Inferior synovial lubrication is a hallmark of osteoarthritis (OA), and synovial fluid (SF) lubrication and composition are variable among OA patients. Hyaluronic acid (HA) viscosupplementation is a widely used therapy for improving SF viscoelasticity and lubrication, but it is unclear how the effectiveness of HA viscosupplements varies with arthritic endotype. The objective of this study was to investigate the effects of the HA viscosupplement, Hymovis®, on the lubricating properties of diseased SF from patients with non-inflammatory OA and inflammatory arthritis (IA). The composition (cytokine, HA, and lubricin concentrations) of the SF was measured as well as the mechanical properties (rheology, tribology) of the SF alone and in a 1:1 mixture with the HA viscosupplement. Using rotational rheometry, no difference in SF viscosity was detected between disease types, and the addition of HA significantly increased all fluids' viscosities. In non-inflammatory OA SF, friction coefficients followed a typical Stribeck pattern and their magnitude was decreased by the addition of HA. While some of the IA SF also showed typical Stribeck behavior, a subset showed more erratic behavior with highly variable and larger friction coefficients. Interestingly, this aberrant behavior was not eliminated by the addition of HA, and it was associated with low concentrations of lubricin. Aberrant SF exhibited significantly lower effective viscosities compared to non-inflammatory OA and IA SF with typical tribological behavior. Collectively, these results suggest that different endotypes of arthritis exist with respect to lubrication, which may impact the effectiveness of HA viscosupplements in reducing friction.

OBJECTIVE:Viscosupplementation has been used for decades to treat mild to moderate osteoarthritis, yet it is unknown if the lubricating function of different pathological synovial fluids (SF) vary, or if they respond differentially to viscosupplementation. The objectives of this study were to (i) evaluate the friction coefficients and induced shear strains in articular cartilage when lubricated with pathological SF, (ii) identify the effect of hyaluronic acid (HA) supplementation on friction coefficients and shear strains, and (iii) identify SF biomarkers that correlate with lubricating function. METHOD:Human pathological SF was grouped by white blood cell count (inflammatory: >2000 cells/mm3, n = 6; non-inflammatory: <2000 cells/mm3, n = 6). Compositional analyses for lubricin and cytokines were performed. Friction coefficients and local tissue shear strain measurements were coupled using new, microscale rheological analyses by lubricating neonatal bovine cartilage explants with SF alone and in a 1:1 ratio with HA (Hymovis®). RESULTS:Friction coefficients were not significantly different between the inflammatory and non-inflammatory pathologies (p = 0.09), and were poorly correlated with peak tissue strains at the cartilage articular surface (R2 = 0.34). A subset of inflammatory SF samples induced higher tissue strains, and HA supplementation was most effective at lowering friction and tissue strains in this inflammatory subset. Across all pathologies there were clear relationships between polymorphonuclear neutrophil (PMN), IL-8, and lubricin concentrations with cartilage tissue strains. CONCLUSION:These results suggest that pathological SF is characterized by distinct tribological endotypes where SF lubricating behaviors are differentially modified by viscosupplementation and are identifiable by biomarkers.

Purpose: Osteoarthritis (OA) is a degenerative disease of the joint leading to chronic pain and disability, and consequently resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder that affects various anatomical structures, including subchondral bone; moreover, periarticular bone abnormalities seem also to be involved in the disease initiation and progression. Hyaluronic Acid (HA), a physiological component of synovial fluid and extracellular matrix, is commonly used as intra-articular viscosupplementation therapy for its viscoelastic and lubricant features. In addition, exogenous HA has been proven to stimulate endogenous HA synthesis, chondrocyte metabolism and synthesis of cartilage matrix components and to inhibit chondrodegenerative enzymes, as well as the inflammatory process. Bisphosphonates (BPs) are anti-resorptive agents that inhibit the recruitment and maturation of osteoclast precursors and the activity of mature osteoclasts in the bone. Although it is still not clear if oral administration of BPs is beneficial in OA therapy, pre-clinical evidences are reported in the literature showing that intra-articular BPs could have an impact in slowing down or reversing OA progression. The combination of the biological and mechanical role of HA as viscosupplement and the antiresorptive effect of Alendronate (ALD) could be an interesting strategy for OA treatment. The present study describes the synthesis and characterization of FID-134, a new chemical derivative of HA, conjugated with ALD by means of a covalent bond, cleavable in physiological condition. The activity of the substance, acting as a macromolecular drug delivery system, was tested in an in vitro model of inflammatory OA. Methods: FID-134 was synthesized starting from 500 kDa HA: the chemical structure and functionalization degree with ALD were investigated by 1H NMR and ICP-OES, respectively. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA and ALD; a 10 kDa cut-off membrane was employed to mimic the synovial membrane. A 20mg/mL FID-134 formulation was investigated for viscoelastic properties, in absence and presence of Ca2+ ions, and compared to HA and HA+ALD mixture formulated at the same concentration. The cytotoxicity of FID-134 was tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes at 24h, 3 and 7 days and compared to free ALD. The efficacy of FID134 was assessed in an in vitro model of inflammatory arthritis, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls. Results: The structure of FID-134 was confirmed by 1H NMR, with diagnostic ALD signals resonating at about 3.0 and 1.7 ppm respectively, and the molar functionalization degree of 20% mol/mol was determined by ICP-OES. Compared to HA+ALD mixture, ALD release assay demonstrated the slow release of the bisphosphonate from FID-134, showing that only about 50% of total bound ALD was released from FID-134 within 7 days. In presence of Ca2+ ions, viscoelastic properties of FID-134 dramatically improved with respect to HA and HA+ALD formulations, which remained unaffected. The cytotoxicity of ALD was evident at the concentration of 100 μM on Saos-2 and primary bovine chondrocytes after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed when FID-134 was added to the cartilage biopsies, with collagen release halved compared to control. Conclusions: The synthesized HA-ALD adduct was demonstrated to act as a drug delivery system and ALD was slowly released during time in a controlled manner. FID-134 opens the door for a new approach for OA treatment, as it combines viscosupplementation and biological effect due to HA presence with the pharmacological activity of BPs, targeting both cartilage and subchondral bone. In vitro results showed reduced cytotoxicity for FID-134, compared to free ALD, towards the main cell populations in the joint: chondrocytes and osteoblasts. Moreover, the beneficial effect of FID-134 against inflammatory conditions in cartilage were demonstrated on an in vitro model of inflammatory OA, through the reduction of collagen degradation. These findings suggest that FID-134 could be beneficial in both cartilage degradation and restoration of subchondral bone function, as previously reported in the literature for HA and ALD. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as aspecificity and long-term toxic side effects.

Purpose: Patients with mild to moderate osteoarthritis (OA) have been treated with hyaluronic acid (HA) viscosupplementation for decades as it functions to increase the viscosity of diseased synovial fluid and improve cartilage lubrication. Viscosupplementation has shown mixed results with reports of no effect and reports of reduced pain up to 6 months after injections. As a result, it is unknown if viscosupplementation may benefit specific OA disease states. Measuring shear strains is a powerful metric for assessing lubrication therapies because increased strains in articular cartilage have been linked to negative cellular responses including apoptosis.

Hyaluronic acid injections have been a mainstay of arthritis treatment for decades. However, much controversy remains about their clinical efficacy and their potential mechanism of action. This approach to arthritis therapy is often called viscosupplementation, a term which is rooted in the elevated viscosity of the injected solutions. This terminology also suggests a mechanical pathway of action and further implies that their efficacy is dependent on viscosity. Notably, previous studies of the relationship between viscous properties of hyaluronic acid solutions and their clinical efficacy have not been definitive. Recently we developed an experimental and analytical framework for studying cartilage lubrication that captures the Stribeck-like behavior of cartilage in an elastoviscous transition curve. Here we apply this framework to study the lubricating behavior of six hyaluronan products currently used for injectable arthritis therapy in the US. Despite the fact that the source and chemical modifications endow these products with a range of lubricating properties, we show that the lubricating effect of all of these materials can be described by this Stribeck-like elastoviscous transition. Fitting this data to the elastoviscous transition model enables the calculation of effective lubricating viscosities for each material, which differ substantially from the viscosities measured using standard rheometry. Further we show that while data from standard rheometry are poor predictors of clinical performance of these materials, measurements of friction coefficient and effective lubricating viscosity correlate well (R2 = 0.77; p < 0.005) with assessments of improved clinical function reported previously. This approach offers both a novel method that can be used to evaluate potential clinical efficacy of hyaluronic acid formulations and provide new insight on their mode of action.

SummaryBoth endogenous lubricin and injectable hyaluronic acid reduced cartilage friction coefficients, but by distinct mechanisms. Lubricin operated in boundary mode and hyaluronic acid shifted lu...

The evaluation of key factors modulating cell homing following injection can provide new insights in the comprehension of unsolved biological questions about the use of cell therapies for osteoarthritis (OA). The main purpose of this in vivo study was to investigate the biodistribution of an intra-articular injection of mesenchymal stromal cells (MSCs) and bone marrow concentrate (BMC) in a rabbit OA model and whether the additional use of sodium hyaluronate (HA) could modulate their migration and delay joint degeneration. OA was surgically induced in adult male New Zealand rabbits. A group of animals was used to test the biodistribution of labeled cells alone or with HA at 7 and 14 days to investigate cell migration. The efficacy of treatments was evaluated in other experimental groups at 2 months. Histology and immunohistochemistry for markers identifying anabolic and catabolic processes in the cartilage and meniscus, or macrophage subset population in the synovial membrane, were performed. Kruskal-Wallis test, followed by post hoc Dunn's test, and Spearman's rank-order correlation method were used. MSCs and BMC preferentially migrate toward tissue areas showing OA features in the meniscus and cartilage and in detail near inflammatory zones in the synovial membrane. The combination with HA contributed to boost cell migration toward articular cartilage. In general, both labeled cells combined with HA were found near cell cluster and fissures in the cartilage and meniscus, respectively, and close to areas of synovial membrane showing mainly anti-inflammatory macrophages. A promotion of joint repair was observed at different levels for all treatments, although BMC-HA treatment resulted as the best strategy to support joint repair. This last, displayed a good protein expression of type II collagen in the cartilage, as well as the presence of anti-inflammatory macrophages in the synovial membrane at 2 months from the treatment. Studies tracking cell biodistribution indicate that priming progenitor cells with HA modulated cell homing favoring not only attachment but also their integration within articular cartilage.

INTRODUCTION: Patients with mild to moderate osteoarthritis have been treated with viscosupplementation using hyaluronic acid (HA) for decades as it functions to increase the viscosity of diseased synovial fluid and improve cartilage lubrication. Clinical efficacy of viscosupplementation has shown mixed results with some patients reporting reduced pain and function up to 6 months after injections and others reporting no change. It is unknown if there are different tribological phenotypes of arthritic synovial fluid, and if viscosupplementation may benefit specific disease states. Assessing friction coefficients of synovial fluid provides global measurements over the shear cycle, but does not account for temporal differences in static and kinetic friction. Few reports have investigated how static friction differs between pathologic human synovial fluid, and if temporal differences in static friction can explain observed strains through the tissue depth. The objective of this study was to investigate the effect of viscosupplementation on friction coefficients and shear strains in articular cartilage between inflammatory and non-inflammatory arthritic phenotypes of human synovial fluid, and identify any temporal differences in friction behavior.

Novel tissue engineered and biomaterial approaches to treat intervertebral disc (IVD) degeneration focus on single aspects of the progressive disease and hence are insufficient repair strategies. In this study, annulus fibrosus (AF) and nucleus pulposus (NP) biomaterial repair strategies were used individually and combined to treat IVD degeneration modeled in ex vivo rat-tail motion segments by annulotomy and nucleotomy. An injectable riboflavin cross-linked high-density collagen gel patched defects in the AF, while NP repair consisted of injections of a modified hyaluronic acid (HA) hydrogel. Qualitative imaging showed the annulotomy and nucleotomy successfully herniated NP material, while the HA NP injections restored intact NP morphology and the collagen AF patches sealed AF defects. Assessed by quantitative T2 magnetic resonance imaging, combined repair treatments yielded disc hydration not significantly different than intact hydration, while AF and NP repairs alone only restored similar to 1/3 of intact hydration. Mechanical testing showed NP injections alone recovered on average similar to 35% and similar to 40% of the effective instantaneous and equilibrium moduli. The combined treatment comprising biomaterial AF and NP repair was effective at increasing NP hydration from NP repair alone, however HA injections alone are sufficient to improve mechanical properties.Statement of SignificanceIntervertebral disc degeneration affects an estimated 90% of individuals throughout their life, and is a candidate pathology for tissue engineered repair. The current standard of clinical care reduces spinal articulation and leads to further degeneration along the spine, hence great interest in a regenerative medicine therapy. Literature studies focused on biomaterial repair strategies for treating degenerated discs have partially restored native disc function, however no studies have reported the use of combined therapies to address multiple aspects of disc degeneration. This initial investigation screened injectable biomaterial repair strategies ex vivo, and through complementary outcome measures showed a combined therapy restores disc function better than individual approaches. This study is the first of its kind to address multiple aspects of disc degeneration, using clinically-oriented biomaterials in a well established animal model. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hyaluronic acid (HA) is widely injected as a viscosupplement in the treatment of osteoarthritis. Despite its extensive use, it is not currently known if cartilage degradation alters how HA-based solutions lubricate the articular surface. In this study we utilized a model of cartilage degradation by IL-1β along with a recently developed framework to study role of cartilage degradation on lubrication by clinically-approved HA-based lubricants with high viscosities. Cartilage explants were cultured up to 8 days with 10 ng/ml IL-1β. After culture, samples were examined histologically, immunohistochemically, biochemically, mechanically, topographically, and tribologically. The tribological testing analyzed both boundary and mixed lubrication modes to assess individual effects of viscosity and boundary lubricating ability. Friction testing was carried out using PBS and two clinically approved HA-based viscosupplements in a cartilage-glass configuration. After culture with IL-1β, boundary mode friction was elevated after both 4 and 8 days. Additionally, friction in mixed mode lubrication, where HA is most effective as a lubricant, was significantly elevated after 8 days of culture. As cartilage became rougher, softer, and more permeable after culture, the boundary mode plateau was extended, and as a result, significantly increased lubricant viscosities or sliding speeds were necessary to achieve effective mixed lubrication. Overall, this study revealed that lubrication of cartilage by HA is degradation-dependent and coincides with changes in mechanics and roughness. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1456-1464, 2018.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial‐scale recombinant production, purification and full characterization, including X‐ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross‐infection.

INTRODUCTION: Hyaluronic acid (HA) injections have been a mainstay of arthritis treatment for decades. However, much controversy remains about their clinical efficacy and their potential mechanism of action. This approach to arthritis therapy is often called viscosupplementation, a term which is rooted in the extremely high viscosity of the injected solutions. This terminology also suggests a mechanical pathway of action and further implies that efficacy is dependent on viscosity. Notably, previous studies of the relationship between viscous properties of hyaluronic acid solutions and their clinical efficacy have not been definitive [1-3]. Recently, we developed an experimental and analytical framework to study high viscosity cartilage lubricants that exploits Stribeck-like behavior [4]. The goals of this study were to characterize commercially available HA products in terms of their rheological and lubricating properties in vitro and to determine the extent to which these mechanical parameters correlate with clinical efficacy of these products reported during previous clinical trials. METHODS: Rheological properties of approved HA products (Monovisc, Synvisc, Supartz, Euflexxa, Hyalgan, and Hymovis) were evaluated using a cone and plate rheometer to determine low shear rate dynamic viscosity (η), storage modulus (G’) and loss modulus (G”) [4]. Lubrication studies were performed on 6 mm diameter articular cartilage cylinders harvested from the patellofemoral groove of neonatal bovids. As described previously [4,5], cartilage samples were mated against a polished glass flat counterface while bathed in lubricant baths consisting of the HA products described above, phosphate buffered saline, or 2 MDa dextran. Samples were compressed to 25% strain and allowed to depressurize for 1 hour resulting in average normal loads of 2.6 N (92 kPa normal stress), and the glass counterface was reciprocated at predetermined speeds ranging from 0.1 to 10 mm/s. Friction coefficients (μ) were recorded as the ratio of shear load to normal load measured by a biaxial load cell for n = 4 samples for each product listed as well as PBS. Friction coefficients were analyzed as a function of Sommerfeld number (S), which was calculated as a product of the sliding speed (v), dynamic viscosity, and contact width (a) divided by the normal load (F) (S = v η a/F). Additionally, friction as a function of S was fit to a Stribeck-like model (i.e, elastoviscious transition curve) as reported previously [4]. This mapping was performed two way: first, using the measured viscosities to calculate values of S for each material; and second by considering viscosity to be a free variable and minimizing the RMS error between the measured friction data for each HA product and the model, which enabled the calculation of an effective lubricating viscosity (ηeff). To determine the extent to which the measured rheological and tribological properties were correlated with clinical outcomes, data from clinical trials of each of the HA products was surveyed. For each product as well as placebo, the maximum improvement in WOMAC score was compared to η, G’, G”, μ measured at 10 mm/s, and ηeff. Using all data on all products and data collected on PBS as placebo, linear correlation analysis was performed between the measurements listed and maximum improvement in WOMAC score.

Gangliosides (sialic acid-containing glycosphingolipids) are abundant in neurons of all animal species and play important roles in many cell physiological processes, including differentiation, memory control, cell signaling, neuronal protection, neuronal recovery, and apoptosis. Gangliosides also function as anchors or entry points for various toxins, bacteria, viruses, and autoantibodies. GM1, a ganglioside component of mammalian brains, is present mainly in neurons. GM1 is one of the best studied gangliosides, and our understanding of its properties is extensive. Simple and rapid procedures are available for preparation of GM1 as a natural compound on a large scale, or as a derivative containing an isotopic radionuclide or a specific probe. Great research interest in the properties of GM1 arose from the discovery in the early 1970s of its role as receptor for the bacterial toxin responsible for cholera pathogenesis.

Purpose: Although inflammatory responses play an important role in both cartilage injury and degeneration related to specific OA phenotypes, it is not fully understood how changes in tissue structure and composition resulting from inflammation alter cartilage lubrication. Consequently, this study evaluated the effect of Interleukin-1β on the lubricating mechanisms of articular cartilage. Specifically, we analyzed elastoviscous transition curves of cartilage that can decouple the effects of boundary and viscous lubricants (e.g., lubricin and hyaluronic acid, respectively) by presenting friction as a function of the Sommerfeld number (sliding speed * lubricant viscosity * contact width / normal load). We hypothesized that changes in viscous and boundary lubrication result from distinct consequences of IL-1β degradative activity on the cartilage extracellular matrix. Methods: Neonatal bovine cartilage explants were cultured in DMEM supplemented with 10ng/ml IL-1β over a period of 4 or 8 days. After culture, samples were tested histologically, biochemically, mechanically, topographically, and tribologically. Friction testing was conducted using a custom-built tribometer with friction coefficients measured of cartilage on glass while bathed in a lubricant. Full elastoviscous transition curves were obtained by altering sliding speed from 0.1 to 10mm/s and by sliding in viscous lubricants spanning 5 orders of magnitude in viscosity (PBS, 600kDa HA 10mg/ml, and HYADD4 8mg/ml). These friction curves provided values for the boundary friction coefficient and quantitative values for the ease of transition away from boundary-mode lubrication (i.e., the transition number). Results: In vitro degradation with IL-1β caused significant loss of proteoglycan content seen histologically (Fig 1A-C) and biochemically (Fig 1D). Consequently, mechanical properties were progressively altered by decreased modulus and increased permeability (data not shown). Further, surface bound lubricin was lost after IL-1β supplementation. Similarly, cartilage surface roughness increased after culture with IL-1β, and after 8 days, the surface roughness was more than twice as high (Fig 2). Elastoviscous transition curves for both 4 and 8 day cultures were different from the curve for control tissue (Fig 3AB). For the 4 day culture, the boundary and minimum friction coefficients were elevated (Fig 3CD), but after 8 days of culture the boundary and minimum friction coefficients remained high and the transition between boundary and minimum friction was also significantly hindered (Fig 3E). Conclusions: This study revealed degradation-dependent alterations in lubrication mechanisms of articular cartilage supplemented with IL-1β. In early stages of degradation, boundary lubrication was inhibited, likely an effect of lubricin loss near the tissue surface. But, after progressive degradation, the transition away from boundary-mode lubrication was inhibited, likely an effect of increased surface roughness and altered mechanical properties. These data indicate that both compositional and structural changes due to degradation initiated by IL-1β have critical effects on cartilage lubrication that may lead to damage progression in a feedback mechanism between the mechanical changes and chemical signaling.Figure 2. (Left) Representative profile scans of cartilage explants. (Right) Surface roughness progressively increased with culture (n=4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3. (AB) Elastoviscous transition curves of cartilage after culture (n=5). Boundary friction (C) and minimum friction (D) rose after 4 days and remained high after 8 days. (E) After 8 days, the transition number increased 10-fold.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Extracellular matrix (ECM) degradation, one of the main features of osteoarthritis, is driven by at least two major classes of enzymes: matrix metalloproteases (MMPs) and hyaluronidases. Among certain glycosaminoglycans, including natural and chemically cross-linked HAs, which are currently used as viscosupplements, the hyaluronic acid (HA) alkyl-amides (Hyadd) were here selected as the strongest MMP and hyaluronidase inhibitors. We used C. histolyticum collagenase (ChC) and bovine testicular hyaluronidase (BTH) as representative models of human MMPs and hyaluronidases, respectively. The role of the alkyl moiety was investigated using HA derivatives with varying alkyl lengths and degrees of derivatization. The selected compound was then screened against 10 different human MMPs in vitro, and the results were validated ex vivo in human synovial fluid. Hyadd-C16, identified as a lead compound, showed the highest inhibition potency against MMP13 and MMP8. The in vitro results were confirmed by the inhibition of human MMP13 (Ki=106.1 μM) and hyaluronidase-2 in the synovial fluid of patients with osteoarthritis. This study demonstrates the unique properties of Hyadd-C16, including its remarkable enzymatic inhibitory activity, which is conferred by the hydrophobic chain, and its high biocompatibility and water solubility of the HA backbone.

Although post-traumatic osteoarthritis accounts for a significant proportion of all osteoarthritis, the understanding of both biological and mechanical phenomena that lead to cartilage degeneration in the years to decades after trauma is still lacking. In this study, we evaluate how cartilage lubrication is altered after a sub-critical impact (i.e., an impact to the cartilage surface that produces surface cracking but not full thickness fissuring). Through utilizing a Stribeck-like framework, the elastoviscous transition, we evaluated changes to both the innate boundary lubricating ability of cartilage after impact and also the effectiveness of high viscosity lubricants to lower friction after impact. Increases in boundary friction coincided with changes in lubricin localization after impact. However, larger increases in friction coefficient were observed in mixed-mode lubrication which can be predicted by increases in surface roughness due to cartilage fissuring. The data here reveal distinct mechanisms of cartilage lubrication that can fail after traumatic impact and may explain a key mechanical phenomenon that predisposes cartilage to development of osteoarthritis after injury.

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.

Osteoarthritis (OA) is characterized by chronic degeneration of joints, involving mainly the articular cartilage and the underlying bone, and severely impairing the quality of life of the patient. Although with limited efficacy, currently available pharmacological treatments for OA aim to control pain and to retard disease progression. Salmon calcitonin (sCT) is a drug which has been shown to have therapeutic effects in experimental arthritis by inhibiting both bone turnover and cartilage degradation and reducing the activities of matrix metalloproteinases (MMP). High molecular weight hyaluronic acid (HA) is used as a lubricant in OA therapy, and, interestingly, HA polymers may normalize the levels of MMP-1, -3 and -13. We demonstrated that sCT rapidly clears from the knee joint of rat animal model, after intra-articular (i.a.) administration, and it induces systemic effects. Here, sCT was conjugated to HA (200kDa) with the aim of prolonging the residence time of the polypeptide in the joint space by reducing its clearance. An aldehyde derivative of HA was used for N-terminal site-selective coupling of sCT. The activity of sCT was preserved, both in vitro and in vivo, after its conjugation and the i.a. injection of HA-sCT did not trigger any systemic effects in rats. The efficacy of HA-sCT treatment was tested in a rabbit OA model and clear chondro-protective effect was proven by macro- and microscopic assessments and histological findings. Our results indicate that HAylation of sCT increases the size of the polypeptide in a stable covalent manner and delays its passage into the blood stream. We conclude that HA conjugation prolongs the anti-catabolic effects of sCT in joint tissues, including the synovial membrane and cartilage.

Summary Both endogenous lubricin and injectable hyaluronic acid reduced cartilage friction coefficients, but by distinct mechanisms. Lubricin operated in boundary mode and hyaluronic acid shifted lubrication to mixed or hydrodynamic mode. Introduction Intra-articular injections of viscous agents and boundary lubricants have been presented as options to mitigate the progression of articular cartilage damage after the onset of osteoarthritis 1,2 . Mechanically, these injections are predicted to lower the friction coefficient within a load bearing joint and consequently slow the propagation of damage at the articular surface. Tribologically, boundary lubricants and viscous agents are hypothesised to be effective through different mechanisms affecting boundary-mode lubrication and transition to mixed-mode lubrication, respectively. By normalizing sliding speeds on a Stribeck curve, this study evaluated the efficacy of injectable hyaluronic acid (HA) supplements and endogenous lubricin to alter tribological properties. Methods Cartilage samples were extracted from the patellofemoral groove of neonatal bovine. A custom-built tribometer was used to measure friction coefficients of cartilage sliding against polished glass while in a lubricant solution. Cartilage samples were compressed to 20% strain and the normal load was allowed to reach a steady-state value before sliding at speeds from 0.1 to 10mm/s. For some samples, endogenous lubricin was removed from the surface as described previously 3 via incubation in 1.5M NaCl in PBS for 20 minutes followed by re-equilibration in PBS for 1hr. Samples were tested in bathing solutions of PBS (control), equine synovial fluid (ESF), 10mg/ml HA, and a hydrophobic HA derivative (HYADD). Results Friction coefficients as a function of sliding speed for some lubricants are presented. Comparisons show that lubricin removal from the tissue surface increases friction coefficients when PBS is used as a lubricant (p −6 m, suggesting that this lubricant may have enabled hydrodynamic lubrication, a phenomenon not noted previously in this system. Lubricin removal increased friction coefficient from 0.16 to 0.25, occurring at slow sliding speeds and Hersey numbers of 10 −12 m Conclusions Endogenous lubricin and injectable hyaluronan both effectively lower friction coefficients, but do so by distinct mechanism. At the same operating conditions (normal loads and sliding speed), lubricin lowers boundary mode friction coefficient, while hyaluronan shifts behavior to mixed mode (HA) or hydrodynamic mode (HYADD). The combination of the presence of lubricin on cartilage surface in the most viscous formulation of HA (HYADD) lowered the friction coefficient of articular cartilage from 0.26 to 0.05.