Osteoarthritis and Cartilage Open (Mar 2022)

A human in vitro 3D neo-cartilage model to explore the response of OA risk genes to hyper-physiological mechanical stress

  • Ritchie G.M. Timmermans,
  • Niek G.C. Bloks,
  • Margo Tuerlings,
  • Marcella van Hoolwerff,
  • Rob G.H.H. Nelissen,
  • Robert J.P. van der Wal,
  • Peter M. van der Kraan,
  • Arjen B. Blom,
  • Martijn H.J. van den Bosch,
  • Yolande F.M. Ramos,
  • Ingrid Meulenbelt

Journal volume & issue
Vol. 4, no. 1
p. 100231

Abstract

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Objective: Due to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up an in vitro 3D neo cartilage pellet model that enables us to explore the responses of OA risk genes to hyper-physiological MS. Design: Human primary chondrocyte neo-cartilage pellets were exposed for 2 days to 2 ​× ​10 ​min of hyper-physiological dynamic MS attained by a 20% strain and a frequency of 5 ​Hz. In order to assess cartilage damage, sulphated glycosaminoglycan (sGAG) content in the neo-cartilage was quantified using Alcian blue staining and a dimethyl methylene blue (DMMB) assay, while cleavage of aggrecan was visualized by immunohistochemical staining of aggrecan neo-epitope NITEGE. In addition, changes in expression levels of catabolic, anabolic and hypertrophic genes, and of three OA risk genes; IL11, MGP and TGFA were determined. Results: Hyper-physiological MS induced cartilage damage, as reflected by decreased sGAG content. mRNA levels of aggrecanase ADAMTS5 were increased, while hypertrophic gene RUNX2 was downregulated. MS increased expression of pro-apoptotic marker NOXA. Furthermore, 20% MS led to increased expression of all three OA risk genes IL11, MGP and TGFA. Conclusions: We established a human in vitro model in which hyper-physiological MS induced cartilage damage and catabolic signalling. Next, we demonstrated its usage to study OA risk genes and their response to the mechanical aspects of OA pathophysiology.

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