EFFECT OF COUNTERFACE ON CARTILAGE BOUNDARY LUBRICATING ABILITY BY HYALURONAN AND PROTEOGLYCAN 4: CARTILAGE-CARTILAGE VS CARTILAGE-GLASS

INTRODUCTION Proteoglycan 4 (PRG4), also known as lubricin, is a mucin-like glycoprotein found in synovial fluid (SF) [1]. Hyaluronan (HA) is another constituent of SF that works synergistically with PRG4 to promote joint lubrication required for joint health [2]. Current in vitro friction tests used to analyze these lubricants have found similar trends but varying friction coefficient (m) magnitudes. These effects may be attributed to different testing protocols, and in particular the various interfaces [3,4,5,6,7,8]. Previous studies have used cartilage-cartilage [7,8] and cartilage-glass [6] friction tests to assess SF lubricants. However, few studies have examined the friction reducing ability of SF lubricants at various velocities and different interfaces. The objective of this study was to determine HA and PRG4’s lubricating ability at cartilage-glass and cartilage-cartilage biointerfaces at various velocities.   METHODS HA (1.5 MDa, Lifecore Biomedical) was prepared at 3.3 mg/mL with phosphate buffered saline (PBS) [9]. PRG4 was purified from media conditioned bovine cartilage explants, and prepared at 450 μg/mL in PBS [9]. Two sets of tests were conducted using a modified boundary lubrication test protocol [7]. For cartilage-glass, a 6mm radius glass piece, with a root mean square surface roughness of 6.061±0.7554 nm, acted as the base core with a cartilage annuli [7]. Three lubricants were tested over three days, PBS (n=8), HA or PRG4 (n=4), and SF (n=8). Cartilage-cartilage underwent the same test sequence. Both sets of samples underwent the same testing protocol; samples were compressed to 18% of the total cartilage thickness with a 40 minute stress relaxation [9]. Samples were then rotated at effective sliding velocities of 10, 3, 1, 0.3, 0.1, and 0.01 mm/s with a 120s pre-sliding duration. Kinetic μ was calculated with instantaneous (<μkinetic>) load values. A two-factor ANOVA was used to determine effects of lubricant and velocity, with Tukey post-hoc testing. RESULTS At the cartilage-glass interface (Fig. 1A), <μkinetic> varied with test lubricant and velocity (p<0.001), with no interaction (p=0.78). All lubricants were significantly different from each other (p<0.001), except for PBS/HA (p=0.65) and PRG4/SF (p=0.72). Velocities were significantly different from each other when comparing 0.01 mm/s to all other speeds (p<0.02). At the cartilage-cartilage interface (Fig. 1B), <μkinetic> varied with test lubricant and velocity (p<0.01), with no interaction (p=0.82). All lubricants were significantly different from each other (p<0.01), except for HA/SF (p=0.07) and HA/PRG4 (p=0.35). Velocity was only significantly different at 0.01mm/s when compared to 3 and 10 mm/s (p<0.028). DISCUSSION AND CONCLUSIONS These results indicate that different articulating interfaces can result in different trends of <μkinetic>, and also affect the magnitude of the value. This agrees with previous research; showing that stiff and impermeable surfaces versus hydrated and permeable surfaces results in different <μkinetic> [3]. This data illustrates that HA is indeed a cartilage boundary lubricant and reduces friction at the cartilage-cartilage interface, but not at a cartilage-glass set up. Overall, different test systems are suitable for characterizing lubrication properties, but direct <μkinetic> values should not be compared

CARTILAGE BOUNDARY LUBRICATING ABILITY OF PRG4 (LUBRICIN) MONOMERS AND MULTIMERS WITH HYALURONAN

INTRODUCTION Proteoglycan 4 (PRG4), or lubricin, is a glycoprotein that exists in synovial fluid (SF) as monomeric and disulfide-bonded multimeric forms [1,2]. PRG4 functions as a critical boundary lubricant on the surface of articular cartilage [1,3]. Hyaluronan (HA) is another lubricant that lubricates at a cartilage-cartilage interface alone, and synergistically with PRG4 to reduce friction levels near that of SF [4]. Previously PRG4-multimers (PRG4-multi) have been shown to lubricate considerably better than PRG4 monomers (PRG4-mono) using an in vitro cartilage-on-cartilage friction testing system [5]. However, it is unknown if the difference in structure of PRG4-multi/-mono affects the synergism with HA. Therefore, the objective of this study was to evaluate the cartilage boundary lubricating ability of PRG4-multi & PRG4-mono with HA.   METHODS Samples: PRG4 was purified from media conditioned by mature bovine cartilage explants [4]. PRG4 multi-mono was prepared via size exclusion chromatography [5]. HA (1.5MDa) was obtained from Lifecore Biomedical. Cartilage boundary lubrication: Lubricants of interest were assessed using a previously described in vitro cartilage-cartilage friction test [6]. Briefly, bovine osteochondral samples (n=8) were incubated with test lubricants overnight, compressed by 18%, allowed to stress relax then rotated at an effective velocity of 0.3 mm/s with pre-sliding durations (Tps) of 1200, 120, 12, and 1.2s. Phosphate buffered saline (PBS) and bovine SF served as negative and positive controls, respectively. All samples were prepared at a physiological concentration [4]; PRG4 at 450 μg/mL and HA at 3.33 mg/mL, in PBS. Test sequence: PBS, PRG4-mono+HA, PRG4-multi+HA, PRG4+HA, SF. Static (μstatic,Neq) and kinetic (<μkinetic,Neq>) friction coefficients were calculated [4,6]. ANOVA was used to assess the effect of lubricant and Tps, as a repeated factor, on μstatic,Neq and <μkinetic,Neq>, with Tukey post-hoc on <μkinetic,Neq> at Tps=1.2s. RESULTS Cartilage boundary lubrication: PRG4-mono/-multi+HA, functioned as effective friction-reducing cartilage boundary lubricants. However, they did not lubricate as well as PRG4+HA or SF. μstatic,Neq values increased with Tps; they were highest in PBS and lowest in SF, with PRG4-mono+HA, PRG4-multi+HA, and PRG4+HA being intermediate. For clarity, <μkinetic,Neq> values at Tps=1.2s are shown since values at Tps=1.2s were within 13.0±2.7% of those at Tps=1200s. <μkinetic,Neq> values for PRG4-mono+HA and PRG4-multi+HA were similar (p=0.96). PRG4+HA and SF were similar (p=0.53). All other combinations were significantly different from each other (p<0.05). DISCUSSION AND CONCLUSIONS Previous studies have shown that PRG4-multi lubricates better than PRG4-mono [5]. However the results from this study show that PRG4-mono/-multi with HA lubricate in a similar manner. Interestingly, PRG4+HA lubricates better than PRG4-mono/-multi+HA, and close to that of SF. These differences could be due to a potential PRG4+HA “link molecule” [7] being purified from the PRG4-mono/-multi preparations

The Autocrine Role of Proteoglycan-4 (PRG4) in Modulating Osteoarthritic Synoviocyte Proliferation and Expression of Matrix Degrading Enzymes

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 \u3c 0. 05). rhPRG4 (100 μg/ml) reduced p50 and p65 nuclear levels in Prg4+/+ and Prg4-/- synoviocytes (p \u3c 0.001). Similarly, rhPRG4 (200 μg/ml) inhibited NFκB translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p \u3c 0.001) via inhibition of IκBα phosphorylation. IL-1β reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 μg/ml) treatment reversed this effect (p \u3c 0.001). rhPRG4 (200 μg/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p \u3c 0.001). rhPRG4 (200 μg/ml) reduced IL-1β induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p \u3c 0.001). Conclusion: PRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1β-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes

Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1β Secretion by Urate Crystal Stimulated Gout PBMCs

Objectives To compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1β secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes. Methods Acute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1μg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200μg/mL) stimulated IL-1β secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1β, secreted IL-1β, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed. Results Enhanced phagocytic activation of gout monocytes under basal conditions (p\u3c0.001) was associated with ROS generation and MSU stimulated IL-1β secretion (p\u3c0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (p\u3c0.05). rhPRG4 reduced pro-IL-1β content, caspase-1 activity, conversion of pro-IL-1β to mature IL-1β and restored PP2A activity in monocytes (p\u3c0.05). PP2A inhibition reversed rhPRG4’s effects on pro-IL-1β and mature IL-1β in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (p\u3c0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p\u3c0.05). Conclusions MSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1β secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1β secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare

Correction to: Proteoglycan-4 is an Essential Regulator of Synovial Macrophage Polarization and Inflammatory Macrophage Joint Infiltration

Correction to: Arthritis Res Ther 23, 241 (2021) https://doi.org/10.1186/s13075-021-02621-

Proteoglycan-4 is an Essential Regulator of Synovial Macrophage Polarization and Inflammatory Macrophage Joint Infiltration

Background Synovial macrophages perform a multitude of functions that include clearance of cell debris and foreign bodies, tissue immune surveillance, and resolution of inflammation. The functional diversity of macrophages is enabled by distinct subpopulations that express unique surface markers. Proteoglycan-4 (PRG4) is an important regulator of synovial hyperplasia and fibrotic remodeling, and the involvement of macrophages in PRG4’s synovial role is yet to be defined. Our objectives were to study the PRG4’s importance to macrophage homeostatic regulation in the synovium and infiltration of pro-inflammatory macrophages in acute synovitis and investigate whether macrophages mediated synovial fibrosis in Prg4 gene-trap (Prg4GT/GT) murine knee joints. Methods Macrophage phenotyping in Prg4GT/GT and Prg4+/+ joints was performed by flow cytometry using pan-macrophage markers, e.g., CD11b, F4/80, and surface markers of M1 macrophages (CD86) and M2 macrophages (CD206). Characterizations of the various macrophage subpopulations were performed in 2- and 6-month-old animals. The expression of inflammatory markers, IL-6, and iNOS in macrophages that are CD86+ and/or CD206+ was studied. The impact of Prg4 recombination on synovial macrophage populations of 2- and 6-month-old animals and infiltration of pro-inflammatory macrophages in response to a TLR2 agonist challenge was determined. Macrophages were depleted using liposomal clodronate and synovial membrane thickness, and the expression of fibrotic markers α-SMA, PLOD2, and collagen type I (COL-I) was assessed using immunohistochemistry. Results Total macrophages in Prg4GT/GT joints were higher than Prg4+/+ joints (p\u3c0.0001) at 2 and 6 months, and the percentages of CD86+/CD206− and CD86+/CD206+ macrophages increased in Prg4GT/GT joints at 6 months (p\u3c0.0001), whereas the percentage of CD86−/CD206+ macrophages decreased (p\u3c0.001). CD86+/CD206− and CD86+/CD206+ macrophages expressed iNOS and IL-6 compared to CD86−/CD206+ macrophages (p\u3c0.0001). Prg4 re-expression limited the accumulation of CD86+ macrophages (p\u3c0.05) and increased CD86−/CD206+ macrophages (p\u3c0.001) at 6 months. Prg4 recombination attenuated synovial recruitment of pro-inflammatory macrophages in 2-month-old animals (p\u3c0.001). Clodronate-mediated macrophage depletion reduced synovial hyperplasia, α-SMA, PLOD2, and COL-I expressions in the synovium (p\u3c0.0001). Conclusions PRG4 regulates the accumulation and homeostatic balance of macrophages in the synovium. In its absence, the synovium becomes populated with M1 macrophages. Furthermore, macrophages exert an effector role in synovial fibrosis in Prg4GT/GT animals

In vivo printing of growth factor-eluting adhesive scaffolds improves wound healing

Acute and chronic wounds affect millions of people around the world, imposing a growing financial burden on patients and hospitals. Despite the application of current wound management strategies, the physiological healing process is disrupted in many cases, resulting in impaired wound healing. Therefore, more efficient and easy-to-use treatment modalities are needed. In this study, we demonstrate the benefit of in vivo printed, growth factor-eluting adhesive scaffolds for the treatment of full-thickness wounds in a porcine model. A custom-made handheld printer is implemented to finely print gelatin-methacryloyl (GelMA) hydrogel containing vascular endothelial growth factor (VEGF) into the wounds. In vitro and in vivo results show that the in situ GelMA crosslinking induces a strong scaffold adhesion and enables printing on curved surfaces of wet tissues, without the need for any sutures. The scaffold is further shown to offer a sustained release of VEGF, enhancing the migration of endothelial cells in vitro. Histological analyses demonstrate that the administration of the VEGF-eluting GelMA scaffolds that remain adherent to the wound bed significantly improves the quality of healing in porcine wounds. The introduced in vivo printing strategy for wound healing applications is translational and convenient to use in any place, such as an operating room, and does not require expensive bioprinters or imaging modalities

A Two Compartment Pharmacokinetic Model Describes the Intra‐articular Delivery and Retention of rhPRG4 Following ACL Transection in the Yucatan Mini Pig

Treatment of the injured joint with rhPRG4 is based on recent observations that inflammation diminishes expression of native PRG4. Re‐establishing lubrication between pressurized and sliding cartilage surfaces during locomotion promotes the nascent expression of PRG4 and thus intra‐articular (IA) treatment strategies should be supported by pharmacokinetic evidence establishing the residence time of rhPRG4. A total of 21 Yucatan minipigs weighing ∼55 Kg each received 4 mg of 131I‐rhPRG4 delivered by IA injection 5 days following surgical ACL transection. Animals were sequentially euthanized following IA rhPRG4 at 10 mins (time zero), 24, 72 hrs, 6, 13 and 20 days later. The decay of the 131I‐rhPRG4 was measured relative to a non‐injected aliquot and normalized to the weight of cartilage samples, menisci and synovium, and known cartilage volumes from each compartment surface obtained from representative Yucatan minipig knees. Decay of 131I‐rhPRG4 from joint tissues best fit a two‐compartment model with an α half‐life (t1/2α) of 11.28 hours and β half‐life (t1/2β) of 4.81 days. The tibial and femoral cartilage, meniscii and synovium retained 7.7% of dose at 24 hrs. High concentrations of rhPRG4 were found in synovial fluid (SF) that was non‐aspiratable and resided on the articular surfaces, removable by irrigation, at 10 mins following 131I‐rhPRG4 injection. Synovial fluid K21 exceeded K12 and SF t1/2β was 28 days indicating SF is the reservoir for rhPRG4 following IA injection. Clinical Significance: rhPRG4 following IA delivery in a traumatized joint populates articular surfaces for a considerable period and may promote the native expression of PRG4

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in \u3cem\u3ePrg4\u3c/em\u3e Null Mice

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

The TFOS international workshop on contact lens discomfort: report of the contact lens materials, design, and care subcommittee

Jones, L., Brennan, N. A., González-Méijome, J., Lally, J., Maldonado-Codina, C., Schmidt, T. A., … Nichols, J. J. (2013). The TFOS International Workshop on Contact Lens Discomfort: Report of the Contact Lens Materials, Design, and Care Subcommittee. Investigative Opthalmology & Visual Science, 54(11), TFOS37. https://doi.org/10.1167/iovs.13-13215Examining the role of the contact lens material, design, and the care system is fundamental to understanding contact lens discomfort (CLD). However, a systematic review that tries to determine the governing factors is fraught with difficulties. A lack of a validated “instrument” (or single validated questionnaire) for measuring discomfort makes it impossible to compare between studies because reported levels of comfort (or discomfort) are inconsistent. Subject classifications can vary widely, from studies that include only neophytes or asymptomatic contact lens (CL) wearers to studies including only those contact lens–wearing subjects who experience marked dryness or symptoms of discomfort. Also, it is difficult to measure issues of importance in isolation because changing one factor in a contact lens or care solution can invariably affect another. An illustration of this relates to a change in hydrogel water content, which also affects oxygen permeability, oxygen transmissibility, modulus, and possibly lens thickness. Finally, various confounding factors between studies also make true comparisons problematic. Typical examples would include differences between brands of lenses made from the same material (which may have differing geometric designs, edge configuration, or production methods); wearing modality (lenses may be worn on a daily wear [DW] basis, overnight occasionally, or for up to 30 nights on a continuous wear [CW] basis); duration of use prior to replacement, wearing time during the day (from just a few hours to most of the day); and care product differences or exposures (which could range from no exposure in the case of daily disposable [DD] materials to a preserved system that has extensive uptake and release from the contact lens material being examined). The purpose of this report is to summarize evidence-linking associations, mechanistic and etiological factors between contact lens materials, designs, and care solutions with CLD. The potential factors associated with this are many and varied, and graphically display the complexity of this issue