Human Chondrocyte Cultures Research Articles

The effect of favipiravir on histone deacetylase (HDAC) enzyme activity in administered to human primary chondrocyte cultures and an evaluation of its relationship with inflammation

The effect of favipiravir on histone deacetylase (HDAC) enzyme activity in administered to human primary chondrocyte cultures and an evaluation of its relationship with inflammation

The Effects of Seleno-Methionine in Cadmium-Challenged Human Primary Chondrocytes.

Cadmium (Cd) is a potentially toxic element able to interfere with cellular functions and lead to disease or even death. Cd accumulation has been demonstrated in cartilage, where it can induce damage in joints. The aim of this study was to evaluate the effect of CdCl2 on primary cultures of human chondrocytes and the possible protective effect of seleno-methionine (Se-Met). Human primary articular chondrocytes were cultured and treated as follows: control groups, cells challenged with 7.5 μM and 10 μM CdCl2 alone, and cells pretreated with 10 and 20 μM Se-Met and then challenged with 7.5 μM and 10 μM CdCl2. Twenty-four hours after incubation, cell viability, histological evaluation with hematoxylin-eosin stain, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed. Furthermore, reverse transcription-PCR was carried out to evaluate mRNA levels of BAX, BAK1, CASP-3, and CASP-9. After CdCl2 challenge at both doses, a reduced cell viability and an overexpression of BAX, BAK1, CASP-3, and CASP-9 genes, as well as a high number of TUNEL-positive cells, were demonstrated, all parameters becoming higher as the dose of CdCl2 was increased. The pretreatment with Se-Met lowered the expression of all considered genes, improved cell viability and morphological changes, and reduced the number of TUNEL-positive cells. It was concluded that Se-Met plays a protective role against CdCl2-induced structural and functional changes in chondrocytes in vitro, as it improved cell viability and showed a positive role in the context of the apoptotic pathways. It is therefore suggested that a translational, multifaceted approach, with plant-based diets, bioactive functional foods, nutraceuticals, micronutrients, and drugs, is possibly advisable in situations of environmental pollution caused by potentially toxic elements.

Hydrogel Based on Chitosan/Gelatin/Poly(Vinyl Alcohol) for In Vitro Human Auricular Chondrocyte Culture.

Three-dimensional (3D) hydrogels provide tissue-like complexities and allow for the spatial orientation of cells, leading to more realistic cellular responses in pathophysiological environments. There is a growing interest in developing multifunctional hydrogels using ternary mixtures for biomedical applications. This study examined the biocompatibility and suitability of human auricular chondrocytes from microtia cultured onto steam-sterilized 3D Chitosan/Gelatin/Poly(Vinyl Alcohol) (CS/Gel/PVA) hydrogels as scaffolds for tissue engineering applications. Hydrogels were prepared in a polymer ratio (1:1:1) through freezing/thawing and freeze-drying and were sterilized by autoclaving. The macrostructure of the resulting hydrogels was investigated by scanning electron microscopy (SEM), showing a heterogeneous macroporous structure with a pore size between 50 and 500 μm. Fourier-transform infrared (FTIR) spectra showed that the three polymers interacted through hydrogen bonding between the amino and hydroxyl moieties. The profile of amino acids present in the gelatin and the hydrogel was determined by ultra-performance liquid chromatography (UPLC), suggesting that the majority of amino acids interacted during the formation of the hydrogel. The cytocompatibility, viability, cell growth and formation of extracellular matrix (ECM) proteins were evaluated to demonstrate the suitability and functionality of the 3D hydrogels for the culture of auricular chondrocytes. The cytocompatibility of the 3D hydrogels was confirmed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reaching 100% viability after 72 h. Chondrocyte viability showed a high affinity of chondrocytes for the hydrogel after 14 days, using the Live/Dead assay. The chondrocyte attachment onto the 3D hydrogels and the formation of an ECM were observed using SEM. Immunofluorescence confirmed the expression of elastin, aggrecan and type II collagen, three of the main components found in an elastic cartilage extracellular matrix. These results demonstrate the suitability and functionality of a CS/Gel/PVA hydrogel as a 3D support for the auricular chondrocytes culture, suggesting that these hydrogels are a potential biomaterial for cartilage tissue engineering applications, aimed at the regeneration of elastic cartilage.

TARGETING HYPERTROPHIC CHONDROCYTES IN OSTEOARTHRITIS: THE ROLE OF INFLAMMATION AND THE USE OF A MULTI-MODEL APPROACH IN SEARCH OF PHARMACOLOGICAL TREATMENTS

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780).In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis.Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants.In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis.

Elucidating the mechanism of IL-1β-Mediated Piezo1 expression regulation of chondrocyte autophagy and apoptosis via the PI3K/AKT/mTOR signaling Pathway

For the pathogenesis of osteoarthritis (OA), the classical view is that chondrocyte apoptosis is associated with and may cause age-related joint degeneration. Recent observations indicate that Piezo1, a mechanical stress channel expressed in articular cartilage, plays a crucial role in this process. We wanted to investigate whether other conditions activate the expression of Piezo1 in chondrocytes. Therefore, we simulated OA to investigate whether Piezo1 gene expression and channel function were affected by the inflammatory factor,interleukin-1β, and the role of Piezo1 in the regulation of autophagy and apoptosis of chondrocytes. After the primary culture of human chondrocytes, the primary chondrocytes were treated with different concentrations of IL-1β. It was found that IL-1β upregulated the expression of Piezo1 in human chondrocytes. After Piezo1 activation, we analyzed the expression of autophagy and apoptosis of chondrocytes and investigated whether the downstream PI3K/AKT/mTOR pathway mediated the autophagy and apoptosis of chondrocytes. IL-1β activates Piezo1 to inhibit chondrocyte autophagy and promote chondrocyte apoptosis partially, represented by up-regulation of related proteins c-caspase 3, Bax expression, and down-regulation of Bcl2, LC3, p62 expression. Piezo1-siRNA inverted this step partially. Inhibition of the PI3K/AKT/mTOR pathway reduces Piezo1 inhibition of chondrocyte autophagy and activation of chondrocyte apoptosis. Therefore, IL-1β-mediated Piezo1 inhibition of chondrocyte autophagy and promotion of chondrocyte apoptosis partially through the PI3K/AKT/mTOR pathway is considered a novel pathogenesis of osteoarthritis.

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage – an in vitro study

ObjectivesTo compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at different concentrations on primary human articular chondrocytes and cartilage.Materials and methodsPrimary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modified Eagle medium or phosphate-buffered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining.ResultsOctenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures.ConclusionThe degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic effects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50.Clinical relevanceThese data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes.

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Hyaluronic acid and multiwalled carbon nanotubes as bioink additives for cartilage tissue engineering

Articular cartilage and meniscus injuries are prevalent disorders with insufficient regeneration responses offered by available treatment methods. In this regard, 3D bioprinting has emerged as one of the most promising new technologies, offering novel treatment options. Additionally, the latest achievements from the fields of biomaterials and tissue engineering research identified constituents facilitating the creation of biocompatible scaffolds. In this study, we looked closer at hyaluronic acid and multi-walled carbon nanotubes as bioink additives. Firstly, we assessed the minimal concentrations that stimulate cell viability, and decrease reactive oxygen species and apoptosis levels in 2D cell cultures of normal human knee articular chondrocytes (NHAC) and human adipose-derived mesenchymal stem cells (hMSC-AT). In this regard, 0.25 mg/ml of hyaluronic acid and 0.0625 mg/ml of carbon nanotubes were selected as the most optimal concentrations. In addition, we investigated the protective influence of 2-phospho-L-ascorbic acid in samples with carbon nanotubes. Tests conducted on 3D bioprinted constructs revealed that only a combination of components positively impacted cell viability throughout the whole experiment. Gene expression analysis of COL1A1, COL6A1, HIF1A, COMP, RUNX2, and POU5F1 showed significant changes in the expression of all analyzed genes with a progressive overall loss of transcriptional activity in most of them.

WWP2 confers risk to osteoarthritis by affecting cartilage matrix deposition via hypoxia associated genes

To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

The Critical Role of Hypoxia in the Re-Differentiation of Human Articular Chondrocytes.

The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.

Development of meniscus cartilage using polycaprolactone and decellularized meniscus surface modified by gelatin, hyaluronic acid biomacromolecules: A rabbit model

The lack of vascularization in the white-red and white zone of the meniscus causes these zones of tissue to have low self-healing capacity in case of injury and accelerate osteoarthritis (OA). In this study, we have developed hybrid constructs using polycaprolactone (PCL) and decellularized meniscus extracellular matrix (DMECM) surface modified by gelatin (G), hyaluronic acid (HU) and selenium (Se) nanoparticles (PCL/DMECM/G/HU/Se), following by the cross-linking of the bio-polymeric surface. Material characterization has been performed on the fabricated scaffold using scanning electron microscopy (SEM), Fourier transforms infrared (FTIR) spectroscopy, swelling and degradation analyses, and mechanical tests. In Vitro, investigations have been conducted by C28/I2 human chondrocyte culture into the scaffold and evaluated the cytotoxicity and cell/scaffold interaction. For the in vivo study, the scaffolds were transplanted into the defect sites of female New Zealand white rabbits. Good regeneration was observed after two months. We have concluded that the designed PCL/DMECM/G/HU construct can be a promising candidate as a meniscus tissue engineering scaffold to facilitate healing.

Selected physico-chemical properties of composite scaffolds of sintered submicrocrystalline corundum and bioglass

Presented paper contains description and interpretation of the results of selected physicochemical and structural properties of two types of composite sinters. They were constituted of a mixture of sintered microcrystalline corundum and bioglass CaO-P2O5-SiO2-Na2O system intended for scaffolds to cell culture of human chondrocytes. The composites contained a mixture of both above-mentioned components in the volumetric proportion of 50:50 (W5) and 30:70 (W7). They were obtained using powder metallurgy by free sintering in air atmosphere. Phase analysis of composites and verification of theoretical identification using X-ray diffraction were performed. The same phases were found in both cases (Al2O3 SiO2 CaAl2Si2O8, Ca3 (PO4)2, Ca2Al4O7 and NaAlSiO4). Microscopic tests of composite surfaces were performed and some differences were found. W5 sample was not completely covered with bioglass, whilst W7 sample was completely covered with bioglass with few fine pores. Tests of surface topography confirmed the presence of large and small pores. Composite surfaces immersed for 30 days in artificial blood plasma were tested and then electron microscopy analysis was performed. It was found that no significant changes occurred on the surface of the W5 composite, probably partial corrosion of the glass happened. Spherical forms characteristic of HA-hydroxyapatites were observed on the surface of sample W7. Human articular chondrocyte cells were seeded on both types of sinters and proliferation assay was performed. Results indicate that tested scaffolds support cellular attachment and proliferation of chondrocytes.

Metabolic reprogramming in chondrocytes to promote mitochondrial respiration reduces downstream features of osteoarthritis

Metabolic dysfunction in chondrocytes drives the pro-catabolic phenotype associated with osteoarthritic cartilage. In this study, substitution of galactose for glucose in culture media was used to promote a renewed dependence on mitochondrial respiration and oxidative phosphorylation. Galactose replacement alone blocked enhanced usage of the glycolysis pathway by IL1β-activated chondrocytes as detected by real-time changes in the rates of proton acidification of the medium and changes in oxygen consumption. The change in mitochondrial activity due to galactose was visualized as a rescue of mitochondrial membrane potential but not an alteration in the number of mitochondria. Galactose-replacement reversed other markers of dysfunctional mitochondrial metabolism, including blocking the production of reactive oxygen species, nitric oxide, and the synthesis of inducible nitric oxide synthase. Of more clinical relevance, galactose-substitution blocked downstream functional features associated with osteoarthritis, including enhanced levels of MMP13 mRNA, MMP13 protein, and the degradative loss of proteoglycan from intact cartilage explants. Blocking baseline and IL1β-enhanced MMP13 by galactose-replacement in human osteoarthritic chondrocyte cultures inversely paralleled increases in markers associated with mitochondrial recovery, phospho-AMPK, and PGC1α. Comparisons were made between galactose replacement and the glycolysis inhibitor 2-deoxyglucose. Targeting intermediary metabolism may provide a novel approach to osteoarthritis care.

Using melt-electrowritten microfibres for tailoring scaffold mechanics of 3D bioprinted chondrocyte-laden constructs

Moulded hydrogels reinforced with melt-electrowritten (MEW) microfibres to tailor mechanical properties show promise in the articular cartilage regeneration field. We aim to transfer this knowledge to bioprinted constructs of volumetric dimensions towards real anatomical shapes, using a previously established alginate methylcellulose (algMC) blend. We show the potential of algMC to be a bioink for MEW fibre-reinforced 3D printed constructs that have tailorable mechanical properties and support long-term culture of bioprinted human chondrocytes. It is hoped that these composite algMC-PCL scaffolds have potential for use in auricular (ear) cartilage regeneration as an alternative to current reconstructive treatments using autografted rib cartilage or high-density polyethylene implants. Scaffolds of different designs were assessed to determine the effect of the hybrid structure on compressive strength, as well as an initial in vitro experiment with human chondrocytes to assess cell viability, DNA, sGAG, and collagen II content. The inclusion of MEW PCL scaffolds showed up to a 7.5-fold increase in the maximum compressive stress after 7 days of incubation in DMEM compared to algMC only scaffolds. In vitro work showed there was no significant differences in the cell viability between groups after 21 days of culture. However, DNA and sGAG content (relative to cell number) showed an increase in algMC scaffolds reinforced with MEW PCL sheets with 750 μm pores suggesting a more favourable stiffness for chondrocyte extracellular matrix (ECM) deposition. Future work will investigate how the scaffold stiffness changes over time as materials degrade and ECM is deposited as well as further in depth analysis of the biological performance with relevance to auricular cartilage. This microfibre reinforced algMC scaffold provides a promising way to tailor the mechanical properties of bioprinted structures for chondrocyte delivery at clinically relevant dimensions.

A novel placental tissue biologic, PTP-001, inhibits inflammatory and catabolic responses in vitro and prevents pain and cartilage degeneration in a rat model of osteoarthritis

Characterization of a novel human placental tissue-derived biologic, PTP-001, which is in development as a candidate therapeutic for the treatment of osteoarthritis symptoms and pathophysiology. Human placental tissues from healthy donors were prepared as a particulate formulation, PTP-001. PTP-001 extracts were assayed for the presence of disease-relevant biofactors which could have beneficial effects in treating osteoarthritis. PTP-001 eluates were tested in human chondrocyte cultures to determine effects on the production of a key collagen-degrading matrix metalloproteinase, MMP-13. PTP-001 eluates were also assessed for anti-inflammatory potential in human monocyte/macrophage cultures, as well as for growth-stimulating anabolic effects in human synoviocytes. The in vivo effects of PTP-001 on joint pain and histopathology were evaluated in a rat model of osteoarthritis induced surgically by destabilization of the medial meniscus. PTP-001 was found to contain an array of beneficial growth factors, cytokines and anti-inflammatory molecules. PTP-001 eluates dose-dependently inhibited the production of chondrocyte MMP-13, and the secretion of proinflammatory cytokines from monocyte/macrophage cultures. PTP-001 eluates also promoted proliferation of cultured synovial cells. In a rat osteoarthritis model, PTP-001 significantly reduced pain responses throughout 6 weeks post-dosing. The magnitude and duration of pain reduction following a single intraarticular treatment with PTP-001 was comparable to that observed for animals treated with a corticosteroid (active control). For rats dosed twice with PTP-001, significant reductions in cartilage histopathology scores were observed. PTP-001 represents a promising biologic treatment for osteoarthritis, with a multi-modal mechanism of action that may contribute to symptom management and disease modification.

S-allylcysteine inhibits chondrocyte inflammation to reduce human osteoarthritis via targeting RAGE, TLR4, JNK, and Nrf2 signaling: comparison with colchicine.

The discovery of new pharmacological agents is needed to control the progression of osteoarthritis (OA), characterized by joint cartilage damage. Human OA chondrocyte (OAC) cultures were either applied to S-allylcysteine (SAC), a sulfur-containing amino acid derivative, or colchicine, an ancient anti-inflammatory therapeutic, for 24 h. SAC or colchicine did not change viability at 1 nM-10 µM but inhibited p-JNK/pan-JNK. While SAC seems to be more effective, both agents inhibited reactive oxygen species (ROS), 3-nitrotyrosine (3-NT), lipid hydroperoxides (LPO), advanced lipoxidation end-products (ALEs as 4-hydroxy-2-nonenal, HNE), advanced glycation end-products (AGEs), and increased glutathione peroxidase (GPx) and type-II-collagen (COL2). IL-1β, IL-6, and osteopontin (OPN) were more strongly inhibited by SAC than by colchicine. In contrast, TNF-α was inhibited only by SAC, and COX2 was only inhibited by colchicine. Casp-1/ICE, GM-CSF, receptor for advanced glycation end-products (RAGE), and toll-like receptors (TLR4) were inhibited by both agents, but bone morphogenetic protein 7 (BMP7) was partially inhibited by SAC and induced by colchicine. Nuclear factor erythroid 2-related factor 2 (Nrf2) was induced by SAC; in contrast, it was inhibited by colchicine. Although they exert opposite effects on TNF-α, COX2, BMP7, and Nrf2, SAC and colchicine exhibit anti-osteoarthritic properties in OAC by modulating redox-sensitive inflammatory signaling.

MiR-214-3p, a novel possible therapeutic target for the pathogenesis of osteoarthritis

MiR-214-3p, a novel possible therapeutic target for the pathogenesis of osteoarthritis

In Vitro Effects of Low Doses of β-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation

β-caryophyllene (BCP), a plant-derived sesquiterpene, has been reported to have anti-inflammatory and antioxidant effects. The purpose of this study is to evaluate the effects of BCP in combination with ascorbic acid (AA) and d-glucosamine (GlcN) against macrophage-mediated inflammation on in vitro primary human chondrocytes. Changes in cell viability, intracellular ROS generation, gene expression of pro-inflammatory mediators, metalloproteinases (MMPs), collagen type II and aggrecan were analyzed in primary human chondrocytes exposed to the conditioned medium (CM) of activated U937 monocytes and subsequently treated with BCP alone or in combination with AA and GlcN. The CM-induced chondrocyte cytotoxicity was reduced by the presence of low doses of BCP alone or in combination with AA and GlcN. The exposure of cells to CM significantly increased IL-1β, NF-κB1 and MMP-13 expression, but when BCP was added to the inflamed cells, alone or in combination with AA and GlcN, gene transcription for all these molecules was restored to near baseline values. Moreover, chondrocytes increased the expression of collagen type II and aggrecan when stimulated with AA and GlcN alone or in combination with BCP. This study showed the synergistic anti-inflammatory and antioxidative effects of BCP, AA and GlcN at low doses on human chondrocyte cultures treated with the CM of activated U937 cells. Moreover, the combination of the three molecules was able to promote the expression of collagen type II and aggrecan. All together, these data could suggest that BCP, AA and GlcN exert a chondro-protective action.

Integrin \u03b12\u03b21 plays an important role in the interaction between human articular cartilage-derived chondrocytes and atelocollagen gel

Although atelocollagen gel is used as a scaffold for culturing human articular cartilage-derived chondrocytes, little is known about cell–gel interactions. In this study, we investigated the mechanism via which atelocollagen gel affects human articular cartilage-derived chondrocytes. Two types of three-dimensional cultures of human articular cartilage-derived chondrocytes (i.e., with and without atelocollagen gel) were compared. While the amount of atelocollagen gel in culture gradually decreased with time, it promoted the expression of matrix metalloproteinases (MMPs) during the early stages of culture. Genome-wide differential gene expression analysis revealed that cell membrane- and extracellular matrix-related genes were highly ranked among up- and down-regulated groups in cells cultured in the presence of atelocollagen gel. Among the integrin family of genes, the expression of integrin subunit alpha 2 and integrin subunit alpha 10 was significantly increased in the presence of atelocollagen gel. Blocking α2β1 integrin with the specific inhibitor BTT 3033 had a significant effect on cell proliferation, MMP expression, and cell shape, as well as on the response to mechanical stimulation. Taken together, our findings indicate that the α2β1 integrin pathway plays an important role in the interaction of atelocollagen gel with human articular cartilage-derived chondrocytes and may be a potential therapeutic target for articular cartilage disorders.

New “Tribo-bioreactor” for In-situ Monitoring of the Mechanical Stress Transmission at the Cellular Level: Application to Cartilage Tissue Engineering

The mechanotransduction is an important aspect to provide suitable conditions for the cartilage engineering process. Therefore, a new bioreactor was developed to apply different mechanical stresses to cell/organ culture. It allows performing cell culture in situ with appropriate mechanical constraints according to the real-time evolution of the physical parameters with the possibility of microscopic observation. In addition to the two-dimensional mechanical stimulation of a scaffold, the originality of this device is in situ monitoring of the cells and rheological measurements. This study presents the design of the new prototype with the validation of its different functions. First, the mechanical characterization of different non-biological samples is performed to calibrate the rheological measurement. Then, the monitoring of fluorescent beads in different scaffolds is carried out to estimate the mechanical stress transmitted to the cells. Finally, the ability of the device to handle a cell culture of human chondrocytes seeded in a scaffold is evaluated. The combined functions of the new bioreactor open the possibility to develop new scaffolds with optimal mechanical stress transmission, which is the basis of tissue engineering of cartilage.