Figure 1.

Chondrocytes secrete extracellular matrix proteins and proteoglycans that are heterogeneous and can be visualized with metabolic labeling. A) Schematic of nascent protein and proteoglycan metabolic labeling. The azide-modified methionine analog (azidohomoalanine) or mannose analog (N-azidoacetylmannosamine tetraacylated) are added to the culture media and incorporated into nascent proteins (for example, collagen type II and collagen type VI) or proteoglycans (for example, aggrecan core protein and its associated chondroitin sulfate residues), respectively. The biorthogonal Cu(I)-free strain-promoted cyclo-addition between the azide and DBCO-modified fluorophore (DBCO-488) enables visualization of either the nascent proteins or proteoglycans. B) Representative images (magnifications on the right) of nascent proteins (white, co-stained with either collagen type II and VI) or proteoglycans (cyan, co-stained with aggrecan and chondroitin sulfate) deposited by bovine chondrocytes encapsulated in norbornene-modified hyaluronic acid (NorHA) hydrogels and cultured for 7 d in chondrogenic media (scale bars 10 μm). C) Representative images of nascent proteins (white, left) and proteoglycans (cyan, right) for a panel of chondrocytes (red) after 7 d of culture (scale bars 10 μm). D) Radial profiles of fluorescence intensities of nascent proteins (gray) and nascent proteoglycans (cyan) after 7 d of culture. Lines represent median intensity profiles, shaded areas represent standard deviation (n = 40 cells per group from two independent biological experiments).