Synovial mesenchymal stem cells secrete more lubricin than adipose mesenchymal stem cells after injection into rat osteoarthritis knees.

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.

Prg4-Expressing Chondroprogenitor Cells in the Superficial Zone of Articular Cartilage.

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.

Recombinant manufacturing of multispecies biolubricants.

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.

Dissolvable Immunomodulatory Microneedles for Treatment of Skin Wounds.

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.

Calcium ions have a detrimental impact on the boundary lubrication property of hyaluronic acid and lubricin (PRG-4) both alone and in combination.

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.

The Actin Cytoskeleton as a Regulator of Proteoglycan 4.

OBJECTIVE: The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro. There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies. MATERIALS AND METHODS: A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed. RESULTS: PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro. Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling. CONCLUSION: Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics.

PRG4 deficiency in mice alters skeletal structure, mechanics, and calvarial osteoclastogenesis, and rhPRG4 inhibits in vitro osteoclastogenesis.

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.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Optimizing Bioink Composition for Human Chondrocyte Expression of Lubricin.

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage regeneration.

CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Computational and experimental characterizations of the spatiotemporal activity and functional role of TGF-ß in the synovial joint.

TGF-ß is a prominent anabolic signaling molecule associated with synovial joint health. Recent work has uncovered mechanochemical mechanisms that activate the latent form of TGF-ß (LTGF-ß) in the synovial joint-synovial fluid (SF) shearing or cartilage compression-pointing to mechanobiological phenomena, whereby enhanced TGF-ß activity occurs during joint stimulation. Here, we implement computational and experimental models to better understand the role of mechanochemical-activated TGF-ß (aTGF-ß) in regulating the functional biosynthetic activities of synovial joint tissues. Reaction-diffusion models describe the pronounced role of extracellular chemical reactions-load-induced activation, reversible ECM-binding, and cell-mediated internalization-in modulating the spatiotemporal distribution of aTGF-ß in joint tissues. Of note, aTGF-ß from SF shearing predominantly acts on cells in peripheral tissue regions (superficial zone [SZ] chondrocytes and synoviocytes) and aTGF-ß from cartilage compression acts on chondrocytes through all cartilage layers. Further, ECM reversible binding sites in cartilage act to modulate the temporal delivery of aTGF-ß to cells, creating a dynamic where short durations of joint activity give rise to extended periods of aTGF-ß exposure at moderated doses. Ex vivo tissue models were subsequently utilized to characterize the influence of physiologic aTGF-ß activity regimens in regulating functional biosynthetic activities. Physiologic exposure regimens of aTGF-ß in SF induce strong 4-fold to 9-fold enhancements in the secretion rate of the synovial biolubricant, PRG4, from SZ cartilage and synovium explants. Further, aTGF-ß inhibition in cartilage over 1-month culture leads to a pronounced loss of GAG content (30-35% decrease) and tissue softening (60-65% EY reduction). Overall, this work advances a novel perspective on the regulation of TGF-ß in the synovial joint and its role in maintaining synovial joint health.

Advanced bioactive glue tethering Lubricin/PRG4 to promote integrated healing of avascular meniscus tears.

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 (TGFß3) 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.

Biological injection therapy with leukocyte-poor platelet-rich plasma induces cellular alterations, enhancement of lubricin, and inflammatory downregulation in vivo in human knees: A controlled, prospective human clinical trial based on mass spectrometry imaging analysis.

Objective: To investigate the in vivo biological effects of leukocyte-poor platelet-rich plasma (LpPRP) treatment in human synovial layer to establish the cellular basis for a prolonged clinical improvement. Methods: Synovial tissues (n = 367) were prospectively collected from patients undergoing arthroscopic surgery. Autologous-conditioned plasma, LpPRP, was injected into the knees of 163 patients 1-7 days before surgery to reduce operative trauma and inflammation, and to induce the onset of regeneration. A total of 204 patients did not receive any injection. All samples were analyzed by mass spectrometry imaging. Data analysis was evaluated by clustering, classification, and investigation of predictive peptides. Peptide identification was done by tandem mass spectrometry and database matching. Results: Data analysis revealed two major clusters belonging to LpPRP-treated (LpPRP-1) and untreated (LpPRP-0) patients. Classification analysis showed a discrimination accuracy of 82%-90%. We identified discriminating peptides for CD45 and CD29 receptors (receptor-type tyrosine-protein phosphatase C and integrin beta 1), indicating an enhancement of musculoskeletal stem cells, as well as an enhancement of lubricin, collagen alpha-1-(I) chain, and interleukin-receptor-17-E, dampening the inflammatory reaction in the LpPRP-1 group following LpPRP injection. Conclusions: We could demonstrate for the first time that injection therapy using "autologic-conditioned biologics" may lead to cellular changes in the synovial membrane that might explain the reported prolonged beneficial clinical effects. Here, we show in vivo cellular changes, possibly based on muscular skeletal stem cell alterations, in the synovial layer. The gliding capacities of joints might be improved by enhancing of lubricin, anti-inflammation by activation of interleukin-17 receptor E, and reduction of the inflammatory process by blocking interleukin-17.

Flexor Tendon Adhesion Formation: Current Concepts.

Over the years, various physical and chemical/biological methods of inhibiting adhesion formation have been developed, focusing on how to suppress healing around the tendon and not inhibit healing within the tendon. Unfortunately, however, these methods are accompanied by drawbacks, both large and small, and no absolute antiadhesion method capable of maintaining tendon repair strength has yet been developed. Recent innovations in biomaterials science and tissue engineering have produced new antiadhesion technologies, such as barriers combined with cytokines and cells, which have improved outcomes in animal models, and which may find clinical relevance in the future.

The Role of Lubricin, Irisin and Exercise in the Prevention and Treatment of Osteoarthritis.

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.

Friction reducing ability of a poly-l-lysine and dopamine modified hyaluronan coating for polycaprolactone cartilage resurfacing implants.

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.

Rapid Point-of-Care Electrochemical Sensor for the Detection of Cancer Tn Antigen Carbohydrate in Whole Unprocessed Blood.

Improving outcomes for cancer patients during treatment and monitoring for cancer recurrence requires personalized care which can only be achieved through regular surveillance for biomarkers. Unfortunately, routine detection for blood-based biomarkers is cost-prohibitive using currently specialized laboratories. Using a rapid self-assembly sensing interface amenable to methods of mass production, we demonstrate the ability to detect and quantify a small carbohydrate-based cancer biomarker, Tn antigen (αGalNAc-Ser/Thr) in a small volume of blood, using a test format strip reminiscent of a blood glucose test. The detection of Tn antigen at picomolar levels is achieved through a new transduction mechanism based on the impact of Tn antigen interactions on the molecular dynamic motion of a lectin cross-linked lubricin antifouling brush. In tests performed on retrospective blood plasma samples from patients presenting three different tumor types, differentiation between healthy and diseased patients was achieved, highlighting the clinical potential for cancer monitoring.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

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.

Noncovalent hyaluronan crosslinking by TSG-6: Modulation by heparin, heparan sulfate, and PRG4.

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.

Recombinant lubricin improves anti-adhesive, wear protection, and lubrication of collagen II surface.

Camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) is a joint disease caused by a lack of lubricin, resulting in failed lubrication and abnormal deposition at the cartilage surface. Injection of recombinant lubricin (R-LUB) is a promising approach to improve the symptoms of the disease. However, the antifouling and lubrication properties of R-LUB on cartilage surfaces have not yet been studied. Here, the adsorption and lubrication behavior of a type II collagen (COL II) mimicking the cartilage surface upon R-LUB injection was followed by surface plasmon resonance spectroscopy and surface forces apparatus. The results indicated R-LUB can bind well on a COL II surface and COL II/R-LUB complex layer exhibited ultralow nonspecific adsorption of BSA (3.25 ng/cm2) and LYS (0.26 ng/cm2) compared to those of the COL II layer (32.7 ng/cm2, 7.26 ng/cm2). Normal force measurements indicated there was always a repulsive force between COL II/R-LUB complex and different surfaces with -COO-, -NH3+, and -CH3 groups. Likewise, COL II had a high coefficient of friction (∼0.48) with surface damage at 2 µm/s and a wear pressure of 1.56 MPa, while that of COL II/R-LUB complex was down to ∼0.008-0.13 with surface damage at 13 µm/s and a wear pressure of 11.96 MPa, which was 7.7 times higher than for COL II. Hence, R-LUB may act as an anti-adhesive and lubrication layer adsorbed on COL II surfaces to prevent direct contact. Our findings provide fundamental insights into the adsorption and lubrication behavior for understanding biological lubrication, especially the potential supplementation of R-LUB for treating CACP disease.