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.

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.

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).

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.

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.

Active and passive drug release by self-assembled lubricin (PRG4) anti-fouling coatings.

Electroactive polymers (EAPs) have been investigated as materials for use in a range of biomedical applications, ranging from cell culture, electrical stimulation of cultured cells as well as controlled delivery of growth factors and drugs. Despite their excellent drug delivery ability, EAPs are susceptible to biofouling thus they often require surface functionalisation with antifouling coatings to limit unwanted non-specific protein adsorption. Here we demonstrate the surface modification of para toluene sulfonate (pTS) doped polypyrrole with the glycoprotein lubricin (LUB) to produce a self-assembled coating that both prevents surface biofouling while also serving as a high-capacity reservoir for cationic drugs which can then be released passively via diffusion or actively via an applied electrical potential. We carried out our investigation in two parts where we initially assessed the antifouling and cationic drug delivery ability of LUB tethered on a gold surface using quartz crystal microbalance with dissipation monitoring (QCM) to monitor molecular interactions occurring on a gold sensor surface. After confirming the ability of tethered LUB nano brush layers on a gold surface, we introduced an electrochemically grown EAP layer to act as the immobilisation surface for LUB before subsequently introducing the cationic drug doxorubicin hydrochloride (DOX). The release of cationic drug was then investigated under passive and electrochemically stimulated conditions. High-performance liquid chromatography (HPLC) was then carried out to quantify the amount of DOX released. It was shown that the amount of DOX released from nano brush layers of LUB tethered on gold and EAP surfaces could be increased by up to 30% per minute by applying a positive electrochemically stimulating pulse at 0.8 V for one minute. Using bovine serum albumin (BSA), we show that DOX loaded LUB tethered on para toluene sulfonic acid (pTS) doped polypyrrole retained antifouling ability of up to 75% when compared to unloaded tethered LUB. This work demonstrates the unique, novel ability of tethered LUB to actively participate in the delivery of cationic therapeutics on different substrate surfaces. This study could lead to the development of versatile multifunctional biomaterials for use in wide range of biomedical applications, such as dual drug delivery and lubricating coatings, dual drug delivery and antifouling coatings, cellular recording and stimulation.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants.

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-ß 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-ß 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-ß 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.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

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.

Lubricin-Inspired Loop Zwitterionic Peptide for Fabrication of Superior Antifouling Surfaces.

Biofouling represents great challenges in many applications, and zwitterionic peptides have been a promising candidate due to their biocompatibility and excellent antifouling performance. Inspired by lubricin, we designed a loop-like zwitterionic peptide and investigated the effect of conformation (linear or loop) on the antifouling properties using a combination of surface plasma resonance (SPR), surface force apparatus (SFA), and all atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that the loop-like zwitterionic peptides perform better in resisting the adsorption of proteins and bacteria. SFA measurements show that the loop-like peptides reduce the adhesion between the modified surface and the modeling foulant lysozyme. All atomistic MD simulations reveal that the loop-like zwitterionic peptides are more rigid than the linear-like zwitterionic peptides and avoid the penetration of the terminus into the foulants, which lower the interaction between the zwitterionic peptides and foulants. Besides, the loop-like zwitterionic peptides avoid the aggregation of the chains and bind more water, improving the hydrophilicity and antifouling performance. Altogether, this study provides a more comprehensive understanding of the conformation effect of zwitterionic peptides on their antifouling properties, which may contribute to designing novel antifouling materials in various biomedical applications.

Mucin-Like Glycoproteins Modulate Interfacial Properties of a Mimetic Ocular Epithelial Surface.

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.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

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-1ß-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-1ß 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-1ß 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.

The lubricating effect of iPS-reprogrammed fibroblasts on collagen-GAG scaffolds for cartilage repair applications.

Tissue engineering products, like collagen-glycosaminoglycan scaffolds, have been successfully applied to chondrogenic defects. Inducible Pluripotent Stem cell (iPS) technology allows reprograming of somatic cells into an embryonic-like state, allowing for redifferentiation. We postulated that a fibroblast cell line (BJ cells - 'pre-iPSF') cycled through iPS reprogramming and redifferentiated into fibroblasts (post-iPSF) could lubricate collagen-glycosaminoglycan scaffolds; fibroblasts are known to produce lubricating molecules (e.g., lubricin) in the synovium. Herein, we quantified the coefficient of friction (CoF) of collagen-glycosaminoglycan scaffolds seeded with post-iPSF; tested whether cell-free scaffolds made of post-iPSF derived extracellular matrix had reduced friction vs. pre-iPSF; and assessed lubricin quantity as a possible protein responsible for lubrication. Post-iPSF seeded CG had 6- to 10-fold lower CoF versus pre-iPSF. Scaffolds consisting of a collagen and pre-/post-iPSF extracellular matrix blend outperformed these cell-seeded scaffolds (~5-fold lower CoF), yielding excellent CoF values close to synovial fluid. Staining revealed an increased presence of lubricin within post-iPSF scaffolds (confirmed by western blotting) and on the surface of iPSF-seeded collagen-glycosaminoglycan scaffolds. Interestingly, when primary cells from patient biopsy-derived fibroblasts were used, iPS reprogramming did not further reduce the already low CoF of these cells and no lubricin expression was found. We conclude that iPS reprogramming activates lubricating properties in iPS-derived cells in a source cell-specific manner. Additionally, lubricin appears to play a lubricating role, yet other proteins also contribute to lubrication. This work constitutes an important step for understanding post-iPSF lubrication of scaffolds and its potential for cartilage tissue engineering.

Lubricin expression in the lumbar endplate and its association with Modic changes.

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 â€‹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- 1ß (IL-1ß) 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-1ß 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.

Lubricin (PRG4) Antiadhesive Coatings Mitigate Electrochemical Impedance Instabilities in Polypyrrole Bionic Electrodes Exposed to Fouling Fluids.

Surface fouling is a major problem faced by bionic implants (e.g., cochlear implants, pacemakers), where the adsorption of unwanted biomolecules has a detrimental effect on interfacial charge transfer processes, which severely impairs their capacity to sense and transmit electrical signals with high fidelity. Polypyrrole (PPy) is a conductive polymer whose naturally high impedance, ionic and electric conductivity, mechanical "softness", and biocompatibility make it a leading candidate for next-generation neural electrode interfaces. However, PPy (and related conductive polymer) surfaces are susceptible to surface fouling upon exposure to biological fluids (e.g., blood, perilymph, saliva), which compromises performance and shortens its expected working lifespan. Here, we report the ability of lubricin (LUB) coatings, a rapidly self-assembling, biological antiadhesive glycoprotein, to mitigate the harmful electrochemical effects caused by the surface fouling of electrochemically grown PPy films. LUB, a biological antiadhesive glycoprotein, undergoes rapid self-assembly and adheres strongly to most interfaces, including PPy, resulting in an easy-to-apply and highly efficacious coating. The LUB-coated PPy electrodes are electrochemically characterized, and its antifouling properties are assessed against concentrated solutions of bovine serum albumin (BSA) and following long-term exposure to artificial perilymph (AP). Periodic impedance measurement conducted over 6 days in AP solution demonstrates the high stability and capacity of the LUB coatings to maintain stable impedance values under real-world mimicking conditions.

Temporal changes in synovial fluid composition and elastoviscous lubrication in the equine carpal fracture model.

The objective of this study was to examine temporal variations in synovial fluid composition and lubrication following articular fracture. Post-traumatic osteoarthritis (PTOA) was induced by creating an osteochondral fracture in the middle carpal joint of four horses while the contralateral limb served as a sham-operated control. Horses were exercised on a high-speed treadmill, and synovial fluid was collected pre-operatively and at serial timepoints until 75 days post-operatively. Lubricin and hyaluronic acid (HA) concentrations were measured using sandwich ELISAs, and the molecular weight distribution of HA was analyzed via gel electrophoresis. Synovial fluid viscosity and cartilage friction coefficients across all modes of lubrication were measured on days 0, 19, 33, and 61 using a commercial rheometer and a custom tribometer, respectively. HA concentrations were significantly decreased post-operatively, and high molecular weight HA (>6.1MDa) did not recover to pre-operative values by the study termination at day 75. Lubricin concentrations increased after surgery to a greater extent in the OA as compared to sham-operated limbs. Viscosity was significantly reduced after surgery. While boundary and elastoviscous mode friction coefficients did not vary, the transition number, representing the shift between these modes, was lower. Although more pronounced in the OA limbs, similar derangements in HA, HA molecular weight distribution, viscosity, and transition number were observed in the sham-operated limbs, which may be explained by synovial fluid washout during arthroscopy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Moderate Physical Activity as a Prevention Method for Knee Osteoarthritis and the Role of Synoviocytes as Biological Key.

The purpose of this study was to investigate the influence of moderate physical activity (MPA) on the expression of osteoarthritis (OA)-related (IL-1ß, IL-6, TNF-α, MMP-13) and anti-inflammatory and chondroprotective (IL-4, IL-10, lubricin) biomarkers in the synovium of an OA-induced rat model. A total of 32 rats were divided into four groups: Control rats (Group 1); rats performing MPA (Group 2); anterior cruciate ligament transection (ACLT)-rats with OA (Group 3); and, ACLT-rats performing MPA (Group 4). Analyses were performed using Hematoxylin & Eosin (H & E) staining, histomorphometry and immunohistochemistry. In Group 3, OA biomarkers were significantly increased, whereas, IL-4, IL-10, and lubricin were significantly lower than in the other experimental groups. We hypothesize that MPA might partake in rescuing type B synoviocyte dysfunction at the early stages of OA, delaying the progression of the disease.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.