Computational and experimental investigation of the role of hyaluronic acid-protein interactions in the rheology of synovial fluid
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Hyaluronic acid (HA) is the key component of synovial fluid responsible for lubrication in human joints. It has a concentration of 3 mg/mL in healthy synovial fluid. It behaves as a lubricant and a shock absorber in synoial fluid [9,10,11]. This glycosaminoglycan (GAG) is a very important biomacromolecule on account of its role in various biological processes like signal transduction and cell motility . It is widely used in the treatment of inflammatory and degenerative joint diseases . Intra-articular injections of HA are administered to patients of osteoarthritis (OA)  for healing and regeneration of the cartilage, and are known to provide temporary relief from pain in the knee joint. However, the concept behind this treatment for OA is not clear. If the cause of lubrication in synovial fluid can be found out, it can help significantly in the treatment of arthritis. Studies by Oates and coworkers  point towards protein-HA aggregation playing an important role in the viscoelastic properties of synovial fluid. This, however, is in contrast with their earlier work  where they suggest HA-protein repulsion. The mechanism of lubrication needs to be explored further and the components of synovial fluid responsible for its viscoelastic properties need to be studied. Studies on viscoelastic properties of HA  and artificial synovial fluid  have been carried out, but these studies do not focus on the role of proteins on account of their interaction with HA. They do not discuss the individual effects of proteins (serum albumin, ã-globulin) on the viscoelastic properties of HA. There is a need to look into influence of individual proteins on HA viscoelasticity due to possible structure formation. To delve deeper into this aspect, we undertake rheological measurements on samples of HA and HA+ proteins. These measurements are taken at three different temperatures (20, 25, 37 °C) on the TA instruments AR-2000 rheometer. All samples contain 10 mg/mL Sodium Hyaluronate (NaHA). Bovine serum albumin (BSA) and ã-globulin concentrations of 12 mg/mL and 2.4 mg/mL have been used. These correspond to the concentrations in normal synovial fluid. Both steady flow and oscillatory shear measurements have been performed on these samples. The properties studied are zero shear viscosities from steady flow measurements, and G' (storage modulus) and G" (loss modulus) from oscillatory shear measurements. Characteristic relaxation time is determined from the crossover of G' and G". Molecular dynamics can help in studying the intermolecular interactions between various components of the synovial fluid. We undertake molecular dynamics simulations on systems of HA and water as well as HA-proteins-water. These simulations are analyzed for hydrogen bond formation and radial distribution functions. Addition of proteins to HA is found to affect both steady flow and dynamic mechanical properties of HA solutions. Addition of proteins is found to increase the zero shear viscosity of HA. Oscillatory shear properties show that both G' and G" values increase with angular frequency for addition of proteins to HA. Characteristic relaxation time determined from the crossover of G' and G" also increases with addition of proteins. The changes in viscoelastic properties have been found to be significant due to the addition of ã-globulin than BSA, although ã-globulin is at a lower concentration than BSA. Molecular dynamics simulations carried out in this work on a system containing HA, proteins and water shows that HA is in more spatial proximity to proteins, than water. Another MD simulation of a HA decamer and water shows that HA does not associate with water. Another MD simulation of HA trimers and water has shown that HA trimers form aggregates among themselves rather than binding with water. The Hydrogen bond calculations for systems of HA-water and HA-protein-water show that the hydrogen bond formation between HA and water molecules is disrupted due to addition of the protein (BSA). The decrease in the number of Hydrogen bonds between HA-water due to addition of BSA confirms that interaction exists between HA and BSA. Thus, addition of proteins is seen to affect both the structure and rheology of HA solutions. Rheological studies on samples of synovial fluids with different protein concentrations from patients of OA would throw more light on the effect of HA-protein interactions. Identification of HA-protein binding sites could help analyze the HA-protein-water MD simulations in a better way. This work on HA-protein interaction shows that it is not just HA, but other components of synovial fluid also, which are responsible for rheological properties of synovial fluid.