Final Report Summary - LUBRIJOINT (Molecular Mechanism of Synovial Joint Lubrication)
Osteoarthritis (OA) is a chronic joint disorder, which is associated with increasing wear of the joint cartilage, causing stiffness and pain. OA is one of the most frequent and rapidly growing causes of permanent disability across the world, with major socio-economic impact on western societies. A major contributor to OA is impaired lubrication. There is a strong evidence as to the role phospholipids (PLs) play in proper lubrication of synovial joints. It was shown that the level of PLs in the OA synovial fluid is reduced. PLs are also believed to perform as a carrier of lubricin. It is therefore of high importance to understand the molecular processes taking place between articulating cartilage layers. This could lead to better strategies for alleviating breakdown of lubrication in natural joints, as well as to strategies for improving lubrication and wear.
The main objective of this project was to study the possible molecular mechanisms of the efficient lubrication of synovial joints. In particular, the aim of the current work was to characterize the interactions between articulating surfaces in the presence of some of the biologically-active components present on the native articular cartilage surface, e.g. surface-active PLs and hyaluronic acid (HA), by correlating their physicochemical properties with their ability to lubricate. We have used native cartilage surfaces to measure their lubricating capabilities in the presence of externally added PLs and HA, replacing the natural synovial fluid. To this aim, we have developed an experimental setup sourced with native cartilage, which was used to measure friction forces between articulating cartilage surfaces in this study.
Here, we have focused on PLs and lubricin, which are believed to play a key role in lubrication. Specifically, we have synthesized and tested few dimyristol phosphatidylcholine (DMPC)-based PLs, as these are the dominant PLs residing on the surface of articulating cartilage. In addition to DMPC, which was originally suggested in the work plan, we have synthesized several other DMPC-based formulations (DMPC/PEG and DMPC/cholesterol) differing in their physicochemical properties and studied their lubricating capabilities. This was carried out by introducing them into the interface between two articulating cartilage surfaces and measuring the dynamic friction coefficients (u), using the cartilage-on-cartilage experimental setup we developed. The following parameters were tested: (i) Ionic state: Ionic and nonionic PLs were synthesized and compared. Ionic PLs (DMPC) were better lubricants (u=0.011) than nonionic PLs (DMPC/PEG) with u=0.025; (ii) Physical transition phase: PLs in the liquid-disorder phase (DMPC), which is softer and compressible, were better lubricants than PLs in the liquid ordered-phase (DMPC/cholesterol), which is drier and less compressible with u=0.036. The behavior of DMPC as a lubricating supplement was tested. We have shown the superior lubricating ability of osteoarthritic synovial fluid (OASF) enriched with DMPC (u=0.014) over OASF alone (u=0.046) or HA alone (u=0.023). The latter, which is clinically used to treat OA was shown to have less favorable lubricating capabilities.
In order to understand the role of lubricin, which is considered a key player in lubrication, it was important to use its intact, biologically-active form. We have used various methods to purify lubricin from synovial fluid and cartilage. All the non-specific chromatographic methods used resulted in highly fragmented lubricin. We assumed therefore, that a shorter, more specific methodology was required. To this aim, we have been working towards the development of a novel bioaffinity-based method for the purification of lubricin. The new procedure involved the choice and activation of the resin, chemical attachment of the monoclonal anti-lubricin to the activated resin and characterization of the eluted samples. Intact lubricin was detected using immunoblotting and mass spectroscopy, however, in small amounts (2%). Currently, efforts towards scaling up the process for subsequent force measurements and study the lubricating forces in the absence and presence of PLs are been carried out.
In parallel and based on the fact that aggrecan is a biomarker for the onset of OA and other cartilaginous pathologies, we have exploited the expertise and knowledge gained throughout this study to develop a needle micro-osmometer which can determine aggrecan level as part of the diagnostic process of pathologies like OA and disc degeneration. To this aim, methods for extracting aggrecan were developed. Aggrecan of different molecular weights was extracted and its physicochemical properties (e.g. ionic state and swell) were assessed using isotropic osmotic stress and radioactive tracer techniques. This part of the work was carried out with the collaboration of scientist from Hungary, UK and Israel.
The results of this study affect the current treatment of OA and may therefore have an enormous socio-economic impact in the following manner:
On the basis of the results obtained in this study, showing the superior lubricating ability of OASF enriched with DMPC over OASF alone, or HA alone, we propose that DMPC be used as a lubricating supplement (instead of HA) in intra-articular injections as part of the treatment of OA and other joint dysfunctions. Our finding that HA, which is widely used in the treatment of OA, has low lubricating properties when used alone challenges current treatment of OA. Furthermore, the fact that DMPC preparation is simple, reproducible on a large scale basis and economical, as well as chemically and physically stable makes them attractive for further clinical development as lubricants for synovial joints.
Taken altogether, the results of this project may be used to (i) improve our understanding of the relationships between physicochemical properties and lubrication capability of some DMPC-based PLs; (ii) test the potential clinical relevance of DMPC-based PLs as intra-articular lubricating supplement in OA patients; (iii) suggest ways to improve OA diagnosis by using the needle-micro-osmometer developed and (iv) improve lubrication and wear properties between articulating surfaces in vivo and also in joint implants.
The main objective of this project was to study the possible molecular mechanisms of the efficient lubrication of synovial joints. In particular, the aim of the current work was to characterize the interactions between articulating surfaces in the presence of some of the biologically-active components present on the native articular cartilage surface, e.g. surface-active PLs and hyaluronic acid (HA), by correlating their physicochemical properties with their ability to lubricate. We have used native cartilage surfaces to measure their lubricating capabilities in the presence of externally added PLs and HA, replacing the natural synovial fluid. To this aim, we have developed an experimental setup sourced with native cartilage, which was used to measure friction forces between articulating cartilage surfaces in this study.
Here, we have focused on PLs and lubricin, which are believed to play a key role in lubrication. Specifically, we have synthesized and tested few dimyristol phosphatidylcholine (DMPC)-based PLs, as these are the dominant PLs residing on the surface of articulating cartilage. In addition to DMPC, which was originally suggested in the work plan, we have synthesized several other DMPC-based formulations (DMPC/PEG and DMPC/cholesterol) differing in their physicochemical properties and studied their lubricating capabilities. This was carried out by introducing them into the interface between two articulating cartilage surfaces and measuring the dynamic friction coefficients (u), using the cartilage-on-cartilage experimental setup we developed. The following parameters were tested: (i) Ionic state: Ionic and nonionic PLs were synthesized and compared. Ionic PLs (DMPC) were better lubricants (u=0.011) than nonionic PLs (DMPC/PEG) with u=0.025; (ii) Physical transition phase: PLs in the liquid-disorder phase (DMPC), which is softer and compressible, were better lubricants than PLs in the liquid ordered-phase (DMPC/cholesterol), which is drier and less compressible with u=0.036. The behavior of DMPC as a lubricating supplement was tested. We have shown the superior lubricating ability of osteoarthritic synovial fluid (OASF) enriched with DMPC (u=0.014) over OASF alone (u=0.046) or HA alone (u=0.023). The latter, which is clinically used to treat OA was shown to have less favorable lubricating capabilities.
In order to understand the role of lubricin, which is considered a key player in lubrication, it was important to use its intact, biologically-active form. We have used various methods to purify lubricin from synovial fluid and cartilage. All the non-specific chromatographic methods used resulted in highly fragmented lubricin. We assumed therefore, that a shorter, more specific methodology was required. To this aim, we have been working towards the development of a novel bioaffinity-based method for the purification of lubricin. The new procedure involved the choice and activation of the resin, chemical attachment of the monoclonal anti-lubricin to the activated resin and characterization of the eluted samples. Intact lubricin was detected using immunoblotting and mass spectroscopy, however, in small amounts (2%). Currently, efforts towards scaling up the process for subsequent force measurements and study the lubricating forces in the absence and presence of PLs are been carried out.
In parallel and based on the fact that aggrecan is a biomarker for the onset of OA and other cartilaginous pathologies, we have exploited the expertise and knowledge gained throughout this study to develop a needle micro-osmometer which can determine aggrecan level as part of the diagnostic process of pathologies like OA and disc degeneration. To this aim, methods for extracting aggrecan were developed. Aggrecan of different molecular weights was extracted and its physicochemical properties (e.g. ionic state and swell) were assessed using isotropic osmotic stress and radioactive tracer techniques. This part of the work was carried out with the collaboration of scientist from Hungary, UK and Israel.
The results of this study affect the current treatment of OA and may therefore have an enormous socio-economic impact in the following manner:
On the basis of the results obtained in this study, showing the superior lubricating ability of OASF enriched with DMPC over OASF alone, or HA alone, we propose that DMPC be used as a lubricating supplement (instead of HA) in intra-articular injections as part of the treatment of OA and other joint dysfunctions. Our finding that HA, which is widely used in the treatment of OA, has low lubricating properties when used alone challenges current treatment of OA. Furthermore, the fact that DMPC preparation is simple, reproducible on a large scale basis and economical, as well as chemically and physically stable makes them attractive for further clinical development as lubricants for synovial joints.
Taken altogether, the results of this project may be used to (i) improve our understanding of the relationships between physicochemical properties and lubrication capability of some DMPC-based PLs; (ii) test the potential clinical relevance of DMPC-based PLs as intra-articular lubricating supplement in OA patients; (iii) suggest ways to improve OA diagnosis by using the needle-micro-osmometer developed and (iv) improve lubrication and wear properties between articulating surfaces in vivo and also in joint implants.