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OBJECTIVE: The clinical success of focal metallic resurfacing implants depends largely on the friction between implant and opposing cartilage. Therefore, the present study determines the lubricating ability of the synovial fluid components hyaluronic acid (HA), proteoglycan 4 (PRG4) and a surface-active phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), on the articulation between cartilage and a Cobalt Chromium Molybdenum (CoCrMo) implant surface, compared with two cartilage surfaces. METHODS: A ring-on-disk geometry was used to perform repeated friction measurements at physiologically relevant velocities (6 and 60 mm/s) using lubricants with an increasing number of components present. Shear measurements were performed in order to evaluate the viscosity. To ensure that it is clinically relevant to explore the effect of these components, the presence of PRG4 in synovial fluid obtained from primary and revision knee and hip implant surgeries was examined. RESULTS: PRG4 in the presence of HA was found to significantly reduce the coefficient of friction for both cartilage-cartilage and cartilage-CoCrMo interface. This is relevant, as it was also demonstrated that PRG4 is still present at the time of revision surgeries. The addition of POPC had no effect for either configurations. HA increased the viscosity of the lubricating fluid by one order of magnitude, while PRG4 and POPC had no effect. CONCLUSION: The present study demonstrates the importance of selecting the appropriate lubrication solution to evaluate implant materials with biotribology tests. Because PRG4 is a key component for reducing friction between cartilage and an opposing surface, developing coatings which bind PRG4 is recommended for cartilage resurfacing implants. ispartof: Osteoarthritis And Cartilage vol:29 issue:6 pages:894-904 ispartof: location:England status: Published online

Summary Objective Lubricin is increasingly being evaluated as an outcome measure in studies investigating post-traumatic and naturally occurring osteoarthritis. However, there are discrepancies in results, making it unclear as to whether lubricin is increased, decreased or unchanged in osteoarthritis. The purpose of this study was to review all papers that measured lubricin in joint injury or osteoarthritis in order to draw conclusions about lubricin regulation in joint disease. Design A systematic search of the Pubmed, Web of Knowledge, and EBSCOhost databases for papers was performed. Inclusion criteria were in vivo studies that measured lubricin in humans or animals with joint injury, that investigated lubricin supplementation in osteoarthritic joints, or that described the phenotype of a lubricin knock-out model. A methodological assessment was performed. Results Sixty-two studies were included, of which thirty-eight measured endogenous lubricin in joint injury or osteoarthritis. Nineteen papers found an increase or no change in lubricin and nineteen reported a decrease. Papers that reported a decrease in lubricin were cited four times more often than those that reported an increase. Fifteen papers described lubricin supplementation, and all reported a beneficial effect. Eleven papers described lubricin knock-out models. Conclusions The human literature reveals similar distributions of papers reporting increased lubricin as compared to decreased lubricin in osteoarthritis. The animal literature is dominated by reports of decreased lubricin in the rat anterior cruciate ligament transection model, whereas studies in large animal models report increased lubricin. Intra-articular lubricin supplementation may be beneficial regardless of whether lubricin increases or decreases in OA.

Summary Objective We previously identified 3′-phosphoadenosine 5′-phosphosulfate synthase 2 ( PAPSS2 ) as a transcriptional target of transforming growth factor β (TGF-β) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-β responsive gene, proteoglycan 4/lubricin ( PRG4 ). In this study, TGF-β-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. Design Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3 . Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. Results Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-β treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-β signaling. Furthermore, Sox9 was required for full TGF-β-mediated induction of Papss2 . In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-β, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-β. Conclusions TGF-β stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-β-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-β-mediated gene regulation in chondrocytes.

Summary Objective The goals of this study were (1) to quantify proteoglycan 4 ( PRG4 ) gene expression; (2) to assess lubricin immunostaining; and (3) to measure synovial fluid lubricin concentrations in clinical and experimental models of equine carpal osteoarthritis (OA). Design Lubricin synovial fluid concentrations and cartilage and synovial membrane PRG4 expression were analyzed in research horses undergoing experimental OA induction ( n = 8) and in equine clinical patients with carpal OA ( n = 58). Lubricin concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay, and PRG4 expression was quantified using qRT-PCR. Lubricin immunostaining was assessed in synovial membrane and osteochondral sections in the experimental model. Results Lubricin concentrations increased in synovial fluid following induction of OA, peaking at 21 days post-operatively in OA joints vs sham-operated controls (331 ± 69 μg/mL vs 110 ± 19 μg/mL, P = 0.001). Lubricin concentrations also increased in horses with naturally occurring OA as compared to control joints (152 ± 32 μg/mL vs 68 ± 4 μg/mL, P = 0.003). Synovial membrane PRG4 expression increased nearly 2-fold in naturally occurring OA ( P = 0.003), whereas cartilage PRG4 expression decreased 2.5-fold ( P = 0.025). Lubricin immunostaining was more pronounced in synovial membrane from OA joints as compared to controls, with intense lubricin localization to sites of cartilage damage. Conclusions Although PRG4 gene expression decreases in OA cartilage, synovial membrane PRG4 expression, synovial fluid lubricin concentrations and lubricin immunostaining all increase in an equine OA model. Lubricin may be elevated to protect joints from post-traumatic OA.

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.

Summary Objective This study investigated the ability of endogenous lubricin secretion to restore joint health following a brief Prg4 gene trap (GT) mouse under the control of cre-recombinase. Previously we showed that re-expression of lubricin at 21 days was partly restorative of joint lubrication. Design The tibio-femoral joints of adult C57BL/6J mice containing lubricin, lacking lubricin, and postnatally lacking lubricin until restoration of lubricin expression at 7 days or 14 days of age were evaluated ex vivo . At 8-weeks of age, whole joint coefficient of friction (COF), and caspase-3 activation were measured and the tibial-femoral joints histologically analyzed for degenerative changes, following progressive cyclic loading. The peroxynitrite content of femoral head cartilage from these mice prior to cyclic loading was measured. Results Mice that underwent gene recombination at 7 and 14 days of age did not reestablish low COF as joint cycling time increased and were histopathologically indistinguishable from the joints of lubricin-null littermates. However, cartilage from tibio-femoral joints that underwent recombination at 7 and 14 days of age had significantly fewer caspase-3 positive cells and significantly reduced peroxynitrite content compared to lubricin-null littermates. Conclusions The biological effects of lubricin, which include limiting inflammation via peroxynitrite production and caspase-3 activation, may be achieved without completely restituting low COF. However, fully recapitulating low COF may require undamaged cartilage surfaces or absence of biofouling, which may interfere with the activity of lubricin.

Summary Objectives (1) assess the molecular weight dependence of hyaluronan's (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA). Methods The cartilage boundary lubricating ability of a broad range of MW HA (20kDa, 132kDa, 780kDa, 1.5MDa, and 5MDa) at 3.33mg/ml, both alone and in combination with PRG4 at 450μg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, μ static, Neq , and kinetic, kinetic, Neq >, were calculated. An EMSA was conducted with PRG4 and monodisperse 150kDa and 1,000kDa HA. Results Friction coefficients were reduced by HA, in a MW-dependent manner. Values of kinetic, Neq > in 20kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780kDa, 0.080 (0.072, 0.088), and 5MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both μ static, Neq and kinetic, Neq >, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of kinetic, Neq > in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4+various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150kDa and 1,000kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios. Conclusions These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.

Summary Objectives To determine (1) if interleukin-1 alpha (IL-1α), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-β1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion. Methods Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10ng/ml IL-1α, 300ng/ml IGF-I, or 10ng/ml TGF-β1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot. Results PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1α, stimulated by TGF-β1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion. Conclusions Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair.

Summary Objective To investigate the effect of surface motion on the gene expression of proteoglycan 4 (PRG4), hyaluronan synthases (HAS1, HAS2) and on the hyaluronan (HA) and proteoglycan 4 (PRG4) release of chondrocytes from different zones of bovine articular cartilage. Design Superficial zone, deep zone, full thickness, and superficial/deep 1:1 mixed chondrocytes were seeded into 3D polyurethane scaffolds and stimulated using our bioreactor that approximates kinematics and surface motion characteristics of natural joints. One hour of surface motion superimposed on cyclic compression was applied twice a day over 3 consecutive days. Scaffolds were cut into top and bottom sections and analyzed for gene expression of PRG4, HAS1, and HAS2. Results Depending on the cell population, the gene expression levels increased within 8 days of culture in unloaded scaffolds, with a stronger increase in the top compared to the bottom sections. Mechanical loading further enhanced the messenger RNA (mRNA) levels in all cell types, with most pronounced up-regulations observed for the PRG4 expression in deep zone and the HAS2 expression in superficial zone cells. The effect of the biochemical and biomechanical environment appeared to be additive, resulting in highest mRNA levels in the top sections of loaded constructs. Bioreactor stimulation also enhanced the HA release in all cell populations. Full thickness chondrocytes experienced the greatest effect on HAS1 mRNA expression and HA release, indicating that the interaction between cell populations may promote HA synthesis compared to subpopulations alone. Conclusions Reciprocating sliding can be an efficient tool for generating tissue-engineered constructs from various chondrocyte populations by providing a functional cartilage–synovial interface.

digestion by trypsin and reversed phase separations coupled to tandem mass spectrometry for the identification and targeted quantification of peptides by multiple reaction monitoring (MRM). The tissue was sliced from synovial surface to bone in 10mm thin sections and the full thickness cartilage was divided into 100mm pools including superficial, intermediate and deep zones. Results: Previous known distribution of proteoglycan 4 (PRG4, lubricin, superficial zone protein) was confirmed but also novel findings were observed e.g. asporin which was predominantly present in the superficial layers. In total 40 proteins were quantified showing distinct patterns in normal tissue. In addition we observed altered protein distribution patterns in preclinical (macroscopically normal) tissue from a joint with early fibrillation (OA-lesions) on the opposite surface. Conclusions: As an alternative to immunohistochemistry we used proteomics technology to study the protein abundance across full thickness articular cartilage. The advantages of this approach are that it allows multiple targets to be studied simultaneously, it is independent of antibody availability and circumvent some antibody-related artifacts. Other advantages include unambiguous identifications and improved quantifications. The work shows novel information on the differences that exist in different layers of cartilage that is of value in understanding changes in early pathology.

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S17 immuno-histological staining was analyzed using an arbitrary scale. Osteophyte formation was measured using digital image analysis. Bone marrow derived cells were differentiated into macrophages and preincubated with oxLDL or LDL for 24 hours, after which they were stimulatedwith the endogenous toll-like receptor 4 ligand S100A8. RNA was analyzed for gene expression. Data are depicted as mean SEM. Results: WT mice receiving a normal diet developed moderate cartilage destruction (6.1 1.5), synovial thickening (1.4 0.2) and osteophyte formation (32.4mm2 14.6). SerumLDL levelswere significantly higher in LDLr-/-micecompared toWTmice (7.33mmol/L 0.46and0.54mmol/L 0.04 respectively; p

Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. ----- ----- Objectives: (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis.----- ----- Methods: A bioreactor was developed to apply CPM to bovine stifle joints in vitro. Effects of 24 h of CPM on PRG4 biosynthesis were determined.----- ----- Results: PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without.----- ----- Conclusions: Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation. (C) 2006 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Summary Objective Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. Methods The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage. Results SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-β in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1β. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-β. Conclusions These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.

Summary Objectives The lubrication of articulating cartilage surfaces in joints occurs through several distinct modes. In the boundary mode of lubrication, load is supported by surface-to-surface contact, a feature that makes this mode particularly important for maintenance of the normally pristine articular surface. A boundary mode of lubrication is indicated by a kinetic friction coefficient being invariant with factors that influence formation of a fluid film, including sliding velocity and axial load. The objectives of this study were to (1) implement and extend an in vitro articular cartilage-on-cartilage lubrication test to elucidate the dependence of the friction properties on sliding velocity, axial load, and time, and establish conditions where a boundary mode of lubrication is dominant, and (2) determine the effects of synovial fluid (SF) on boundary lubrication using this test. Methods Fresh bovine osteochondral samples were analyzed in an annulus-on-disk rotational configuration, maintaining apposed articular surfaces in contact, to determine static ( μ static and μ static , N eq ) and kinetic (〈 μ kinetic 〉 and 〈 μ kinetic , N eq 〉 ) friction coefficients, each normalized to the instantaneous and equilibrium ( N eq ) normal loads, respectively. Results With increasing pre-sliding durations, μ static and μ static , N eq were similar, and increased up to 0.43±0.03 in phosphate buffered saline (PBS) and 0.19±0.01 in SF, whereas 〈 μ kinetic 〉 and 〈 μ kinetic , N eq 〉 were steady. Over a range of sliding velocities of 0.1–1mm/s and compression levels of 18% and 24%, 〈 μ kinetic 〉 was 0.072±0.010 in PBS and 0.014±0.003 in SF, and 〈 μ kinetic , N eq 〉 was 0.093±0.005 in PBS and 0.018±0.002 in SF. Conclusions A boundary mode of lubrication was achieved in a cartilage-on-cartilage test configuration. SF functioned as an effective friction-lowering boundary lubricant for native articular cartilage surfaces.

SummaryObjectiveWe hypothesize that chondrocytes from distinct zones of articular cartilage respond differently to compressive loading, and that zonal chondrocytes from osteoarthritis (OA) patients can benefit from optimized compressive stimulation. Therefore, we aimed to determine the transcriptional response of superficial (S) and middle/deep (MD) zone chondrocytes to varying dynamic compressive strain and loading duration. To confirm effects of compressive stimulation on overall matrix production, we subjected zonal chondrocytes to compression for 2 weeks.DesignHuman S and MD chondrocytes from osteoarthritic joints were encapsulated in 2% alginate, pre-cultured, and subjected to compression with varying dynamic strain (5, 15, 50% at 1 Hz) and loading duration (1, 3, 12 h). Temporal changes in cartilage-specific, zonal, and dedifferentiation genes following compression were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The benefits of long-term compression (50% strain, 3 h/day, for 2 weeks) were assessed by measuring construct glycosaminoglycan (GAG) content and compressive moduli, as well as immunostaining.ResultsCompressive stimulation significantly induced aggrecan (ACAN), COL2A1, COL1A1, proteoglycan 4 (PRG4), and COL10A1 gene expression after 2 h of unloading, in a zone-dependent manner (P