  10  −2  10  −2  10  −1  10  0  1  10  10  2  10  3  10  −1  Shear Rate (s )  Figure 2.12: Shear viscosity as a function of shear rate for two OA SF samples before and after centrifugation before and after storage for two weeks.  2.3.5  Total Protein Concentration  The protein concentration was determined for 33 OA SF. The concentration ranged between 6 and 56 mg/mL. The average concentration ± the standard deviation was 24 ± 11 mg/mL. There was a wide variation between the samples’ protein concentration. Complete results are given in Appendix Table A.12. Our results show that the protein concentration in OA SF was higher than that of healthy SF reported in previous studies [17, 93]. The protein concentration of OA SF had been found to be between 25 and 35 mg/mL by Mathieu et al. [67]. However, their method does not measure the total protein concentration. The increase in the protein concentration of OA SF is attributed to the reduction of molecular weight and concentration of HA which forms a barrier in a healthy SF 38  Storage Modulus G′ and Loss Modulus G′′(Pa)  0  10  SF #15L G′ SF #15L G′ After 2 Weeks SF #15L G′′ SF #15L G′′ After 2 Weeks SF #10 G′ SF #10 G′ After 2 Weeks SF #10 G′′ SF #10 G′′ After 2 Weeks −1  0  10  10  1  10  Frequency of Oscillation (Hz)  Figure 2.13: The dynamic rheological properties as a function of frequency for two OA SF samples before and after storage for two weeks. that resists the influx of protein and other particles [31, 64].  2.3.6  Correlations between Total Protein Concentration and Rheological Properties  The rheological properties of OA SF samples were correlated with its protein concentration using Spearman’s rank correlation. No significant correlations were found between the protein concentration and the following rheological properties: λ , a, cross-over frequency, dynamic moduli at a cross-over frequency, and time dependent behaviour. Negative correlations were found between the protein concentration and η0 (p = 0.0384, r = −0.3621), and G0.5Hz (p = 0.0342, r = −0.3697). Moderate negative correlations were found between the protein concentration and G0.5Hz (p = 39  0.0165, r = −0.4143), G2.5Hz (p = 0.0168, r = −0.4133), and G2.5Hz (p = 0.0172, r = −0.4118). In addition, a weak positive correlation between the protein concentration and n (p = 0.0242, r = 0.3916) was found. Clearly, there is a weak to moderate correlation between the protein concentration and rheological properties of SF. The addition of protein may play a small role on the rheological properties of SF [90].  2.4  Conclusion  All OA SF samples exhibited a non-Newtonian shear thinning behaviour. There was a wide variation between samples’ rheological behaviour. Most of OA SF samples from this study are less viscous than healthy SF; however, some of OA SF samples showed rheological properties similar to those reported for healthy SF. The dynamic moduli and cross-over frequencies of some of the OA SF samples were similar to that of healthy SF reported in the literature. Those samples exhibited viscoelastic behaviour, and their cross-over frequencies took place at frequencies lower than that for walking. However, some samples did not show a cross-over frequency in the investigated frequency range. Thus, OA SF loses its shock absorption capability. Additionally, the Carreau-Yasuda model was found to be a suitable model to represent the rheological behaviour of OA SF and VS. Time dependent tests revealed that OA SF demonstrated a quick structural recovery and, it exhibited rheopectic behaviour. At constant shear or oscillation, the shear viscosity and dynamic moduli of SF increased with time. This means that SF at a constant low shear or oscillation becomes stiffer with time. Viscosupplement exhibited thixotropic behaviour. The difference between the time dependent behaviour of SF and VS might be related to the protein content in SF and the lack of it in VS. Protein aggregation was hypothesized to cause the rheopectic behaviour of SF [80]. The test temperature and pre-treatments modified the rheological properties of OA SF. Viscosupplement was only affected by the temperature. This suggests that neither centrifugation nor storage altered the compositions or structure of VS, and that storage and centrifugation may alter SF composition and HA-protein structure. Therefore, careful consideration should be taken if any of these pre-treatments is  40  required for future studies. The protein concentration of OA SF samples had a wide variation. Weak to moderate correlations between some of the rheological properties of OA SF (η0 , G0.5Hz , G0.5Hz , G2.5Hz , G2.5Hz and n) and the protein concentration were found. Nevertheless, further analysis of SF composition is needed to understand the protein contribution to the rheological properties of SF. The current work will lead to a better understanding of the rheological properties of SF. Moreover, the values of the Carreau-Yasuda model parameters could be used for further simulation studies of SF lubrication abilities. Further studies of healthy SF using modern rheometry are required to evaluate their rheological properties. The characterization of SF rheology will assist in developing a better viscosupplement or/and pseudo SF.  41  Chapter 3  Osteoarthritic Synovial Fluid and Viscosupplement Extensional Rheology 3.1  Introduction  Steady state and dynamic shear flow cannot fully describe the rheological behaviour of SF in joint movement [2]. During joint movements, SF is subjected to shear, compression and extension. Extensional flow can significantly stretch HA molecules, which result in a significant increase in the elastic forces and extensional viscosity [11, 74]. The extensional viscosity is potentially dominant at high shear rates (500 s−1 ) [2]. Thus, measurement of the extensional rheology of SF is important. There are limited techniques to measure the extensional rheology of polymer solutions. Moreover, early designs of extensional rheometers required a large sample volume. Thus, studies were only conducted on HA solutions [2, 11, 74]. Capillary breakup extensional rheometer (CaBER) is one reliable technique that has attracted attention by many research groups to study the extensional rheological properties [6, 23, 30, 72]. Capillary breakup extensional rheometer was used by Bing¨ol et al. [23] to study five post mortem human SF samples, a synovial fluid  42  model and NaHA solutions. However, extensional rheological characterization of SF is still required. Moreover, there is a need to understand the effect of CaBER configurations on the extensional rheology measurements. In this chapter, the optimal CaBER configurations for OA SF and VS are investigated by studying the influence of CaBER endplates diameter and step-stretch parameters on capillary breakup extensional rheology measurements. Furthermore, the extensional rheological properties of different OA SF samples and VS are discussed. Additionally, the extensional rheological properties are correlated with the protein concentration. The materials and methods will be presented first. Subsequently, the results of the extensional rheological tests and correlations with the protein concentration will be presented and discussed. Lastly, the conclusions of this part of the study will be presented.  3.2 3.2.1  Materials and Methods Materials  Synovial Fluid In this part of the study, extensional rheological measurements were done on 21 OA SF samples. The samples were obtained from 20 subjects (10 males and 10 females) and were labelled SF#1 - SF#32 (R or L was used when two samples were collected from the same subject to indicate right or left knee respectively). The average age was 65 years. Density measurements were conducted immediately after collecting SF samples. The samples were stored at - 20 °C for 1 to 2 days before conducting extensional rheology experiments and surface tension measurements.  3.2.2  Methods  Capillary Breakup Extensional Rheometer Extensional rheological properties of each SF sample were determined using a CaBER 1 extensional rheometer (Thermo Electron, Karlsruhe, Germany) using 4 43  and 6 mm circular endplates diameter, D. A small volume of the sample was applied between two plates forming a cylindrical liquid bridge with an initial distance hi of 2 mm equivalent to an initial aspect ratio, Λi =  hi D,  of 0.33. After loading the  sample, an extensional step strain was applied by moving the upper plate to a predefined final axial height h f with a linear increasing separation profile. The final aspect ratio, Λ f =  hf D,  used for this study ranged between 1.4 and 2. The separation  times used for this study were 50 ms, 60 ms, and 70 ms. Extensional measurements started once the upper plate reached its final position, corresponding to time t = 0. The evolution in time of the midpoint diameter was monitored using a laser micrometer. The filament tinning is driven by the material surface tension, and it is resisted by the viscous and elastic forces [102]. The filament thinning for a viscoelastic fluid like SF consists of two stages [102]. Initially, the filament decreases exponentially over time. Then, it decreases linearly over time in at a later stage. Monitoring the filament diameter changes during the first stage reveals the longest relaxation time for the material. This is the time that polymer molecules need to constrict after stretching [103]. For polymer solutions, it can be calculated using the following equation [103]: D(t) = D0 e  − 3λt  E  (3.1)  where D(t) is the filament diameter change with time, D0 is the initial value of filament diameter, t is the time, and λE is the longest relaxation time. The later stage of the filament thinning reveals the terminal extensional viscosity of the material. This occurs when the maximum extension of the polymer molecules has been reached. The following equation was used to calculate the terminal extensional viscosity [103]: D(t) = D0 −  σ t ηE,t  (3.2)  where ηE,t is the terminal extensional viscosity. Phantom V611 high speed camera (Vision Research, Inc., Stuart, FL) recording images at 1000 frames per second at a resolution of 800 × 600 pixel equipped with a 18-108/2.5 Navitar Zoom 7000 (Technical Instruments, San Francisco, CA) 44  macro zoom lens was used to capture temporal evolution in filament profiles. Electroluminescent sheet (Electro Luminescence Inc., Aromas, CA) was used as a light source and was placed behind the sample. Capillary breakup extensional rheometer has a linear motor with a resolution of 0.02 mm and a laser micrometer with a resolution of 0.01 mm. The laser micrometer was calibrated using an optical fibers of known size. Tensiometer Material surface tension was measured at room temperature using a FTA 1000 Drop Shape Analysis instrument (First Ten Angstroms, Portsmouth, VA). A drop from the sample was formed by the apparatus which was then captured by video image. The shape of the drop was then fitted to the Young-Laplace equation which associates inter-facial tension to drop shape [111]. The Young-Laplace equation required the drop to be distorted into a pendant shape to show the effects of gravity distortion balanced against the restoring force of surface tension [111]. 500 µL syringes (Hamilton, Reno, Nevada) were used to load samples into the system. The pump out rate was set at 0.8967 µL/s for OA SF and 0.2 µL/s for VS to form 10 µL drop. The decrease of surface tension with time was observed until a plateau was reached (time required ca. 5 min). In order to verify the accuracy of the measurements, the surface tension of distilled water was measured before the samples measurements. The apparatus has a resolution of 0.01 mN/m. Density Measurements The density of the samples were measured by dividing the material mass to the volume. 1 mL of each sample was pipetted using PIPETMAN P1000 (Gilson Inc., Middleton, WI). Then, the mass of 1 mL of each sample was measured by GR-200 analytical semi-microbalance (A and D Co., ltd, Japan). The combined error was +/- 0.008 mg/mL.  45  Correlation between Synovial Fluid Protein Concentration and Flow Properties The protein concentration was correlated with the extensional rheological properties: the terminal extensional viscosity and the longest relaxation time. The correlation tests were done using Spearman’s rank test.  3.3 3.3.1  Results and Discussion Surface Tension  Prior to CaBER measurements, the surface tension of OA SF samples and VS were measured. The surface tension of OA SF ranged between 46.8 and 52 mN/m (49.2 ± 1.43). The variations between OA SF samples’ surface tension were small. The difference between the surface tension of the samples could be due to the variation between the compositions of those samples. The surface tension of VS is 66.8 mN/m. It is higher than OA SF, possibly due to the fact that VS does not contain protein as OA SF [23].  3.3.2  Influence of Endplates Diameter and Final Aspect Ratio  Although CaBER is fairly easy to operate, careful consideration should be taken for reliable measurements. Some parameters are user selectable. Those parameters are the endplate diameter, the step strain duration, the initial aspect ratio Λi = and the final aspect ratio Λ f =  hf D.  hi D  We aimed to determine the optimal CaBER  configuration which is suitable for testing a wide range of OA SF samples. Initially, four OA SF samples were tested using different CaBER configurations. A high speed camera was used to ensure that the filament thinning of OA SF followed trends similar to those of other polymer fluids reported in the literature [5, 29, 92, 102]. Figure 3.1 shows different frames from video images starting at t = 0 and ending when the filament is ruptured at t = tevent (using 6 mm endplate, hi = 2 mm, h f = 10 mm, and separation time = 50 ms). The initial plate separation hi should be below or equal to the capillary length lcap =  σ /ρg (σ is the surface  tension and ρ is the density) in order to keep the initial loading cylindrical and to minimize sagging [92]. The capillary length lcap ranged between 2.3 and 2.4 mm 46  t= 0  t= 0.2 t  event  t= 0.4 t  t= 0.6 t  event  event  t= 0.8 t  event  t= t  event  Figure 3.1: Temporal evolution in filament profiles for SF#32 using 6 mm endplate, hi = 2 mm, h f = 10 mm, and separation time = 50 ms. (t = 0 is the time when the upper plate reached its final position, and t = tevent is the time when the filament ceased). for OA SF, and it was 2.6 mm for VS. Therefore, an initial height of hi = 2 mm was used in this study. Using an initial separation hi of 3 mm similar to [23] caused an axial sagging. Three different endplate diameters (4, 6, and 8 mm) can be used with CaBER. The common and standard geometry for CaBER is a 6 mm plate diameter. This geometry has been used for a variety of polymers [23, 30, 92]. Samples were tested using two endplate diameters (4 and 6 mm), and using four final aspect ratios (Λ f = 1.4, 1.6, 1.8 and 2). Eight mm endplate diameter was not chosen due to the limited sample volume and to avoid sagging due to the gravity. Bond number (Bo = ρgD2 /4σ ) gives an estimation of the relative magnitude of the sagging effect compared with the capillary forces [5]. The Bond number for OA SF ranged between 0.7 and 0.8 for 4 mm endplates and between 1.6 and 1.7 for 6 mm endplates. Bond numbers for both 4 and 6 mm endplates were low enough to do CaBER experiments successfully [5]. Figure 3.2 shows the filament evolution for an OA SF sample using 4 mm endplates, 50 ms extension time and different final aspect ratios Λ f (1.4, 1.6, 1.8 and 2). Table 3.1 shows the extensional rheological properties for four OA SF samples corresponding to the same final aspect ratios Λ f . When final aspect ratios Λ f of 1.4 and 1.6 were used, the filament thinning curve did not provide a good fit with Equation 3.1 and Equation 3.2. Figure 3.3 shows the filament evolution for an OA SF sample using 6 mm  47  1  10  1.4 1.6 1.8 2  0  Diameter (mm)  10  −1  10  −2  10  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  Time (s)  Figure 3.2: Filament diameter thinning over time for SF#2 using 4 mm endplates and different final aspect ratios Λ f (1.4, 1.6, 1.8 and 2). (initial plate separation hi = 2mm, and separation time = 50 ms)  Table 3.1: Extensional rheological properties for four OA SF samples using 4 mm endplates at different final aspect ratios. (initial plate separation hi = 2 mm, and separation time = 50 ms). SF#1  SF#2  SF#3R  SF#3L  Λf  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  1.4 1.6 1.8 2  87 76 82  93 44 57  52 52 59  58 42 35  306 219 247  126 135 122  96 96 105  128 54 68  48  endplates, 50 ms extension time and different final aspect ratios Λ f (1.4, 1.6, 1.8 and 2). Table 3.2 shows the extensional rheological properties for four OA SF samples corresponding to the same final aspect ratios Λ f . For SF#3R, there was not enough sample to conduct the test using Λ f = 2. Inertial oscillations of the filament was observed when 4 mm endplates were used. Moreover, due to the nature of SF samples, it was difficult to load the samples. On the contrary, inertial oscillations of the filament were not observed, and sample loading was easier when the 6 mm endplates were used. Furthermore, varying the final aspect ratio Λ f did not result in a large difference between the measurements compared to 4 mm endplates. Thus, 6 mm endplates were chosen for all subsequent testing. It is worth noting that due to the small volume of OA SF samples, the preliminary testing was carried once for each sample. When the test was repeated for OA SF samples, the rheological properties varied slightly from one measurement to another. Variations were more visible in the measurements of the terminal extensional viscosity. Although the shear rheology measurements were repeatable, the CaBER measurements were not. Due to the small sample volume used for CaBER, small particles that were found in the samples could have influenced the extensional rheological measurements. Centrifugation of the samples might have removed the small particles; thus, centrifugation might have resulted in more repeatable results. Nevertheless, studying OA SF with those particles is still relevant because it reflects the current state of SF in the joint. Table 3.3 shows the result of repeated testing done on SF#30 using the following configuration: plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm, and separation time = 70 ms. For all subsequent testing, samples were measured by repeating the test at least three times and the average was reported.  3.3.3  Influence of Step-stretch Parameters  Osteoarthritic Synovial Fluid We further investigated the effect of step-stretch parameters on CaBER measurements. The influence of the final aspect ratio using a constant extension rate, and  49  1  10  1.4 1.6 1.8 2  0  Diameter (mm)  10  −1  10  −2  10  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  Time (s)  Figure 3.3: Filament diameter thinning over time for SF#2 using 6 mm endplates and different final aspect ratios Λ f (1.4, 1.6, 1.8 and 2). (initial plate separation hi = 2mm, and separation time = 50 ms)  Table 3.2: Extensional rheological properties for four OA SF samples using 6 mm endplates at different final aspect ratios. (initial plate separation hi = 2 mm, and separation time = 50 ms) SF#1  SF#2  SF#3R  SF#3L  Λf  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  1.4 1.6 1.8 2  104 107 105 100  56 60 59 58  73 75 75 79  40 42 39 38  270 279 283 -  101 198 206 -  97 139 135 142  47 55 65 53  50  Table 3.3: Results from repeated CaBER experiments for SF#30. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm, and separation time = 70 ms) Trial #  λE (ms)  ηE,t (Pa·s)  1 2 3 4 5  77 74 73 75 75  33 30 27 31 33  Table 3.4: Extensional rheological properties for five OA SF samples and VS using three different final heights h f . (Plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm, and imposed extension rate = 80 s−1 ) SF#27  SF#28  SF#30  SF#31  SF#32  VS  hf  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  9 mm 10 mm 11 mm  105 109 118  52 52 56  70 73 71  33 32 30  66 76 81  31 32 29  78 86 92  34 36 36  54 66 58  20 23 22  47 44 36  51 50 51  the effect of the step-strain time on CaBER measurements were studied. Figure 3.4 shows the filament midpoint diameter for SF#33 as a function of time at three different final heights: 9, 10, and 11 mm (corresponding to the final aspect ratios Λ f : 1.5, 1.67 and 1.83, respectively) using a constant imposed extension rate (80 s−1 ). This experiment was carried out on five OA SF samples. Their extensional rheological properties are presented in Table 3.4. Figure 3.5 shows the filament midpoint diameter for SF#33 as a function of time using three different step-strain durations: 50, 60, and 70 ms, using a constant final plate separation (10 mm). Table 3.5 presents CaBER results for six samples. Osteoarthritic synovial fluid samples were also not affected by varying step-strain duration. Similar to our results, Miller et al. [72] found that varying step-strain parameters should not influence the rheological properties for polymer solutions. Thus, both step-stretch parameters (final height and separation time) were only  51  1  10  hf = 9 mm hf = 10 mm hf = 11 mm  0  Diameter (mm)  10  −1  10  −2  10  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  Time (s)  Figure 3.4: Filament diameter thinning over time for SF#33 using three different final plate separation. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, and imposed extension rate = 80 s−1 ) found to change the extensional rheological properties of SF slightly. Viscosupplement Viscosupplement only contains HA, and it does not contain small particles like OA SF. In this study, VS was mainly tested to investigate the repeatability of CaBER measurements and to investigate the influence of step-stretch parameters on CaBER measurements. Viscosupplement was tested using three different final separations (9 mm, 10 mm, 11 mm) at fixed imposed stretch rate (80 s−1 ). Figure 3.6 shows the filament diameter evolution; two final separation heights, 9 and 10 mm, almost resulted in the same profile. However, using 11 mm as a final height resulted in a distinct 52  1  10  50 ms 60 ms 70 ms  0  Diameter (mm)  10  −1  10  −2  10  0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  Time (s)  Figure 3.5: Filament diameter thinning over time for SF#33 using three different extension times. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, and final plate separation h f = 10 mm)  Table 3.5: Extensional rheological properties for six OA SF samples and VS using three different extension rates. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, and final plate separation h f = 10 mm) SF#20R  SF#22  SF#26  SF#28  SF#30  SF#31  VS  Extension time  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  λE (ms)  ηE,t (Pa·s)  50 ms 60 ms 70 ms  121 126 114  47 50 45  125 104 111  46 33 40  114 114 118  45 48 47  73 69 68  32 32 31  69 71 75  27 33 31  86 84 87  36 32 34  44 44 43  50 50 50  53  1  10  hf = 9 mm hf = 10 mm hf = 11 mm  0  Diameter (mm)  10  −1  10  −2  10  0  0.05  0.1  0.15  0.2  0.25  0.3  0.35  0.4  Time (s)  Figure 3.6: Filament diameter thinning versus time for VS using three different final plate separation. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, and imposed extension rate = 80 s−1 ) profile from those for 9 and 10 mm. Table 3.4 presents the extensional rheological properties of VS. Changing the final height did not change the terminal extensional viscosity; however, it influenced the relaxation time. Increasing the final separation height reduced the longest relaxation time. Viscosupplement was also tested using three different step-strain durations: 50 ms, 60 ms, and 70ms. Figure 3.7 shows the filament diameter evolution. All of the filament diameters thinning profiles are almost identical. The terminal extensional properties are presented in Table 3.5. The extensional rheological properties of VS were not influenced by the step-strain duration. Table 3.6 shows the result of repeated testing done on VS using the following configuration: plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate 54  1  10  50 ms 60 ms 70 ms  0  Diameter (mm)  10  −1  10  −2  10  0  0.05  0.1  0.15  0.2  0.25  0.3  0.35  0.4  Time (s)  Figure 3.7: Filament diameter thinning versus time for VS using three different extension times. (Plate diameter = 6mm, initial plate separation hi = 2mm, and final plate separation h f = 10mm) separation h f = 10 mm, and separation time = 50 ms. The repeatability of CaBER measurements were better for VS than OA SF. This observation supports the hypothesis that small particles inside SF might influence the extensional rheological measurements as discussed earlier.  3.3.4  Extensional Rheological Properties  The optimal setup for CaBER to study the extensional rheological properties of OA SF was selected based on the observations in Section 3.3.2 and Section 3.3.3. The following configuration was chosen: 6 mm plate diameter, hi = 2 mm, h f = 10 mm, and an extension time of 50 ms. As mentioned in Section 3.3.2, 6 mm endplates provided more consistent results compared to 4 mm endplates, and 55  Table 3.6: Results from repeated CaBER experiments for VS. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm, and separation time = 50 ms) Trial #  λE (ms)  ηE,t (Pa·s )  1 2 3 4 5  43 44 44 44 43  51 50 50 49 50  inertial oscillations of the filament were not observed. Moreover, loading samples were easier with 6 mm endplates. The final height was chosen to be within the previous investigated range in Section 3.3.3 . Figure 3.8 shows the diameter evolution for five OA SF samples. There was a wide variation between the rheological properties of the samples. As illustrated by the diameter evolution in Figure 3.8, the lowest viscous sample breakup earlier. The reduction in the extensional rheological properties is probably due to the reduction in the molecular weight and concentration of HA [2, 3, 11, 23, 74]. The extensional rheological properties of VS were investigated using the following configuration: 6 mm plate diameter, hi = 2 mm, h f = 10 mm, and an extension time of 50 ms . The diameter evolution for VS is presented in Figure 3.9. The relaxation time for VS was 44 ms, and the terminal extensional viscosity was 50 Pa·s. A clear dependency between the terminal extensional viscosity and relaxation time was also observed as shown in Figure 3.10. This can be used as guidance for estimating terminal viscosity if relaxation time is known or vice versa. The relationship between the terminal extensional viscosity and relaxation time can also be used to determine the polymer flexibility as proposed by Stelter et al. [103]. They found two limiting ranges for flexible and rigid polymer behaviour. Different polymer solutions that they studied fell into one of the two limiting cases. Figure 3.10 shows that OA SF is lower than those two limiting cases for flexible and rigid like behaviour. However, VS agreed with the behaviour of rigid polymers that was observed by Stelter et al. [103]. Protein content and other molecules may increase 56  1  10  SF#3R SF#11 SF#15L SF#15R SF#25  0  Diameter (mm)  10  −1  10  −2  10  0  0.5  1  1.5  2  2.5  Time (s)  Figure 3.8: Filament diameter thinning over time for five OA SF samples. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm extension time: 50 ms) the rigidity of SF.  3.3.5  Correlation between Total Protein Concentration and Extensional Rheology  The protein concentration of OA SF samples have been determined and presented in Section 2.3.5. The steady state and dynamic rheological properties of OA SF were correlated with the protein concentration. Correlations were found between the shear rheological properties and the protein concentration. Protein contents contribution on the rheological properties of SF is not totally clear. Oates et al. [79, 80] suggests that protein aggregations cause the rheopectic behaviour of SF. The protein concentration usually increases with OA. This is attributed to the reduction 57  Table 3.7: Extensional rheological properties for 21 OA SF samples. (Plate diameter = 6mm, initial plate separation hi = 2mm, final plate separation h f = 10mm, and extension time = 50 ms) Sample #  σ (mN/m)  ηE,t (Pa·s)  λE (ms)  SF#3R SF#9 SF#10 SF#11 SF#12 SF#13 SF#14 SF#15R SF#15L SF#20R SF#22 SF#23 SF#24 SF#25 SF#26 SF#27 SF#28 SF#29 SF#30 SF#31 SF#32  49.2 49.1 47.1 49.1 48.9 46.8 48.2 50.1 48.4 50 48.3 48.9 47.3 49.3 49.8 48.4 52.8 49.7 52 50 49.7  280 67 104 58 119 114 128 145 200 121 128 107 77 101 116 109 73 118 76 86 66  196 30 61 33 44 45 50 60 93 47 53 51 33 46 48 52 32 48 32 36 23  of the molecular weight and concentration of HA which forms a barrier in healthy SF that resists the influx of protein and other particles [31, 64]. In the current work, the correlation between the extensional rheological properties of OA SF and the protein concentration were investigated. However, no significant correlation between the protein concentration and the terminal extensional viscosity and the longest relaxation time were found.  58  1  10  VS  0  Diameter (mm)  10  −1  10  −2  10  0  0.05  0.1  0.15  0.2  0.25  0.3  0.35  Time (s)  Figure 3.9: Filament diameter thinning over time for VS. (Plate diameter = 6 mm, initial plate separation hi = 2 mm, final plate separation h f = 10 mm extension time: 50 ms)  59  4  10  Terminal Extensional Viscosity η  E,t  (Pa.s)  OA SF Experimental Data VS Experimental Data Flexible Behaviour Rigid Behaviour  3  10  2  10  η E,t = 432.78λ E + 0.099 1  10 −2 10  −1  10  Longest Relaxation Time (s)  Figure 3.10: The terminal extensional viscosity vs. the relaxation time.  60  0  10  3.4  Conclusion  In this study, CaBER was used to study the extensional rheological properties of OA SF and VS. The effect of different CaBER configurations on the extensional rheological properties was investigated first to find the optimal CaBER setup. 6 mm endplate was found to produce more consistent results compared to 4 mm endplates when the samples were tested using different final aspect ratios. The influence of step-stretch parameters (final separation at a fixed extension rate of stretch and separation time) on CaBER measurements was investigated. Both step-stretch parameters were found to change the extensional rheological properties of SF slightly. Viscosupplement was not affected by varying the extension rate of stretch; however, varying the final separation caused a pronounced difference. The extensional rheological properties of 21 OA SF samples were studied using the optimal setup for CaBER (6 mm plate diameter, hi = 2 mm, h f = 10 mm, and an extension time of 50 ms). There was a wide variation between the rheological properties of the samples. Additionally, a clear dependency between the terminal extensional viscosity and relaxation time was observed. Capillary breakup extensional rheometer measurements were not repeatable for OA SF; however, it was more repeatable for VS. The small particles in OA SF could influence the repeatability of CaBER measurements. Consequently, averages were taken for at least three measurements. No correlation was found between the extensional rheological properties and the protein concentration of OA SF. The current work leads to a better understanding of the rheological properties of SF under extensional flow. However, there is still a need for more studies of the extensional rheological properties of healthy SF. Complete rheological studies of healthy and OA SF will also assist in developing a better viscosupplement that mimic the rheological properties of SF under both shear and extensional flows.  61  Chapter 4  Conclusions 4.1  Shear Rheology  All OA SF samples exhibited a non-Newtonian shear thinning behaviour. There was a wide variation between samples’ rheological behaviour. Most of OA SF samples from this study are less viscous than healthy SF; however, some of OA SF samples showed rheological properties similar to those reported for healthy SF. The dynamic moduli and cross-over frequencies of some of the OA SF samples were similar to that of healthy SF reported in the literature. Those samples exhibited viscoelastic behaviour, and their cross-over frequencies took place at frequencies lower than that for walking. However, some samples did not show a cross-over frequency in the investigated frequency range. Thus, OA SF loses its shock absorption capability. Additionally, the Carreau-Yasuda model was found to be a suitable model to represent the rheological behaviour of OA SF and VS. Time dependent tests revealed that OA SF demonstrated a quick structural recovery and, it exhibited rheopectic behaviour. At constant shear or oscillation, the shear viscosity and dynamic moduli of SF increased with time. This means that SF at a constant low shear or oscillation becomes stiffer with time. Viscosupplement exhibited thixotropic behaviour. The difference between the time dependent behaviour of SF and VS might be related to the protein content in SF and the lack of it in VS. Protein aggregation was hypothesized to cause the rheopectic behaviour of SF [80]. 62  The test temperature and pre-treatments modified the rheological properties of OA SF. Viscosupplement was only affected by the temperature. This suggests that neither centrifugation nor storage altered the compositions or structure of VS, and that storage and centrifugation may alter SF composition and HA-protein structure. Therefore, careful consideration should be taken if any of these pre-treatments is required for future studies.  4.2  Extensional Rheology  In this study, CaBER was used to study the extensional rheological properties of OA SF and VS. The effect of different CaBER configurations on the extensional rheological properties was investigated first to find the optimal CaBER setup. 6 mm endplate was found to produce more consistent results compared to 4 mm endplates when the samples were tested using different final aspect ratios. The influence of step-stretch parameters (final separation at a fixed extension rate of stretch and separation time) on CaBER measurements was investigated. Both step-stretch parameters were found to change the extensional rheological properties of SF slightly. Viscosupplement was not affected by varying the extension rate of stretch; however, varying the final separation caused a pronounced difference. The extensional rheological properties of 21 OA SF samples were studied using the optimal setup for CaBER (6 mm plate diameter, hi = 2 mm, h f = 10 mm, and an extension time of 50 ms). There was a wide variation between the rheological properties of the samples. Additionally, a clear dependency between the terminal extensional viscosity and relaxation time was observed. Capillary breakup extensional rheometer measurements were not repeatable for OA SF; however, it was more repeatable for VS. The small particles in OA SF could influence the repeatability of CaBER measurements. Consequently, averages were taken for at least three measurements.  63  4.3  Correlations between Rheological Properties and Protein Concentration  The protein concentration of OA SF samples had a wide variation. Weak to moderate correlations between some of the rheological properties of OA SF (η0 , G0.5Hz , G0.5Hz , G2.5Hz , G2.5Hz and n) and the protein concentration were found. Nevertheless, further analysis of SF composition is needed to understand the protein contribution to the rheological properties of SF. No correlation was found between the extensional rheological properties and the protein concentration of OA SF.  4.4  Contributions to the Research • The time-dependent rheological properties of SF where investigated by oscillatory shear flow for the first time. • To the best of my knowledge, this study was the first to use the CarreauYasuda model to fit the experimental rheological results of SF. • The current work provided an extensive correlation of the rheological properties of SF with the protein concentration. • The extensional rheological properties of OA SF were characterized for the first time, to the best of my knowledge. • This study investigated the influence of different CaBER configurations on the extensional rheological measurements for SF. • The dependency between the terminal extensional viscosity and relaxation time was determined. • In the current work, the pre-treatments (centrifugation and storage) effect on the rheological properties of SF were investigated.  4.5  Limitations to Study • The rheological measurements of SF do not reflect the in vivo physiological condition of a human joint. 64  • The limited sample volume of SF prohibited the use of a larger geometry for shear and oscillatory rheology which is more suitable for low viscous samples. • The small sample volume of SF did not allow us to perform all the required tests. • Extension rate in CaBER is instantaneous, and it is not user controlled.  4.6  Recommendations for Future Work • Previous rheological studies of healthy SF were conducted in the ’80s and early ’90s. Since then, there have been advancements in the rheometry measurements. Therefore, there is a need for a more recent study for the rheological properties of healthy SF using modern technology. • A complete rheological characterization of healthy and OA SF with its chemical composition is still required. • A thorough rheological test using CaBER accompanied by the fluid composition is needed. • A systemic evaluation of viscosupplements commercially available using CaBER is needed to determine if any of them can mimic the extensional rheological properties of SF. • There is a need for a design of pseudo-synovial fluid that mimics SF rheological properties.  65  Bibliography [1] N. Adam and P. Ghosh. Hyaluronan molecular weight and polydispersity in some commercial intra-articular injectable preparations and in synovial fluid. Inflammation Research, 50(6):294–299, 2001. → pages 36 [2] S. al Assaf, J. Meadows, G. O. Phillips, and P. A. Williams. 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Sample  Age  Gender  Right/Left  SF Appearance  SF#1  72  M  Right  Yellow  SF#2  56  F  Left  Yellow  SF#3R  67  M  Right  Yellow  SF#3L  67  M  Left  Yellow  SF#4  73  F  Right  Red  SF#5  56  F  Left  Red  SF#6  74  M  Right  Light Yellow  SF#7  58  F  Right  Dark Yellow  SF#8  71  M  Left  Light Yellow  SF#9  62  M  Right  Light Yellow  SF#10  63  M  Left  Red  SF#11  54  F  Right  Light Yellow  SF#12  75  M  Right  Yellow  SF#13  58  F  Right  Yellow  SF#14  64  M  Left  Yellow  SF#15R  54  M  Right  Yellow  SF#15L  54  M  Left  Yellow  SF#16  52  M  Right  Yellow  SF#17  49  F  Right  Yellow-Orange  SF#18  72  M  Left  Yellow  SF#19  44  M  Right  Yellow-Light Orange  SF#20R  68  M  Right  Red  SF#20L  68  M  Left  Yellow  SF#21  65  F  Right  Yellow  SF#22  69  F  Left  Yellow-Hint of Orange  SF#23  70  F  Right  Yellow  SF#24  59  F  Right  Yellow-Hint of Orange  SF#25  59  F  Right  Yellow Continued on next page  78  Table A.1 – continued from previous page Sample  Age  Gender  Right/Left  SF Appearance  SF#26  73  F  Right  Yellow  SF#27  71  F  Left  Yellow  SF#28  71  F  Right  Yellow  SF#29  57  M  Left  Yellow  SF#30  74  M  Right  Yellow  SF#31  69  M  Left  Yellow  SF#32  58  F  Left  Yellow  Table A.2: Steady state rheological properties for OA SF samples at 37 °C.  Sample  η0 (Pa.s)  η0 /η300  SF#1  0.71  SF#2  Carreau-Yasuda Parameters η0 (Pa.s)  λ (s)  a  n  30  0.67  1.74  0.800  0.470  0.18  12  0.19  0.92  0.532  0.561  SF#3R  14.40  260  16.45  12.76  0.790  0.311  SF#3L  0.81  32  0.87  2.74  0.965  0.475  SF#4  0.53  25  0.56  1.91  0.821  0.491  SF#5  0.05  5  0.05  0.29  1.376  0.659  SF#6  2.22  82  2.44  5.54  0.760  0.395  SF#7  1.23  39  1.20  1.21  0.917  0.385  SF#8  0.59  29  0.60  2.36  0.826  0.485  SF#9  0.21  10  0.22  0.59  0.848  0.547  SF#10  1.90  68  2.00  3.90  0.918  0.398  SF#11  0.21  12  0.24  0.96  0.981  0.542  SF#12  1.02  31  1.27  1.66  0.679  0.414  SF#13  3.31  74  3.49  2.07  0.852  0.324  SF#14  2.23  69  2.38  3.48  0.985  0.383  SF#15R  2.03  63  2.10  3.51  0.987  0.402  Continued on next page 79  Table A.2 – continued from previous page Sample  η0 (Pa.s)  η0 /η300  SF#15L  5.63  SF#16  Carreau-Yasuda Parameters η0 (Pa.s)  λ (s)  a  n  122  6.23  6.45  0.815  0.353  1.10  32  1.11  1.78  0.819  0.450  SF#17  0.14  8  0.15  0.72  0.827  0.592  SF#18  1.87  73  1.98  5.47  0.944  0.413  SF#19  0.63  25  0.68  1.79  0.781  0.475  SF#20R  2.95  101  4.50  10.28  0.605  0.376  SF#20L  0.85  30  0.92  1.58  0.831  0.440  SF#21  8.70  289  9.56  18.78  1.036  0.333  SF#22  1.56  40  1.69  2.07  0.813  0.417  SF#23  1.28  49  1.34  3.02  0.582  0.426  SF#24  0.29  12  0.31  0.77  0.870  0.525  SF#25  0.26  15  0.27  1.07  1.090  0.522  SF#26  1.07  35  1.14  1.84  0.808  0.429  SF#27  0.61  26  0.66  1.62  0.904  0.465  SF#28  0.23  11  0.24  0.66  0.940  0.540  SF#29  0.80  30  0.85  2.15  1.000  0.467  SF#30  0.35  16  0.36  0.79  0.918  0.489  SF#31  0.48  22  0.49  1.37  0.765  0.765  SF#32  0.28  12  0.32  0.42  0.768  0.471  Average  1.73  51  1.93  3.09  0.862  0.463  Table A.3: Dynamic rheological properties for OA SF samples at 37 °C. Sample  G0.5Hz  G0.5Hz  G2.5Hz  G2.5Hz  fc  Gc  (Pa)  (Pa)  (Pa)  (Pa)  (Hz)  (Pa)  SF#1  0.47  0.59  1.31  1.18  1.55  0.94  SF#2  0.16  0.21  0.45  0.56  No  N/A  Continued on next page 80  Table A.3 – continued from previous page Sample  G0.5Hz  G0.5Hz  G2.5Hz  G2.5Hz  fc  Gc  (Pa)  (Pa)  (Pa)  (Pa)  (Hz)  (Pa)  SF#3R  4.00  2.58  7.32  3.46  0.07  1.51  SF#3L  0.46  0.54  1.18  1.11  1.10  0.76  SF#4  0.41  0.51  1.08  1.05  2.76  1.03  SF#5  0.05  0.11  0.13  0.34  No  N/A  SF#6  1.03  0.92  2.31  1.49  0.26  0.74  SF#7  1.00  1.18  2.72  2.00  0.89  1.45  SF#8  0.43  0.49  1.06  0.96  1.94  0.84  SF#9  0.23  0.36  0.74  0.95  No  N/A  SF#10  1.09  0.98  2.32  1.55  0.35  0.89  SF#11  0.28  0.37  0.71  0.86  No  N/A  SF#12  0.81  0.95  2.17  1.81  1.17  1.35  SF#13  2.10  1.99  4.88  2.99  0.42  1.87  SF#14  1.33  1.19  2.90  1.81  0.32  1.02  SF#15R  1.18  1.08  2.61  1.76  0.41  1.04  SF#15L  2.22  1.77  4.54  2.62  0.18  1.25  SF#16  0.87  0.95  2.15  1.89  1.19  1.39  SF#17  0.11  0.24  0.40  0.68  No  N/A  SF#18  1.07  0.86  2.18  1.38  0.15  0.57  SF#19  0.38  0.54  1.11  1.15  3.96  1.27  SF#20R  1.43  1.08  2.89  1.71  0.06  0.46  SF#20L  0.61  0.75  1.66  1.49  1.66  1.25  SF#21  2.03  1.16  3.45  1.52  0.06  0.69  SF#22  1.01  1.14  2.64  2.18  1.00  1.50  SF#23  0.61  0.69  1.56  1.30  1.05  0.94  SF#24  0.25  0.43  0.83  1.04  No  N/A  SF#25  0.25  0.39  0.72  0.78  No  N/A  SF#26  0.74  0.90  1.92  1.61  1.17  1.23  SF#27  0.54  0.65  1.36  1.21  1.65  1.02  Continued on next page 81  Table A.3 – continued from previous page Sample  G0.5Hz  G0.5Hz  G2.5Hz  G2.5Hz  fc  Gc  (Pa)  (Pa)  (Pa)  (Pa)  (Hz)  (Pa)  SF#28  0.22  0.36  0.65  0.89  No  N/A  SF#29  0.75  0.75  1.65  1.34  0.52  0.76  SF#30  0.33  0.53  1.04  1.13  5.00  1.42  SF#31  0.35  0.50  0.93  0.99  3.98  1.13  SF#32  0.23  0.51  0.96  1.20  No  N/A  Average  0.83  0.81  1.90  1.43  1.26  1.09  Table A.4: Relative viscosity change for OA SF when applying constant 0.05 s−1 , for 1800 s. Sample  Relative Viscosity Change (ηt=1800s − ηt=0s ) · 100/ηt=0s  SF#10 SF#15L SF#20L SF#22 SF#25  20.88 18.15 10.37 11.22 33.78  Average  18.88  82  Table A.5: Relative change in storage modulus G , loss modulus G , and viscosity η when applying the oscillation/rotation/oscillation profile presented in Figure 2.1. Relative G Change  Relative G Change  Relative η Change  (Gt=1410 −Gt=210 )·100 Gt=210  (Gt=1410 −Gt=210 )·100 Gt=210  (ηt=1410 −ηt=210 )·100 ηt=210  SF#1 SF#2 SF#3R SF#3L SF#4 SF#5 SF#6 SF#7 SF#8 SF#9 SF#10 SF#12 SF#13 SF#14 SF#15L  14 43 8 5 10 42 9 6 7 16 4 27 -6 4 6  11 1 7 5 2 12 5 8 8 -3 6 13 2 4 4  12 12 8 5 6 14 8 7 8 2 5 20 -5 4 5  Average  13  6  7  Sample  Table A.6: Steady state rheological properties for OA SF samples at 25 °C. Carreau-Yasuda Parameters η0 (Pa.s) λ (s) a n  Sample  η0 (Pa.s)  η0 /η300  SF#12 SF#13 SF#14 SF#15R SF#15L SF#20L  1.61 5.80 4.51 3.40 9.85 1.51  59 112 120 92 193 45  2.35 6.15 4.83 3.82 11.34 1.67  3.12 3.45 6.65 6.29 10.97 2.81  0.627 0.849 0.908 0.917 0.811 0.820  0.627 0.314 0.362 0.387 0.334 0.422  Average  4.45  104  5.03  5.55  0.822  0.408  83  Table A.7: Dynamic rheological properties for OA SF samples at 25 °C. Sample  G0.5Hz (Pa)  G0.5Hz (Pa)  G2.5Hz (Pa)  G2.5Hz (Pa)  fc (Hz)  Gc (Pa)  SF#12 SF#13 SF#14 SF#15R SF#15L SF#20L  2.56 5.95 3.52 3.02 5.39 2.17  1.11 2.30 1.36 1.21 1.97 0.93  2.56 5.95 3.52 3.02 5.39 2.17  1.95 3.21 1.97 1.87 2.75 1.67  0.71 0.26 0.19 0.21 0.09 0.71  1.27 1.91 1.01 0.91 1.21 1.06  Average  3.77  1.48  3.77  2.24  0.36  1.23  Table A.8: Steady state rheological properties for OA SF samples after centrifugation. Carreau-Yasuda Parameters η0 (Pa.s) λ (s) a n  Sample  η0 (Pa.s)  η0 /η300  SF#12 SF#13 SF#14 SF#15L SF#16  0.91 2.10 2.08 3.59 0.77  28 56 64 95 24  1.01 2.32 2.40 3.91 0.84  1.37 1.73 3.45 5.21 1.37  0.809 0.845 0.949 0.855 0.899  0.434 0.342 0.383 0.371 0.460  Average  1.89  53  2.10  2.62  0.872  0.398  Table A.9: Dynamic rheological properties for OA SF samples after centrifugation. Sample  G0.5Hz (Pa)  G0.5Hz (Pa)  G2.5Hz (Pa)  G2.5Hz (Pa)  fc (Hz)  Gc (Pa)  SF#12 SF#13 SF#14 SF#15L SF#16  0.69 1.57 1.29 1.69 0.70  0.90 1.55 1.15 1.40 0.86  2.02 3.84 2.89 3.59 1.86  1.78 2.46 1.83 2.18 1.75  1.26 0.47 0.31 0.21 1.29  1.31 1.51 0.97 1.03 1.26  Average  1.19  1.17  2.84  2.00  0.71  1.22  84  Table A.10: Steady state rheological properties for OA SF samples after storage for 2 weeks at -20 °C. Carreau-Yasuda Parameters η0 (Pa.s) λ (s) a n  Sample  η0 (Pa.s)  η0 /η300  SF#10 SF#15L SF#20L SF#22 SF#23 SF#28 SF#30 SF#31  1.41 2.95 0.71 1.14 0.58 0.25 0.28 0.41  53 80 26 32 26 12 13 19  1.52 3.19 0.77 1.23 0.60 0.27 0.29 0.43  3.11 4.63 1.40 1.69 1.58 0.73 0.68 1.15  1.007 0.868 0.831 0.826 0.949 0.903 0.960 0.960  0.411 0.385 0.449 0.431 0.468 0.534 0.506 0.494  Average  0.97  33  1.04  1.87  0.913  0.460  Table A.11: Dynamic rheological properties for OA SF samples after storage for 2 weeks at -20 °C. Sample  G0.5Hz (Pa)  G0.5Hz (Pa)  G2.5Hz (Pa)  G2.5Hz (Pa)  fc (Hz)  Gc (Pa)  SF#10 SF#15L SF#20L SF#22 SF#23 SF#28 SF#30 SF#31  0.87 1.73 0.56 0.90 0.60 0.16 0.15 0.36  0.81 1.46 0.72 1.07 0.72 0.32 0.29 0.53  1.55 2.62 1.52 2.33 1.53 0.60 0.53 1.04  1.34 2.26 1.43 2.01 1.31 0.80 0.72 1.05  0.40 0.24 2.25 1.31 1.47 No No 3.11  0.73 1.15 1.31 1.55 1.08 N/A N/A 1.07  Average  0.67  0.74  1.47  1.37  1.46  1.15  85  Table A.12: Total protein concentration in OA SF samples. Sample  Protein Concentration (mg/mL)  SF#1  31  SF#2  30  SF#3R  12  SF#3L  33  SF#4  37  SF#5  30  SF#6  22  SF#7  9  SF#8  15  SF#9  21  SF#10  29  SF#11  25  SF#12  18  SF#13  7  SF#14  16  SF#15R  15  SF#15L  14  SF#16  56  SF#17  39  SF#18  21  SF#20R  16  SF#21  23  SF#22  22  SF#23  27  SF#24  31  SF#25  45  SF#26  29  SF#27  25 Continued on next page  86  Table A.12 – continued from previous page Sample  Protein Concentration (mg/mL)  SF#28  21  SF#29  36  SF#30  6  SF#31  8  SF#32  10  Average  24  87  

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