The Potential Role of Ionic Liquid as a Multifunctional Dental Biomaterial.

In craniofacial research and routine dental clinical procedures, multifunctional materials with antimicrobial properties are in constant demand. Ionic liquids (ILs) are one such multifunctional intelligent material. Over the last three decades, ILs have been explored for different biomedical applications due to their unique physical and chemical properties, high task specificity, and sustainability. Their stable physical and chemical characteristics and extremely low vapor pressure make them suitable for various applications. Their unique properties, such as density, viscosity, and hydrophilicity/hydrophobicity, may provide higher performance as a potential dental material. ILs have functionalities for optimizing dental implants, infiltrate materials, oral hygiene maintenance products, and restorative materials. They also serve as sensors for dental chairside usage to detect oral cancer, periodontal lesions, breath-based sobriety, and dental hard tissue defects. With further optimization, ILs might also make vital contributions to craniofacial regeneration, oral hygiene maintenance, oral disease prevention, and antimicrobial materials. This review explores the different advantages and properties of ILs as possible dental material.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Biochemical Markers of Musculoskeletal Health and Aging to be Assessed in Clinical Trials of Drugs Aiming at the Treatment of Sarcopenia: Consensus Paper from an Expert Group Meeting Organized by the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) and the Centre Académique de Recherche et d'Expérimentation en Santé (CARES SPRL), Under the Auspices of the World Health Organization Collaborating Center for the Epidemiology of Musculoskeletal Conditions and Aging.

In clinical trials, biochemical markers provide useful information on the drug's mode of action, therapeutic response and side effect monitoring and can act as surrogate endpoints. In pharmacological intervention development for sarcopenia management, there is an urgent need to identify biomarkers to measure in clinical trials and that could be used in the future in clinical practice. The objective of the current consensus paper is to provide a clear list of biochemical markers of musculoskeletal health and aging that can be recommended to be measured in Phase II and Phase III clinical trials evaluating new chemical entities for sarcopenia treatment. A working group of the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) proposed classifying biochemical markers into 2 series: biochemical markers evaluating musculoskeletal status and biochemical markers evaluating causal factors. For series 1, the group agreed on 4 biochemical markers that should be assessed in Phase II or Phase III trials (i.e., Myostatin-Follistatin, Brain Derived Neurotrophic Factor, N-terminal Type III Procollagen and Serum Creatinine to Serum Cystatin C Ratio - or the Sarcopenia Index). For series 2, the group agreed on 6 biochemical markers that should be assessed in Phase II trials (i.e., the hormones insulin-like growth factor-1 (IGF-I), dehydroepiandrosterone sulphate, and cortisol, and the inflammatory markers C-reactive protein (CRP), interleukin-6 and tumor necrosis factor-α), and 2 in Phase III trials (i.e., IGF-I and CRP). The group also proposed optional biochemical markers that may provide insights into the mode of action of pharmacological therapies. Further research and development of new methods for biochemical marker assays may lead to the evolution of these recommendations.

Therapeutic Perspectives for the Clinical Application of Umbilical Cord Hematopoietic and Mesenchymal Stem Cells: Overcoming Complications Arising After Allogeneic Hematopoietic Stem Cell Transplantation.

This review focuses on the therapeutic features of umbilical cord blood (UCB) cells as a source for allogeneic hematopoietic stem cell transplantation (aHSCT) in adult and child populations to treat malignant and nonmalignant hematologic diseases, genetic disorders, or pathologies of the immune system, when standard treatment (e.g., chemotherapy) is not effective or clinically contraindicated. In this article, we summarize the immunological properties and the advantages and disadvantages of using UCB stem cells and discuss a variety of treatment outcomes using different sources of stem cells from different donors both in adults and pediatric population. We also highlight the critical properties (total nucleated cell dose depending on HLA compatibility) of UCB cells that reach better survival rates, reveal the advantages of double versus single cord blood unit transplantation, and present recommendations from the most recent studies. Moreover, we summarize the mechanism of action and potential benefit of mesenchymal umbilical cord cells and indicate the most common posttransplantation complications.

Taurine mitigates the development of pulmonary inflammation, oxidative stress, and histopathological alterations in a rat model of bile duct ligation.

Lung injury is a significant complication associated with cholestasis/cirrhosis. This problem significantly increases the risk of cirrhosis-related morbidity and mortality. Hence, finding effective therapeutic options in this field has significant clinical value. Severe inflammation and oxidative stress are involved in the mechanism of cirrhosis-induced lung injury. Taurine (TAU) is an abundant amino acid with substantial anti-inflammatory and antioxidative properties. The current study was designed to evaluate the role of TAU in cholestasis-related lung injury. For this purpose, bile duct ligated (BDL) rats were treated with TAU (0.5 and 1% w: v in drinking water). Significant increases in the broncho-alveolar lavage fluid (BALF) level of inflammatory cells (lymphocytes, neutrophils, basophils, monocytes, and eosinophils), increased IgG, and TNF-α were detected in the BDL animals (14 and 28 days after the BDL surgery). Alveolar congestion, hemorrhage, and fibrosis were the dominant pulmonary histopathological changes in the BDL group. Significant increases in the pulmonary tissue biomarkers of oxidative stress, including reactive oxygen species formation, lipid peroxidation, increased oxidized glutathione levels, and decreased reduced glutathione, were also detected in the BDL rats. Moreover, significant myeloperoxidase activity and nitric oxide levels were seen in the lung of BDL rats. It was found that TAU significantly blunted inflammation, alleviated oxidative stress, and mitigated lung histopathological changes in BDL animals. These data suggest TAU as a potential protective agent against cholestasis/cirrhosis-related lung injury.

Exploring the translational potential of clusterin as a biomarker of early osteoarthritis.

BACKGROUND: Clusterin (CLU; also known as apolipoprotein J) is an ATP-independent holdase chaperone that prevents proteotoxicity as a consequence of protein aggregation. It is a ∼60 kDa disulfide-linked heterodimeric protein involved in the clearance of cellular debris and the regulation of apoptosis. CLU has been proposed to protect cells from cytolysis by complement components and has been implicated in Alzheimer's disease due to its ability to bind amyloid-ß peptides and prevent aggregate formation in the brain. Recent studies suggest that CLU performs moonlighting functions. CLU exists in two major forms: an intracellular form and a secreted extracellular form. The intracellular form of CLU may suppress stress-induced apoptosis by forming complexes with misfolded proteins and facilitates their degradation. The secreted form of CLU functions as an extracellular chaperone that prevents protein aggregation. METHODS: In this review, we discuss the published literature on the biology of CLU in cartilage, chondrocytes, and other synovial joint tissues. We also review clinical studies that have examined the potential for using this protein as a biomarker in synovial and systemic fluids of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). RESULTS: Since CLU functions as an extracellular chaperone, we propose that it may be involved in cytoprotective functions in osteoarticular tissues. The secreted form of CLU can be measured in synovial and systemic fluids and may have translational potential as a biomarker of early repair responses in OA. CONCLUSION: There is significant potential for investigating synovial and systemic CLU as biomarkers of OA. Future translational and clinical orthopaedic studies should carefully consider the diverse roles of this protein and its involvement in other comorbidities. Therefore, future biomarker studies should not correlate circulating CLU levels exclusively to the process of OA pathogenesis and progression. Special attention should be paid to CLU levels in synovial fluid. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: There is significant potential for investigating synovial and systemic CLU as a predictive biomarker of osteoarthritis (OA) progression and response to novel treatments and interventions. Given that CLU plays diverse roles in other comorbidities such as rheumatoid arthritis (RA) and obesity, future translational and clinical orthopaedic biomarker studies should not directly correlate circulating CLU levels to the process of OA pathogenesis and progression. However, special attention should be paid to CLU levels in synovial fluid. The cytoprotective properties of CLU may support the implementation of regenerative strategies and new approaches for developing targeted therapeutics for OA.

Minimally Invasive Cell-Based Therapy for Symptomatic Bone Marrow Lesions of the Knee: A Prospective Clinical Study at 1 Year.

Bone marrow lesions (BMLs) are typical findings in magnetic resonance imaging present in different pathologies, such as spontaneous insufficiency fractures, osteonecrosis, transient BML syndromes, osteoarthritis, and trauma. The etiology and evolution of BMLs in multiple conditions remain unclear. There is still no gold standard protocol for the treatment of symptomatic BMLs in the knee. The biologic augmentation by Osteo Core Plasty™ is a new treatment modality showing promising results reducing pain with the aim to stop the progression of the disease. The purpose of this prospective study is to report the clinical outcomes and safety of Osteo Core Plasty for the treatment of symptomatic BMLs in the knee. Fifteen patients with symptomatic BMLs of the knee treated with the Osteo Core Plasty technique were included and followed prospectively for a minimum of 12 months. Each patient was evaluated before the surgery and respectively at 6 and 12 months using the Tegner Score, Marx Score, the International Knee Documentation Committee, the Knee Injury and Osteoarthritis Outcome Score divided in pain, activity daily living and quality of life subscale, and the Visual Analog Scale for pain. All clinical scores except Tegner and Marx score showed an overall statistically significant improvement through the entire follow-up (P < 0.05) and a significant improvement (P < 0.05) between each follow-up period (T0 vs. T1; T0 vs. T2; T1 vs. T2). No complications were reported. These preliminary results confirm that biological subchondral bone augmentation by Osteo Core Plasty technique is a safe and effective minimally invasive treatment option for symptomatic BMLs in the knee at 1-year follow-up. There is still a need for high-quality randomized controlled trials studies and systematic reviews in the future to enhance further treatment strategies in preventing or treating BMLs of the knee.

Diagnosis and treatment of the most common neuropathies following knee injuries and reconstructive surgery - A narrative review.

The main nerves in the knee region are the tibial nerve, the common peroneal nerve, and the saphenous nerve. These three nerves innervate the lower leg and foot, providing sensory and motor function. The large sciatic nerve splits just above the knee to form the tibial and common peroneal nerves. The tibial nerve travels down in the posterior region, while the common peroneal nerve runs around the lateral side of the knee and runs down the front of the leg to the foot. Although all these nerves can be affected by injuries of the knee, the infrapatellar branch of the saphenous nerve (IPBSN) and the common peroneal nerve (CPN) are most affected. In this narrative review we focus on neuropathies associated with nerves located in the region of the knee joint in the context of their injuries and possible iatrogenic damage during reconstructive surgery.

The Effect of Platelet-Rich Plasma on the Intra-Articular Microenvironment in Knee Osteoarthritis.

Knee osteoarthritis (KOA) represents a clinical challenge due to poor potential for spontaneous healing of cartilage lesions. Several treatment options are available for KOA, including oral nonsteroidal anti-inflammatory drugs, physical therapy, braces, activity modification, and finally operative treatment. Intra-articular (IA) injections are usually used when the non-operative treatment is not effective, and when the surgery is not yet indicated. More and more studies suggesting that IA injections are as or even more efficient and safe than NSAIDs. Recently, research to improve intra-articular homeostasis has focused on biologic adjuncts, such as platelet-rich plasma (PRP). The catabolic and inflammatory intra-articular processes that exists in knee osteoarthritis (KOA) may be influenced by the administration of PRP and its derivatives. PRP can induce a regenerative response and lead to the improvement of metabolic functions of damaged structures. However, the positive effect on chondrogenesis and proliferation of mesenchymal stem cells (MSC) is still highly controversial. Recommendations from in vitro and animal research often lead to different clinical outcomes because it is difficult to translate non-clinical study outcomes and methodology recommendations to human clinical treatment protocols. In recent years, significant progress has been made in understanding the mechanism of PRP action. In this review, we will discuss mechanisms related to inflammation and chondrogenesis in cartilage repair and regenerative processes after PRP administration in in vitro and animal studies. Furthermore, we review clinical trials of PRP efficiency in changing the OA biomarkers in knee joint.

Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.

Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-ß3.

BACKGROUND: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor ß (TGF-ß3) and activin A. METHODS: MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-ß3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. RESULTS: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-ß3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-ß3 alone were more pronounced in BMMSCs in vitro. CONCLUSION: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

Dickkopf-3 (DKK3) Signaling in IL-1α-Challenged Chondrocytes: Involvement of the NF-κB Pathway.

OBJECTIVE: Osteoarthritis (OA) is an age-related biomechanical and low-grade inflammometabolic disease of the joints and one of the costliest and disabling forms of arthritis. Studies on matrix-degrading enzymes such as metalloproteases, which are implicated in the increased catabolism of extracellular matrix, are of paramount relevance. DKK3 is a member of DKK family and is best known for its role in cancer. Although there is some information about the participation of DKK3 in cartilage pathophysiology and on metalloproteases regulation, in particular, little is known about DKK3 signaling mechanisms. Thus, the aim of this study is to explore how DKK3 regulates matrix metalloproteinase-13 (MMP-13) expression. DESIGN: Gene, protein expression and protein phosphorylation in primary human chondrocytes and ATDC5 mouse cells were assessed by RT-qPCR and Western blot analysis. Further studies on DKK3 activity were performed by targeting DKK3 gene with a specific siRNA. RESULTS: DKK3 expression was found to be higher in OA human chondrocytes than healthy cells, being its expression decreased in interleukin-1α (IL-1α)-stimulated cells. DKK3 knockdown increased the induction of MMP-13 elicited by IL-1α in human and mouse chondrocytes and after the analysis of different signalling pathways, we observed that NF-κB pathway was involved in the regulation of MMP-13 expression by DKK3. CONCLUSIONS: Herein we have demonstrated, for the first time, that DKK3 gene silencing exacerbated NF-κB activation, resulting in an increased IL-1α-driven induction of MMP-13. Our results further confirm that DKK3 may play a protective role in OA by attenuating NF-κB activation and the subsequent production of metalloproteases.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

TissueGene-C promotes an anti-inflammatory micro-environment in a rat monoiodoacetate model of osteoarthritis via polarization of M2 macrophages leading to pain relief and structural improvement.

Osteoarthritis (OA) is the most common form of arthritis, characterized by cartilage destruction, pain and inflammation in the joints. Existing medications can provide relief from the symptoms, but their effects on the progression of the disease are limited. TissueGene-C (TG-C) is a novel cell and gene therapy for the treatment of OA, comprising a mixture of human allogeneic chondrocytes and irradiated cells engineered to overexpress transforming growth factor-ß1 (TGF-ß1). This study aims to investigate the efficacy and mechanism of action of TG-C in a rat model of OA. Using the monosodium-iodoacetate (MIA) model of OA, we examined whether TG-C could improve OA symptoms and cartilage structure in rats. Our results showed that TG-C provided pain relief and cartilage structural improvement in the MIA OA model over 56 days. In parallel with these long-term effects, cytokine profiles obtained on day 4 revealed increased expression of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the synovial lavage fluid. Moreover, the increased levels of TGF-ß1 and IL-10 caused by TG-C induced the expression of arginase 1, a marker of M2 macrophages, and decreased the expression of CD86, a marker of M1 macrophages. These results suggest that TG-C exerts a beneficial effect on OA by inducing a M2 macrophage-dominant micro-environment. Cell therapy using TG-C may be a promising strategy for targeting the underlying pathogenic mechanisms of OA, reducing pain, improving function, and creating a pro-anabolic micro-environment. This environment supports cartilage structure regeneration and is worthy of further evaluation in future clinical trials.

Cohort profile: The Applied Public-Private Research enabling OsteoArthritis Clinical Headway (IMI-APPROACH) study: a 2-year, European, cohort study to describe, validate and predict phenotypes of osteoarthritis using clinical, imaging and biochemical markers.

PURPOSE: The Applied Public-Private Research enabling OsteoArthritis Clinical Headway (APPROACH) consortium intends to prospectively describe in detail, preselected patients with knee osteoarthritis (OA), using conventional and novel clinical, imaging, and biochemical markers, to support OA drug development. PARTICIPANTS: APPROACH is a prospective cohort study including 297 patients with tibiofemoral OA, according to the American College of Rheumatology classification criteria. Patients were (pre)selected from existing cohorts using machine learning models, developed on data from the CHECK cohort, to display a high likelihood of radiographic joint space width (JSW) loss and/or knee pain progression. FINDINGS TO DATE: Selection appeared logistically feasible and baseline characteristics of the cohort demonstrated an OA population with more severe disease: age 66.5 (SD 7.1) vs 68.1 (7.7) years, min-JSW 2.5 (1.3) vs 2.1 (1.0) mm and Knee injury and Osteoarthritis Outcome Score pain 31.3 (19.7) vs 17.7 (14.6), except for age, all: p<0.001, for selected versus excluded patients, respectively. Based on the selection model, this cohort has a predicted higher chance of progression. FUTURE PLANS: Patients will visit the hospital again at 6, 12 and 24 months for physical examination, pain and general health questionnaires, collection of blood and urine, MRI scans, radiographs of knees and hands, CT scan of the knee, low radiation whole-body CT, HandScan, motion analysis and performance-based tests.After two years, data will show whether those patients with the highest probabilities for progression experienced disease progression as compared to those wit lower probabilities (model validation) and whether phenotypes/endotypes can be identified and predicted to facilitate targeted drug therapy. TRIAL REGISTRATION NUMBER: NCT03883568.

Serum NT/CT SIRT1 ratio reflects early osteoarthritis and chondrosenescence.

OBJECTIVE: Previous work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA). METHODS: We developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium. RESULTS: Wild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA. CONCLUSIONS: Increased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.

BACKGROUND: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. RESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. CONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Nanotechnological Strategies for Osteoarthritis Diagnosis, Monitoring, Clinical Management, and Regenerative Medicine: Recent Advances and Future Opportunities.

PURPOSE OF REVIEW: In this review article, we discuss the potential for employing nanotechnological strategies for the diagnosis, monitoring, and clinical management of osteoarthritis (OA) and explore how nanotechnology is being integrated rapidly into regenerative medicine for OA and related osteoarticular disorders. RECENT FINDINGS: We review recent advances in this rapidly emerging field and discuss future opportunities for innovations in enhanced diagnosis, prognosis, and treatment of OA and other osteoarticular disorders, the smart delivery of drugs and biological agents, and the development of biomimetic regenerative platforms to support cell and gene therapies for arresting OA and promoting cartilage and bone repair. Nanotubes, magnetic nanoparticles, and other nanotechnology-based drug and gene delivery systems may be used for targeting molecular pathways and pathogenic mechanisms involved in OA development. Nanocomposites are also being explored as potential tools for promoting cartilage repair. Nanotechnology platforms may be combined with cell, gene, and biological therapies for the development of a new generation of future OA therapeutics. Graphical Abstract.

Cell and Gene Therapy for Spine Regeneration: Mammalian Protein Production Platforms for Overproduction of Therapeutic Proteins and Growth Factors.

This article provides an evidence-based personal perspective on the future of cell and gene therapy for degenerative diseases of the intervertebral disc. This paper focuses on how mammalian protein production platforms and transfected and irradiated protein packaging cell lines may be used as "cellular factories" for overproduction of therapeutic proteins and proanabolic growth factors, particularly in the context of regenerative therapies. This paper also speculates on future opportunities and challenges in this area of research and how new innovations in biotechnology affect cell and gene therapy for degenerative diseases.

The Antihypertensive Drug Nifedipine Modulates the Metabolism of Chondrocytes and Human Bone Marrow-Derived Mesenchymal Stem Cells.

Aging is associated with the development of various chronic diseases, in which both cardiovascular disorders and osteoarthritis are dominant. Currently, there is no effective treatment for osteoarthritis, whereas hypertension is often treated with L-type voltage-operated calcium channel blocking drugs, nifedipine being among the most classical ones. Although nifedipine together with other L-type voltage-operated calcium channel inhibitors plays an important role in controlling hypertension, there are unresolved questions concerning its possible effect on cartilage tissue homeostasis and the development of osteoarthritis. The aim of this study was to analyse the effects of nifedipine on metabolic processes in human chondrocytes and bone marrow mesenchymal stem cells. To better understand whether the metabolic effects are mediated specifically through L-type voltage-operated calcium channel, effects of the agonist BayK8644 were analyzed in parallel. Nifedipine downregulated and mitochondrial respiration and ATP production in both cell types. Analysis of cartilage explants by electron microscopy also suggested that a small number of chondrocyte mitochondria's lose their activity in response to nifedipine. Conversely, nifedipine enhanced glycolytic capacity in chondrocytes, suggesting that these cells have the capacity to switch from oxidative phosphorylation to glycolysis and alter their metabolic activity in response to L-type voltage-operated calcium channel inhibition. Such a metabolic switch was not observed in bone marrow mesenchymal stem cells. Nitric oxide activity was upregulated by nifedipine in bone marrow mesenchymal stem cells and particularly in chondrocytes, implying its involvement in the effects of nifedipine on metabolism in both tested cell types. Furthermore, stimulation with nifedipine resulted in elevated production of collagen type II and glycosaminoglycans in micromass cultures under chondrogenic conditions. Taken together, we conclude that the antihypertensive drug nifedipine inhibits mitochondrial respiration in both chondrocytes and bone marrow mesenchymal stem cells and that these effects may be associated with the increased nitric oxide accumulation and pro-inflammatory activity. Nifedipine had positive effects on the production of collagen type II and proteoglycans in both cell types, implying potentially beneficial anabolic responses in articular cartilage. These results highlight a potential link between antihypertensive drugs and cartilage health.