# Protocol to study the contribution of biomechanics in osteoarthritis development using a cartilage-on-chip model

## TL;DR

This paper provides a detailed protocol for studying how biomechanics and calcification contribute to osteoarthritis using a 3D cartilage-on-chip model.

## Contribution

The novelty lies in the adaptable protocol for investigating cartilage degeneration mechanisms under controlled biomechanical conditions.

## Key findings

- A 3D hydrogel system enables culturing human chondrocytes under physiological loading.
- Calcification and gene/protein changes can be analyzed using alizarin red S, qPCR, immunofluorescence, and Olink assays.
- The protocol allows controlled exploration of osteoarthritis mechanisms in a cartilage-on-chip model.

## Abstract

Here, we present a protocol to study biomechanics and calcification in osteoarthritis using a cartilage-on-chip model. We describe steps for culturing human chondrocytes in 3D hydrogels and subjecting them to physiological loading. We then detail procedures for assessing spontaneous calcification via alizarin red S staining, gene expression using qPCR, protein expression by immunofluorescence, and protein secretome analysis through the Olink proximity extension assay. This protocol is adaptable and allows exploration of cartilage degeneration mechanisms under controlled conditions.

•Procedure for assessing effects of mechanical loading in a 3D cartilage-on-chip system•Steps for imaging pathologic calcification of chondrocytes•Procedures to analyze gene expression by RT-qPCR and protein levels by Olink

Procedure for assessing effects of mechanical loading in a 3D cartilage-on-chip system

Steps for imaging pathologic calcification of chondrocytes

Procedures to analyze gene expression by RT-qPCR and protein levels by Olink

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to study biomechanics and calcification in osteoarthritis using a cartilage-on-chip model. We describe steps for culturing human chondrocytes in 3D hydrogels and subjecting them to physiological loading. We then detail procedures for assessing spontaneous calcification via alizarin red S staining, gene expression using qPCR, protein expression by immunofluorescence, and protein secretome analysis through the Olink proximity extension assay. This protocol is adaptable and allows exploration of cartilage degeneration mechanisms under controlled conditions.