Tracking Spatiotemporal Extracellular Matrix Evolution and Tissue Fusion in 3D Microtissues via Click Chemistry-Based...

The extracellular matrix (ECM) is a complex network of macromolecules that forms the cellular microenvironment. Proper regulation of ECM formation is crucial for maintaining tissue homeostasis, and understanding the intricate processes involved in tissue growth, maturation, and degeneration is vital for developing functional regenerative solutions. However, investigating large-scale ECM dynamics within cell-dense engineered 3-dimensional (3D) tissues remains a challenge. To address this, we introduce a technique to metabolically label nascent proteins in 3D cartilage microtissues and track the spatiotemporal evolution of ECM. Non-canonical amino acids (L-azidohomoalanine) are incorporated into newly secreted proteins and fluorescently labeled, enabling visualization of the nascent matrix. Labeling cartilage microtissues at early (Day 1), mid (Day 7) or late (Day 14) time points during microtissue formation revealed increased nascent protein deposition at the microtissue periphery. Comparing the protein secretion over time, we observed significant protein deposition at late times compared to minimal ECM formation at early time points. Furthermore, protein deposition during microtissue fusion was studied using a 3D bioassembly model, enabling tracking of tissue integration over time. Ultimately, this approach is a powerful tool for uncovering mechanisms involved in tissue formation in dynamic 3D microenvironments at a clinically relevant scale, with potential implications for new regenerative strategies.