Boundary Lubrication Of Cartilage Research Articles

Lubricant Strategies in Osteoarthritis Treatment: Transitioning from Natural Lubricants to Drug Delivery Particles with Lubricant Properties.

Osteoarthritis (OA) is a debilitating joint disease characterized by cartilage degradation, leading to pain and functional impairment. A key contributor to OA progression is the decline in cartilage lubrication. In physiological conditions, synovial fluid (SF) macromolecules like hyaluronic acid (HA), phospholipids, and lubricin play a crucial role in the boundary lubrication of articular cartilage. In early OA, cartilage damage triggers inflammation, altering SF composition and compromising the lubrication layer. This increases friction between mating interfaces, worsening cartilage degradation and local inflammation. Therefore, early-stage restoration of lubrication (by injecting in the joint different classes of compounds and formulations) could alleviate, and potentially reverse, OA progression. In the light of this, a broad variety of lubricants have been investigated for their ability to reduce friction in OA joints and promote cartilage repair in clinical and preclinical studies. This review examines recent advancements in lubricant-based therapy for OA, focusing on natural, bioinspired, and alternative products. Starting from the currently applied therapy, mainly based on natural lubricants as HA, we will present their modified versions, either in hydrogel form or with specific biomimetic moieties with the aim of reducing their clearance from the joint and of enhancing their lubricating properties. Finally, the most advanced and recent formulation, represented by alternative strategies, will be proposed. Particular emphasis will be placed on those ones involving new types of hydrogels, microparticles, nanoparticles, and liposomes, which are currently under investigation in preclinical studies. The potential application of particles and liposomes could foster the transition from natural lubricants to Drug Delivery Systems (DDSs) with lubricant features; transition which could provide more complete OA treatments, by simultaneously providing lubrication replacement and sustained release of different payloads and active agents directly at the joint level. Within each category, we will examine relevant preclinical studies, highlighting challenges and future prospects.

Proteoglycan 4 (PRG4)/Lubricin and the Extracellular Matrix in Gout

Proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes, released into synovial fluid, and adsorbed on cartilage and synovial surfaces. PRG4′s roles include cartilage boundary lubrication, synovial homeostasis, immunomodulation, and suppression of inflammation. Gouty arthritis is mediated by monosodium urate (MSU) crystal phagocytosis by synovial macrophages, with NLRP3 inflammasome activation and IL-1β release. The phagocytic receptor CD44 mediates MSU crystal uptake by macrophages. By binding CD44, PRG4 limits MSU crystal uptake and downstream inflammation. PRG4/CD44 signaling is transduced by protein phosphatase 2A, which inhibits NF-κB, decreases xanthine oxidoreductase (XOR), urate production, and ROS-mediated IL-1β secretion. PRG4 also suppresses MSU crystal deposition in vitro. In contrast to PRG4, collagen type II (CII) alters MSU crystal morphology and promotes the macrophage uptake of MSU crystals. PRG4 deficiency, mediated by imbalance in PRG4-degrading phagocyte proteases and their inhibitors, was recently implicated in erosive gout, independent of hyperuricemia. Thus, dysregulated extracellular matrix homeostasis, including deficient PRG4 and increased CII release, may promote incident gout and progression to erosive tophaceous joint disease. PRG4 supplementation may offer a new therapeutic option for gout.

Insight into the assembly of lipid-hyaluronan complexes in osteoarthritic conditions.

Interactions between molecules in the synovial fluid and the cartilage surface may play a vital role in the formation of adsorbed films that contribute to the low friction of cartilage boundary lubrication. Osteoarthritis (OA) is the most common degenerative joint disease. Previous studies have shown that in OA-diseased joints, hyaluronan (HA) not only breaks down resulting in a much lower molecular weight (MW), but also its concentration is reduced ten times. Here, we have investigated the structural changes of lipid-HA complexes as a function of HA concentration and MW to simulate the physiologically relevant conditions that exist in healthy and diseased joints. Small angle neutron scattering and dynamic light scattering were used to determine the structure of HA-lipid vesicles in bulk solution, while a combination of atomic force microscopy and quartz crystal microbalance was applied to study their assembly on a gold surface. We infer a significant influence of both MW and HA concentrations on the structure of HA-lipid complexes in bulk and assembled on a gold surface. Our results suggest that low MW HA cannot form an amorphous layer on the gold surface, which is expected to negatively impact the mechanical integrity and longevity of the boundary layer and could contribute to the increased wear of the cartilage that has been reported in joints diseased with OA.

Commentary on the Lamellar-Repulsive-Slippage Lubrication

In this article, we evaluated surface topographical images of the bovine (cartilage/cartilage) pair friction. In healthy joints, the cartilage (AC) surface coated with phospholipid multi-bilayers is activated by the lamellar-repulsive-slippage lubrication mechanism. Hydrophilic and negatively charged (--) natural cartilage surface is covered by phospholipid bilayers. These phospholipids have been demonstrated to exert highly desirable characteristics on the surface of articular cartilage such as efficient lubrication, load processing, and semi-permeability for nutrient transport. We attempt to demonstrate phospholipids involvement in boundary lubrication of articular cartilage by: 1) the surface amorphous layer (SAL); 2) negatively charged surface; 3) lamellar-repulsive lubrication; and 4) lamellar-slippage mechanism in (cartilage/cartilage) pair lubrication. The secret of the super low friction and wear between the cartilage-bearing surfaces is lamellar-repulsive and slippage mechanism of lubrication. We also present the evidence that the superficial phospholipid bilayer covering the articular surface of cartilage has a primary function of creating a hydrophilic surface with wetting properties, and hence, of controlling interfacial properties under 7.4 pH values. We conclude that lamellar bilayers slippage, as well as the short-range repulsion between the interfaces of the negatively charged (-) cartilage surfaces, is a primary determinant of the low frictional properties of the joint.

Interactions Between Bilayers of Phospholipids Extracted from Human Osteoarthritic Synovial Fluid

Duncan Dowson, whom this issue commemorates, was a world leader in the field of biotribology, with prolific contributions both in fluid-based and boundary lubrication of biological tissues, in particular articular cartilage, a central issue in biotribology due to its importance for joint homeostasis. Here we explore further the issue of cartilage boundary lubrication, which has been attributed to phospholipid (PL)-exposing layers at the cartilage surface in part. A surface force balance (SFB) with unique sensitivity is used to investigate the normal and frictional interactions of the boundary layers formed by PLs extracted from osteoarthritic (OA) human synovial fluid (hSF). Our results reveal that vesicles of the OA-hSF lipids rupture spontaneously to form bilayers on the mica substrate (which, like the in-vivo articular cartilage surface in synovial joints, is negatively-charged) which then undergo hemifusion at quite low pressures in the SFB, attributed to the large heterogeneity of the hSF lipids. Nanometric friction measurements reveal friction coefficients μ ≈ 0.03 across the hemifused bilayer of these lipids, indicating residual hydration lubrication at the lipid-headgroup/substrate interface. Addition of calcium ions causes an increase in friction to μ ≈ 0.2, attributed either to calcium-bridging attraction of lipid headgroups to the negatively-charged substrate, or a shift of the slip plane to the more dissipative hydrophobic-tail/hydrophobic-tail interface. Our results suggest that the heterogeneity and composition of the OA-hSF lipids may be associated with higher friction at the cartilage boundary layers – and thus a connection with greater wear and degradation – due to hemifusion of the exposed lipid bilayers.

The Role of Hyaluronic Acid in Cartilage Boundary Lubrication.

Hydration lubrication has emerged as a new paradigm for lubrication in aqueous and biological media, accounting especially for the extremely low friction (friction coefficients down to 0.001) of articular cartilage lubrication in joints. Among the ensemble of molecules acting in the joint, phosphatidylcholine (PC) lipids have been proposed as the key molecules forming, in a complex with other molecules including hyaluronic acid (HA), a robust layer on the outer surface of the cartilage. HA, ubiquitous in synovial joints, is not in itself a good boundary lubricant, but binds the PC lipids at the cartilage surface; these, in turn, massively reduce the friction via hydration lubrication at their exposed, highly hydrated phosphocholine headgroups. An important unresolved issue in this scenario is why the free HA molecules in the synovial fluid do not suppress the lubricity by adsorbing simultaneously to the opposing lipid layers, i.e., forming an adhesive, dissipative bridge between them, as they slide past each other during joint articulation. To address this question, we directly examined the friction between two hydrogenated soy PC (HSPC) lipid layers (in the form of liposomes) immersed in HA solution or two palmitoyl–oleoyl PC (POPC) lipid layers across HA–POPC solution using a surface force balance (SFB). The results show, clearly and surprisingly, that HA addition does not affect the outstanding lubrication provided by the PC lipid layers. A possible mechanism indicated by our data that may account for this is that multiple lipid layers form on each cartilage surface, so that the slip plane may move from the midplane between the opposing surfaces, which is bridged by the HA, to an HA-free interface within a multilayer, where hydration lubrication is freely active. Another possibility suggested by our model experiments is that lipids in synovial fluid may complex with HA, thereby inhibiting the HA molecules from adhering to the lipids on the cartilage surfaces.

Proteoglycan-4 and hyaluronan composition in synovial fluid and serum from clinical equine subjects: relationship to cartilage boundary lubrication and viscosity of synovial fluid

ABSTRACT Purpose: In experimental models of equine joint-injury and osteoarthritis synovial fluid (SF) composition (proteoglycan-4, hyaluronan) can vary, along with changes to SF mechanical function (lubrication, viscosity). The study hypotheses were a) clinical equine joint-injury and disease results in altered SF composition and diminished mechanical function, and b) serum composition (proteoglycan-4 or hyaluronan) changes concurrently. The objectives were to characterize composition (proteoglycan-4, hyaluronan), and function of SF and serum from normal horses compared to clinical groups: osteoarthritis, acute-joint-injury, and osteochondrosis. Materials and Methods: Equine samples of SF (from various joints) and blood were collected at the point-of-care. Proteoglycan-4 concentrations were measured by amplified-luminescence-proximity-assay and enzyme-linked-immunosorbent-assay in SF and serum, respectively. Molecular-weight of hyaluronan was characterized by agarose-gel-electrophoresis, and concentrations were measured by enzyme-linked-immunosorbent-assay kit. Biomechanical function of SF was characterized by an in vitro cartilage-on-cartilage friction test, and viscosity test. Results: SF proteoglycan-4 concentration increased in acute-joint-injury (1185 ± 276 versus normal 205 ± 106 µg/mL, µ± SEM, p < 0.01), with increased percentage of lower molecular-weight hyaluronan in acute-joint-injury and osteochondrosis. SF and serum proteoglycan-4 concentrations were correlated in normal horses (r2 = 0.85, p < 0.05), but not in clinical groups. Cartilage-lubricating ability was unchanged, although steady-shear viscosity of acute-joint-injury SF decreased from normal. Conclusion: Composition of SF from cases of equine acute-joint-injury changed; both proteoglycan-4 concentration and hyaluronan molecular-weight were altered, with decreased SF viscosity, but no associated changes to serum. Serum proteoglycan-4 and hyaluronan concentrations alone may not be useful biomarkers for equine joint-injury or disease.

Influence of Block Length on Articular Cartilage Lubrication with a Diblock Bottle-Brush Copolymer.

We report how the tribological properties of a class of diblock copolymers with architecture and function inspired by the lubricating glycoprotein lubricin correlate to chemical composition. This class of diblock copolymers, consisting of a cationic cartilage-binding block and a brush-lubricating block, demonstrates that boundary lubrication of articular cartilage more strongly depends on the cartilage-binding block than the lubrication block. Specifically, the cartilage-binding functional groups (tertiary or quaternary amines) and cartilage-binding block length significantly influence the degree of lubrication under boundary mode experimental conditions. An optimal number (∼24 in this case) of cartilage-binding groups led to the lowest coefficient of friction, and an increase or decrease in the number of cations in the binding block led to partial (>24, and between 12 and 24) or complete (=12) loss of lubricating ability. The length of the lubricating block (DP = 200 or 400) chosen in this study had no effect on the degree of lubrication. These results are put into context in terms of binding affinity to the cartilage and the spatial packing density of the polymer on the cartilage surface and can serve as a useful guide for future designs of synthetic lubricants that rival the efficacy of natural lubricants.

Lubricin binds cartilage proteins, cartilage oligomeric matrix protein, fibronectin and collagen II at the cartilage surface

Lubricin, a heavily O-glycosylated protein, is essential for boundary lubrication of articular cartilage. Strong surface adherence of lubricin is required given the extreme force it must withstand. Disulfide bound complexes of lubricin and cartilage oligomeric matrix protein (COMP) have recently been identified in arthritic synovial fluid suggesting they may be lost from the cartilage surface in osteoarthritis and inflammatory arthritis. This investigation was undertaken to localise COMP-lubricin complexes within cartilage and investigate if other cartilage proteins are involved in anchoring lubricin to the joint. Immunohistochemical analysis of human cartilage biopsies showed lubricin and COMP co-localise to the cartilage surface. COMP knockout mice, however, presented with a lubricin layer on the articular cartilage leading to the further investigation of additional lubricin binding mechanisms. Proximity ligation assays (PLA) on human cartilage biopsies was used to localise additional lubricin binding partners and demonstrated that lubricin bound COMP, but also fibronectin and collagen II on the cartilage surface. Fibronectin and collagen II binding to lubricin was confirmed and characterised by solid phase binding assays with recombinant lubricin fragments. Overall, COMP, fibronectin and collagen II bind lubricin, exposed on the articular cartilage surface suggesting they may be involved in maintaining essential boundary lubrication.

Ultra-low friction between boundary layers of hyaluronan-phosphatidylcholine complexes.

Can designed biomaterials emulate the unique lubrication ability of articular cartilage, and thus provide potential alleviation to friction-related joint diseases? This is the motivation behind the present study. The principles of cartilage lubrication have attracted considerable attention for decades, and several models have been proposed to elucidate it, however, the mechanism of this ultralow friction is still not clear. In this paper we explore the recent suggestion that its efficient lubrication arises from boundary layers of hyaluronan-lipid complexes at its surface, in particular exploring a range of different phosphatidylcholines (PCs) mimicking the wide range of PCs in synovial joints. The present study suggests a synergistic lubricating behavior of the different lipids in living joints, and potential treatment directions using such biomaterial complexes for widespread cartilage-friction-related diseases such as osteoarthritis.

Effect of disulfide bonding and multimerization on proteoglycan 4’s cartilage boundary lubricating ability and adsorption

ABSTRACTPurpose: The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. Materials and methods: PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi−), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi− was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi− were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants. Results: Separation into enriched populations of PRG4Multi+ and PRG4Multi− was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi− both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi−. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi− did not. R/A PRG4Multi− lubricated similar to NR PRG4Multi−. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi− appeared to have less intensity. Conclusions: These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure–lubrication function relationship will further contribute to the understanding of PRG4’s role in diarthrodial joint homeostasis and disease.

Cartilage boundary lubrication synergism is mediated by hyaluronan concentration and PRG4 concentration and structure

BackgroundProteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4.MethodsStatic and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively.ResultsThis study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important.ConclusionsCollectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.

Superficial Zone Extracellular Matrix Extracts Enhance Boundary Lubrication of Self-Assembled Articular Cartilage.

Previous work has shown that increasing the production of boundary lubricant, superficial zone protein (SZP), did not reduce the friction coefficient of self-assembled articular cartilage constructs and was possibly due to poor retention of the lubricant. The aim of this investigation was to reduce the friction coefficient of self-assembled articular cartilage constructs through enhancing SZP retention by the exogenous addition of extracellular matrix (ECM) extracted from the superficial zone of native articular cartilage. Superficial zone cartilage was shaved from juvenile bovine femoral condyles using a dermatome, minced finely with razor blades, extracted with 4 M guanidine-hydrochloride, buffer exchanged with culture medium, and added directly to the culture medium of self-assembled articular cartilage constructs at low (10 µg/mL) and high (100 µg/mL) concentrations for 4 weeks. Biochemical and biomechanical properties were determined at the conclusion of 4 weeks culture. ECM treatment increased compressive and tensile stiffness of self-assembled articular cartilage constructs and decreased the friction coefficient. Glycosaminoglycan content decreased and collagen content increased significantly in self-assembled constructs by the ECM treatment. Friction coefficients of self-assembled articular cartilage constructs were reduced by adding extracted superficial zone ECM into the culture medium of self-assembled articular cartilage constructs.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study

To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS.

Synovial Fluid Lubricant Properties Are Transiently Deficient After Arthroscopic Articular Cartilage Defect Repair With Platelet-Enriched Fibrin Alone and With Mesenchymal Stem Cells

Background:Following various types of naturally occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan, and proteoglycan-4 (PRG4). However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown.Hypothesis:Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin + mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and PRG4 composition.Study Design:Controlled laboratory study.Methods:Equine synovial fluid was aspirated from normal joints at a preinjury state (0 days) and at 10 days and 3 months following fibrin or fibrin + mesenchymal stem cell repair of full-thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and PRG4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high–molecular weight hyaluronan to restore lubrication function.Results:Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to preinjury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months, while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (−64%) and shifted toward lower molecular weight forms, while PRG4 concentration was increased (+210%) in 10-day samples relative to preinjury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high–molecular weight hyaluronan. Addition of high–molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0 mg/mL restored kinetic friction to preinjury levels.Conclusion:Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high–molecular weight hyaluronan.Clinical Relevance:Synovial fluid lubrication is deficient shortly after arthroscopic cartilage repair surgery, and supplementation with high–molecular weight hyaluronan may be beneficial.

Effects of Stress Deprivation on Lubricin Synthesis and Gliding of Flexor Tendons in a Canine Model in Vivo

Lubricin facilitates boundary lubrication of cartilage. The synthesis of lubricin in cartilage is regulated by mechanical stimuli, especially shear force. Lubricin is also found in flexor tendons. However, little is known about the effect of mechanical loading on lubricin synthesis in tendons or about the function of lubricin in flexor tendons. The purpose of this study was to investigate the relationship of mechanical loading to lubricin expression and gliding resistance of flexor tendons. Flexor tendons were harvested from canine forepaws that had been suspended without weight-bearing for twenty-one days and from the contralateral forepaws that had been allowed free motion. Lubricin expression in each flexor tendon was investigated with real-time RT-PCR (reverse transcription polymerase chain reaction) and immunohistochemistry. Lubricin in the flexor tendon was extracted and quantified with ELISA (enzyme-linked immunosorbent assay). The friction between the flexor tendon and the proximal pulley was measured. The non-weight-bearing flexor tendons had a 40% reduction of lubricin expression (p < 0.01) and content (p < 0.01) compared with the flexor tendons in the contralateral limb. However, the gliding resistance of the tendons in the non-weight-bearing limb was the same as that of the tendons on the contralateral, weight-bearing side. Mechanical loading affected lubricin expression in flexor tendons, resulting in a 40% reduction of lubricin content, but these changes did not affect the gliding resistance of the flexor tendons.

Diminished cartilage‐lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation

The purposes of this study were 1) to quantify the proteoglycan 4 (PRG4) and hyaluronan (HA) content in synovial fluid (SF) from normal donors and from patients with chronic osteoarthritis (OA) and 2) to assess the cartilage boundary-lubricating ability of PRG4-deficient OA SF as compared to that of normal SF, with and without supplementation with PRG4 and/or HA. OA SF was aspirated from the knee joints of patients with symptomatic chronic knee OA prior to therapeutic injection. PRG4 concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay (ELISA), and HA concentrations were measured using a commercially available ELISA. The molecular weight distribution of HA was measured by agarose gel electrophoresis. The cartilage boundary-lubricating ability of PRG4-deficient OA SF, PRG4-deficient OA SF supplemented with PRG4 and/or HA, and normal SF was assessed using a cartilage-on-cartilage friction test. Two friction coefficients (μ) were calculated: static (μ(static, Neq) ) and kinetic (<μ(kinetic, Neq) >) (where N(eq) represents equilibrium axial load and angle brackets indicate that the value is an average). The mean ± SEM PRG4 concentration in normal SF was 287.1 ± 31.8 μg/ml. OA SF samples deficient in PRG4 (146.5 ± 28.2 μg/ml) as compared to normal were identified and selected for lubrication testing. The HA concentration in PRG4-deficient OA SF (mean ± SEM 0.73 ± 0.08 mg/ml) was not significantly different from that in normal SF (0.54 ± 0.09 mg/ml). In PRG4-deficient OA SF, the molecular weight distribution of HA was shifted toward the lower range. The cartilage boundary-lubricating ability of PRG4-deficient OA SF was significantly diminished as compared to normal (mean ± SEM <μ(kinetic, Neq) > = 0.043 ± 0.008 versus 0.025 ± 0.002; P < 0.05) and was restored when supplemented with PRG4 (<μ(kinetic, Neq) > = 0.023 ± 0.003; P < 0.05). These results indicate that some OA SF may have decreased PRG4 levels and diminished cartilage boundary-lubricating ability as compared to normal SF and that PRG4 supplementation can restore normal cartilage boundary lubrication function to these OA SF.

Cartilage boundary lubricating ability of aldehyde modified proteoglycan 4 (PRG4-CHO)

Proteoglycan 4 (PRG4), also known as lubricin1 and superficial zone protein2, is a mucinous glycoprotein that is present in synovial fluid (SF) and at the surface of articular cartilage, where it functions as a critical boundary lubricant necessary for joint health3. High friction and high wear are amongst many factors that may contribute to cartilage degeneration4. PRG4 is critical in reducing friction and minimising surface tissue shear5 at the surface of cartilage, thus preventing the degradation of cartilage through boundary lubrication. Indeed, an increased coefficient of friction in PRG4-knockout mice is associated with increased wear of the articular surface6. Furthermore, intra-articular injection of PRG4 has been shown to prevent cartilage degradation in post-injury rat models of osteoarthritis (OA)7. A recent study, motivated by diminished PRG4 levels in SF associated with early OA, demonstrated aldehyde modification of PRG4 (PRG4-CHO) significantly enhanced its binding to a depleted articular surface8. Such modification may contribute to an improved biotherapeutic treatment of early OA with PRG4 through enhanced residence time within the joint and/or binding to the articular cartilage surface. However, it remains to be determined if PRG4-CHO maintains the friction-reducing ability of unmodified PRG4. Therefore, the objective of this study was to assess the cartilage boundary lubricating ability of PRG4-CHO versus unmodified PRG4 using a previously described in-vitro cartilage-cartilage friction test5,9. The findings of this study indicate that aldehyde modification does not significantly affect the protein structure or lubricating function of PRG4, and PRG4-CHO is an effective friction reducing cartilage boundary lubricant. These results, together with previously published data8, collectively suggest PRG4-CHO may be useful in molecular resurfacing strategies for tissue surfaces requiring lubrication, and potentially other biointerfaces or biomaterials as well.

Effects of equine joint injury on boundary lubrication of articular cartilage by synovial fluid: Role of hyaluronan

To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [μ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the μ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. The μ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had μ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the μ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the μ(kinetic) to a value near that of normal equine SF. In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high μ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.