A human in vitro 3D neo-cartilage model to explore the response of OA risk genes to hyper-physiological mechanical stress | Osteoarthritis and Cartilage Open| 4(1) (2022) 100231 | doi: 10.1016/j.ocarto.2021.100231

R.G.M. Timmermans, N.G.C. Bloks, M. Tuerlings, M. van Hoolwerff, R.G.H.H. Nelissen, R.J.P. van der Wal, P.M. van der Kraan, A.B. Blom, M.H.J. van den Bosch, Y.F.M. Ramos, I. Meulenbelt

Abstract

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.