Development of a Microphysiological Cartilage-on-Chip Platform for Dynamic Biomechanical Stimulation of Three-Dimensional Encapsulated Chondrocytes in Agarose Hydrogels

Valtteri Peitso, Zahra Sarmadian, João Henriques, Elsa Lauwers, Carlo Alberto Paggi, Ali Mobasheri
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Abstract

Osteoarthritis (OA) is one of the most prevalent joint diseases globally, characterized by the progressive breakdown of articular cartilage, resulting in chronic pain, stiffness, and loss of joint function. Despite its significant socioeconomic impact, therapeutic options remain limited, largely due to an incomplete understanding of the molecular mechanisms driving cartilage degradation and OA pathogenesis. Recent advances in in vitro modeling have revolutionized joint tissue research, transitioning from simplistic two-dimensional cell cultures to sophisticated three-dimensional (3D) constructs that more accurately mimic the physiological microenvironment of native cartilage. Over the last decade, organ-on-chip technologies have emerged as transformative tools in tissue engineering, offering microphysiological platforms with precise control over biomechanical and biochemical stimuli. These platforms are providing novel insights into tissue responses and disease progression and are increasingly integrated into the early stages of drug screening and development. In this article, we present a detailed experimental protocol for constructing a cartilage-on-chip system capable of delivering controlled dynamic biomechanical stimulation to 3D-encapsulated chondrocytes in an agarose hydrogel matrix. Our protocol, optimized for both bovine and human chondrocytes, begins with Basic Protocol 1, detailing the preparation and injection of cell-laden hydrogels into the microdevice. Basic Protocol 2 describes the application of dynamic mechanical loading using a calibrated pressurized pump. Finally, Basic Protocols 3 and 4 focus on the retrieval of the hydrogel and RNA extraction for downstream molecular analyses. This platform represents a critical advancement for in vitro studies of cartilage biology, enabling more precise modeling of OA pathophysiology and evaluation of experimental therapeutics. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Cartilage-on-chip injection

Basic Protocol 2: Cartilage-on-chip actuation

Basic Protocol 3: Cartilage-on-chip agarose hydrogel removal

Basic Protocol 4: Preparation of cartilage-on-chip for RNA extraction

Abstract Image

骨关节炎(OA)是全球最常见的关节疾病之一,其特点是关节软骨逐渐破坏,导致慢性疼痛、僵硬和关节功能丧失。尽管这种疾病对社会经济有重大影响,但治疗方案仍然有限,这主要是由于人们对驱动软骨降解和 OA 发病机制的分子机制了解不全面。体外建模的最新进展彻底改变了关节组织研究,从简单的二维细胞培养过渡到复杂的三维(3D)构建,更准确地模拟了原生软骨的生理微环境。在过去十年中,片上器官技术已成为组织工程领域的变革性工具,它提供了可精确控制生物力学和生物化学刺激的微物理平台。这些平台提供了有关组织反应和疾病进展的新见解,并越来越多地融入药物筛选和开发的早期阶段。在本文中,我们介绍了构建片上软骨系统的详细实验方案,该系统能够向琼脂糖水凝胶基质中的三维封装软骨细胞提供可控的动态生物力学刺激。我们的方案针对牛软骨细胞和人软骨细胞进行了优化,首先是基本方案 1,详细说明了细胞载体水凝胶的制备和注入微装置的过程。基本程序 2 描述了使用校准加压泵进行动态机械加载的应用。最后,基本程序 3 和 4 重点介绍了水凝胶的回收和用于下游分子分析的 RNA 提取。该平台代表了软骨生物学体外研究的重要进步,能够更精确地模拟 OA 病理生理学和评估实验疗法。© 2024 作者。当前协议》由 Wiley Periodicals LLC 出版。基本方案 1:片上软骨注射 基本方案 2:片上软骨驱动 基本方案 3:片上软骨琼脂糖水凝胶去除 基本方案 4:制备片上软骨以提取 RNA。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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