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Surface-enhanced Raman scattering (SERS) is a powerful optical sensing platform that amplifies the target signals by Raman scattering. Despite SERS enabling a meager detection limit, even at the single-molecule level, SERS also tends to equally enhance unwanted molecules due to the non-specific binding of noise molecules in clinical samples, which complicates its use in complex samples such as bodily fluids, environmental water, or food matrices. To address this, we developed a novel non-fouling biomimetic SERS sensor by self-assembling an anti-adhesive, anti-fouling, and size-selective Lubricin (LUB) coating on gold nanoparticle (AuNP) functionalized glass slide surfaces via a simple drop-casting method. Compared to a conventional AuNPs-SERS substrate, the biomimetic SERS meets the requirements of simple preparation and enables direct droplet detection without any sample pre-treatment. Atomic force microscopy was used to confirm the self-assembled Lubricin coating on the AuNP surface, acting as an anti-fouling and size-selective protection layer. A series of Raman spectra were collected using melamine as the target analyte, which was spiked into 150 mM NaCl solution or undiluted milk. It was demonstrated that the LUB coating effectively prevents the detrimental fouling generated by the proteins and fats in milk, ensuring the clear detection of melamine. Our sensor showed high selectivity and could detect melamine in milk at concentrations as low as 1 ppm. Given that the EU/US legal limit for melamine in food is 2.5 ppm, this sensor offers a promising, cost-effective solution for routine screening and has potential applications for detecting food adulteration in the food safety, environmental monitoring, aquaculture, and biomedical fields. [ABSTRACT FROM AUTHOR]

Osteoporosis is a chronic disease characterized by reduced bone mass and increased fracture risk, estimated to affect over 10 million people in the United States alone. Drugs used to treat bone loss often come with significant limitations and/or long‐term safety concerns. Proteoglycan‐4 (PRG4, also known as lubricin) is a mucin‐like glycoprotein best known for its boundary lubricating function of articular cartilage. In more recent years, it has been shown that PRG4 has anti‐inflammatory properties, contributes to the maintenance of subchondral bone integrity, and patients with PRG4 mutations are osteopenic. However, it remains unknown how PRG4 impacts mechanical and material properties of bone. Therefore, our objective was to perform a phenotyping study of bone in a Prg4 gene trap (GT) mouse (PRG4 deficient). We found that femurs of Prg4 GT mice have altered mechanical, structural, and material properties relative to wildtype littermates. Additionally, Prg4 GT mice have a greater number of calvarial osteoclasts than wildtype mice, but do not have a notable inflammatory serum profile. Finally, Prg4 GT mice do not have an altered rate of bone formation, and exogenous recombinant human PRG4 (rhPRG4) administration inhibited osteoclastogenesis in vitro, suggesting that the skeletal phenotype may be due to changes in bone resorption. Overall, this work demonstrates that PRG4 deficiency affects several integral properties of bone structure, mechanics, and skeletal cell activity, and provides the foundation and insight toward future work evaluating PRG4 as a potential therapeutic target in treating bone loss. [ABSTRACT FROM AUTHOR]

Joint-resident chondrogenic precursor cells have become a significant therapeutic option due to the lack of regenerative capacity in articular cartilage. Progenitor cells are located in the superficial zone of the articular cartilage, producing lubricin/Prg4 to decrease friction of cartilage surfaces during joint movement. Prg4-positive progenitors are crucial in maintaining the joint's structure and functionality. The disappearance of progenitor cells leads to changes in articular hyaline cartilage over time, subchondral bone abnormalities, and the formation of ectopic ossification. Genetic labeling cell technology has been the main tool used to characterize Prg4-expressing progenitor cells of articular cartilage in vivo through drug injection at different time points. This technology allows for the determination of the origin of progenitor cells and the tracking of their progeny during joint development and cartilage damage. We endeavored to highlight the currently known information about the Prg4-producing cell population in the joint to underline the significance of the role of these cells in the development of articular cartilage and its homeostasis. This review focuses on superficial progenitors in the joint, how they contribute to postnatal articular cartilage formation, their capacity for regeneration, and the consequences of Prg4 deficiency in these cells. We have accumulated information about the Prg4+ cell population of articular cartilage obtained through various elegantly designed experiments using transgenic technologies to identify potential opportunities for further research. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman scattering (SERS) is a powerful optical sensing platform that amplifies the target signals by Raman scattering. Despite SERS enabling a meager detection limit, even at the single-molecule level, SERS also tends to equally enhance unwanted molecules due to the non-specific binding of noise molecules in clinical samples, which complicates its use in complex samples such as bodily fluids, environmental water, or food matrices. To address this, we developed a novel non-fouling biomimetic SERS sensor by self-assembling an anti-adhesive, anti-fouling, and size-selective Lubricin (LUB) coating on gold nanoparticle (AuNP) functionalized glass slide surfaces via a simple drop-casting method. Compared to a conventional AuNPs-SERS substrate, the biomimetic SERS meets the requirements of simple preparation and enables direct droplet detection without any sample pre-treatment. Atomic force microscopy was used to confirm the self-assembled Lubricin coating on the AuNP surface, acting as an anti-fouling and size-selective protection layer. A series of Raman spectra were collected using melamine as the target analyte, which was spiked into 150 mM NaCl solution or undiluted milk. It was demonstrated that the LUB coating effectively prevents the detrimental fouling generated by the proteins and fats in milk, ensuring the clear detection of melamine. Our sensor showed high selectivity and could detect melamine in milk at concentrations as low as 1 ppm. Given that the EU/US legal limit for melamine in food is 2.5 ppm, this sensor offers a promising, cost-effective solution for routine screening and has potential applications for detecting food adulteration in the food safety, environmental monitoring, aquaculture, and biomedical fields.

ObjectivePathology of the facet and sacroiliac (SI) joints contributes to 15% to 45% and 10% to 27% of lower back pain cases, respectively. Although tissue engineering may offer novel treatment options to patients suffering from cartilage degeneration in these joints, the tribological characteristics of the facet and SI joints have not been studied in either the human or relevant large animal models, which hinders the development of joint-specific cartilage implants.DesignCartilage was isolated from the knee, cervical facet, thoracic facet, lumbar facet, and SI joints of 6 skeletally mature Yucatan minipigs (Sus scrofa). Tribological characteristics were assessed via coefficient of friction testing, interferometry, and immunohistochemistry for lubricin organization.ResultsCompared with the knee, the coefficient of friction was higher by 43% in the cervical facet, 77% in the thoracic facet, 37% in the lumbar facet, and 28% in the SI joint. Likewise, topographical features of the facet and SI joints varied significantly, ranging from a 114% to 384% increase and a 48% to 107% increase in global and local surface roughness measures, respectively, compared with the knee. Additionally, the amount of lubricin in the SI joint was substantially greater than in the knee. Statistical correlations among the various tribological parameters revealed that there was a significant correlation between local roughness and coefficient of friction, but not global roughness or the presence of lubricin.ConclusionThese location-specific tribological characteristics of the articular cartilages of the spine will need to be taken into consideration during the development of physiologically relevant, functional, and durable tissue-engineered replacements for these joints.

Meniscus injuries are extremely common with approximately one million patients undergoing surgical treatment annually in the U.S. alone, but no regenerative therapy exist. Previously, we showed that controlled applications of connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) via fibrin-based bio-glue facilitate meniscus healing by inducing recruitment and stepwise differentiation of synovial mesenchymal stem/progenitor cells. Here, we first explored the potential of genipin, a natural crosslinker, to enhance fibrin-based glue's mechanical and degradation properties. In parallel, we identified the harmful effects of lubricin on meniscus healing and investigated the mechanism of lubricin deposition on the injured meniscus surface. We found that the pre-deposition of hyaluronic acid (HA) on the torn meniscus surface mediates lubricin deposition. Then we implemented chemical modifications with heparin conjugation and CD44 on our bioactive glue to achieve strong initial bonding and integration of lubricin pre-coated meniscal tissues. Our data suggested that heparin conjugation significantly enhances lubricin-coated meniscal tissues. Similarly, CD44, exhibiting a strong binding affinity to lubricin and hyaluronic acid (HA), further improved the integrated healing of HA/lubricin pre-coated meniscus injuries. These findings may represent an important foundation for developing a translational bio-active glue guiding the regenerative healing of meniscus injuries.

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. [ABSTRACT FROM AUTHOR]

Frictional properties of cartilage resurfacing implants should be sufficiently low to limit damaging of the opposing cartilage during articulation. The present study determines if native lubricious molecule proteoglycan 4 (PRG4) can adsorb onto a layer‐by‐layer bioinspired coating composed of poly‐l‐lysine (PLL) and dopamine modified hyaluronic acid (HADN) and thereby can reduce the friction between implant and articular cartilage. An ELISA was developed to quantify the amount of immobilized human recombinant (rh)PRG4 after exposure to the PLL‐HADN coating. The effect on lubrication was evaluated by comparing the coefficient of friction (CoF) of bare polycaprolactone (PCL) disks to that of PLL‐HADN coated PCL disks while articulated against cartilage using a ring‐on‐disk geometry and a lubricant solution consisting of native synovial fluid components including rhPRG4. The PLL‐HADN coating effectively immobilized rhPRG4. The surface roughness of PCL disks significantly increased while the water contact angle significantly decreased after application of the coating. The average CoF measured during the first minute of bare PCL against cartilage exceeded twice the CoF of the PLL‐HADN coated PCL against cartilage. After 60 min, the CoF reached equilibrium values which were still significantly higher for bare PCL compared to coated PCL. The present study demonstrated that PCL can effectively be coated with PLL‐HADN. Additionally, this coating reduces the friction between PCL and cartilage when a PRG4‐rich lubricant is used, similar to the lubricating surface of native cartilage. This makes PLL‐HADN coating a promising application to improve the clinical success of PCL‐based cartilage resurfacing implants. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a debilitating disease that results in pain and joint stiffness. Currently, steroidal and nonsteroidal anti-inflammatory drugs and supplements aimed at restoring lubrication to the affected joint are the most successful with respect to improving patient comfort. Due to the success in lubricating therapies, there exists a keen interest to develop better therapies that mimic how lubrication occurs naturally in the joint. Here we describe the results obtained using a chondroitin sulfate chain to which is conjugated peptides that bind to either hyaluronic acid (found in high concentrations in the synovial fluid) or collagen type II (present on the cartilage surface). Our study investigates the effect of binding to the cartilage surface and interacting with hyaluronic acid on lubrication at the cartilage surface. The results described here suggest that binding to the cartilage surface is critical to supporting lubrication and did not require the addition of hyaluronic acid to reduce friction.

This review summarises the material covered during a workshop entitled “Natural Products as Treatments for Urinary Incontinence” that was presented at the online ICS (Melbourne) annual conference held in 2021. The clinical and scientific evidence of the effectiveness of naturally sourced treatments such as traditional Chinese medicines, phytoestrogens and saw palmetto for lower urinary tract symptoms are discussed, and also the use of cranberry and D-mannose for the treatment of bacterial infections of the urinary tract. The workshop and this review finish with a look towards the future and a discussion of potential treatments to repair the barrier function of the urothelium, an action that may be useful in conditions such as interstitial cystitis/bladder pain syndrome.

Osteoarthritis is a chronic degenerative musculoskeletal disease that worsens with age and is defined by pathological alterations in joint components. All clinical treatment recommendations for osteoarthritis promote exercise, although precise molecular pathways are unclear. The purpose of this study was to critically analyze the research on lubricin and irisin and how they relate to healthy and diseased joint tissue. Our research focused specifically on exercise strategies and offered new perspectives for future potential osteoarthritis treatment plans. Although lubricin and irisin have only recently been discovered, there is evidence that they have an impact on cartilage homeostasis. A crucial component of cartilage lubrication and integrity, lubricin is a surface-active mucinous glycoprotein released by the synovial joint. Its expression increases with joint movement. In healthy joints, lubricin molecules cover the cartilage surface to lubricate the boundary of the joint and inhibit protein and cell attachment. Patients with joint trauma, inflammatory arthritis, or genetically mediated lubricin deficiency, who do not produce enough lubricin to protect the articular cartilage, develop arthropathy. Irisin, sometimes known as the "sports hormone", is a myokine secreted primarily by skeletal muscle. It is a physiologically active protein that can enter the circulation as an endocrine factor, and its synthesis and secretion are primarily triggered by exercise-induced muscle contraction. We searched PubMed, Web of Science, Google Scholar, and Scopus using the appropriate keywords to identify the most recent research. The studies considered advance our knowledge of the role that exercise plays in the fight against osteoarthritis, serve as a valuable resource, and support the advancement of osteoarthritis prevention and therapy. [ABSTRACT FROM AUTHOR]

Abstract The superficial zone of articular cartilage contributes to smooth joint motion through the production of proteoglycan 4 (PRG4), also known as lubricin. Recent studies indicate novel effects of PRG4 as a signaling molecule, other than a simple extracellular matrix protein. Additionally, the accumulating evidence displays that various molecules and signaling pathways are involved in regulating the superficial zone and PRG4 expression. In addition, Prg4-expressing cells include a progenitor population of articular chondrocytes. Several non-clinical and clinical studies have shown that PRG4 and related molecules are promising candidates for disease-modifying drugs for treating osteoarthritis. Since PRG4 is also expressed in the synovium, tendons, and ligaments, further studies of PRG4-related pathways and PRG4-positive cells may elucidate the mechanisms underlying joint homeostasis.

Abstract Background Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage. Methods Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared. Results Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes. Conclusions In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED. [ABSTRACT FROM AUTHOR]

The size, conformation, and organization of the glycosaminoglycan hyaluronan (HA) affect its interactions with soluble and cell surface-bound proteins. HA that is induced to form stable networks has unique biological properties relative to unmodified soluble HA. AlphaLISA assay technology offers a facile and general experimental approach to assay protein-mediated networking of HA in solution. Connections formed between two end-biotinylated 50 kDa HA (bHA) chains can be detected by signal arising from streptavidin-coated donor and acceptor beads being brought into close proximity when the bHA chains are bridged by proteins. We observed that incubation of bHA with the protein TSG-6 (tumor necrosis factor alpha stimulated gene/protein 6, TNFAIP/TSG-6) leads to dimerization or higher order multimerization of HA chains in solution. We compared two different heparin (HP) samples and two heparan sulfate (HS) samples for the ability to disrupt HA crosslinking by TSG-6. Both HP samples had approximately three sulfates per disaccharide, and both were effective in inhibiting HA crosslinking by TSG-6. HS with a relatively high degree of sulfation (1.75 per disaccharide) also inhibited TSG-6 mediated HA networking, while HS with a lower degree of sulfation (0.75 per disaccharide) was less effective. We further identified Proteoglycan 4 (PRG4, lubricin) as a TSG-6 ligand, and found it to inhibit TSG-6-mediated HA crosslinking. The effects of HP, HS, and PRG4 on HA crosslinking by TSG-6 were shown to be due to HP/HS/PRG4 inhibition of HA binding to the Link domain of TSG-6. Using the AlphaLISA platform, we also tested other HA-binding proteins for ability to create HA networks. The G1 domain of versican (VG1) effectively networked bHA in solution but required a higher concentration than TSG-6. Cartilage link protein (HAPLN1) and the HA binding protein segment of aggrecan (HABP, G1-IGD-G2) showed only low and variable magnitude HA networking effects. This study unambiguously demonstrates HA crosslinking in solution by TSG-6 and VG1 proteins, and establishes PRG4, HP and highly sulfated HS as modulators of TSG-6 mediated HA crosslinking.

Abstract Background Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage. Results Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage. Conclusions Synovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Intra-articular injection of mesenchymal stem cells (MSCs) is envisioned as a solution for knee osteoarthritis (OA). Although synovial MSCs (SyMSCs) are promising for cartilage regeneration, the clinical choice is usually adipose MSCs (AdMSCs). However, the similarities/differences in the mode of action between SyMSCs and AdMSCs remain unclear. Here, we compared factors secreted by human SyMSCs and AdMSCs after injection into OA knees. Human SyMSCs or AdMSCs were injected into the knees of rat partial meniscectomy models. The next day, the knee joints were collected to analyze the distribution of injected MSCs and transcriptome changes in the human MSCs and rat synovium. Non-injected MSCs were mixed with rat synovium as a control. After injection, no difference was apparent in intra-articular distribution of the SyMSCs or AdMSCs. RNA sequencing demonstrated an enrichment of cytokine-cytokine receptor interaction-related genes in both human SyMSCs and AdMSCs after injection. Differentially expressed genes (DEGs) specific to SyMSCs were associated with cartilage matrix synthesis and homeostasis. PCR analysis of the matrisome-related DEGs showed significantly higher expression of PRG4 in SyMSCs than in AdMSCs after injection. Immunostaining also confirmed a significantly greater expression of lubricin by SyMSCs than by AdMSCs. These findings indicate that SyMSCs will be a more promising treatment for OA. • The yield of MSCs is greater from the synovium than from adipose tissue. • SyMSCs and AdMSCs change their secretion profiles after injection into OA knees. • SyMSCs show a specific upregulation of chondroprotective factor PRG4/lubricin. • SyMSCs will be a more promising treatment for OA than AdMSCs. [ABSTRACT FROM AUTHOR]

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 posttraumatic osteoarthritis. In this study, 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. 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. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before 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 vs. AAV-GFP: n = 7–14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9–10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. 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. [ABSTRACT FROM AUTHOR]

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4—PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4−/− (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4. [ABSTRACT FROM AUTHOR]

Background: Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.Methods: Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.Results: Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.Conclusions: In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a debilitating disease that results in pain and joint stiffness. Currently, steroidal and nonsteroidal anti-inflammatory drugs and supplements aimed at restoring lubrication to the affected joint are the most successful with respect to improving patient comfort. Due to the success in lubricating therapies, there exists a keen interest to develop better therapies that mimic how lubrication occurs naturally in the joint. Here we describe the results obtained using a chondroitin sulfate chain to which is conjugated peptides that bind to either hyaluronic acid (found in high concentrations in the synovial fluid) or collagen type II (present on the cartilage surface). Our study investigates the effect of binding to the cartilage surface and interacting with hyaluronic acid on lubrication at the cartilage surface. The results described here suggest that binding to the cartilage surface is critical to supporting lubrication and did not require the addition of hyaluronic acid to reduce friction.

Objective: To explore the expression of lubricin in the lumbar endplate and its association with Modic changes (MCs). Methods: Human endplate specimens harvested from patients undergoing surgery for thoracolumbar spine fractures ​or lumbar interbody fusion were divided into two groups: MCs group and normal group. Lubricin expression was examined by immunohistochemistry, and differences between the groups were analysed using quantitative polymerase chain reaction (qPCR). Lubricin expression and differences between endplates with MCs and normal endplates were confirmed using a rabbit model. In a final experiment, rabbit endplate chondrocytes were cocultured with Propionibacteria acnes (P. acnes) supernatant, and the expression of lubricin and endplate degeneration related genes were evaluated. In addition, the expression of matrix metalloproteinase 1(MMP-1), A disintegrin-like and metalloproteinase with thrombospondin type 5 motif (ADAMTS5) and inflammatory factors (Interleukin- 1β (IL-1β) and Interleukin-6 (IL-6)) were evaluated after lubricin overexpression. Results: Lubricin was found in human lumbar endplates and its expression was lower in the MCs group compared to the normal group. In the rabbit model, lubricin was also found in the endplate. In rabbits injected with P. acnes (the MCs group), lubricin expression of endplate decreased compared to the normal group. In the culture of rabbit endplate chondrocytes with P. acnes supernatant, the expression of lubricin, aggrecan, sox9 and collagen type-II decreased significantly, while that of MMP-1 and ADAMTS5 increased significantly. Moreover, lubricin overexpression could downregulate the expression of MMP-1, ADAMTS5 and inflammatory factors (IL-1β and IL-6) compared to negative control. Conclusion: Lubricin is present in the lumbar endplate where it may have an anti-inflammatory role. P. acnes infection inhibits lubricin expression by cartilage endplate cells and this may facilitate the progression of MCs and endplate degeneration. The translational potential of this article: Lubricin may have an anti-inflammatory role. P. acnes infection inhibits lubricin expression by cartilage endplate cells and this may facilitate the progression of MCs and endplate degeneration.

Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls’ tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucinlike glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time. [ABSTRACT FROM AUTHOR]

BackgroundPlatelet-rich plasma (PRP) contains high concentrations of autologous growth factors that originate from platelets. Intra-articular injections of PRP have the potential to ameliorate the symptoms of osteoarthritis in the knee. Superficial zone protein (SZP) is a boundary lubricant in articular cartilage and plays an important role in reducing friction and wear and therefore is critical in cartilage homeostasis.PurposeTo determine if PRP influences the production of SZP from human joint-derived cells and to evaluate the lubricating properties of PRP on normal bovine articular cartilage.Study designControlled laboratory study.MethodsCells were isolated from articular cartilage, synovium, and the anterior cruciate ligament (ACL) from 12 patients undergoing ACL reconstruction. The concentrations of SZP in PRP and culture media were measured by enzyme-linked immunosorbent assay. Cellular proliferation was quantified by determination of cell numbers. The lubrication properties of PRP from healthy volunteers on bovine articular cartilage were investigated using a pin-on-disk tribometer.ResultsIn general, PRP stimulated proliferation in cells derived from articular cartilage, synovium, and ACL. It also significantly enhanced SZP secretion from synovium- and cartilage-derived cells. An unexpected finding was the presence of SZP in PRP (2.89 ± 1.23 μg/mL before activation and 3.02 ± 1.32 μg/mL after activation). In addition, under boundary mode conditions consisting of high loads and low sliding speeds, nonactivated and thrombin-activated PRP decreased the friction coefficient (μ = 0.012 and μ = 0.015, respectively) compared with saline (μ = 0.047, P < .004) and high molecular weight hyaluronan (μ = 0.080, P < .006). The friction coefficient of the cartilage with PRP was on par with that of synovial fluid.ConclusionPRP significantly stimulates cell proliferation and SZP secretion by articular cartilage and synovium of the human knee joint. Furthermore, PRP contains endogenous SZP and, in a functional bioassay, lubricates bovine articular cartilage explants.Clinical relevanceThese findings provide evidence to explain the biochemical and biomechanical mechanisms underlying the efficacy of PRP treatment for osteoarthritis or damage in the knee joint.

Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls’ tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.

Abstract Dry eye disease (DED) has high personal and societal costs, but its pathology remains elusive due to intertwined biophysical and biochemical processes at the ocular surface. Specifically, mucin deficiency is reported in a subset of DED patients, but its effects on ocular interfacial properties remain unclear. Herein a novel in vitro mucin‐deficient mimetic ocular surface (Mu‐DeMOS) with a controllable amount of membrane‐tethered mucin molecules is developed to represent the diseased ocular surfaces. Contact angle goniometry on mimetic ocular surfaces reveals that high surface roughness, but not the presence of hydrophilic mucin molecules, delivers constant hydration over native ocular surface epithelia. Live‐cell rheometry confirms that the presence of mucin‐like glycoproteins on ocular epithelial cells reduces shear adhesive strength at cellular interfaces. Together, optimal surface roughness and surface chemistry facilitate sustainable lubrication for healthy ocular surfaces, while an imbalance between them contributes to lubrication‐related dysfunction at diseased ocular epithelial surfaces. Furthermore, the restoration of low adhesive strength at Mu‐DeMOS interfaces through a mucin‐like glycoprotein, recombinant human lubricin, suggests that increased frictional damage at mucin‐deficient cellular surfaces may be reversible. More broadly, these results demonstrate that Mu‐DeMOS is a promising platform for drug screening assays and fundamental studies on ocular physiology.

Dry eye disease (DED) has high personal and societal costs, but its pathology remains elusive due to intertwined biophysical and biochemical processes at the ocular surface. Specifically, mucin deficiency is reported in a subset of DED patients, but its effects on ocular interfacial properties remain unclear. Herein a novel in vitro mucin‐deficient mimetic ocular surface (Mu‐DeMOS) with a controllable amount of membrane‐tethered mucin molecules is developed to represent the diseased ocular surfaces. Contact angle goniometry on mimetic ocular surfaces reveals that high surface roughness, but not the presence of hydrophilic mucin molecules, delivers constant hydration over native ocular surface epithelia. Live‐cell rheometry confirms that the presence of mucin‐like glycoproteins on ocular epithelial cells reduces shear adhesive strength at cellular interfaces. Together, optimal surface roughness and surface chemistry facilitate sustainable lubrication for healthy ocular surfaces, while an imbalance between them contributes to lubrication‐related dysfunction at diseased ocular epithelial surfaces. Furthermore, the restoration of low adhesive strength at Mu‐DeMOS interfaces through a mucin‐like glycoprotein, recombinant human lubricin, suggests that increased frictional damage at mucin‐deficient cellular surfaces may be reversible. More broadly, these results demonstrate that Mu‐DeMOS is a promising platform for drug screening assays and fundamental studies on ocular physiology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage. Results: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage. Conclusions: Synovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity. [ABSTRACT FROM AUTHOR]

Lubricin encoded by the proteoglycan 4 (Prg4) gene is produced from superficial zone (SFZ) cells of articular cartilage and synoviocytes, which is indispensable for lubrication of joint surfaces. Loss‐of‐function of human and mouse Prg4 results in early‐onset arthropathy accompanied by lost SFZ cells and hyperplastic synovium. Here, we focused on increases in the thickness of articular cartilage in Prg4‐knockout joints and analyzed the underlying mechanisms. In the late stage of articular cartilage development, the articular cartilage was thickened at 2 to 4 weeks and the SFZ disappeared at 8 weeks in Prg4‐knockout mice. Similar changes were observed in cultured Prg4‐knockout femoral heads. Cell tracking showed that Prg4‐knockout SFZ cells at 1 week of age expanded to deep layers after 1 week. In in vitro experiments, overexpression of Prg4 lacking a mucin‐like domain suppressed differentiation of ATDC5 cells markedly, whereas pellets of Prg4‐knockout SFZ cells showed enhanced differentiation. RNA sequencing identified matrix metalloproteinase 9 (Mmp9) as the top upregulated gene by Prg4 knockout. Mmp9 expressed in the SFZ was further induced in Prg4‐knockout mice. The increased expression of Mmp9 by Prg4 knockout was canceled by IκB kinase (IKK) inhibitor treatment. Phosphorylation of Smad2 was also enhanced in Prg4‐knockout cell pellets, which was canceled by the IKK inhibitor. Expression of Mmp9 and phosphorylated Smad2 during articular cartilage development was enhanced in Prg4‐knockout joints. Lubricin contributes to homeostasis of articular cartilage by suppressing differentiation of SFZ cells, and the nuclear factor‐kappa B‐Mmp9‐TGF‐β pathway is probably responsible for the downstream action of lubricin. © 2020 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Sustained inflammation can halt or delay wound healing, and macrophages play a central role in wound healing. Inflammatory macrophages are responsible for the removal of pathogens, debris, and neutrophils, while anti-inflammatory macrophages stimulate various regenerative processes. Recombinant human Proteoglycan 4 (rhPRG4) is shown to modulate macrophage polarization and to prevent fibrosis and scarring in ear wound healing. Here, dissolvable microneedle arrays (MNAs) carrying rhPRG4 are engineered for the treatment of skin wounds. The in vitro experiments suggest that rhPRG4 modulates the inflammatory function of bone marrow-derived macrophages. Degradable and detachable microneedles are developed from gelatin methacryloyl (GelMA) attach to a dissolvable gelatin backing. The developed MNAs are able to deliver a high dose of rhPRG4 through the dissolution of the gelatin backing post-injury, while the GelMA microneedles sustain rhPRG4 bioavailability over the course of treatment. In vivo results in a murine model of full-thickness wounds with impaired healing confirm a decrease in inflammatory biomarkers such as TNF-α and IL-6, and an increase in angiogenesis and collagen deposition. Collectively, these results demonstrate rhPRG4-incorporating MNA is a promising platform in skin wound healing applications., (© 2024 Wiley‐VCH GmbH.)

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O -glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general., Competing Interests: CONFLICT OF INTEREST Cornell University has filed patents related to the lubricin sequences and processes described in this manuscript. M.J.C., H.L.R., and M.J.P. are listed as inventors.

Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within synovial joints. For the horse, changes to proteoglycan-4 concentration and function are notable in acute joint injury and osteoarthritis. Proteoglycan-4 (also known as Lubricin) is present in the blood, however the effect of exercise on equine serum levels is unknown. The overall objective of this study was, therefore, to investigate the effect of intense exercise on serum proteoglycan-4 in thoroughbred horses.Methods: Samples of blood were taken from thoroughbreds (n = 12) during a chuckwagon racing event (Alberta, Canada). The chuckwagon race is a sprint racing event where teams of horses pull a combined 1,325 lbs (601 kg) of wagon and driver around a 5/8th mile (1 km) of dirt track, racing at full gallop to the finish. Blood samples were collected 30-min before the race start, and several timepoints post-race: 5-min, 90-min, 3-h, 12-h, and 23-h. Proteoglycan-4 concentrations in serum were quantified by enzyme-linked-immunosorbent-assay using recombinant-human proteoglycan-4 standards and anti-proteoglycan-4 mAb 9G3. The molecular weight of immunoreactive proteoglycan-4 in serum was assessed by western blot.Results: Proteoglyan-4 in serum demonstrated the expected high MW immunoreactivity to mAb 9G3, consistent with that of full length PRG4. Serum proteoglycan-4 decreased five-minutes post-race from baseline concentration (0.815 ± 0.175 to 0.466 ± 0.090 μg/mL, μ ± SEM, p < 0.01).Conclusions: The concentration of serum proteoglycan-4 in horses decreased significantly five min post-exercise. A potential explanation for this finding could be increased proteoglycan-4 clearance from the circulation. Further investigations could extend to complete the detailed characterization of proteoglycan-4 structure and its potential function within the blood as it relates to joint health and exercise.

Abstract Background Gout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo. Methods THP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4 +/+ and Prg4 −/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined. Results rhPRG4 reduced MSU crystal phagocytosis at 4 h (p

Abstract Background Lubricin is chondrocyte-secreted glycoprotein that primarily conducts boundary lubrication between joint surfaces. Besides its cytoprotective function and extracellular matrix (ECM) attachment, lubricin is recommended as a novel biotherapeutic protein that restore functional articular cartilage. Likewise, malfunction of lubrication in damaged articular cartilage caused by complex and multifaceted matter is a major concern in the field of cartilage tissue engineering. Main body Although a noticeable progress has been made toward cartilage tissue regeneration through numerous approaches such as autologous chondrocyte implantation, osteochondral grafts, and microfracture technique, the functionality of engineered cartilage is a challenge for complete reconstruction of cartilage. Thus, delicate modulation of lubricin along with cell/scaffold application will expand the research on cartilage tissue engineering. Conclusion In this review, we will discuss the empirical analysis of lubricin from fundamental interpretation to the practical design of gene expression regulation.

Graphene oxide (GO) and other 2D-nanosheet materials exhibit a range of useful physical, chemical, electrical, and optical properties that may usher in the next generation of biomedical, bioimaging, or sensing technologies. One limitation of GO is its poor stability in concentrated electrolytes and other complex fluids that requires steric stabilizers, including surface grafted polymers, to overcome attractive van der Waals interactions. Here, we describe a simple, rapid, and highly effective method of modifying GO and other 2D-nanosheets with thick, grafted (bio)polymer brushes via the solution self-assembly of lubricin (LUB; a.k.a. PRG4), an antiadhesive glycoprotein. Atomic force microscopy (AFM) imaging and force measurements were used to characterize the morphology and nanomechanical response of these LUB–GO, 2D-nanosheet complexes (2D-NSC). These characterization studies reveal a strong correlation between the GO surface area and the thickness (i.e., molecular extension) of the grafted LUB brush caused by edge free volume effects. Likewise, this edge free volume influences the extension of the LUB brush structure more than 300 nm away from the edge, resulting in a transition region of increasing brush extension before reaching a fully extended state within the central regions of the 2D-NSC. Fitting AFM normal force measurements using an adapted Alexander–de Gennes polymer brush model also indicate that the edge free volume leads to a mechanical softening of the LUB brush due to the lateral spreading and/or deflection of LUB molecules under compression. Finally, the stability studies of 2D-NSCs dispersed in concentrated electrolyte solutions demonstrate the effectiveness of the grafted LUB brushes at inhibiting aggregation even in the harshest environments. 2D-NSCs thus represent a simple solution to modifying nanosheets with thick, multifunctional brushes with promising application in biosensing, bioimaging, catalysis, and biolubrication, where nanosheets must perform in concentrated electrolytes or complex fluids. [ABSTRACT FROM AUTHOR]

Proteoglycan 4 (PRG4; lubricin) is a member of two gene co-expression network modules associated with human vein graft failure. However, little is known about PRG4 and the vascular system. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation in human veins. Effects of rhPRG4 on cell migration, proliferation, and neointima formation were determined in human venous tissue and cultured venous smooth muscle cells (SMCs), adventitial cells, and endothelial cells. Expression of PRG4 by cultured human saphenous veins, failed vein grafts, and varicose veins was determined by immunostaining or Western blotting. Limited expression of PRG4 in fresh saphenous veins was dramatically increased around medial SMCs after culture ex vivo. rhPRG4 inhibited the migration of cultured SMCs, adventitial cells, and endothelial cells, as well as the proliferation of endothelial cells. rhPRG4 also inhibited the migration of SMCs and adventitial cells from tissue explants, but there was no effect on cell proliferation or neointima formation in ex vivo whole veins. Finally, PRG4 was largely absent in two examples of venous pathology, that is, failed human vein grafts and varicose veins. Although rhPRG4 can inhibit the migration of venous SMCs, endothelial cells, and adventitial cells, and the proliferation of endothelial cells, PRG4 was only increased around medial SMCs in veins after ex vivo culture. PRG4 was not observed around medial SMCs in failed human vein grafts and varicose veins, suggesting the possibility that a failure of PRG4 upregulation may promote these pathologies. [ABSTRACT FROM AUTHOR]

The electrochemical sensing of clonazepam in samples of unprocessed saliva is reported, using commercially available reduced graphene oxide (rGO) screen printed electrodes (SPEs). The rGO electrodes were modified with self‐assembled lubricin (LUB) antiadhesive coatings that function as a size‐selective molecular sieve that partitions small molecule analytes from large fouling molecules at the electrode‐fluid interface. By mitigating the adverse effects of protein fouling, these LUB coatings enable direct, reliable, and quantitative electrochemical measurements of clonazepam concentrations in untreated saliva, which was not possible with uncoated rGO electrodes in identical tests. The proposed assay design, which is suitable for disposable point‐of‐care diagnostics requires no sample pre‐treatment and shows a linear detection response over a concentration range of 25–250 nM with a correlation coefficient of R2=0.987 and a limit of detection (LoD) of 16.67 nM. The swab‐based sample acquisition method paired with the antifouling properties of the LUB coatings provide a cost effective and practical biosensing platform for assessing electroactive drug concentrations in saliva in a point‐of‐care setting. [ABSTRACT FROM AUTHOR]

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

Introduction: Osteoarthritis (OA) is a degenerative disease which primarily affects hyaline cartilage, leading to pain, stiffness and loss of mobility of the entire articulation. Diagnosis is commonly based on symptoms and radiographs, but there is a growing interest in detecting novel biomarkers, in serum, urine and synovial fluid, which can be predictors of disease onset and progression. Areas covered: This review provides an overview of the main biomarkers currently used in OA clinical practice, with a focus on lubricin, a surface glycoprotein secreted in the synovial fluid that lubricates the cartilage and reduces the coefficient of friction within the joint. Key findings of the last years are presented throughout the article. Expert opinion: Analysis of biomarkers might suggest personalized protocols of treatment, guide the classification of OA phenotypes, contribute to precision medicine, avoid further unnecessary exams, facilitate drug discovery or refine treatment guidelines. For all these reasons, the investigation of novel cartilage-based biomarker of osteoarthritis needs to be promoted and improved. [ABSTRACT FROM AUTHOR]

Background: Lubricin, a mucinous glycoprotein, plays a chondroprotective role as a constituent of synovial fluid. Structural analogs have been synthesized to mimic the structure and function of native lubricin in an effort to recapitulate this effect with the goal of delaying progression of osteoarthritis (OA). Purpose: To investigate the efficacy of intra-articular injections of lubricin mimetics in slowing or preventing the progression of posttraumatic OA by using a rat anterior cruciate ligament transection model. Study Design: Controlled laboratory design. Methods: Four lubricin mimetics were investigated, differing from one another in their binding orientations and steric interactions. Eighty skeletally mature Sprague-Dawley rats underwent bilateral anterior cruciate ligament transections and were randomly allocated to receive intra-articular injections (50 µL/injection) of 1 of the 4 mimetics in the right knee and equal volumes of saline injection in the contralateral knee (control). All rats were euthanized 8 weeks postoperatively and assessed via biomechanical analysis, which evaluated comparative friction coefficients across the 4 groups, and histological evaluation of articular cartilage, osteophytes, and synovitis. The Osteoarthritis Research Society International (OARSI) histopathological assessment system was used to evaluate the degree of articular cartilage degeneration and osteophytes, while synovitis was assessed through a semiquantitative scoring system. Binding efficacy of the 4 mimetics was assessed in vitro and in vivo through the immunohistochemical localization of polyethylene glycol. Articular cartilage degeneration and synovitis scoring data analyses were performed with generalized estimating equation modeling. Results: Injection of the group 3 mimetic (random 24 + 400 + 30) directly correlated with improved OARSI scores for femoral articular cartilage degeneration when compared with saline-injected contralateral control knees (P =.0410). No lubricin mimetic group demonstrated statistically significant differences in OARSI scores for tibial articular cartilage degeneration. Injection of the group 4 mimetic (AB 24 + 400 + 30) led to a statistically significant difference in osteophyte OARSI score (P =.0019). None of the 4 lubricin mimetics injections incited an additive synovial inflammatory response. Immunohistochemical staining substantiated the binding capacity of all 4 mimetics, while in vivo experimentation revealed that the group 1 and 3 mimetics were still retained within the joint 4 weeks after injection. There were no differences in friction coefficients between any pair of groups and no significant trends based on lubricin mimetic structure. Conclusion: We demonstrated that the tribosupplementation of a traumatically injured knee with a specific lubricin structural analog may attenuate the natural progression of OA. Clinical Relevance: The current lack of efficacious clinical options to counter the onset and subsequent development of OA suggests that further investigation into the synthesis and behavior of lubricin analogs could yield novel translational applications. [ABSTRACT FROM AUTHOR]

Advanced biomaterials and tissue engineered constructs have been developed to improve tissue repair; nevertheless, their clinical translation has been hampered, also by the lack of reliable in vitro models suitable for preclinical screening of new implants and compounds mimicking the in vivo situation. Tissue regeneration is strongly influence by the mechanical properties and behavior of biomaterials, which can be completely different when tested in "isolation" or in a biological context. Therefore, it is important to evaluate the performance of such advance biomaterials in in vitro models, which reproduce closely the in vivo tissue status. To such end, we have developed several complex organ models (here, cartilage) which include, not only the tissue part, but the tissue is cultured within a bioreactor, reproducing loading patterns similar to the in vivo microenvironment. Here, we will focus on bioreactor systems that transmit a mechanical stimulus, as this is a key parameter in the homeostasis of various musculoskeletal tissues, such as bone, cartilage, tendon, and intervertebral disc. By testing regenerative therapies under conditions that are closer to the ones encountered in vivo, bioreactors can provide a useful screening tool and standardization opportunities for the evaluation of various biomaterials, but as well as cell types, drugs, or tissue engineered products. This will allow to reduce the number of samples for the final in vivo evaluation, allowing the 3R philosophy approach to be implemented. [ABSTRACT FROM AUTHOR]

Cartilage demineralisation in Osteoarthritis (OA) patients can elevate calcium ion levels in synovial fluid, as evidenced by the prevalence of precipitated calcium phosphate crystals in OA synovial fluid. Although it has been reported that there is a potential connection between elevated concentrations of calcium ions and a deterioration in the lubrication and wear resistance of cartilage tissues, the mechanism behind the strong link between calcium ion concentration and decreased lubrication performance is unclear. In this work, the AFM friction, imaging, and normal force distance measurements were used to investigate the lubrication performances of hyaluronic acid (HA), Lubricin (LUB), and HA-LUB complex in the presence of calcium ions (5 mM, 15 mM, and 30 mM), to understand the possible mechanism behind the change of lubrication property. The results of AFM friction measurements suggest that introducing calcium ions to the environment effectively eliminated the lubrication ability of HA and HA-LUB, especially with relatively low loading applied. The AFM images indicate that it is unlikely that structural or morphological changes in the surface-bound layer upon calcium ions addition are primarily responsible for the friction results demonstrated. Further, the poor correlation between the effect of calcium ions on the adhesion forces and its impact on friction suggests that the decrease in the lubricating ability of both layers is likely a result of changes in the hydration of the HA-LUB surface bound layers than changes in intermolecular or intramolecular binding. This work provides the first experimental evidence lending towards the relationship between bone demineralisation and articular cartilage degradation at the onset of OA and the mechanism through which elevated calcium levels in the synovial fluid act on joint lubrication. [Display omitted] • Introducing Ca2+ significantly impacts HA and HA-LUB complex's lubrication properties. • Ca2+ directly reduced the degree of hydration within the HA and LUB layers. • Experiments associated bone demineralisation and articular cartilage degradation. [ABSTRACT FROM AUTHOR]

Abstract Background Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions. Methods Cytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4 +/+ and Prg4 -/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50–200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours. Results Prg4 -/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4 +/+ synoviocytes (p

The camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) is a rare autosomal recessive disease characterized by congenital or early-onset camptodactyly, childhood-onset non-inflammatory arthropathy associated with synovial hyperplasia, constrictive pericarditis and coxa vara. This rare disease is caused by mutations in PRG4 Gene which encodes for lubricin, a protein present in synovial fluid responsible for lubrication. The case we are reporting is of 9 and half years old boy who presented to us in pediatric Out Patient Department with early onset camptodactyly and non-inflammatory arthropathy. Synovial biopsy shows synovial hyperplasia with numerous CD68 positive multinucleated giant cells. He was discharged and physiotherapy was advised with regular follow ups.