Microstructure characterization with scanning electron microscopy (SEM) was carried out by adapting the protocols in [35 (link),41 (link)]. Briefly, COL1, HC-L-ECM and LC-L-ECM thin structures were fixed for 48 h in 4% PFA in PBS and then washed three times in 0.1 M phosphate buffer (PB) for 10 min. Resulting samples were incubated in 4% osmium tetroxide for 90 min followed by successive washes in deionized water until there was absence of osmium tetroxide. Then, samples were dehydrated in increasing concentrations of ethanol solutions and preserved in absolute ethanol at 4 °C and critical point dried using an autosamdri-815 critical point dryer (Tousimis, Rockville, MD, USA). Samples were mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA) for imaging and were carbon coated before imaging with a JSM-6510 (JEOL, Tokyo, Japan) scanning electron microscope at 15 kV. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to process the SEM images. Density of fibers per area was obtained by applying a threshold to isolate the fibers in a specific plane to quantify them, while diameter of fibers was estimated as the average of ten randomly-selected fibers in three different zones of each sample.
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