Computational protocol: Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin

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Protocol publication

[…] After 21 days of culture, scaffolds were cut in half by a scalpel and fixed in 4% paraformaldehyde (PFA)/4.6% D-Mannitol for 15 min and subsequently stored in 1% PFA/4.6% D-Mannitol until further processing. Fixed scaffolds were submitted to heat-induced epitope retrieval by heating to 95°C in 0.05% citraconic anhydride in milliQ water (pH 7.4) for 15 min, incubated with monoclonal mouse anti-human collagen type I antibody (I-8H5, MP Biomedicals, Santa Ana, CA, USA) diluted to 1 µg/mL in 1.25% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with 0.2% Triton X for 1 h at room temperature, followed by incubation for 30 min at room temperature in Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted in 2.5% BSA/0.05% Tween-20/PBS at a concentration of 2 mg/mL. Cell-seeded whole mount stained scaffolds were counterstained using 4′,6-diamidino-2-phenylindole (DAPI), placed on a coverslip and covered with Dako fluorescent mounting medium (Dako, Glostrup, Denmark). Confocal laser scanning microscopy was performed on a FluoView 1000 confocal laser scanning microscope (CLSM) (Olympus, Center Valley, PA, USA). The scaffold surfaces were visualized using the CLSM in reflection mode. Images were analysed using ImageJ (NIH, Bethesda, MD, USA). [...] The data obtained by cytotoxicity, ALP activity, gene expression, protein and calcium secretion analyses were compared between the groups using Holm-Sidak test following a parametric one-way analysis of variance (ANOVA). Where the equal variance and/or the normality test failed, a Kruskal–Wallis one-way ANOVA on ranks was performed (SigmaPlot 12.3, Systat Software, San Jose, CA, USA). A probability of ≤0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Simvastatin