Development of a DNA damage-induced senescence model in osteoarthritic chondrocytes.

Senescent cells (SnCs) have been described to accumulate in osteoarthritis (OA) joint tissues in response to injury, thereby participating in OA development and progression. However, clinical therapeutic approaches targeting SnCs using senolysis, although promising in preclinical OA models, have not yet proven their efficacy in patients with knee OA. This pitfall may be due to the lack of understanding of the mechanisms underlying chondrocyte senescence. Therefore, our study aimed to generate models of chondrocyte senescence. This study used etoposide, to induce DNA damage-related senescence or chronic exposure to IL-1ß to entail inflammation-related senescence in human OA chondrocytes. Several hallmarks of cellular senescence, such as cell cycle arrest, expression of cyclin-dependent kinase inhibitors, DNA damages, and senescence-associated secretory profile were evaluated. Chronic exposure to IL-1ß induces only partial expression of senescence markers and does not allow us to conclude on its ability to induce senescence in chondrocytes. On the other hand, etoposide treatment reliably induces DNA damage-related senescence in human articular chondrocytes evidenced by loss of proliferative capacity, DNA damage accumulation, and expression of some SASP components. Etoposide-induced senescence model may help investigate the initiation of cellular senescence in chondrocytes, and provide a useful model to develop therapeutic approaches to target senescence in OA.

Osteoarthritic chondrocytes undergo a glycolysis-related metabolic switch upon exposure to IL-1b or TNF.

BACKGROUND: Osteoarthritis is an age-related disease that currently faces a lack of symptomatic treatment. Inflammation, which is mainly sustained by pro-inflammatory cytokines such as IL-1b, TNF, and IL-6, plays an important role in osteoarthritis progression. In this context, pro-inflammatory cytokines are widely used to mimic the inflammatory component of osteoarthritis in vitro. However, the therapeutic failures of clinical trials evaluating anti-cytokines drugs highlight the lack of overall understanding of the effects of these cytokines on chondrocytes. METHODS: Here, we generated a comprehensive transcriptomic and proteomic dataset of osteoarthritic chondrocytes treated with these cytokines to describe their pro-inflammatory signature and compare it to the transcriptome of non-osteoarthritic chondrocytes. Then, the dysregulations highlighted at the molecular level were functionally confirmed by real-time cellular metabolic assays. RESULTS: We identified dysregulation of metabolic-related genes in osteoarthritic chondrocytes but not in non-osteoarthritic chondrocytes. A metabolic shift, toward increased glycolysis at the expense of mitochondrial respiration, was specifically confirmed in osteoarthritic chondrocytes treated with IL-1b or TNF. CONCLUSION: These data show a strong and specific association between inflammation and metabolism in osteoarthritic chondrocytes, which was not found in non-osteoarthritic chondrocytes. This indicates that the link between inflammation and metabolic dysregulation may be exacerbated during chondrocyte damage in osteoarthritis. Video Abstract.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

PPARß/δ-Interfering Peptide Enhanced Mesenchymal Stromal Cell Immunoregulatory Properties.

Background: Mesenchymal stem/stromal cells (MSCs) have been widely used for their therapeutic properties in many clinical applications including osteoarthritis. Despite promising preclinical results showing the ability of MSC to reduce the clinical severity of osteoarthritis (OA) in experimental animal models, the benefits of intra-articular injection of MSC in OA patients are limited to the short term. In this regard, it is anticipated that improving the properties of MSC may collectively enhance their long-term beneficial effects on OA. Methods and Results: Recently, we have shown that PPARß/δ inhibition using a commercially available antagonist in murine MSC increases their immunoregulatory potential in vitro as well as their therapeutic potential in an experimental murine arthritis model. Here, we relied on an innovative strategy to inhibit PPARß/δ:NF-κB TF65 subunit interaction in human MSC by designing and synthesizing an interfering peptide, referred to PP11. Through RT-qPCR experiments, we evidenced that the newly synthesized PP11 peptide reduced the expression level of PDK4, a PPARß/δ target gene, but did not modify the expression levels of ACOX1 and CPT1A, PPARα target genes, and FABP4, a PPARγ target gene compared with untreated human MSC. Moreover, we showed that human MSCs pretreated with PP11 exhibit a significantly higher capacity to inhibit the proliferation of activated PBMC and to decrease the frequency of M1-like macrophages. Conclusions: We designed and synthesized an interfering peptide that potently and specifically blocks PPARß/δ activity with concomitant enhancement of MSC immunoregulatory properties.

Gait Alteration Due to Haemophilic Arthropathies in Patients with Moderate Haemophilia.

Some patients with moderate haemophilia (PWMH) report joint damage potentially responsible for gait disorders. Three-dimensional gait analysis (3DGA) is a relevant tool for the identification of complex musculoskeletal impairment. We performed an evaluation with 3DGA of 24 PWMH aged 44.3 ± 16.1 according to their joint status [Haemophilia Joint Health Score (HJHS) < 10 or HJHS ≥ 10] and assessed the correlation with the radiological and clinical parameters. Sixteen had HJHS < 10 (group 1) and eight had HJHS ≥ 10 (group 2). They were compared to 30 healthy subjects of a normative dataset. Both knee and ankle gait variable scores were increased in group 2 compared to the controls (p = 0.02 and p = 0.04, respectively). The PWMH of group 2 had a significant increase in their stance phase, double support duration, and stride width compared to the controls and group 1 (p < 0.01). Very low correlations were found for the ankle gait variable score with the ankle Pettersson sub-score (r2 = 0.250; p = 0.004) and ankle HJHS sub-score (r2 = 0.150; p = 0.04). For the knee, very low correlation was also found between the knee gait variable score and its HJHS sub-score (r2 = 0.290; p < 0.0001). Patients with moderate haemophilia presented a gait alteration in the case of poor lower limb joint status.

Posttraumatic Osteoarthritis Damage in Mice: From Histological and Micro-Computed Tomodensitometric Changes to Gait Disturbance.

OBJECTIVES: Osteoarthritis is a painful joint disease responsible for walking impairment. Its quantitative assessment by gait analysis in mice may be a relevant and noninvasive strategy to assess the disease severity. In this study, we aimed to determine the severity of osteoarthritis at the tissular and gait levels in unilateral and bilateral posttraumatic murine osteoarthritis. METHODS: Twenty-four C57BL/6 male mice were randomly assigned to 3 groups (n = 8/group): controls, unilateral surgery, and bilateral surgery. Posttraumatic osteoarthritis was induced unilaterally or bilaterally by destabilization of the medial meniscus. Gait analysis was performed weekly with the CatWalkTM XT system until the 16th week after surgery. After animal sacrifices, histological and micro-computed tomographic assessment was performed. RESULTS: Operated knees showed a significant increase in the histological score compared with controls (P < 0.001). Calcified anterior medial meniscal bone volume was higher on the ipsilateral side after unilateral destabilization of the medial meniscus (P < 0.001) and on both sides after bilateral intervention (P < 0.01). One week after surgery, the mice mean speed decreased significantly in both operated groups (P < 0.001 and P < 0.05). In the unilateral group, a significant increase in the contralateral hind print area appeared from week 4 to week 16. CONCLUSIONS: While bilateral destabilization of the medial meniscus induced no detectable gait modification except 1 week after surgery, unilateral model was responsible for a gait disturbance on the contralateral side. Further studies are needed to better define the place of the CatWalkTM in the evaluation of mouse models of osteoarthritis.

Osteoarthritis: From upcoming treatments to treatments yet to come.

Osteoarthritis affects hundreds of millions of people worldwide, and its prevalence is constantly increasing. While there is currently no treatment that can alter the course of the disease, promising therapeutic strategies and novel targets are being investigated. Innovative cell therapies are already reaching clinical trials, and recent progress in our understanding of the disease is opening new routes for gene therapy. In the long term, the development of new biofabrication tools, such as 3D bioprinting, may pave the way for personalized mini-joint models that could be used to screen drugs and to personalize treatments. This review provides an overview of the most promising therapeutic approaches in the field of osteoarthritis, from upcoming treatments to those that are yet to be discovered.

Isokinetic knee strength deficit in patients with moderate haemophilia.

INTRODUCTION: Knee joint bleedings are responsible for quadriceps atrophy and strength deficit in patients with severe haemophilia. Little is known about patients with moderate haemophilia (PWMH). AIM: To evaluate isokinetic quadriceps and hamstrings strength in PWMH and to assess correlation with radiological and clinical parameter. METHODS: 18 PWMH aged 37.1 ± 11.4 and 18 healthy age-, weight- and height-matched controls performed a knee isokinetic test at 180°/s to assess quadriceps and hamstrings strength. In the PWMH group, knee Pettersson's score was pursued and Haemophilia Joint Health Score 2.1 (HJHS) was performed to determine unaffected knees (knee HJHS = 0) and affected ones (knee HJHS >0). RESULTS: Affected knees had a decrease of quadriceps strength compared to controls, 1.26 ± 0.47 vs 1.64 ± 0.27 Nm/kg and a decrease of hamstring strength, 0.60 ± 0.29 vs 1.03 ± 0.21 Nm/kg, (P < 0.001). Unaffected knees also had a decrease of quadriceps strength compared to controls, 1.36 ± 0.31 vs 1.64 ± 0.27 Nm/kg and a decrease of hamstring strength, 0.69 ± 0.18 vs 1.03 ± 0.21 Nm/kg, (P < 0.001). The conventional hamstring-to-quadriceps ratio was significantly decreased in affected knees compared to controls, 0.46 ± 0.15 vs 0.64 ± 0.13 (P < 0.001) but also in unaffected knees, 0.53 ± 0.16 vs 0.64 ± 0.13 (P = 0.02).No correlation was found between strength and HJHS or Pettersson's score. CONCLUSION: PWMH have a significant knee strength deficit, both on the quadriceps and the hamstrings, which is responsible for an important muscle imbalance.

A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs.

Articular cartilage (AC) may be affected by many injuries including traumatic lesions that predispose to osteoarthritis. Currently there is no efficient cure for cartilage lesions. In that respect, new strategies for regenerating AC are contemplated with interest. In this context, we aim to develop and characterize an injectable, self-hardening, mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropymethyl cellulose (Si-HPMC) mixed with silanised chitosan. The in vitro cytocompatibility of Si-HPCH was tested using human adipose stromal cells (hASC). In vivo, we first mixed Si-HPCH with hASC to observe cell viability after implantation in nude mice subcutis. Si-HPCH associated or not with canine ASC (cASC), was then tested for the repair of osteochondral defects in canine femoral condyles. Our data demonstrated that Si-HPCH supports hASC viability in culture. Moreover, Si-HPCH allows the transplantation of hASC in the subcutis of nude mice while maintaining their viability and secretory activity. In the canine osteochondral defect model, while the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without cASC, revealed a significant osteochondral regeneration. To conclude, Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC as well as the regeneration of osteochondral defects in dogs when implanted alone or with ASC.

Chondroprotective Properties of Human-Enriched Serum Following Polyphenol Extract Absorption: Results from an Exploratory Clinical Trial.

Polyphenols are widely acknowledged for their health benefits, especially for the prevention of inflammatory and age-related diseases. We previously demonstrated that hydroxytyrosol (HT) and procyanidins (PCy), alone or in combination, drive preventive anti-osteoathritic effects in vivo. However, the lack of sufficient clinical evidences on the relationship between dietary phytochemicals and osteoarthritis remains. In this light, we investigated in humans the potential osteoarticular benefit of a grapeseed and olive extract (OPCO) characterized for its hydroxytyrosol (HT) and procyanidins (PCy) content. We first validated, in vitro, the anti-inflammatory and chondroprotective properties of the extract on primary cultured human articular chondrocytes stimulated by interleukin-1 beta (IL-1 ß). The sparing effect involved a molecular mechanism dependent on the nuclear transcription factor-kappa B (NF-κB) pathway. To confirm the clinical relevance of such a nutritional strategy, we designed an innovative clinical approach taking into account the metabolites that are formed during the digestion process and that appear in circulation after the ingestion of the OPCO extract. Blood samples from volunteers were collected following ingestion, absorption, and metabolization of the extract and then were processed and applied on human primary chondrocyte cultures. This original ex vivo methodology confirmed at a clinical level the chondroprotective properties previously observed in vitro and in vivo.

Chasing Chimeras - The elusive stable chondrogenic phenotype.

The choice of the best-suited cell population for the regeneration of damaged or diseased cartilage depends on the effectiveness of culture conditions (e.g. media supplements, three-dimensional scaffolds, mechanical stimulation, oxygen tension, co-culture systems) to induce stable chondrogenic phenotype. Herein, advances and shortfalls in in vitro, preclinical and clinical setting of various in vitro microenvironment modulators on maintaining chondrocyte phenotype or directing stem cells towards chondrogenic lineage are critically discussed. Chondrocytes possess low isolation efficiency, limited proliferative potential and rapid phenotypic drift in culture. Mesenchymal stem cells are relatively readily available, possess high proliferation potential, exhibit great chondrogenic differentiation capacity, but they tend to acquire a hypertrophic phenotype when exposed to chondrogenic stimuli. Embryonic and induced pluripotent stem cells, despite their promising in vitro and preclinical data, are still under-investigated. Although a stable chondrogenic phenotype remains elusive, recent advances in in vitro microenvironment modulators are likely to develop clinically- and commercially-relevant therapies in the years to come.

Role of the Inflammation-Autophagy-Senescence Integrative Network in Osteoarthritis.

Osteoarthritis is the most common musculoskeletal disease causing chronic disability in adults. Studying cartilage aging, chondrocyte senescence, inflammation, and autophagy mechanisms have identified promising targets and pathways with clinical translatability potential. In this review, we highlight the most recent mechanistic and therapeutic preclinical models of aging with particular relevance in the context of articular cartilage and OA. Evidence supporting the role of metabolism, nuclear receptors and transcription factors, cell senescence, and circadian rhythms in the development of musculoskeletal system degeneration assure further translational efforts. This information might be useful not only to propose hypothesis and advanced models to study the molecular mechanisms underlying joint degeneration, but also to translate our knowledge into novel disease-modifying therapies for OA.

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype.

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.

Polysaccharide Hydrogels Support the Long-Term Viability of Encapsulated Human Mesenchymal Stem Cells and Their Ability to Secrete Immunomodulatory Factors.

While therapeutically interesting, the injection of MSCs suffers major limitations including cell death upon injection and a massive leakage outside the injection site. We proposed to entrap MSCs within spherical particles derived from alginate, as a control, or from silanized hydroxypropyl methylcellulose (Si-HPMC). We developed water in an oil dispersion method to produce small Si-HPMC particles with an average size of about 68 µm. We evidenced a faster diffusion of fluorescein isothiocyanate-dextran in Si-HPMC particles than in alginate ones. Human adipose-derived MSCs (hASC) were encapsulated either in alginate or in Si-HPMC, and the cellularized particles were cultured for up to 1 month. Both alginate and Si-HPMC particles supported cell survival, and the average number of encapsulated hASC per alginate and Si-HPMC particle (7102 and 5100, resp.) did not significantly change. The stimulation of encapsulated hASC with proinflammatory cytokines resulted in the production of IDO, PGE2, and HGF whose concentration was always higher when cells were encapsulated in Si-HPMC particles than in alginate ones. We have demonstrated that Si-HPMC and alginate particles support hASC viability and the maintenance of their ability to secrete therapeutic factors.

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them.

Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content.

Update on hypoxia-inducible factors and hydroxylases in oxygen regulatory pathways: from physiology to therapeutics.

The "Hypoxia Nantes 2016" organized its second conference dedicated to the field of hypoxia research. This conference focused on "the role of hypoxia under physiological conditions as well as in cancer" and took place in Nantes, France, in October 6-7, 2016. The main objective of this conference was to bring together a large group of scientists from different spheres of hypoxia. Recent advances were presented and discussed around different topics: genomics, physiology, musculoskeletal, stem cells, microenvironment and cancer, and oxidative stress. This review summarizes the major highlights of the meeting.

Biomaterial-assisted cell therapy in osteoarthritis: From mesenchymal stem cells to cell encapsulation.

Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects the cartilage, subchondral bone, and joint tissues. Although current drug therapies can provide a degree of symptomatic relief from pain, they fail to prevent joint damage. Mesenchymal stem/stromal cells (MSCs) have generated significant interest in terms of medical applications because they exert their therapeutic properties by secretion of bioactive factors that have potent immunomodulatory, antiapoptotic, antifibrotic, and anti-inflammatory effects. However, intra-articular injection of MSCs has major limitations including cell death upon injection and massive leakage from the injection site. Encapsulation of MSCs has therefore been developed as a way to overcome these limitations and to deliver therapeutic bioactive factors in several pathologies. In this review, we first briefly highlight the main therapeutic properties of MSCs and their applications in OA treatment. We then focus on MSC encapsulation and the current advances this strategy offers for the treatment of OA.

Olive and grape seed extract prevents post-traumatic osteoarthritis damages and exhibits in vitro anti IL-1ß activities before and after oral consumption.

Polyphenols exert a large range of beneficial effects in the prevention of age-related diseases. We sought to determine whether an extract of olive and grape seed standardized according to hydroxytyrosol (HT) and procyanidins (PCy) content, exerts preventive anti-osteoathritic effects. To this aim, we evaluated whether the HT/PCy mix could (i) have in vitro anti-inflammatory and chondroprotective actions, (ii) exert anti-osteoarthritis effects in two post-traumatic animal models and (iii) retain its bioactivity after oral administration. Anti-inflammatory and chondroprotective actions of HT/PCy were tested on primary cultured rabbit chondrocytes stimulated by interleukin-1 beta (IL-1ß). The results showed that HT/PCy exerts anti-inflammatory and chondroprotective actions in vitro. The preventive effect of HT/PCy association was assessed in two animal models of post-traumatic OA in mice and rabbits. Diet supplementation with HT/PCy significantly decreased the severity of post-traumatic osteoarthritis in two complementary mice and rabbit models. The bioavailability and bioactivity was evaluated following gavage with HT/PCy in rabbits. Regular metabolites from HT/PCy extract were found in sera from rabbits following oral intake. Finally, sera from rabbits force-fed with HT/PCy conserved anti-IL-1ß effect, suggesting the bioactivity of this extract. To conclude, HT/PCy extract may be of clinical significance for the preventive treatment of osteoarthritis.

Osteoarthritis: from pathogenic mechanisms and recent clinical developments to novel prospective therapeutic options.

Osteoarthritis (OA) is a degenerative joint disease that, despite recent progress, has no curative treatment. Considerable research has recently been initiated to identify new potential therapeutic targets. In this review, we will set forth some of the major discoveries in the past 5 years, notably those dealing with the identification of pathogenic factors [hypoxia-inducible factors (HIFs), complement, transforming growth factor (TGF)-ß and zinc-ZIP8]. New drugs and concepts currently in clinical development [anti-nerve growth factor (NGF), mesenchymal stromal cells and fibroblast growth factor (FGF)-18] will then be addressed. Finally, we will consider prospective avenues that could lead to mid-to-long-term developments of novel therapeutic concepts, notably those dealing with autophagy regulation and induced pluripotent stem cells.