Graduate Thesis Or Dissertation
 

Molecular and rheological characterization of sodium hyaluronate (HA) and equine synovial fluid

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  • Sodium hyaluronate (HA) is a polysaccharide found in all parts of the body Although it performs important functions in the eye, the coagulation process and other parts of the body, its contribution to synovial fluid is particularly important. As the major component of synovial fluid, HA is responsible for the viscoelastic properties important in joint lubrication and cartilage protection. In this thesis, molecular and rheological characterization techniques were used to study; i) commercial HA materials and HA synovial fluid supplements; ii) equine synovial fluid from different joints of both live and deceased horses; iii) equine synovial fluid from a clinical study of intraarticular HA supplementation in the hock joints of a group of six horses. Commercial HA materials and HA intended for intra-articular, intravenous and oral supplementation were studied using size exclusion chromatography combined with inline multi-angle laser light scattering (SEC-MALLS), dilute solution capillary viscometry to obtain intrinsic viscosity ([η]), and steady shear and dynamic oscillatory shear rheology. The molecular weight range of the HA samples was 2.88x10⁵ to 1.96x10⁶ Da. The molecular weight and intrinsic viscosity were correlated and a Mark-Houwink-Sakurada equation for HA in phosphate buffer solution (PBS) was found to be [η] = 0.17 Mw⁰`⁶⁸. The "a" value of 0.68 indicates HA behaves as a random coil in PBS which is consistent with values reported in literature. Zero shear viscosities of the samples at a concentration of 2.5 mg/ml ranged from approximately 0.06 to 0.5 Pa-s and were found to have a nearly linear relationship with the product of molecular weight and concentration (η ∝ cMw¹.⁰⁸). All samples exhibited both viscous and elastic properties in the dynamic oscillatory shear tests. The correlation between molecular weight and rheological properties of pure HA indicates that these techniques may be used in the future to characterize HA materials and possibly to discern the connection between molecular properties of HA and their lubrication properties in synovial fluid. The techniques used to characterize pure sodium hyaluronate samples were then applied to equine synovial fluid from a number of live and deceased horses (euthanized for reasons unrelated to this study) in an attempt to elucidate the role of HA in joint lubrication. The concentration and molecular weight of the HA-protein complex in synovial fluid was measured using SECMALLS. Steady shear and dynamic oscillatory shear tests were also performed on synovial fluid samples. The investigation of the properties of synovial fluid in normal equine joints reported in this thesis is the beginning of an attempt to establish baseline values for comparison with diseased joints. The molecular weight of HA in synovial fluid ranged from 1.5x10⁶ to 6.5x10⁶ Dalton (Da) and concentration ranged from 0.11 to 0.84 mg/mI, both are in the range of values reported in literature. The steady shear viscosity of synovial fluid samples ranged from about 0.001 to 1 Pa-s, with slight upturns at low shear rates in some samples, indicative of aggregation. The molecular characterization is difficult due to the complexity of the fluid. Although each sample exhibits unique rheological properties, at this time changes in viscosity or elasticity cannot be correlated to changes in either molecular weight or concentration of HA. Finally, a preliminary study of the intra-articular injection of HA was completed using six healthy horses and rheological characterization of synovial fluid from tarsocrural hock joints. The horses were divided into three groups: the experimental group (2 ml of Hyvisc, an HA supplement, in each hock); the positive control group (2 ml of Lactated Ringers Solution (LRS) in each hock); and the negative control group (no treatment). The horses received the above treatments after aspiration of synovial fluid for the samples that were treated as the baseline value for each hock joint. Synovial fluid samples were also taken one and two weeks after treatment. Cytology, including total protein and nucleated cell counts, was performed to monitor the health of the joints throughout the study. Rheological properties of synovial fluid in the experimental group increased one week after treatment compared to the control groups. This could be due to exogenous HA remaining in the joint after treatment, indicating that it takes longer than one week for exogenous HA to be cleared from the joint. Two weeks after treatment all test groups returned to the pre-treatment state. This was most likely due to the fact that the joints were aspirated one week after treatment, negating both treatment and non-treatment effects by the second week of the study.
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