RESUMEN

Adipose-derived stem cells (ADSCs) comprise a promising therapy for osteoarthritis (OA). The therapeutic potential of ELIXCYTE®, an allogeneic human ADSC (hADSC) product, was demonstrated in a phase I/II OA clinical trial. However, the exact mechanism underlying such effects is not clear. Moreover, studies suggest that interleukin-11 (IL-11) has anti-inflammatory, tissue-regenerative, and immune-regulatory functions. Our aim was to unravel the mechanism associated with the therapeutic effects of ELIXCYTE® on OA and its relationship with IL-11. We cocultured ELIXCYTE® with normal human articular chondrocytes (NHACs) in synovial fluid obtained from individuals with OA (OA-SF) to investigate its effect on chondrocyte matrix synthesis and degradation and inflammation by assessing gene expression and cytokine levels. NHACs exposed to OA-SF exhibited increased MMP13 expression. However, coculturing ELIXCYTE® with chondrocytes in OA-SF reduced MMP13 expression in chondrocytes and downregulated PTGS2 and FGF2 expression in ELIXCYTE®. ELIXCYTE® treatment elevated anti-inflammatory cytokine (IL-1RA, IL-10, and IL-13) levels, and the reduction in MMP13 was positively correlated with IL-11 concentrations in OA-SF. These findings indicate that IL-11 in OA-SF might serve as a predictive biomarker for the ELIXCYTE® treatment response in OA, emphasizing the therapeutic potential of ELIXCYTE® to mitigate OA progression and provide insights into its immunomodulatory effects.

RESUMEN

Human small extracellular vesicles (sEVs) derived from adipose-derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium-derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium-derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically-defined media (CDM). The quantity and purity of ASC-derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1-3 contained detectable particles, while there was no visible contamination of culture media-derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4-sEVs) exhibited the highest purity and yield. Notably, the CDM4-sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti-apoptotic activity of hACs. Furthermore, CDM4-sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

Asunto(s)

Vesículas Extracelulares , Células Madre Mesenquimatosas , Osteoartritis , Animales , Ratones , Humanos , Condrocitos , Osteoartritis/terapia , Modelos Animales de Enfermedad

RESUMEN

OBJECTIVE: In autologous chondrocyte implantation (ACI), there is no consensus about used bioscaffolds. The aim of this study was to perform an in vitro comparative analysis of 2 clinically applied biomaterials for cartilage lesion treatment. DESIGN: Monolayer expanded human chondrocytes (n = 6) were embedded in a collagen scaffold (CS) and a hyaluronic acid-based hydrogel (HA). Cells were cultured in chondropermissive medium supplemented with and without interleukin-10 (IL-10) and bone morphogenetic protein-2 (BMP-2). Gene expression of chondrogenic markers (COL1A1, COL2A1, COL10A1, ACAN, SOX9) was detected via quantitative real-time-polymerase chain reaction (RT-qPCR). Biosynthesis of matrix compounds, cell viability, morphology as well as migration from surrounding native bovine cartilage into cell-free scaffolds were analyzed histologically. Adhesion of the material to adjacent cartilage was investigated by a custom-made push-out test. RESULTS: The shift of COL1/2 ratio toward COL2A1 was more pronounced in HA, and cells displayed a more spherical morphology compared with CS. BMP-2 and IL-10 significantly increased COL2A1, SOX9, and ACAN expression, which was paralleled by enhanced staining of glycosaminoglycans (GAGs) and type 2 collagen in histological sections of CS and HA. COL10A1 was not significantly expressed in HA and CS. Better interfacial integration and enhanced cell invasion was observed in CS. Push-out tests using CS showed higher bonding strength to native cartilage. CONCLUSION: HA-based hydrogel revealed a more chondrocyte-like phenotype but only allowed limited cell invasion, whereas CS were advantageous in terms of cellular invasion and interfacial adhesion. These differences may be clinically relevant when treating cartilaginous or osteochondral defects.

Asunto(s)

Condrocitos , Hidrogeles , Animales , Bovinos , Humanos , Condrocitos/metabolismo , Interleucina-10 , Materiales Biocompatibles/farmacología , Andamios del Tejido , Células Cultivadas , Colágeno/metabolismo

RESUMEN

Irisin is a recently discovered cytokine, better known as an exercise-induced myokine, produced primarily in skeletal muscle tissue as a response to exercise. Although the skeleton was initially identified as the main target of Irisin, its action is also proving effective in many other tissues. Physical activity determines a series of beneficial effects on health, including the possibility of counteracting the damage that is caused by arthritis to the cartilage of people suffering from osteoarthritis. Nevertheless, up to now, the studies that have taken into consideration the possible involvement of Irisin on the well-being of cartilage tissue are particularly limited. In this study, we postulated that the protective effect of physical activity on cartilage tissue may depend on the paracrine action of Irisin secreted during exercise; therefore, we analyzed the effects of Irisin, in vitro, on chondrogenic differentiation. To achieve this goal, three-dimensional cultures of commercially available human articular chondrocytes (HACs) were treated with the molecule under study. Our results revealed new crosstalk mechanisms between muscle and cartilage tissue. Furthermore, the confirmation of Irisin ability to induce chondrogenic differentiation could favor the development of exercise-mimetic drugs, with application relevance for patients who cannot perform physical activity.

RESUMEN

The in vitro effects of four nutraceuticals, catechin hydrate, gallic acid, α-tocopherol and ascorbic acid, on the ability of human osteoarthritic chondrocytes of two female obese groups to form articular cartilage (AC) tissues and to reduce inflammation were investigated. Group 1 represented thirteen females in the 50-69 years old range, an average weight of 100 kg and an average body mass index (BMI) of 34⋅06 kg/m2. Group 2 was constituted of three females in the 70-80 years old range, an average weight of 75 kg and an average BMI of 31⋅43 kg/m2. The efficacy of nutraceuticals was assessed in monolayer cultures using histological, colorimetric and mRNA gene expression analyses. AC engineered tissues of group 1 produced less total collagen and COL2A1 (38-fold), and higher COL10A1 (2⋅7-fold), MMP13 (50-fold) and NOS2 (15-fold) mRNA levels than those of group 2. In comparison, engineered tissues of group 1 had a significant decrease in NO levels from day 1 to day 21 (2⋅6-fold), as well as higher mRNA levels of FOXO1 (2-fold) and TNFAIP6 (16-fold) compared to group 2. Catechin hydrate decreased NO levels significantly in group 1 (1⋅5-fold) while increasing NO levels significantly in group 2 (3⋅8-fold). No differences from the negative control were observed in the presence of other nutraceuticals for either group. In conclusion, engineered tissues of the younger but heavier patients responded better to nutraceuticals than those from the older but leaner study participants. Finally, cells of group 2 formed better AC tissues with less inflammation and better extracellular matrix than cells of group 1.

Asunto(s)

Condrocitos/efectos de los fármacos , Suplementos Dietéticos , Osteoartritis , Anciano , Anciano de 80 o más Años , Ácido Ascórbico/farmacología , Catequina/farmacología , Células Cultivadas , Femenino , Ácido Gálico/farmacología , Humanos , Inflamación , Persona de Mediana Edad , Osteoartritis/tratamiento farmacológico , ARN Mensajero , alfa-Tocoferol/farmacología

RESUMEN

BACKGROUND: Osteoarthritis (OA) is characterized by joint pain and joint function limitation. Hsa_circ_0045714 (circ_0045714) is a novel OA-related circular RNA. However, its repertoire remains to be further clarified in joint chondrocytes. METHODS: RNA and protein expression levels and inflammatory factor levels were detected by real-time quantitative polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. Cell proliferation and apoptosis were determined by colony formation assay, cell counting kit-8 assay and apoptosis assay. Direct interaction was predicted by bioinformatics method and confirmed by dual-luciferase reporter assay. RESULTS: Expression of circ_0045714 and phosphoinositide-3-kinase (PI3K) regulatory subunit 3 (PIK3R3) was declined, and microRNA (miR)-331-3p was promoted in knee articular cartilages and cells from OA patients, as well as interleukin (IL)-1ß-challenged human articular chondrocytes (HAC) cell line. In stimulation of IL-1ß, HAC cells showed a loss of colony formation ability, cell viability and expression of Bcl-2 and Collagen II, allied with an increase in apoptosis rate and levels of IL-6, IL-8 and tumor necrosis factor-α, Bcl-2-associated X protein, cleaved caspase-3, and ADAM with thrombospondin motif-5. Noticeably, overexpressing circ_0045714 and inhibiting miR-331-3p could suppress IL-1ß-evoked these effects, and both were through up-regulating PIK3R3, a key gene in PI3K/AKT signaling pathway. Mechanically, circ_0045714 functioned as competing endogenous RNA (ceRNA) for miR-331-3p and further regulated expression of the downstream target gene PIK3R3. CONCLUSION: There was a novel circ_0045714/miR-331-3p/PIK3R3 ceRNA axis in HAC, and its inhibition might be one mechanism of HAC injury in OA.

Asunto(s)

MicroARNs , Osteoartritis , Condrocitos , Humanos , MicroARNs/genética , Osteoartritis/genética , Fosfatidilinositol 3-Quinasas , ARN Circular

RESUMEN

The inflammatory cytokine interleukin-26 (IL-26) is highly expressed in the serum and synovial fluid of patients with inflammatory arthritis. The effect of IL-26 on human articular chondrocytes (HACs) remains unclear. Obesity is associated with disability of patients with rheumatoid arthritis and disease activity in those with ankylosing spondylitis. The saturated free fatty acid palmitate with IL-1ß can synergistically induce catabolic effects in HACs. The aim of this study was to evaluate the effects of IL-26 and palmitate in HACs. In this study, palmitate markedly synergizes the IL-26-induced proinflammatory effects and matrix protease, including COX-2, IL-6, and MMP-1, in HACs via the toll-like receptor 4 (TLR4)-ERK1/2-c-Jun signal transduction pathway. The synergistic catabolic effects of palmitate and IL-26 were attenuated by inhibitors of TLR4 (TAK242), ERK1/2 (U0126), or c-Jun (SP600125) in HACs and cartilage matrix. In addition, metformin, a potential inhibitor of TLR4, also decreased expression of COX-2 and IL-6 induced by co-incubation with IL-26 and palmitate. IL-26 and palmitate synergistically induced expression of inflammatory and catabolic mediators, resulting in articular cartilage matrix breakdown. The present study also revealed a possible mechanism and therapeutic targets against articular cartilage degradation by increased saturated fatty acids in patients with inflammatory arthritis.

Asunto(s)

Condrocitos/metabolismo , Interleucinas/metabolismo , Palmitatos/metabolismo , Artritis/inmunología , Artritis/metabolismo , Artritis/fisiopatología , Artritis Reumatoide/metabolismo , Cartílago Articular/metabolismo , Condrocitos/fisiología , Genes jun/fisiología , Humanos , Interleucinas/inmunología , Sistema de Señalización de MAP Quinasas/fisiología , Metabolismo/fisiología , Osteoartritis/metabolismo , Transducción de Señal/genética , Membrana Sinovial/metabolismo , Taiwán , Receptor Toll-Like 4/metabolismo

RESUMEN

Mesenchymal stem cells (MSCs) on injectable hydrogels are mostly used to regenerate articular cartilage, which would have a variety of outcomes. Chondrocyte extracellular vesicles (EVs) have attracted many attentions for their chondrogenic differentiation capacity; however, the roles of EVs in both chondrogenic differentiation of MSCs and cartilage regeneration are poorly understood yet. In the current study, to investigate the differentiation effects of human articular chondrocyte EVs on adipose-derived MSCs, they were cultured in injectable chitosan-hyaluronic acid (CS-HA) hydrogel and then treated with chondrocyte EVs for 21 days. The continuous treatment of EVs performed on MSCs increased chondrogenic genes' expressions ofSOX9andCOL2A1and induced expression of Col II protein. In addition, glycosaminoglycans secretion was detected in the EV-treated MSCs after about 14 days. The therapeutic efficiency of this hydrogel and EVs was studied in a rabbit osteochondral defect model. MRI results revealed that the cartilage regeneration capacity of EV-treated MSCs with CS-HA hydrogel was greater than the untreated MSCs or the EV-treated MSCs without hydrogel. Moreover, histological results showed hyaline-like cartilage in the CS-HA/MSC and CS-HA/EV/MSC groups in the cartilage defect sites. These findings suggested that the chondrocyte-EVs and CS-HA hydrogel could provide the preferable niche for chondrogenic differentiation of MSCs and cartilage regeneration in osteoarthritis cartilage injuries.

Asunto(s)

Quitosano , Condrocitos/citología , Vesículas Extracelulares , Células Madre Mesenquimatosas , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Cartílago Articular/citología , Cartílago Articular/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quitosano/química , Quitosano/farmacología , Condrogénesis/efectos de los fármacos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/trasplante , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Conejos , Andamios del Tejido

RESUMEN

Several studies suggest that inflammation has a pivotal role during the progression of osteoarthritis (OA) and cytokines have been identified as the main process mediators. This study aimed to explore the ability to modulate the main OA pro-inflammatory biomarkers of novel gels (H-HA/BC) based on high molecular weight hyaluronan (H-HA) and unsulfated biotechnological chondroitin (BC). For the first time, BC was tested also in combination with H-HA on human primary cells isolated from pathological knee joints. Specifically, the experiments were performed using an OA in vitro model based on human chondrocytes and synoviocytes. To evaluate the anti-inflammatory effects of H-HA/BC in comparison with H-HA and BC single gels, NF-kB, COMP-2, MyD88, MMP-13 and a wide range of cytokines, known to be specific biomarkers in OA (e.g., IL-6, IL-8, and TNF-α), were evaluated. In addition, cell morphology and proliferation occurring in the presence of either H-HA/BC or single components were assessed using time-lapse video microscopy. It was shown that synovial fluids and cells isolated from OA suffering patients, presented a cytokine pattern respondent to an ongoing inflammation status. H-HA and BC significantly reduced the levels of 23 biomarkers associated with cartilage damage. However, H-HA/BC decreased significantly 24 biological mediators and downregulated 19 of them more efficiently than the single components. In synoviocytes cultures, cytokine analyses proved that H-HA/BC gels re-established an extracellular environment more similar to a healthy condition reducing considerably the concentration of 11 analytes. Instead, H-HA and BC significantly modulated 7 (5 only with a longer treatment) and 8 biological cytokines, respectively. Our results suggest that H-HA/BC beyond the viscosupplementation effect typical for HA-based gels, can improve the inflammation status in joints and thus could be introduced as a valid protective and anti-inflammatory intraarticular device in the field of Class III medical devices for OA treatments.

RESUMEN

OBJECTIVE: Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs. DESIGN: Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus. RESULTS: Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface. CONCLUSION: Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.

Asunto(s)

Cartílago Articular/cirugía , Condrocitos , Ingeniería de Tejidos , Células Cultivadas , Condrogénesis , Colágeno Tipo II , Humanos

RESUMEN

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-ß1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

Asunto(s)

Cartílago Articular , Condrocitos , Diferenciación Celular , Células Cultivadas , Condrogénesis , Humanos , Rejuvenecimiento , Ingeniería de Tejidos

RESUMEN

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved.

Asunto(s)

Fibroblastos , Polimorfismo de Nucleótido Simple , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Secuenciación del Exoma

RESUMEN

INTRODUCTION: Osteoarthritis (OA) is a severe joint degeneration disease in elderly people described by the advanced degradation of articular cartilage, which ultimately leads to chronic pain. Trans-cinnamaldehyde (TCA) exerted its anti-inflammatory function in numerous disease syndromes; however, its role in the pathogenesis of OA remains unknown. The current research aimed to explore the potential protective impact of TCA in the progression of osteoarthritis in vitro. MATERIAL AND METHODS: Human knee articular chondrocytes were treated with 10 ng/ml IL-1b alone for 24 h or in a combination in a pretreatment with TCA at different concentrations (2, 5, 10 µg/mL, 24 h). The viability and cell apoptosis were determined by CCK-8 assay and flow cytometry methods. The protein levels of IL-8, PGE2, and TNF-a and the levels of phosphorylated AKT and PI3K were evaluated using ELISA assay. Moreover, RT-qPCR was used to measure the relative mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 in IL-1b-induced chondrocytes. RESULTS: Our results revealed that the treatment with TCA had no effect on chondrocytes' proliferation and apoptosis. Moreover, the protein levels of IL-8, TNF-a, and PGE2 were considerably reduced in IL-1b-induced chondrocytes treated with different concentrations of TCA. Furthermore, the mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 and the phosphorylation of AKT and PI3K were markedly reduced in IL-1b-induced chondrocytes with the increase in the concentration of TCA. CONCLUSIONS: Trans-cinnamaldehyde inhibited the inflammation induced by IL-1b in chondrocytes through the PI3K/AKT pathway, which suggests that TCA might serve as a potential therapeutic agent for osteoarthritis treatment.

Asunto(s)

Acroleína/análogos & derivados , Inflamación/prevención & control , Osteoartritis/prevención & control , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Acroleína/farmacología , Acroleína/toxicidad , Células Cultivadas , Condrocitos/efectos de los fármacos , Dinoprostona/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Interleucina-1beta , Interleucina-8/metabolismo , Osteoartritis/inducido químicamente , Osteoartritis/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Sustancias Protectoras/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Rheumatoid arthritis (RA) is an autoimmune disease that affects the synovial cavity of joints, and its pathogenesis is associated with an increased expression of pro-inflammatory cytokines, namely tumour necrosis factor-alpha (TNF-α). It has been clinically shown to have an adequate response to systemic administration of TNF-α inhibitors, although with many shortcomings. To overcome such limitations, the immobilization of a TNF-α antibody on a nanofibrous substrate to promote a localized action is herein proposed. By using this approach, the antibody has its maximum therapeutic efficacy and a prolonged therapeutic benefit, avoiding the systemic side-effects associated with conventional biological agents' therapies. To technically achieve such a purpose, the surface of electrospun nanofibers is initially activated and functionalized, allowing TNF-α antibody immobilization at a maximum concentration of 6 µg/mL. Experimental results evidence that the biofunctionalized nanofibrous substrate is effective in achieving a sustained capture of soluble TNF-α over time. Moreover, cell biology assays demonstrate that this system has no deleterious effect over human articular chondrocytes metabolism and activity. Therefore, the developed TNF-capturing system may represent a potential therapeutic approach for the local management of severely affected joints.

RESUMEN

The association between osteoarthritis (OA), obesity and metabolic syndrome suggests an interrelation between OA and diabetes mellitus (DM). Little is known about the role of anti-inflammatory cytokine interleukin (IL)-10 in the interrelation between OA and DM. Hence, the effects of IL-10 under hyperglycemia (HG) and hyperinsulinemia (HI) in human articular chondrocytes (hAC) and chondrosarcoma cell line Okayama University Medical School (OUMS)-27 were examined. HAC and OUMS-27, cultured in normoglycemic (NG) and HG conditions were stimulated with insulin and/or IL-10. Cell survival, metabolic activity, proliferation and extracellular matrix (ECM) synthesis were immunocytochemically examined. No significant differences in vitality of hAC neither in pure NG (NGw/o) nor HG (HGw/o) conditions were found. Applying HI and/or IL-10 in both conditions reduced significantly the vitality of hAC but not of OUMS-27. HG impaired significantly hAC metabolism. When combined with HI + IL-10 or IL-10 alone it decreased also significantly hAC proliferation compared to NGw/o. In OUMS-27 it induced only a trend of impaired proliferation compared to NGw/o. hAC but not OUMS-27 reduced significantly their collagen type (col) I, SOX9 and proteoglycan (PG) synthesis in HG combined with HI +/- IL-10 compared to NGw/o. IL-10 could not moderate HI and HG effects. In contrast to hAC OUMS-27 showed limited sensitivity as DM model.

Asunto(s)

Diabetes Mellitus/tratamiento farmacológico , Interleucina-10/farmacología , Osteoartritis/tratamiento farmacológico , Anciano , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Diabetes Mellitus/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Hiperinsulinismo/tratamiento farmacológico , Hiperinsulinismo/metabolismo , Interleucina-10/metabolismo , Masculino , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/metabolismo , Osteoartritis/metabolismo

RESUMEN

Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor-ß1 [T]), a biophysical agent (chondroitinase-ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase-like 2 [L]) are efficacious in forming three-dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5-fold and 3-fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5-fold and 3-fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.

Asunto(s)

Aminoácido Oxidorreductasas/farmacología , Cartílago/metabolismo , Condrocitos/metabolismo , Condroitina ABC Liasa/farmacología , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta1/farmacología , Adulto , Cartílago/citología , Condrocitos/citología , Humanos , Masculino , Resistencia a la Tracción

RESUMEN

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30 µg/ml as tested with no effect on proliferation. TGFß3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14 days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.

Asunto(s)

Cartílago Articular/citología , Diferenciación Celular , Movimiento Celular , Condrocitos/citología , Fibronectinas/sangre , Adulto , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Femenino , Fibronectinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Metaloproteinasas de la Matriz/metabolismo , Proteoglicanos/metabolismo

RESUMEN

INTRODUCTION: In this article, we explored the hypothesis that the long noncoding RNA, Nespas, promotes osteoarthritis (OA) by supporting abnormal lipid metabolism in human chondrocytes. MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritis patients were used and expression level of Nespas were determined by real-time polymerase chain reaction. Introduction of Nespas and Nespas-associated genes/miRNAs were performed by using a lentiviral system. The effect of Nespas on mitochondrial function was determined by staining mitochondria and analyzing mitopotential and mitochondrial genes. Moreover, to identify the responsible molecules in Nespas-induced pathogenesis, profiling of peroxisomal genes and miRNAs were applied and interactome analysis was performed. RESULTS: Highly elevated levels of Nespas and Acyl-CoA synthetase 6 (ACSL6) were observed in OA patients. Both Nespas overexpression and ACSL6 upregulation into human chondrocytes induced typical OA characteristics, such as downregulation of type II collagen; upregulation of type I collagen, metalloproteinase 13, and caspase-1 and -3; and dysfunction of mitochondria and peroxisome. Co-expression of Nespas and ACSL6 siRNA reduced caspase-1 and -3 levels. Moreover, Nespas overexpression significantly suppressed levels of miR-291a-3p, -196a-5p, -23a-3p, -24-3p, and let-7a-5p, and these miRs are known to potentially target ACSL6 according to ingenuity pathway analysis. We also confirmed that these miRs were significantly suppressed in human OA chondrocytes. Overexpression of miR-291a-3p, -196a-5p, -23a-3p, -24-3p, or let-7a-5p in the presence of Nespas suppressed levels of ACSL6, caspase-1 and -3. DISCUSSION: Overall, we suggest that elevated Nespas level in OA are associated with OA pathogenesis by suppressing miRs targeting ACSL6 and subsequent ACSL6 upregulation.

Asunto(s)

Condrocitos/metabolismo , Cromograninas/genética , Coenzima A Ligasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Osteoartritis/genética , ARN Largo no Codificante/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Progresión de la Enfermedad , Regulación hacia Abajo , Humanos , MicroARNs , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Regulación hacia Arriba

RESUMEN

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Asunto(s)

Exoesqueleto/química , Condrocitos/efectos de los fármacos , Matriz Extracelular , Pecten/química , Anciano , Anciano de 80 o más Años , Agrecanos/genética , Agrecanos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Fenotipo

RESUMEN

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-α) and interleukin 1ß (IL-1ß). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-α, IL-1ß, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-α biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1ß agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-α or IL-1ß. The differences in potency of anti-TNF-α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045-1058, 2018.

Asunto(s)

Condrocitos/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Células Madre Mesenquimatosas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Diferenciación Celular/fisiología , Células Cultivadas , Condrocitos/citología , Condrogénesis/fisiología , Humanos , Interleucina-1beta/inmunología , Células Madre Mesenquimatosas/citología , Factor de Necrosis Tumoral alfa/inmunología