Human Articular Chondrocytes Retain Their Phenotype in Sustained Hypoxia While Normoxia Promotes Their Immunomodulatory Potential

Objective To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). Design Chondrocytes were extracted from patients undergoing total knee replacement ( n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. Results NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity ( n = 4) and FC ( n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein ( FRZB), fibroblast growth factor receptor 3 ( FGFR3), and collagen type II ( COL2A1) and downregulated activin receptor-like kinase 1 ( ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. Conclusions Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.

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The influence of donor and hypoxic conditions on the assembly of cartilage matrix by osteoarthritic human articular chondrocytes on Hyalograft matrices

Biomaterials ◽

10.1016/j.biomaterials.2008.09.064 ◽

2009 ◽

Vol 30 (4) ◽

pp. 535-540 ◽

Cited By ~ 21

Author(s):

Theoni Katopodi ◽

Simon R. Tew ◽

Peter D. Clegg ◽

Timothy E. Hardingham

Keyword(s):

Articular Chondrocytes ◽

Cartilage Matrix ◽

Human Articular Chondrocytes ◽

Hypoxic Conditions

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Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture

Cartilage ◽

10.1177/1947603519870862 ◽

2019 ◽

pp. 194760351987086

Author(s):

Natalia Viana Tamiasso ◽

Carla Maria Osório Silva ◽

Amanda Maria Sena Reis ◽

Natália Melo Ocarino ◽

Rogéria Serakides

Keyword(s):

Mtt Assay ◽

Articular Chondrocytes ◽

Quantitative Polymerase Chain Reaction ◽

Periodic Acid ◽

3D Culture ◽

Articular Chondrocyte ◽

Periodic Acid Schiff ◽

3 Dimensional ◽

Synthesis Activity

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

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Effects of Ximoprofen and Acetylsalicylic Acid on Human Articular Chondrocytes in 3-Dimensional Culture

Drug Investigation ◽

10.1007/bf03259222 ◽

1993 ◽

Vol 5 (1) ◽

pp. 11-18

Author(s):

C. Bassleer ◽

Y. Henrotin ◽

P. Franchimont

Keyword(s):

Acetylsalicylic Acid ◽

Articular Chondrocytes ◽

Human Articular Chondrocytes ◽

3 Dimensional

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Concentration-Dependent Effects of Cobalt and Chromium Ions on Osteoarthritic Chondrocytes

Cartilage ◽

10.1177/1947603519889389 ◽

2019 ◽

pp. 194760351988938

Author(s):

Christoph Bauer ◽

Christoph Stotter ◽

Vivek Jeyakumar ◽

Eugenia Niculescu-Morzsa ◽

Bojana Simlinger ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Metabolic Activity ◽

Transcriptional Activity ◽

Articular Chondrocytes ◽

Inflammatory Responses ◽

Quantitative Polymerase Chain Reaction ◽

Human Articular Chondrocytes ◽

Tnf Α ◽

Late Apoptosis ◽

Pro Inflammatory Cytokines

Objective Cobalt and chromium (CoCr) ions from metal implants are released into the joint due to biotribocorrosion, inducing apoptosis and altering gene expression in various cell types. Here, we asked whether CoCr ions concentration-dependently changed viability, transcriptional activity, and inflammatory response in human articular chondrocytes. Design Human articular chondrocytes were exposed to Co (1.02-16.33 ppm) and Cr (0.42-6.66 ppm) ions and cell viability and early/late apoptosis (annexin V and 7-AAD) were assessed in 2-dimensional cell cultures using the XTT assay and flow cytometry, respectively. Changes in chondrocyte morphology were assessed using transmitted light microscopy. The effects of CoCr ions on transcriptional activity of chondrocytes were evaluated by quantitative polymerase chain reaction (qPCR). The inflammatory responses were determined by measuring the levels of released pro-inflammatory cytokines (interleukin-1β [IL-1β], IL-6, IL-8, and tumor necrosis factor–α [TNF-α]). Results CoCr ions concentration-dependently reduced metabolic activity and induced early and late apoptosis after 24 hours in culture. After 72 hours, the majority of chondrocytes (>90%) were apoptotic at the highest concentrations of CoCr ions (16.33/6/66 ppm). SOX9 expression was concentration-dependently enhanced, whereas expression of COL2A1 linearly decreased after 24 hours. IL-8 release was enhanced proportionally to CoCr ions levels, whereas IL-1β, IL-6, and TNF-α levels were not affected by the treatments. Conclusions CoCr ions showed concentration- and time-dependent effects on articular chondrocytes. Fractions of apoptotic articular chondrocytes were proportional to CoCr ion concentrations. In addition, metabolic activity and expression of chondrocyte-specific genes were decreased by CoCr ions. Furthermore, exposure to CoCr ions caused a release of pro-inflammatory cytokines.

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A Novel Cross-Linked Hyaluronic Acid Porous Scaffold for Cartilage Repair

Cartilage ◽

10.1177/1947603515611949 ◽

2015 ◽

Vol 7 (3) ◽

pp. 265-273 ◽

Cited By ~ 10

Author(s):

Christoph Bauer ◽

Manuela Berger ◽

Renate R. Baumgartner ◽

Sonja Höller ◽

Hannes Zwickl ◽

...

Keyword(s):

Cell Proliferation ◽

Hyaluronic Acid ◽

Articular Chondrocytes ◽

Porous Scaffold ◽

Cartilage Regeneration ◽

Cell Number ◽

Cell Content ◽

3 Dimensional ◽

Chondrogenic Phenotype ◽

Differentiation Status

Purpose An important feature of biomaterials used in cartilage regeneration is their influence on the establishment and stabilization of a chondrocytic phenotype of embedded cells. The purpose of this study was to examine the effects of a porous 3-dimensional scaffold made of cross-linked hyaluronic acid on the expression and synthesis performance of human articular chondrocytes. Materials and Methods Osteoarthritic chondrocytes from 5 patients with a mean age of 74 years were passaged twice and cultured within the cross-linked hyaluronic acid scaffolds for 2 weeks. Analyses were performed at 3 different time points. For estimation of cell content within the scaffold, DNA-content (CyQuant cell proliferation assay) was determined. The expression of chondrocyte-specific genes by embedded cells as well as the total amount of sulfated glycosaminoglycans produced during the culture period was analyzed in order to characterize the synthesis performance and differentiation status of the cells. Results Cells showed a homogenous distribution within the scaffold. DNA quantification revealed a reduction of the cell number. This might be attributed to loss of cells from the scaffold during media exchange connected with a stop in cell proliferation. Indeed, the expression of cartilage-specific genes and the production of sulfated glycosaminoglycans were increased and the differentiation index was clearly improved. Conclusions These results suggest that the attachment of osteoarthritic P2 chondrocytes to the investigated material enhanced the chondrogenic phenotype as well as promoted the retention.

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