Human articular chondrocytes retain their phenotype in sustained hypoxia whilst normoxia promotes their immunomodulatory capacity

Mennan, C.; Owen, S.; Garcia, J.; McCarthy, H.; Banerjee, R.; Roberts, S.; Richardson, J.

Abstract

Introduction The maintenance of chondrogenic phenotype in human articular chondrocytes grown in continuous normoxic (21% O2) and hypoxic (2% O2) conditions was assessed, with the aim of finding the best environment for culturing cells destined for cartilage repair.

Materials and Methods Articular cartilage was obtained from patients undergoing total knee replacement. Isolated chondrocytes were cultured at 37°C in ˜21% and 2% O2, termed normoxic chondrocytes (NC) or hypoxic chondrocytes (HC), respectively. Media used for hypoxia was equilibrated to 2% O2. Freshly isolated chondrocytes (FC) were also analysed. Flow cytometry was used to assess cell surface markers (indicative of mesenchymal stromal cells (MSCs), chondrogenic potency and de-differentiation), RT-qPCR was used to assess the expression of chondrogenic genes and immunocytochemistry for collagen II production in NC and HC following 28 day pellet culture.

Results NC were positive (over ˜97%, n = 5) for cell surface markers indicative of MSCs (CD73, 90 and 105). HC did not fit the cell surface marker profile for MSCs (<90% positivity for CD73, 90 and 105, n = 4) and FC produced even lower levels of these markers (<20% positivity for CD73, 90 and <40% for CD105).

The markers CD166 and CD151 (which may indicate de-differentiation) were most highly produced in NC. HC showed much lower production, with the lowest production seen in FC. NC also showed the greatest CD106 production, which is classically associated with MSCs and immunomodulation. RT-qPCR analysis of RNA extracted from NC and HC at P3 showed upregulation of the chondrogenic genes, SOX9, frizzled related protein (FRZB), fibroblast growth factor receptor 3 (FGFR3) and COL2, in hypoxia but to varying degrees between different patients. Activin like kinase (receptor) 1 (ALK1) was downregulated in hypoxia. HC chondrogenic pellets showed positive staining for collagen II whereas NC did not. NC had higher upregulation of the immunomodulatory gene, IDO, after IFN-γ stimulation than HC.

Discussion Hypoxic conditions help maintain a chondrogenic phenotype (with up regulation of key chondrogenic genes) whereas normoxia promotes de-differentiation towards an MSC phenotype (with higher expression of MSC markers and immunomodulatory proteins, IDO and CD106). Hypoxic culture of chondrocytes may be beneficial, prior to returning cells to the patient in Autologous Chondrocyte Implantation.

Citation

Mennan, C., Owen, S., Garcia, J., McCarthy, H., Banerjee, R., Roberts, S., & Richardson, J. (2017, March). Human articular chondrocytes retain their phenotype in sustained hypoxia whilst normoxia promotes their immunomodulatory capacity. Poster presented at British Society for Matrix Biology “The Grey Area – Age and the Extracellular Matrix”

• Presentation Conference Type: Poster
• Conference Name: British Society for Matrix Biology “The Grey Area – Age and the Extracellular Matrix”
• Start Date: Mar 27, 2017
• End Date: Mar 29, 2017
• Deposit Date: Jun 12, 2023
• Publisher URL: https://onlinelibrary.wiley.com/doi/10.1111/iep.12211