Different response of human chondrocytes from healthy looking areas and damaged regions to IL1β stimulation under different oxygen tension

Xiaobin Huang, Leilei Zhong, Jan Hendriks, Janine Nicole Post, Marcel Karperien (Corresponding Author)

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Abstract

Due to its avascular nature, articular cartilage is relatively hypoxic. The aim of this study was to elucidate the functional changes of macroscopically healthy looking areas chondrocytes (MHC) and macroscopically damaged regions chondrocytes (MDC) at a cellular level in response to the inflammatory cytokine IL1β under different oxygen tension levels. In this study, two‐dimensional (2‐D) expanded MHC and MDC were redifferentiated in 3‐D pellet cultures in chondrogenic differentiation medium, supplemented with or without IL1β at conventional culture (normoxia) or 2.5% O2 (hypoxia) for 3 weeks. qPCR, immunohistochemistry and ELISA were used to detect the expression of anabolic and catabolic gene expression. Alcian blue/Safranin O staining and GAG assay were used to measure cartilage matrix production. Cell proliferation and apoptosis were assessed by EdU staining and TUNEL assay, respectively. The results showed that hypoxia enhanced matrix production in both MHC and MDC and this effect was stronger on MDC. Under normoxia, MHC showed higher expression of cartilage markers and lower catabolic genes expression than MDC. Interestingly, hypoxia diminished the difference between MHC and MDC. IL1β potently induced MMPs expression regardless of cell population and oxygen tension. The fold induction of these MMPs in hypoxia was however much higher than in normoxia. In addition, hypoxia promoted the expression of HIF1α and HIF2α in MHC, while it only enhanced HIF1α expression but decreased the HIF2α expression in MDC. We concluded that hypoxia stimulated the redifferentiation of cultured chondrocytes, particularly in MDC derived from macroscopically diseased cartilage. Oxygen tension may profoundly and differentially influence inflammation‐associated cartilage injury and diseases by regulating the expression of HIF1α and HIF2α. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:XX–XX, 2018.
Original languageEnglish
Pages (from-to)84-93
Number of pages10
JournalJournal of orthopaedic research
Volume37
Issue number1
DOIs
Publication statusPublished - 1 Jan 2019

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Chondrocytes
Oxygen
Cartilage Diseases
Matrix Metalloproteinases
Cartilage
Staining and Labeling
Gene Expression
Alcian Blue
In Situ Nick-End Labeling
Articular Cartilage
Hypoxia
Enzyme-Linked Immunosorbent Assay
Immunohistochemistry
Cell Proliferation
Apoptosis
Cytokines
Wounds and Injuries

Keywords

  • UT-Hybrid-D
  • cartilage
  • chondrocyte
  • hypoxia
  • osteoarthritis
  • IL1β

Cite this

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title = "Different response of human chondrocytes from healthy looking areas and damaged regions to IL1β stimulation under different oxygen tension",
abstract = "Due to its avascular nature, articular cartilage is relatively hypoxic. The aim of this study was to elucidate the functional changes of macroscopically healthy looking areas chondrocytes (MHC) and macroscopically damaged regions chondrocytes (MDC) at a cellular level in response to the inflammatory cytokine IL1β under different oxygen tension levels. In this study, two‐dimensional (2‐D) expanded MHC and MDC were redifferentiated in 3‐D pellet cultures in chondrogenic differentiation medium, supplemented with or without IL1β at conventional culture (normoxia) or 2.5{\%} O2 (hypoxia) for 3 weeks. qPCR, immunohistochemistry and ELISA were used to detect the expression of anabolic and catabolic gene expression. Alcian blue/Safranin O staining and GAG assay were used to measure cartilage matrix production. Cell proliferation and apoptosis were assessed by EdU staining and TUNEL assay, respectively. The results showed that hypoxia enhanced matrix production in both MHC and MDC and this effect was stronger on MDC. Under normoxia, MHC showed higher expression of cartilage markers and lower catabolic genes expression than MDC. Interestingly, hypoxia diminished the difference between MHC and MDC. IL1β potently induced MMPs expression regardless of cell population and oxygen tension. The fold induction of these MMPs in hypoxia was however much higher than in normoxia. In addition, hypoxia promoted the expression of HIF1α and HIF2α in MHC, while it only enhanced HIF1α expression but decreased the HIF2α expression in MDC. We concluded that hypoxia stimulated the redifferentiation of cultured chondrocytes, particularly in MDC derived from macroscopically diseased cartilage. Oxygen tension may profoundly and differentially influence inflammation‐associated cartilage injury and diseases by regulating the expression of HIF1α and HIF2α. {\circledC} 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:XX–XX, 2018.",
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author = "Xiaobin Huang and Leilei Zhong and Jan Hendriks and Post, {Janine Nicole} and Marcel Karperien",
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T1 - Different response of human chondrocytes from healthy looking areas and damaged regions to IL1β stimulation under different oxygen tension

AU - Huang, Xiaobin

AU - Zhong, Leilei

AU - Hendriks, Jan

AU - Post, Janine Nicole

AU - Karperien, Marcel

N1 - Wiley deal

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Due to its avascular nature, articular cartilage is relatively hypoxic. The aim of this study was to elucidate the functional changes of macroscopically healthy looking areas chondrocytes (MHC) and macroscopically damaged regions chondrocytes (MDC) at a cellular level in response to the inflammatory cytokine IL1β under different oxygen tension levels. In this study, two‐dimensional (2‐D) expanded MHC and MDC were redifferentiated in 3‐D pellet cultures in chondrogenic differentiation medium, supplemented with or without IL1β at conventional culture (normoxia) or 2.5% O2 (hypoxia) for 3 weeks. qPCR, immunohistochemistry and ELISA were used to detect the expression of anabolic and catabolic gene expression. Alcian blue/Safranin O staining and GAG assay were used to measure cartilage matrix production. Cell proliferation and apoptosis were assessed by EdU staining and TUNEL assay, respectively. The results showed that hypoxia enhanced matrix production in both MHC and MDC and this effect was stronger on MDC. Under normoxia, MHC showed higher expression of cartilage markers and lower catabolic genes expression than MDC. Interestingly, hypoxia diminished the difference between MHC and MDC. IL1β potently induced MMPs expression regardless of cell population and oxygen tension. The fold induction of these MMPs in hypoxia was however much higher than in normoxia. In addition, hypoxia promoted the expression of HIF1α and HIF2α in MHC, while it only enhanced HIF1α expression but decreased the HIF2α expression in MDC. We concluded that hypoxia stimulated the redifferentiation of cultured chondrocytes, particularly in MDC derived from macroscopically diseased cartilage. Oxygen tension may profoundly and differentially influence inflammation‐associated cartilage injury and diseases by regulating the expression of HIF1α and HIF2α. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:XX–XX, 2018.

AB - Due to its avascular nature, articular cartilage is relatively hypoxic. The aim of this study was to elucidate the functional changes of macroscopically healthy looking areas chondrocytes (MHC) and macroscopically damaged regions chondrocytes (MDC) at a cellular level in response to the inflammatory cytokine IL1β under different oxygen tension levels. In this study, two‐dimensional (2‐D) expanded MHC and MDC were redifferentiated in 3‐D pellet cultures in chondrogenic differentiation medium, supplemented with or without IL1β at conventional culture (normoxia) or 2.5% O2 (hypoxia) for 3 weeks. qPCR, immunohistochemistry and ELISA were used to detect the expression of anabolic and catabolic gene expression. Alcian blue/Safranin O staining and GAG assay were used to measure cartilage matrix production. Cell proliferation and apoptosis were assessed by EdU staining and TUNEL assay, respectively. The results showed that hypoxia enhanced matrix production in both MHC and MDC and this effect was stronger on MDC. Under normoxia, MHC showed higher expression of cartilage markers and lower catabolic genes expression than MDC. Interestingly, hypoxia diminished the difference between MHC and MDC. IL1β potently induced MMPs expression regardless of cell population and oxygen tension. The fold induction of these MMPs in hypoxia was however much higher than in normoxia. In addition, hypoxia promoted the expression of HIF1α and HIF2α in MHC, while it only enhanced HIF1α expression but decreased the HIF2α expression in MDC. We concluded that hypoxia stimulated the redifferentiation of cultured chondrocytes, particularly in MDC derived from macroscopically diseased cartilage. Oxygen tension may profoundly and differentially influence inflammation‐associated cartilage injury and diseases by regulating the expression of HIF1α and HIF2α. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:XX–XX, 2018.

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