Human Chondrocyte Cell Line Research Articles

Mitomycin C as an Anti-Persister Strategy against Klebsiella pneumoniae: Toxicity and Synergy Studies.

The combination of several therapeutic strategies is often seen as a good way to decrease resistance rates, since bacteria can more easily overcome single-drug treatments than multi-drug ones. This strategy is especially attractive when several targets and subpopulations are affected, as it is the case of Klebsiella pneumoniae persister cells, a subpopulation of bacteria able to transiently survive antibiotic exposures. This work aims to evaluate the potential of a repurposed anticancer drug, mitomycin C, combined with the K. pneumoniae lytic phage vB_KpnM-VAC13 in vitro and its safety in an in vivo murine model against two clinical isolates of this pathogen, one of them exhibiting an imipenem-persister phenotype. At the same time, we verified the absence of toxicity of mitomycin C at the concentration using the human chondrocyte cell line T/C28a2. The viability of these human cells was checked using both cytotoxicity assays and flow cytometry.

Chondrocyte autophagy mediated by T-2 toxin via AKT/TSC/Rheb/mTOR signaling pathway and protective effect of CSA-SeNP

ObjectiveKashin-Beck disease (KBD) is an endemic, degenerative, and cartilage-damaging disease for which low selenium and T-2 toxin are considered environmental pathogenic factors. This study aimed to investigate the molecular mechanisms of autophagy in cartilage damage caused by T-2 toxin and the protective effect of chondroitin sulfate A nano-elemental selenium (CSA-SeNP) on the cartilage. MethodsKBD chondrocytes and C28/I2 human chondrocyte cell lines were used. T-2 toxin, AKT inhibitor, and CSA-SeNP treatment experiments were conducted separately, with a treatment time of 24 hours. Autophagy was monitored using MDC staining, and mRFP-GFP-LC3 adenovirus, respectively. RT-qPCR and western blotting were used to detect the expression of the relevant genes and proteins. ResultsThe suppression of autophagy observed in KBD chondrocytes was replicated by applying 10 ng/mL T-2 toxin to C28/I2 chondrocytes for 24 hours. The AKT/TSCR/Rheb/mTOR signaling pathway was activated by T-2 toxin, which inhibits autophagy. The supplementation with CSA-SeNP alleviated the inhibition of autophagy by T-2 toxin through the AKT/TSCR/Rheb/mTOR signaling pathway. ConclusionsLoss of autophagy regulated by the AKT/TSCR/Rheb/mTOR signaling pathway plays an important role in cartilage damage caused by T-2 toxin. CSA-SeNP supplementation attenuated inhibition of autophagy in chondrocyte by T-2 toxin by modulating this signaling pathway. These findings provide promising new targets for the prevention and treatment of cartilage disease.

Enhancing 3D poly[ɛ]caprolactone scaffold properties through alkaline hydrolysis for improved chondrocyte culture: Morphological, physicochemical, and biocompatibility evaluations

This study investigates the impact of alkaline hydrolysis on the morphology, physicochemical properties, and mechanical strength of poly[ε]caprolactone (PCL) scaffolds fabricated using a melt-based electrohydrodynamic process. We systematically analyzed how a brief, 5-min alkaline hydrolysis treatment influenced scaffold characteristics compared to longer durations (15 and 30 min). Results demonstrated that the short-duration treatment significantly increased fiber diameter and reduced pore size while modifying the scaffold's surface chemistry. Importantly, the 5-min treatment optimized the water contact angle to 55–65°, enhancing hydrophilicity which facilitated superior cell attachment and proliferation of the C28/I2 immortalized human chondrocyte cell line. Mechanical testing revealed that while alkaline hydrolysis decreased the scaffold's Young's modulus, a 5-min treatment maintained sufficient mechanical integrity for potential tissue engineering applications, in contrast to the more substantial degradation observed with longer treatments.Biocompatibility evaluations showed that the scaffolds, particularly those treated for 5 min, supported cell viability and proliferation without inducing cytotoxic effects. The molecular analysis indicated a favorable upregulation of chondrogenic genes such as SOX9 and COL2A1, and a downregulation of COL1A1, suggesting potential benefits for cartilage tissue engineering. These findings highlight that precise control over the duration of alkaline hydrolysis can significantly enhance PCL scaffold properties, suggesting a scalable and cost-effective method for scaffold optimization in regenerative medicine applications. The results pave the way for further in vivo studies to validate these promising in vitro findings and explore the clinical applicability of the optimized scaffolds.

Synthesis and characterization of pH-sensitive nanocarrier based chitosan-g-poly(itaconic acid) for ciprofloxacin delivery for anti-bacterial application

This study aims to develop pH-sensitive and controlled release of ciprofloxacin from ciprofloxacin-loaded grafted chitosan-coated zinc oxide nanoparticles (Cip@Gchit/Zn-NPs) for the treatment of bacterial infections in the human colon. For this aim, first, the chitosan-g-poly(itaconic acid) [Chit-g-poly (Itac)] was synthesized via grafting of itaconic acid onto chitosan in the presence of cerium ammonium nitrate (CAN) under an inert atmosphere using conventional methods, while zinc oxide nanoparticles (Zn-NPs) were prepared via sol-gel technique. Characterization of the synthesized Cip@Gchit/Zn-NPs was analyzed using XRD, FT-IR, SEM, TGA, and zeta potential analysis. The antibacterial efficacy of Cip@Gchit/Zn-NPs against three pathogenic bacteria, namely Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, was superior to that of tetracycline reference drugs, as evidenced by larger inhibition zones. Cytotoxicity assessment of Cip@Gchit/Zn-NPs on the human chondrocyte cell line C28/I2 via MTT assay revealed 100 % cell viability at a concentration of 500 μg/mL. The loading efficiency of ciprofloxacin into Gchit/Zn-NPs was evaluated at various ratios, demonstrating lower loading efficiency; however, sustained release of ciprofloxacin from Cip@Gchit/Zn-NPs was excellent, with 98.13 % release observed at pH 7.2 over 10 h. Kinetic analysis of ciprofloxacin release followed the first-order kinetic models.

Development of Biphasic Injectable Hydrogels for Meniscus Scaffold from Photocrosslinked Glycidyl Methacrylate-Modified Poly(Vinyl Alcohol)/Glycidyl Methacrylate-Modified Silk Fibroin.

The development of a hydrogel material with a modified chemical structure of poly(vinyl alcohol) (PVA) and silk fibroin (SF) using glycidyl methacrylate (GMA) (denoted as PVA-g-GMA and SF-g-GMA) is an innovative approach in the field of biomaterials and meniscus tissue engineering in this study. The PVA-g-GMA/SF-g-GMA hydrogel was fabricated using different ratios of PVA-g-GMA to SF-g-GMA: 100/0, 75/25, 50/50, 25/75, and 0/100 (w/w of dry substances), using lithium phenyl (2,4,6-trimethylbenzoyl)phosphinate (LAP) as a free radical photoinitiator, for 10 min at a low ultraviolet (UV) intensity (365 nm, 6 mW/cm2). The mechanical properties, morphology, pore size, and biodegradability of the PVA-g-GMA/SF-g-GMA hydrogel were investigated. Finally, for clinical application, human chondrocyte cell lines (HCPCs) were mixed into PVA-g-GMA/SF-g-GMA solutions and fabricated into hydrogel to study the viability of live and dead cells and gene expression. The results indicate that as the SF-g-GMA content increased, the compressive modulus of the PVA-g-GMA/SF-g-GMA hydrogel dropped from approximately 173 to 11 kPa. The degradation rates of PVA-g-GMA/SF-g-GMA 100/0, 75/25, and 50/50 reached up to 15.61%, 17.23%, and 18.93% in 4 months, respectively. In all PVA-g-GMA/SF-g-GMA conditions on day 7, chondrocyte cell vitality exceeded 80%. The PVA-g-GMA/SF-g-GMA 75:25 and 50:50 hydrogels hold promise as a biomimetic biphasic injectable hydrogel for encapsulated augmentation, offering advantages in terms of rapid photocurability, tunable mechanical properties, favorable biological responses, and controlled degradation.

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

BackgroundCRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.MethodsWe screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β).ResultsWe established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.ConclusionsWe showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways.

Osteoarthritis (OA) causes joint pain and disability due to the abnormal production of inflammatory cytokines and reactive oxygen species (ROS) in chondrocytes, leading to cell death and cartilage matrix destruction. Selenium (Se) intake can protect cells against oxidative damage. It is still unknown whether Se supplementation is beneficial for OA. This study investigated the effects of Se on sodium iodoacetate (MIA)-imitated OA progress in human chondrocyte cell line (SW1353 cells) and rats. The results showed that 0.3 μM of Se treatment could protect SW1353 cells from MIA-induced damage by the Nrf2 pathway by promoting the gene expression of glutathione-synthesis-related enzymes such as the glutamate-cysteine ligase catalytic subunit, the glutamate-cysteine ligase modifier subunit, and glutathione synthetase. In addition, glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase expressions are also elevated to eliminate excessive ROS production. Moreover, Se could downregulate NF-κB, leading to a decrease in cytokines, matrix proteases, and glycosaminoglycans. In the rats, MIA-induced cartilage loss was lessened after 2 weeks of Se supplementation by oral gavage; meanwhile, glutathione synthesis was increased, and the expressions of pro-inflammatory cytokines were decreased. These results suggest that Se intake is beneficial for OA due to its effects of decreasing cartilage loss by enhancing antioxidant capacity and reducing inflammation.

Cellulose nanocrystals-reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration.

Improvement of mechanical properties of injectable tissue engineering scaffolds is a current challenge. The objective of the current study is to produce a highly porous injectable scaffold with improved mechanical properties. For this aim, cellulose nanocrystals-reinforced dual crosslinked porous nanocomposite cryogels were prepared using chemically crosslinked methacrylated gelatin (GelMA) and ionically crosslinked hyaluronic acid (HA) through the cryogelation process. The resulting nanocomposites showed highly porous structures with interconnected porosity (>90%) and mean pore size in the range of 130-296 μm. The prepared nanocomposite containing 3%w/v of GelMA, 20 w/w% of HA, and 1%w/v of CNC showed the highest Young's modulus (10 kPa) and excellent reversibility after 90% compression and could regain its initial shape after injection by a 16-gauge needle in the aqueous media. The in vitro results demonstrated acceptable viability (>90%) and migration of the human chondrocyte cell line (C28/I2), and chondrogenic differentiation of human adipose stem cells. A two-month in vivo assay on a rabbit's ear model confirmed that the regeneration potential of the prepared cryogel is comparable to the natural autologous cartilage graft, suggesting it is a promising alternative for autografts in the treatment of cartilage defects.

Graphene oxide activates canonical TGFβ signalling in a human chondrocyte cell line via increased plasma membrane tension.

Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway - TGFβ and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFβ response genes, while the use of a TGFβ signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cell-material interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFβ in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.

Exploring the In Vitro Protective Effects of Green-Lipped Mussel (GLM) Oil Extract against Biomarkers of Glucose Metabolism and Inflammation in Chondrocyte Cells

Chondrocyte behavior is critical in osteoarthritis (OA) progression and cartilage deterioration. Recent studies have shown that green-lipped mussel (GLM) oil extracts obtained through solvent extraction can regulate cytokine secretion in cell lines, potentially reducing inflammation and immune cell activation. This study investigates the effects of these extracts on human chondrocyte cell lines to understand their impact on osteoarthritis development. Chondrocyte cultures exposed to osteoarthritis-related inflammatory factors were treated with various concentrations of GLM oil. The results revealed that mussel oils, particularly a commercial mussel powder extract (SOLV.COM), enhance glucose uptake and protect chondrocyte cells. SOLV.COM effectively manages the release of inflammatory markers like interleukin-6 (IL-6) and matrix metalloproteinases-3 (MMP-3), showing increased deoxyglucose uptake. This study highlights the significant influence of extract choice on managing interleukin-6 (IL-6) secretion and cellular activation pathways, demonstrating the potential of SOLV.COM in managing osteoarthritis by controlling reactive oxygen production, regulating glucose metabolism, and inflammatory markers.

WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway.

Kashin-Beck disease (KBD) is a kind of endemic and chronic osteochondropathy in China. This study aims to explore the functional relevance and potential mechanism of Wnt-inducible signaling pathway protein 1 (WISP1) in the pathogenesis of KBD. KBD and control cartilage specimens were collected for tissue section observation and primary chondrocyte culture. Firstly, the morphological and histopathological observations were made under a light and electron microscope. Then, the expression levels of WISP1 as well as molecular markers related to the autophagy pathway and extracellular matrix (ECM) synthesis were detected in KBD and control chondrocytes by qRT-PCR, Western blot, and immunohistochemistry. Furthermore, the lentiviral transfection technique was applied to make a WISP1 knockdown cell model based on KBD chondrocytes. In vitro intervention experiments were conducted on the C28/I2 human chondrocyte cell line using human recombinant WISP1 (rWISP1). The results showed that the autolysosome appeared in the KBD chondrocytes. The expression of WISP1 was significantly higher in KBD chondrocytes. Additionally, T-2 toxin, a risk factor for KBD onset, could up-regulate the expression of WISP1 in C28/I2. The autophagy markers ATG4C and LC3II were upregulated after the low-concentration treatment of T-2 toxin and downregulated after the high-concentration treatment. After knocking down WISP1 expression in KBD chondrocytes, MAP1LC3B decreased while ATG4C and COL2A1 increased. Moreover, the rWISP1 protein treatment in C28/I2 chondrocytes could upregulate the expression of ATG4C and LC3II at the beginning and downregulate them then. Our study suggested that WISP1 might play a role in the pathogenesis of KBD through autophagy.

Kartogenin-loaded silk fibroin–chondroitin sulfate hybrid hydrogel with tailored β-sheet content: control release studies and biological activity

Silk fibroin (SF)–chondroitin sulfate (CS)-hybrid hydrogels (HHGs) containing kartogenin (KGN) were synthesized by the induction of di-tyrosine bonds, using enzymatic crosslinking in SF/CS blend solution. Then, physical crosslinking was performed to optimize the HHG properties. The results showed that the increasing of the β-sheet content as side effect of physical crosslinking from 10% to 40% affects KGN accumulative release percentage almost three times. Biological investigations using C28/I2 human chondrocyte cell line showed that the presence of KGN, CS, and optimized β-sheets content improved cell supporting behavior of HHG. Finally, KGN loaded HHG might be a promising candidate for cartilage repair.

Gremlin-1 promotes IL-1β-stimulated chondrocyte inflammation and extracellular matrix degradation via activation of the MAPK signaling pathway.

The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1β. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1β with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1β-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1β inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1β-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1β-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1β-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1β-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway.

Adenosine A2A receptor activation reduces chondrocyte senescence.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

RUNX2 stabilization by long non-coding RNAs contributes to hypertrophic changes in human chondrocytes.

Background: Chondrocyte hypertrophy has been implicated in endochondral ossification and osteoarthritis (OA). In OA, hypertrophic chondrocytes contribute to the destruction and focal calcification of the joint cartilage. Although studies in this field have remarkably developed the modulation of joint inflammation using gene therapy and regeneration of damaged articular cartilage using cell therapy, studies that can modulate or prevent hypertrophic changes in articular chondrocytes are still lacking. Methods: In vitro hypertrophic differentiation and inflammation assays were conducted using human normal chondrocyte cell lines, TC28a2 cells. Human cartilage tissues and primary articular chondrocytes were obtained from OA patients undergoing total knee arthroplasty. Long non-coding RNAs (lncRNAs), LINC02035 and LOC100130207, were selected through RNA-sequencing analysis using RNAs extracted from TC28a2 cells cultured in hypertrophic medium. The regulatory mechanism was evaluated using western blotting, real-time quantitative polymerase chain reaction, osteocalcin reporter assay, RNA-immunoprecipitation (RNA-IP), RNA-in situ hybridization, and IP. Results: LncRNAs are crucial regulators of various biological processes. In this study, we identified two important lncRNAs, LINC02035 and LOC100130207, which play important roles in hypertrophic changes in normal chondrocytes, through RNA sequencing. Interestingly, the expression level of RUNX2, a master regulator of chondrocyte hypertrophy, was regulated at the post-translational level during hypertrophic differentiation of the normal human chondrocyte cell line, TC28a2. RNA-immunoprecipitation proved the potential interaction between RUNX2 protein and both lncRNAs. Knockdown (KD) of LINC02035 or LOC100130207 promoted ubiquitin-mediated proteasomal degradation of RUNX2 and prevented hypertrophic differentiation of normal chondrocyte cell lines, whereas overexpression of both lncRNAs stabilized RUNX2 protein and generated hypertrophic changes. Furthermore, the KD of the two lncRNAs mitigated the destruction of important cartilage matrix proteins, COL2A1 and ACAN, by hypertrophic differentiation or inflammatory conditions. We also confirmed that the phenotypic changes raised by the two lncRNAs could be rescued by modulating RUNX2 expression. In addition, the KD of these two lncRNAs suppressed hypertrophic changes during chondrogenic differentiation of mesenchymal stem cells. Conclusion: Therefore, this study suggests that LINC02035 and LOC100130207 contribute to hypertrophic changes in normal chondrocytes by regulating RUNX2, suggesting that these two novel lncRNAs could be potential therapeutic targets for delaying or preventing OA development, especially for preventing chondrocyte hypertrophy.

Long noncoding RNA NAV2-AS5 relieves chondrocyte inflammation by targeting miR-8082/TNIP2 in osteoarthritis

ABSTRACT Osteoarthritis (OA) is a common chronic and frequently occurring orthopedic disease in the middle-aged and elderly individuals. Numerous studies have shown that long noncoding RNAs (lncRNAs) play major roles in various diseases. However, the potential molecular mechanism of action of NAV2-AS5 in OA remains unclear. The present study was designed to explore the influence of NAV2-AS5 on the progression of chondrocyte inflammation and its underlying molecular mechanisms. To simulate the inflammatory environment in OA, the human chondrocyte cell line was treated with LPS. Cell proliferation, cell cycle progression, and apoptosis were assessed using Cell Counting Kit-8, 5-ethynyl-2’-deoxyuridine analysis, and flow cytometry. Proliferation- and cycle-related proteins and the release of inflammatory factors were examined by western blot analysis and enzyme-linked immunosorbent assay. The downstream targets of NAV2-AS5 were determined using bioinformatics and confirmed by a luciferase reporter assay. In our study, patients with OA showed downregulation of NAV2-AS5, upregulation of miR-8082, and downregulation of TNFAIP3 interacting protein 2 (TNIP2). Moreover, we found that both overexpression of NAV2-AS5 and miR-8082 inhibitor promoted cell proliferation, inhibited apoptosis, and released inflammatory cytokines in LPS-treated chondrocytes. MiR-8082 was predicted to be a target of both NAV2-AS5 and TNIP2. In addition, rescue experiments showed that silencing of TNIP2 reversed the effects of the miR-8082 inhibitor on proliferation, cell cycle, apoptosis, and inflammatory factors in sh-NAV2-AS5-treated chondrocytes. In conclusion, these findings indicate that NAV2-AS5 relieves chondrocyte inflammation by targeting miR-8082/TNIP2 in OA, which provides a new theoretical basis for OA therapy.

Metals-triggered compound CDPDP exhibits anti-arthritic behavior by downregulating the inflammatory cytokines, and modulating the oxidative storm in mice models with extensive ADMET, docking and simulation studies.

Triggering through abiotic stress, including chemical triggers like heavy metals, is a new technique for drug discovery. In this research, the effect of heavy metal Nickel on actinobacteria Streptomyces sp. SH-1327 to obtain a stress-derived compound was firstly investigated. A new compound cyclo-(D)-Pro-(D)-Phe (CDPDP) was triggered from the actinobacteria strain SH-1327 with the addition of nickel ions 1mM. The stress compound was further evaluated for its anti-oxidant, analgesic, and anti-inflammatory activity against rheumatoid arthritis through in-vitro and in-vivo assays in albino mice. A remarkable in-vitro anti-oxidant potential of CDPDP was recorded with the IC50 value of 30.06 ± 5.11μg/ml in DPPH, IC50 of 18.98 ± 2.91 against NO free radicals, the IC50 value of 27.15 ± 3.12 against scavenging ability and IC50 value of 28.40 ± 3.14μg/ml for iron chelation capacity. Downregulation of pro-inflammatory mediators (NO and MDA), suppressed levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-Iβ) and upregulation of expressions of anti-oxidant enzymes (GSH, catalase, and GST) unveiled its anti-inflammatory potential. CDPDP was analyzed in human chondrocyte cell line CHON-001 and the results demonstrated that CDPDP significantly increased cell survival, and inhibited apoptosis of IL-1β treated chondrocytes and IL-1β induced matrix degrading markers. In addition, to evaluate the mitochondrial fitness of CHON-001 cells, CDPDP significantly upregulated pgc1-α, the master regulator of mitochondrial biogenesis, indicating that CDPDP provides protective effects in CHON-001 cells. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of the CDPDP showed that CDPDP is safe in cases of hepatotoxicity, cardiotoxicity, and cytochrome inhibition. Furthermore, docking results showed good binding of CDPDP with IL-6-17.4kcal/mol, and the simulation studies proved the stability between ligand and protein. Therefore, the findings of the current study prospect CDPDP as a potent anti-oxidant and a plausible anti-arthritic agent with a strong pharmacokinetic and pharmacological profile.

In Vitro Study of the Therapeutic Potential of Brown Crude Fucoidans in Osteoarthritis Treatment.

Osteoarthritis, one of the most common joint degenerative pathologies, still has no cure, and current treatments, such as nonsteroidal anti-inflammatory drugs, can cause serious adverse effects when taken for a long time. Brown seaweed crude fucoidans are used for the clinical treatment of several pathologies. In this study, the therapeutical potential of these biocompounds was analyzed in primary chondrocytes and the 260TT human chondrocyte cell line. Crude fucoidan from Undaria pinnatifida (Up) and Sargassum muticum (Sm) was obtained by different extraction techniques (microwave-assisted extraction, pressurized hot-water extraction, ultrasound-assisted extraction) and chemically and structurally characterized by Fourier transform infrared spectroscopy, high-performance size-exclusion chromatography, proton nuclear magnetic resonance, and scanning electron microscopy. Once cell viability was confirmed in chondrocytes treated with crude fucoidans, we evaluated their anti-inflammatory effects, observing a significant reduction in IL-6 production stimulated by IL-1β. Findings were confirmed by analysis of IL-6 and IL-8 gene expression, although only fucoidans from Up achieved a statistically significant reduction. Besides this, the antioxidant capacity of crude fucoidans was observed through the upregulation of Nrf-2 levels and the expression of its transcriptional target genes HO-1 and SOD-2, with compounds from Up again showing a more consistent effect. However, no evidence was found that crude fucoidans modulate senescence, as they failed to reduced β-galactosidase activity, cell proliferation, or IL-6 production in chondrocytes stimulated with etoposide. Thus, the findings of this research seem to indicate that the tested crude fucoidans are capable of partially alleviating OA-associated inflammation and oxidative stress, but fail to attenuate chondrocyte senescence.

Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.

Bio-electrospraying assessment toward in situ chondrocyte-laden electrospun scaffold fabrication

Electrospinning has been widely used to fabricate fibrous scaffolds for cartilage tissue engineering, but their small pores severely restrict cell infiltration, resulting in an uneven distribution of cells across the scaffold, particularly in three-dimensional designs. If bio-electrospraying is applied, direct chondrocyte incorporation into the fibers during electrospinning may be a solution. However, before this approach can be effectively employed, it is critical to identify whether chondrocytes are adversely affected. Several electrospraying operating settings were tested to determine their effect on the survival and function of an immortalized human chondrocyte cell line. These chondrocytes survived through an electric field formed by low needle-to-collector distances and low voltage. No differences in chondrocyte viability, morphology, gene expression, or proliferation were found. Preliminary data of the combination of electrospraying and polymer electrospinning disclosed that chondrocyte integration was feasible using an alternated approach. The overall increase in chondrocyte viability over time indicated that the embedded cells retained their proliferative capacity. Besides the cell line, primary chondrocytes were also electrosprayed under the previously optimized operational conditions, revealing the higher sensitivity degree of these cells. Still, their post-electrosprayed viability remained considerably high. The data reported here further suggest that bio-electrospraying under the optimal operational conditions might be a promising alternative to the existent cell seeding techniques, promoting not only cells safe delivery to the scaffold, but also the development of cellularized cartilage tissue constructs.