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Objective Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of post-traumatic osteoarthritis (PTOA). Here we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Methods Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. Results In vitro studies showed that PRG4-GFP has lubricin-like cartilage binding and anti-friction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP prior to cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 versus AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 versus AAV-GFP: n = 9-10) when treatments were administered immediately post-operation, but efficacy was lost when treatment was delayed for 2 weeks. Conclusion AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

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

Osteoarthritis is a huge health burden to our society. Seeking for potential ways to induce regeneration of articular cartilage (AC) that is intrinsically limited, we focused on the interaction between two opposing joints. To evaluate the role of the interaction of opposing regions of AC for joint maturation, we amputated digits at the distal interphalangeal level without injuring the articular surface of the intermediate phalanx (P2) and observed that the zonal organization of AC was defective. We then removed the P2 bone without injuring the articular surface of the proximal phalanx (P1), and the remaining part of the digit was amputated near the distal interphalangeal level. The distribution pattern of type II collagen and proteoglycan 4 (PRG4) suggested that maturation of AC in P1 was delayed. These two experiments suggested that an interaction between the opposing AC in a joint is necessary for maturation of the zonal organization of AC in neonatal digits. To test if an interaction of the joints is sufficient to induce articular cartilage, a proximal fragment of P2 was resected, inverted, and put back into the original location. Newly formed cartilage was induced at the interface region between the AC of the inverted graft and the cut edge of the distal part of P2. Type II collagen and PRG4 were expressed in the ectopic cartilage in a similar manner to normal AC, indicating that neonatal AC can induce ectopic joint-like structures in mice comparable with what has been reported in newts and frogs. These results suggest that the neonatal joint could be a source of inductive signals for regeneration of AC.Lay SummaryIn this study, we experimentally show that neonatal mice appear to have the capacity to regenerate articular cartilage (AC) in digits. It is already known that mice can regenerate a digit tip after amputation, but do not regenerate in response to amputations at more proximal levels. Therefore, it has been thought that mammalian joint structures are non-regenerative. However, we found that normal digit AC can induce AC-like structures in a non-joint region when it is placed next to the cut edge of a bone, suggesting that the normal AC has regenerative capacity in certain situations in neonatal mice.Future WorksJoint disorders are a huge health problem of our society. The results of this study suggest that neonatal AC could be a potential source of inductive signals for regeneration of AC. The discovery of these inductive signals will aid in developing regenerative therapies of a joint in human.

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 (lubric...

The in vivo mechanoresponsive and lubricating changes of the mandibular condylar cartilage (MCC) associated with mandibular lateral shift (MLS) and recovery are poorly understood. Using growing rats, we investigated whether the expression of mechanoresponsive factors, including proteoglycan-4 (PRG4), Indian hedgehog (Ihh) and transforming growth factor-β1 (TGF-β1), would be affected by MLS. We also investigated whether these changes could recover to the control level after a 2-week treatment reversal (TR). The MLS appliances were placed for 2 or 4 weeks in 5-week-old rats and removed from 7-week-old rats in the TR group. The MCC was analysed histomorphometrically by toluidine blue staining. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of PRG4, Ihh, PTHrP (parathyroid hormone-related protein), TGF-β1, Matrix metallopeptidase 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). A thickened superficial layer and an enhanced expression of PRG4 were detected in MLS groups. PTHrP-Ihh expression correlated positively with the up-regulation of PRG4. TGF-β1 expression decreased in the early stage of MLS but recovered to the control level in the TR group. A significantly enhanced expression of MMP-13 in MLS groups was detected. MLS treatment, which acted on the growth stage of rats, affected the morphology and expression of lubrication factor in the MCC. Elimination of this mechanical stimulus may help MCC recover to normal conditions. Our study supports that the adaptive changes of MCC, which are caused by mandibular functional deviation, could be largely recovered by early treatment.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein important for joint health. Mice lacking Prg4 demonstrate degeneration of the cartilage and altered skeletal morphology. The purpose of this study was to examine if Prg4 deficiency leads to subchondral bone defects and if these defects could be mitigated through intra-articular injection of recombinant human PRG4 (rhPRG4). Mice deficient in Prg4 expression demonstrated increased cartilage thickness and increased subchondral bone porosity compared with C57BL/6 controls. While the porosity of the subchondral bone of Prg4-/- mice decreased over time with maturation, intra-articular injection of rhPRG4 was able to forestall the increase in porosity. In contrast, neither hyaluronan (HA) nor methylprednisolone injections had beneficial effects on the subchondral bone porosity in the Prg4 knockout mice. Bone marrow progenitor cells from Prg4-/- mice demonstrated reduced osteogenic differentiation capacity at 4 weeks of age, but not at 16 weeks of age. While most studies on PRG4/lubricin focus on the health of the cartilage, this study demonstrates that PRG4 plays a role in the maturation of the subchondral bone. Furthermore, increasing joint lubrication/viscosupplementation through injection of HA or controlling joint inflammation through injection of methylprednisolone may help maintain the cartilage surface, but had no positive effect on the subchondral bone in animals lacking Prg4. Therefore, alterations in the subchondral bone in models with absent or diminished Prg4 expression should not be overlooked when investigating changes within the articular cartilage regarding the pathogenesis of osteoarthritis/arthrosis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2077-2088, 2019.

Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds. In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. STATEMENT OF SIGNIFICANCE: Articular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term. Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.

The objectives of this study were to evaluate temporal changes in lubricin, hyaluronan (HA), and HA molecular weight (MW) distributions in three distinct models of equine joint injury affecting the carpal (wrist), tarsal (ankle), and femoropatellar (knee) joints. To establish ranges for lubricin, HA, and HA MW distributions across multiple joints, we first evaluated clinically healthy, high-motion equine joints. Synovial fluid was collected from high-motion joints in horses without clinical signs of joint disease (n = 11 horses, 102 joints) and from research horses undergoing carpal osteochondral fragmentation (n = 8), talar cartilage impact injury (n = 7), and femoral trochlear ridge full-thickness cartilage injury (n = 22) prior to and following arthroscopically induced joint injury. Lubricin and HA concentrations were measured via enzyme-linked immunosorbent assays, and gel electrophoresis was performed to evaluate HA MW distributions. Synovial fluid parameters were analyzed via linear regression models, revealing that lubricin and HA concentrations were conserved across healthy, high-motion joints. Lubricin concentrations increased post-injury in all osteoarthritis models (carpal fragmentation P = .001; talar impact P < .001; femoral trochlear ridge cartilage defect P = .03). Sustained loss of HA was noted post-arthroscopy following carpal osteochondral fragmentation (P < .0001) and talar impact injury (P < .001). Lubricin may be elevated to compensate for the loss of HA and to protect cartilage post-injury. Further investigation into the mechanisms regulating lubricin and HA following joint injury and their effects on joint homeostasis is warranted, including whether lubricin has value as a biomarker for post-traumatic osteoarthritis.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFβ1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

Clinical studies have reported an association between low blood levels of 25-hydroxyvitamin D and the progression of osteoarthritis (OA), but the mechanism and effects of vitamin D signaling on articular chondrocytes and cartilage remains unclear. The purpose of this study was to investigate the effects of vitamin D on articular cartilage degeneration using eldecalcitol (ED-71), which is an active vitamin D3 analog. Eight-week old male C57BL/6NCrSlc mice were subjected to experimental surgery to induce OA and local treatments with 10 μL ED-71 (0.5 μg/mL) were administered weekly. Four and 12 weeks after surgery, joints were evaluated using histological scoring systems. In addition, gene expression was analyzed in chondrocytes that were isolated from wildtype neonatal mice, cultured, and treated with ED-71 (10-8 M). Joints treated with ED-71 demonstrated slowed progression of OA at 4 weeks after surgery, but few effects were observed at 12 weeks after surgery. Ets-related gene (Erg) expression was upregulated in OA articular cartilage, and further increased by ED-71 treatment. In primary chondrocytes cultured with ED-71, the gene expression of Erg and lubricin/proteoglycan 4 significantly increased, as compared to that of cells cultured without ED-71. Local treatment with ED-71 reduced degenerative changes to the articular cartilage during the early phase of experimental OA. Regulation of Erg by ED-71 in articular cartilage could confer resistance to early osteoarthritic changes.

Objective The pleckstrin homology domain leucine-rich repeat protein phosphatases (Phlpp1/2) were recently identified as potential therapeutic targets for cartilage regeneration in osteoarthritic joints. Phlpp inhibitors NSC 117079 and NSC 45586 increase chondrocyte proliferation and matrix production, but the pharmacodynamics and pharmacokinetics of these compounds are not known. Design Chondrocytic effects of Phlpp inhibitors, NSC 117079 and NSC 45586, were measured by western blotting of Phlpp substrates, glycosaminoglycan (GAG) assays, and transcriptomic assays. Liquid chromatography/mass spectroscopy assays were established to measure NSC 117079 and NSC 45586 in vitro and in vivo. The effects of NSC 117079 and NSC 45586 on articular cartilage structure in vivo after intra-articular injection were determined by histology. Results The Phlpp inhibitors NSC 117079 and NSC 45586 were highly stable in vitro and stimulated GAG, Sox9, proteoglycan 4 and collagen 2 production in maturing but not more differentiated chondrocytes in vitro. Both molecules reduced Phlpp1/2 levels and suppressed matrix degradation to functionally extend their inhibitory effect on these phosphatases. In vivo, NSC 117079 was eliminated from the bloodstream within 4 ​h after intravenous injection, while NSC 45586 was eliminated in 8 ​h and had a higher volume distribution. Both molecules increased articular cartilage area on lateral and medial tibial plateaus and femoral condyles by 15% in C57Bl/6 mice between four and five weeks of age. Conclusion These data advance our understanding of how Phlpp inhibitors promote and preserve cartilage formation and provide a basis for understanding their safety and activity in vivo.

A boundary lubricant attaches and protects sliding bearing surfaces by preventing interlocking asperity-asperity contact. Proteoglycan-4 (PRG4) is a boundary lubricant found in the synovial fluid that provides chondroprotection to articular surfaces. Inflammation of the diarthrodial joint modulates local PRG4 concentration. Thus, we measured the effects of inflammation, with Interleukin-1α (IL-1α) incubation, upon boundary lubrication and PRG4 expression in bovine cartilage explants. We further aimed to determine whether the addition of exogenous human recombinant PRG4 (rhPRG4) could mitigate the effects of inflammation on boundary lubrication and PRG4 expression in vitro. Cartilage explants, following a 7 day incubation with IL-1α, were tested in a disc-on-disc configuration using either rhPRG4 or saline (PBS control) as a lubricant. Following mechanical testing, explants were studied immunohistochemically or underwent RNA extraction for real-time polymerase chain reaction (RT-PCR). We found that static coefficient of friction (COF) significantly decreased to 0.14 ± 0.065 from 0.21 ± 0.059 (p = 0.014) in IL-1α stimulated explants lubricated with rhPRG4, as compared to PBS. PRG4 expression was significantly up regulated from 30.8 ± 19 copies in control explants lubricated with PBS to 3330 ± 1760 copies in control explants lubricated with rhPRG4 (p

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.

The purpose of this study was to investigate the effects of trauma and subsequent articulation on adult human ankle cartilage subjected to an injurious impact. Trauma was initiated through impaction on talar cartilage explants. Articulation and loading were applied in a joint bioreactor over 5 consecutive days. The early (24 h) effects of impaction included a reduced chondrocytes viability (51% vs. 81% for non-impacted; p = 0.03), increased levels of apoptosis (43% vs. 27%; p = 0.03), and an increase in the histopathology score (4.4 vs. 1.7; p = 0.02) as compared to non-impacted cartilage explants. One of the key findings was that damage also stimulated the PRG4 release (2.2 vs. 1.5 μg/ml). Subsequent articulation for 5 days did not lead to further changes in tissue histopathology and cell viability, neither for injured nor non-injured samples. However, articulation led to an increased apoptosis in the injured samples (p = 0.03 for the interaction term). Articulation also caused a significant increase of PG/GAG release into the culture medium (p = 0.04) for both injured and non-injured samples; however, the synthesis of PG was not affected by articulation (p = 0.45) though the PG synthesis was higher in injured samples (p < 0.01). With regard to the PRG4 release, impacted samples continued to show higher amounts (p = 0.01), adding articulation led to a reduction (p = 0.02). The current study demonstrated that adult human talar cartilage increases both the PRG4 release and biosynthetic activity as an immediate cellular response to injury. Articulation played a less contributing role to biosynthesis and remodeling, behaving mostly neutral, in that no further damage emerged. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:667-676, 2017.

Purpose: One of the essential requirements in maintaining the normal joint motor function is the perfect tribological property of the articular cartilage. Many cartilage regeneration strategies have been developed for treatment in early stages of osteoarthritis, but there is little information on how repaired articular cartilage regains durability. The identification of biomarkers that can predict wear resistant property is critical to advancing the success of cartilage regeneration therapies. Proteoglycan 4 (PRG4) is a macromolecule distributing on the chondrocyte surface that contributes to lubrication. In this study, we investigate if PRG4 expression is associated with tribological properties of regenerated cartilage, and is able to predict its wear resistant status. Methods: Two different strategies including bone marrow enrichment plus microfracture (B/BME-MFX) and microfracture alone (B-MFX) of cartilage repair in sheep were used. PRG4 expression and a series of tribological parameters on regenerated cartilage were rigorously examined and compared. Results: Highly and continuously expression of PRG4 in regenerated cartilage surface was negatively correlated with each tribological parameter (P

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa’s articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.

Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging and FoxO expression in cartilage is reduced with aging and in OA. To investigate FoxO in cartilage function, single and triple FoxO1/3/4 floxed mice were crossed with Col2-Cre mice to generate Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO). Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was significantly thicker than in control mice at 1 or 2 months. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months when Col2Cre-FoxO3 KO mice had more severe spontaneous OA. Autophagy and anti-oxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of triple FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased severity of OA induced by surgical knee instability or treadmill running. The superficial zone of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased Prg4. In vitro, ectopic FoxO1 expression increased Prg4 and synergized with TGFβ stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators, cartilage-degrading enzymes, increased protective genes and antagonized IL-1β effects. Our observations suggest that FoxOs play a key role in postnatal cartilage development, maturation and homeostasis and protects against OA-associated cartilage damage.

Osteoarthritis (OA), a degenerative joint disease characterized by progressive cartilage degeneration, is one of the leading causes of disability worldwide owing to the limited regenerative capacity of adult articular cartilage. Currently, there are no disease-modifying pharmacological or surgical therapies for OA. Fetal mammals, in contrast to adults, are capable of regenerating injured cartilage in the first two trimesters of gestation. A deeper understanding of the properties intrinsic to the response of fetal tissue to injury would allow us to modulate the way in which adult tissue responds to injury. In this study, we employed secretome proteomics to compare fetal and adult protein regulation in response to cartilage injury using an ovine cartilage defect model. The most relevant events comprised proteins associated with the immune response and inflammation, proteins specific for cartilage tissue and cartilage development, and proteins involved in cell growth and proliferation. Alarmins S100A8, S100A9 and S100A12 and coiled-coil domain containing 88A (CCDC88A), which are associated with inflammatory processes, were found to be significantly upregulated following injury in adult, but not in fetal animals. By contrast, cartilage-specific proteins like proteoglycan 4 were upregulated in response to injury only in fetal sheep postinjury. Our results demonstrate the power and relevance of the ovine fetal cartilage regeneration model presented here for the first time. The identification of previously unrecognized modulatory proteins that plausibly affect the healing process holds great promise for potential therapeutic interventions., Summary: Secretome proteomics identifies differential regulation of inflammation modulators during fetal and adult articular cartilage defect healing, offering novel strategies for therapy.

The objective of this study was to determine the effect of different sliding interface materials (counterface) on the cartilage lubricating ability of proteoglycan 4 (PRG4) and hyaluronan (HA) by measuring the kinetic coefficient of friction on cartilage-glass and cartilage-cartilage interfaces over a wide range of sliding velocities. The lubrication properties of PRG4 and HA were assessed at cartilage-glass and cartilage-cartilage interfaces using a previously described test setup with a stationary area of contact. Samples were articulated at varying effective sliding velocities of 10, 3, 1, 0.3, 0.1, and 0.01 mm/s. The response of PRG4 and HA as effective friction-reducing cartilage boundary lubricants was varied and was dependent primarily on the test counterface. At a physiological cartilage-cartilage interface both HA and PRG4 effectively reduced friction compared to PBS at slower speeds while at higher speeds PRG4 was similar to PBS, and HA similar to SF. Conversely, at a cartilage-glass interface HA demonstrated no friction reducing ability compared to PBS, and PRG4 appeared just as effective as SF. Cartilage-glass friction coefficients were also significantly greater than cartilage-cartilage friction coefficients. These results indicate the in vitro friction coefficient of putative cartilage boundary lubricants can be affected by the test counterface and suggest that use of synthetic surfaces in studying cartilage boundary lubrication may not always be appropriate for all molecules of interest. As such, care should be taken when interpreting such data, specifically when comparing to in vitro data obtained at a cartilage-cartilage interface, and especially when extrapolating to in vivo situations. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2923-2931, 2018.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage–cartilage friction test was used to assess rhPRG4’s cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.

Lubricin is a surface-active mucinous glycoprotein secreted in the synovial joint that plays an important role in cartilage integrity. In healthy joints, lubricin molecules coat the cartilage surface, providing boundary lubrication and preventing cell and protein adhesion. Arthropathy occurring in patients with joint trauma, inflammatory arthritis or genetically mediated lubricin deficiencies have insufficient lubricin to prevent damage to articular cartilage. Recent studies in lubricin null joints indicate that lubricin (Prg4) plays a role in preventing damage to the superficial zone and preservation of chondrocytes. Progress in the production of recombinant forms of lubricin and the successes of lubricin supplementation in small animal models identify rhPRG4 as a potential therapeutic for patients with transient lubricin deficiency in the setting of trauma or autoimmune arthritis.

Summary Objective Previous studies have shown that Transforming growth factor-β (TGF-β)/TGFβRII-Smad3 signaling is involved in articular cartilage homeostasis. However, the role of TGF-β/ALK5 signaling in articular cartilage homeostasis has not been fully defined. In this study, a combination of in vitro and in vivo approaches was used to elucidate the role of ALK5 signaling in articular cartilage homeostasis and the development of osteoarthritis (OA). Design Mice with inducible cartilage-specific deletion of Alk5 were generated to assess the role of ALK5 in OA development. Alterations in cartilage structure were evaluated histologically. The expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR, western blotting and immunohistochemistry. The chondrocyte apoptosis was detected by TUNEL staining and immunohistochemistry. In addition, the molecular mechanism underlying the effects of TGF-β/ALK5 signaling on articular cartilage homeostasis was explored by analyzing the TGF-β/ALK5 signaling-induced expression of proteoglycan 4 (PRG4) using specific inhibitors. Results Postnatal cartilage-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia, osteophyte formation, subchondral sclerosis, as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. In addition, the expressions of PRG4 mRNA and protein were decreased in Alk5 conditional knockout mice. Furthermore, our results showed, for the first time, that TGF-β/ALK5 signaling regulated PRG4 expression partially through the protein kinase A (PKA)-CREB signaling pathway. Conclusions TGF-β/ALK5 signaling maintains articular cartilage homeostasis, in part, by upregulating PRG4 expression through the PKA-CREB signaling pathway in articular chondrocytes.

Active lifestyles increase the risk of meniscal injury. A permanent meniscus implant of polycarbonate urethane (PCU) is a promising treatment to postpone/prevent total knee arthroplasty. Study of the changes in articular cartilage tribology in the presence of PCU is essential in developing the optimum meniscus implant. Therefore, a cartilage-meniscus reciprocating, sliding model was developed in vitro, mimicking the stance and swing phases of the gait cycle. The meniscus was further replaced with PCU and surface modified PCUs (with C18 chains, mono-functional polydimethylsiloxane groups and mono-functional polytetrafluoroethylene groups) to study the changes. The coefficient of friction (COF) was calculated, and cartilage wear was determined and quantified histologically. The cartilage-meniscus sliding resulted in low COF during both stance and swing (0.01

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.

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.

Background: osteoarthritic human articular cartilage (AC)-derived cartilage cells (CCs) with same-donor bone marrow (BMSCs) and adipose tissue (ASCs)-derived mesenchymal stem cells were compared, in terms of stemness features, and secretory and immunomodulatory responses to inflammation. Methods: proteoglycan 4 (PRG4) presence was evaluated in AC and CCs. MSCs and CCs (n = 8) were cultured (P1 to P4) and characterized for clonogenicity, nanog homeobox (NANOG), and POU class 5 homeobox 1 (POU5F1) expression, immunotypification, and tri-lineage differentiation. Their basal and interleukin-1&beta, (IL-1&beta, )-stimulated expression of matrix metalloproteases (MMPs), tissue inhibitors (TIMPs), release of growth factors, and cytokines were analyzed, along with the immunomodulatory ability of CCs. Results: PRG4 was mainly expressed in the intact AC surface, whereas shifted to the intermediate zone in damaged cartilage and increased its expression in CCs upon culture. All cells exhibited a similar phenotype and stemness maintenance over passages. CCs showed highest chondrogenic ability, no adipogenic potential, a superior basal secretion of growth factors and cytokines, the latter further increased after inflammatory stimulation, and an immunomodulatory behavior. All stimulated cells shared an increased MMP expression without a corresponding TIMP production. Conclusion: based on the observed features, CCs obtained from pathological joints may constitute a potential tissue-specific therapeutic target or agent to improve damaged cartilage healing, especially damage caused by inflammatory/immune mediated conditions.

Proteoglycan 4 (PRG4), first identified in synovial fluid, is an extracellular matrix structural protein in the joint implicated in reducing shear at the cartilage surface as well as controlling adhesion-dependent synovial growth and regulating bulk protein deposition onto the cartilage. However, recent evidence suggests that it can bind to and effect downstream signaling of a number of cell surface receptors implicated in regulating the inflammatory response. Therefore, we pose the hypothesis: Does PRG4 regulate the inflammatory response and maintain tissue homeostasis? Based on these novel findings implicating PRG4 as an inflammatory signaling molecule, we will present and discuss several hypotheses regarding potential mechanisms by which PRG4 may be able to regulate inflammation. If future studies can demonstrate that PRG4 is a potent inflammatory mediator, this will change current paradigms in the musculoskeletal and ophthalmological fields regarding the how the inflammatory microenvironment is regulated in these tissues and potentially others.

The objective of this study was to determine changes in (1) proteoglycan 4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, and (3) cartilage boundary lubricating ability of synovial fluid (SF) from surgical sham (SHAM), anterior cruciate ligament (ACL)/medial collateral ligament (MCL) transection, and lateral meniscectomy (MEN) in a post-knee surgery ovine model. Ovine SF (oSF) was collected at euthanization 20 weeks after surgery, with the contralateral joint serving as the non-operative control. PRG4 and HA concentration in oSF was measured by sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubricating ability of oSF was measured by a cartilage-cartilage friction test. PRG4 and HA concentration in SHAM, ACL/MCL, and MEN oSF were similar in comparison to the contralateral control (CTRL) oSF. The HA MW distribution in the operated oSF for all ranges were similar to the respective CTRL oSF. The kinetic coefficients of friction in operated and CTRL oSF were similar in all groups, and were significantly lower than saline. These results indicate oSF lubricant composition and function at 20 weeks post-knee surgery were similar to contralateral CTRL, and suggest earlier time points post surgery warrant further investigation.

We developed a novel testing system that allows quantification of joint loading and permits analysis of changes in total protein and PRG4 contents in joint fluid of intact knees in live mice. A sequence of 15 repeat, isometric muscular contractions of "low" intensity (less than 50% of the maximal isometric muscular force), and "high" intensity (greater than 55% of maximal) were applied repeatedly (up to five times with a 15 min rest between contractions) to the mouse knee. Increases in knee joint loading were accompanied with significant increases in total protein (p

The long-term efficacy of osteochondral allografts is due to the presence of viable chondrocytes within graft cartilage. Chondrocytes in osteochondral allografts, especially those at the articular surface that normally produce the lubricant proteoglycan-4 (PRG4), are susceptible to storage-associated death. The hypothesis of this study was that the loss of chondrocytes within osteochondral grafts leads to decreased PRG4 secretion, after graft storage and subsequent implant. The objectives were to determine the effect of osteochondral allograft treatment (FROZEN vs. FRESH) on secretion of functional PRG4 after (i) storage, and (ii) 6 months in vivo in adult goats. FROZEN allograft storage reduced PRG4 secretion from cartilage by ∼85% compared to FRESH allograft storage. After 6 months in vivo, the PRG4-secreting function of osteochondral allografts was diminished with prior FROZEN storage by ∼81% versus FRESH allografts and by ∼84% versus non-operated control cartilage. Concomitantly, cellularity at the articular surface in FROZEN allografts was ∼96% lower than FRESH allografts and non-operated cartilage. Thus, the PRG4-secreting function of allografts appears to be maintained in vivo based on its state after storage. PRG4 secretion may be not only a useful marker of allograft performance, but also a biological process protecting the articular surface of grafts following cartilage repair.

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.

Musculoskeletal tissue damage is an increasing problem with a growing aging population. Biomaterials are emerging as extracellular-mimicking platforms designed to provide instructive cues to control cell behavior and ultimately, be applied as therapies to repair, improve, or maintain tissue function. Here, the focus will be on treatment of musculoskeletal disease and injury. Specifically, (a) development of tissue-engineered alternatives and identification of structure–property relationships for musculoskeletal tissue repair in vivo and (b) modification of proteins and application of natural and synthetic materials for targeted therapeutic delivery to tissue surfaces will be discussed. Efficacy of cell-laden implants for cartilage repair can be aided by both the persistence of implanted cells as well as the depth-associated cell organization of superficial and middle cells in implanted constructs. In the case of mild or early osteoarthritis, delivery of functionalized PRG4 to altered cartilage surfaces may be applied in delaying progression of the disease. Modification of biological surfaces with synthetic peptides is also addressed. Future work in the area should focus on the role of microenvironment in and the development of biomaterials for controlling cell fate.

Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

PurposeThe 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 methodsPRG4 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.ResultsSeparation 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.ConclusionsThese 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.

Objective 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. Methods 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 [μkinetic]). 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 μkinetic of equine SF samples from normal joints and joints with acute injury was determined. Results The μkinetic 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 μkinetic, PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the μkinetic 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 μkinetic to a value near that of normal equine SF. Conclusion In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high μkinetic. HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.

Proteoglycan-4 (Prg4) protects synovial joints from arthropathic changes by mechanisms that are incompletely understood. Parathyroid hormone (PTH), known for its anabolic actions in bone, increases Prg4 expression and has been reported to inhibit articular cartilage degeneration in arthropathic joints. To investigate the effect of Prg4 and PTH on articular cartilage, 16-week-old Prg4 mutant and wild-type mice were treated with intermittent PTH (1-34) or vehicle control daily for six weeks. Analyses included histology of the knee joint, micro-CT of the distal femur, and serum biochemical analysis of type II collagen fragments (CTX-II). Compared to wild-type littermates, Prg4 mutant mice had an acellular layer of material lining the surfaces of the articular cartilage and menisci, increased articular cartilage degradation, increased serum CTX-II concentrations, decreased articular chondrocyte apoptosis, increased synovium SDF-1 expression, and irregularly contoured subchondral bone. PTH-treated Prg4 mutant mice developed a secondary deposit overlaying the acellular layer of material lining the joint surfaces, but PTH-treatment did not alter signs of articular cartilage degeneration in Prg4 mutant mice. The increased joint SDF-1 levels and irregular subchondral bone found in Prg4 mutant mice introduce novel candidate mechanisms by which Prg4 protects articular cartilage.