Supplementary Material for: A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1β exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-β1 pathway. Notably, TGF-β1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1β exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

关节软骨（articular cartilage）在发育、健康状态及疾病进程中，均受到机械载荷的关键调控作用。然而，目前学界对于可促进工程化软骨（engineered cartilage）成熟或组织修复的机械载荷条件，认知仍存在显著不足。当前可精准调控局部机械环境的体外模型（in vitro model），却因通量极低而存在极大局限——通常单次实验仅能处理少量样本。为突破这一研究瓶颈，本团队开发了一款可对组织构建体进行高通量压缩加载的新型装置：软骨工程高通量机械激活仪（High Throughput Mechanical Activator for Cartilage Engineering, HiT-MACE），其单次可处理的样本量是现有技术的6倍。借助HiT-MACE，单次实验即可对至多24个样本施加生理范围内（如等同于行走及日常活动）与超生理范围内（如损伤性冲击或过度加载）的循环载荷。本研究中，我们将关节软骨对生理及超生理机械载荷的早期应答，与白介素1β（IL-1β）刺激后的应答进行了对比——IL-1β刺激是一种常用但较为基础的软骨骨关节炎体外模型。生理载荷可快速上调转化生长因子β1（TGF-β1）通路下合成代谢标志物的基因表达，值得注意的是，本次实验的培养基中未添加TGF-β1或血清。超生理载荷仅引发轻度的分解代谢应答，而IL-1β刺激则驱动了快速的合成代谢转变。这一结果与近期研究发现高度吻合，后者表明过度加载是一种更具生理逼真性的软骨退变模拟模型。综上，本研究结果表明，借助HiT-MACE可使用更大规模的样本量生成更高通量的数据，从而有效开展软骨力学生物学（mechanobiology）研究，这将助力开发针对退行性软骨病变的更高效修复与康复策略。