Hydrogel constructs were cultured in glutamine, cystine, and methionine-free high glucose DMEM (Life Technologies) supplemented with 10% FBS, 100 μg/mL sodium pyruvate, 0.201 mM cystine, 75 μM azidohomoalanine, 25 μM methionine, 1% penicillin/streptomycin, and osteogenic supplement (R&D System). Blocking of nascent fibronectin adhesion was performed using a monoclonal antibody against human fibronectin (2.5–10 μg/mL HFN7.1, Developmental Studies Hybridoma Bank)19 (link),53 (link).
For nascent ECM labeling, hydrogels were washed twice in PBS with 2% BSA, followed by 30 min incubation in 30 μM DBCO-488 (Click Chemistry Tools) at 37 °C/5% CO2. Following three washes (3 min each) with PBS/2% BSA, hydrogels were fixed in 10% formalin for 30 min at room temperature and washed with PBS three times. For imaging, z-stack images at 20 × 0.75 NA and 100 × 1.4 NA were acquired using a Nikon A1R Confocal Microscope. Local ECM thickness was determined by creating binary masks of z-stack images in ImageJ with Otsu’s thresholding, and the ImageJ plugin “BoneJ” was used to calculate the average local ECM thickness per slice19 (link),54 . (Nascent ECM thickness: BoneJ Plugin ImageJ (×180, 0.13 μm/pixel), 3–6 slices measured and averaged for each cell)
For nascent ECM labeling, hydrogels were washed twice in PBS with 2% BSA, followed by 30 min incubation in 30 μM DBCO-488 (Click Chemistry Tools) at 37 °C/5% CO2. Following three washes (3 min each) with PBS/2% BSA, hydrogels were fixed in 10% formalin for 30 min at room temperature and washed with PBS three times. For imaging, z-stack images at 20 × 0.75 NA and 100 × 1.4 NA were acquired using a Nikon A1R Confocal Microscope. Local ECM thickness was determined by creating binary masks of z-stack images in ImageJ with Otsu’s thresholding, and the ImageJ plugin “BoneJ” was used to calculate the average local ECM thickness per slice19 (link),54 . (Nascent ECM thickness: BoneJ Plugin ImageJ (×180, 0.13 μm/pixel), 3–6 slices measured and averaged for each cell)
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