Department of Mechanical Engineering, University of Delaware, Newark, Delaware 19716, United States.
School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104, United States.
ACS Biomater Sci Eng. 2022 Jun 13;8(6):2564-2573. doi: 10.1021/acsbiomaterials.2c00024. Epub 2022 May 13.
During traumatic joint injuries, impact overloading can cause mechanical damage to the cartilage. In the following inflammation phase, excessive inflammatory cytokines (., interleukin-1β (IL-1β)) can act on chondrocytes, causing over-proliferation, apoptosis, and extracellular matrix (ECM) degradation that can lead to osteoarthritis. This study investigated the combined effects of traumatic overloading and IL-1β challenge on the metabolic activities of chondrocytes. Bovine cartilage explants underwent impact overloading followed by IL-1β exposure at a physiologically relevant dosage (1 ng/mL). New click chemistry-based methods were developed to visualize and quantify the proliferation of chondrocytes in a nondestructive manner without the involvement of histological sectioning or antibodies. Click chemistry-based methods were also employed to measure the ECM synthesis and degradation in cartilage explants. As the click reactions are copper-free and bio-orthogonal, ., with negligible cellular toxicity, cartilage ECM was cultured and studied for 6 weeks. Traumatic overloading induced significant cell death, mainly in the superficial zone. The high number of dead cells reduced the overall proliferation of chondrocytes as well as the synthesis of glycosaminoglycan (GAG) and collagen contents, but overloading alone had no effects on ECM degradation. IL-1β challenge had little effect on cell viability, proliferation, or protein synthesis but induced over 40% GAG loss in 10 days and 61% collagen loss in 6 weeks. For the overloaded samples, IL-1β induced greater degrees of degradation, with 68% GAG loss in 10 days and 80% collagen loss in 6 weeks. The results imply a necessary immediate ease of inflammation after joint injuries when trauma damage on cartilage is present. The new click chemistry methods could benefit many cellular and tissue engineering studies, providing convenient and sensitive assays of metabolic activities of cells in native three-dimensional (3D) environments.
在创伤性关节损伤中，冲击过载会导致软骨的机械损伤。在随后的炎症阶段，过量的炎症细胞因子（例如白细胞介素-1β（IL-1β））可以作用于软骨细胞，导致过度增殖、凋亡和细胞外基质（ECM）降解，从而导致骨关节炎。本研究调查了创伤性过载和 IL-1β 挑战对软骨细胞代谢活性的联合影响。牛软骨外植体经历了冲击过载，随后以生理相关剂量（1ng/mL）暴露于 IL-1β。开发了新的点击化学方法，以非破坏性的方式可视化和定量检测软骨细胞的增殖，而无需进行组织学切片或抗体。还采用点击化学方法测量软骨外植体中的 ECM 合成和降解。由于点击反应是无铜和生物正交的，对细胞毒性可忽略不计，因此可以培养和研究软骨 ECM 长达 6 周。创伤性过载导致大量细胞死亡，主要发生在浅层。大量死亡细胞减少了软骨细胞的整体增殖以及糖胺聚糖（GAG）和胶原蛋白含量的合成，但单独过载对 ECM 降解没有影响。IL-1β 挑战对细胞活力、增殖或蛋白质合成几乎没有影响，但在 10 天内诱导超过 40%的 GAG 丢失，在 6 周内诱导 61%的胶原蛋白丢失。对于过载样品，IL-1β 诱导了更大程度的降解，在 10 天内 GAG 丢失 68%，在 6 周内胶原蛋白丢失 80%。结果表明，当软骨存在创伤损伤时，关节损伤后立即需要缓解炎症。新的点击化学方法可以有益于许多细胞和组织工程研究，为天然三维（3D）环境中细胞的代谢活性提供方便和敏感的测定。
