Viability Assay of hADMSCs on Scaffolds

Cell viability was monitored using the Viability/Cytotoxicity Assay Kit for Animal Live and Dead Cells (#3000, Biotium, CA, USA), according to the manufacturer’s instructions. Scaffolds seeded with hADMSCs were evaluated for cell viability and colonization after 1 week of proliferation in MSC expansion medium. In addition, visualization of the live cells attached to the UP scaffolds was examined after 2, 5 and 8 weeks culture in osteogenic differentiation medium. The constructs were captured as z-stack images using the EZ—C1 3.20 software. For live/dead staining, hADMSCs/scaffold constructs were doubly stained with calcein AM and ethidium homodimer, staining living and dead cells, respectively. For each scaffold, ten serial sections were taken with a step/section set at 50 μm in the Z direction. In total, an area of 500 μm in height was analyzed for each scaffold at each time point, so both cell attachment and penetration along the z-axis could be visualized. Fluorescence observations were performed by a confocal upright fluorescence microscope (Nikon D-Eclipse 80i C1). Quantification of fluorescence intensity was determined using Image J software (NIH, Bethesda, MD, USA). Corrected total cell fluorescence (CTCF) was calculated using the formula CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings) [77 (link)].