Browsing by Author "Schmidt, Tannin A"

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    Cartilage boundary lubrication and rheology of proteoglycan 4 + hyaluronan solutions and synovial fluid
    (2014-06-12) Ludwig, Taryn Elaine; Schmidt, Tannin A
    Synovial fluid (SF) is the viscous fluid present within articular joints that contributes to load bearing and lubrication functions. Proteoglycan 4 (PRG4) and hyaluronan (HA) in SF contribute synergistically to cartilage boundary lubrication. However, changes in SF PRG4 and HA content with osteoarthritis (OA) and associated effects on cartilage boundary lubricating function are not fully understood. Furthermore, the effects of PRG4+HA interaction on solution viscosity have not been thoroughly characterized. The objectives of this thesis were to 1) investigate the relationship between PRG4 and HA composition and boundary lubricating function of normal and OA SF, and 2) to investigate how the concentration and structure of PRG4 contributes to interactions with itself and HA, and subsequently the boundary lubricating and rheological properties of SF. Novel and previously characterized biochemical and biomechanical methods were used to evaluate boundary lubricant composition and lubricating ability of SF. While not all OA SF samples had low PRG4, samples that had low PRG4 concentration and decreased HA molecular weight (MW) demonstrated decreased cartilage boundary lubricating ability in vitro, which could be restored by addition of PRG4. SF aspirated after a flare reaction to intra-articular injection that had low PRG4 and an approximately normal HA MW distribution demonstrated normal cartilage boundary lubricating ability. In purified solutions of PRG4 and HA, decreased PRG4 or decreased high MW HA limited cartilage boundary lubricating ability. PRG4 and recombinant human PRG4 increased the viscosity of HA solutions at low concentrations, but decreased the viscosity of high concentration HA solutions. The intra- and inter-molecular disulfide bonded structure of PRG4 was observed to be important for its contributions to both PRG4+HA cartilage boundary lubricating ability and PRG4+HA solution viscosity. These results demonstrate that alterations in both PRG4 and HA content in SF may have negative effects on SF cartilage boundary lubricating and rheological function, and are consistent with a non-covalent, crowding mechanism of interaction. They suggest that maintaining PRG4 and HA content in SF during injury and disease, through the development of new PRG4±HA biotherapeutic treatments, may be able to both protect cartilage from degeneration and restore SF viscosity in vivo.
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    Investigating the effect of proteoglycan 4 on hyaluronan solution properties using confocal fluorescence recovery after photobleaching
    (2019-02-26) Bloom, Adam K; Samsom, Michael L; Regmi, Suresh C; Steele, Bridgett L; Schmidt, Tannin A
    Abstract Background The objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions. Methods Confocal FRAP was used to determine the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran tracer (Dt) through 1500 kDa and 500 kDa HA solutions (0–3.3 mg/ml) ± PRG4 or a control protein, bovine serum albumin (BSA), at physiological (450 μg/ml) or pathophysiological (45 μg/ml) concentrations. The effect of PRG4 or R/A PRG4 on 1500 kDa HA solutions was also investigated. Empirical constants obtained from fitting data to the universal scaling equation were used to calculate the average distribution of apparent mesh sizes. Results PRG4 at both 45 and 450 μg/ml slowed the diffusion of the FITC-dextran tracer for all concentrations of HA and caused a decrease in the apparent mesh size within the HA solution. This effect was specific to PRG4, not observed with BSA, but not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4. Conclusions These results demonstrate that PRG4 can significantly alter the solution properties of HA; PRG4 essentially reduced the permeability of the HA network. This effect may be due to PRG4 entangling HA molecules through binding and/or HA crowding PRG4 molecules into a self-assembled network. Collectively these findings contribute to the understanding of PRG4 and HA interaction(s) in solution and therefore the function of SF in diarthroidal joints.
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    Molecular Basis of Articular Cartilage Boundary Lubrication: Role of PRG4 Structure & Multimerisation
    (2015-06-15) Abubacker, Saleem; Schmidt, Tannin A; Matyas, John R.
    Proteoglycan 4 (PRG4) is a mucin-like glycoprotein found in synovial fluid (SF) and at the articular cartilage surface, where it is required for joint lubrication and health. Hyaluronan (HA), a glycosaminoglycan polymer, is another SF constituent that contributes to SF’s viscosity and cartilage lubrication properties. PRG4 and HA function effectively as friction reducing boundary lubricants at a cartilage-cartilage interface, though both have been studied at other interfaces with varying results. PRG4 can exist in SF as disulfide-bonded multimers, a structurally determinant characteristic of mucins, which may be necessary for its cartilage adsorption and boundary lubricating ability. A recently developed full-length recombinant human PRG4 (rhPRG4) has demonstrated appropriate higher order structure, O-linked glycosylations, and boundary lubricating ability at the ocular surface. However, it remains unclear if this rhPRG4 is able to adsorb to and function as a cartilage boundary lubricant. The objectives of this thesis were to (1) determine the effect of different sliding interface materials on the lubricating ability of PRG4 and HA by measuring the kinetic coefficient of friction, (2i) assess the cartilage adsorption and boundary lubricating ability of disulfide-bonded PRG4 multimers and PRG4 monomers, (2ii) evaluate the cartilage boundary lubricating ability of PRG4 multimers and PRG4 monomers with HA, and (3) assess the cartilage adsorption of rhPRG4 and the in vitro cartilage boundary lubricating properties of rhPRG4, with and without HA. PRG4 demonstrated boundary lubricating function at both cartilage-cartilage and cartilage-glass interfaces, while HA demonstrated friction reducing ability only at the cartilage-cartilage interface. The inter-molecular disulfide-bonded multimeric structure of PRG4 was important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. Finally, rhPRG4 demonstrated cartilage adsorption and boundary lubricating function, with and without HA, equivalent to native PRG4. Collectively, these results demonstrate the effectiveness of putative cartilage boundary lubricants can be affected by the counterface, contribute to a greater understanding of the molecular basis of articular cartilage boundary lubrication of PRG4, and provide the foundation and motivation for future clinical evaluation of rhPRG4 as a biotherapeutic treatment for osteoarthritis.
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    The Role of Proteoglycan 4 in the Rheology of Synovial Fluid
    (2021-09-14) Martin-Alarcon, Leonardo; Schmidt, Tannin A; Trifkovic, Milana; Furst, Eric M; Egberts, P; Wood, David H; Martinuzzi, Robert; Ponnurangam, Sathish
    The free motion of joints in the body is rendered safe by the biomechanical properties of articular cartilage and synovial fluid (SF). Cartilage is an elastic tissue capable of withstanding large compressive forces, whereas SF is a lubricating fluid with remarkable viscoelastic properties. The high molecular weight polysaccharide hyaluronan (HA) and the mucin-like glycoprotein proteoglycan 4 (PRG4 or lubricin) are crucial macromolecules in SF that lubricate cartilage synergistically; however, there is an ongoing debate on how their interactions in influence SF rheology. This thesis examined the relationship between the microstructure and the rheological properties of physiologically relevant formulations of HA and recombinant human PRG4 (rhPRG4) to elucidate on the interactions between them in SF. A succinct fractional framework was adapted to effectively describe the linear and nonlinear viscoelastic behaviour of HA+rhPRG4 solutions at both macroscopic and microscopic length scales. It was found that the bulk rheology of HA solutions increased with the addition of rhPRG4 in a dose-dependent manner, but the extent of these enhancements depended on the degree of rhPRG4 glycosylation. Afterwards, an optical tweezers-based methodology was modified using our fractional framework to characterize the rheology of the microstructure in our solutions. Contrary to previous studies, no evidence was found of a favourable physical interaction between HA and rhPRG4 which could functionally alter the macroscopic rheology of the solutions. At the microscopic level, rhPRG4 was shown to cluster into stiff gel-like aggregates that did not interact with the surrounding continuous phase of HA polymers. Furthermore, the interfacial adsorption of rhPRG4 molecules at the air-water interface of the rotational rheometer was shown to play a considerable effect in the interpretation of macrorheological data. Finally, clinically relevant non-ionic surfactants used in the stabilization of rhPRG4 formulations were shown to both suppress the interfacial adsorption of rhPRG4 in rotational rheometers and disrupt the aggregation behaviour of rhPRG4 in the bulk. Taken together, these results suggest that, in SF and potential HA+PRG4 biotherapeutic supplements designed for osteoarthritic joints, HA governs the bulk rheology of the fluid whereas the impact of PRG4 is restricted to its tribological performance.