Smz 10 stereomicroscope

Total collagen production by MC3T3-E1 pre-osteoblasts on 3D-printed scaffolds was visualized and quantified using a picrosirius red stain kit (Chondrex, Inc., Redmond, WA, United States). After 7, 14, 21, and 28 days of culture, one part (1/8th) of the cell/scaffold construct was washed with PBS thrice and fixed in 4% formaldehyde. Fixed constructs were stained for 2 h with picrosirius red at room temperature. Then, constructs were washed twice with acidified water (5 ml acetic acid/L distilled water) to remove the unbound stain, and collagen production was visualized using a Nikon SMZ-10 stereomicroscope (Nikon, Tokyo, Japan) and a Leica inverted microscope (Leica Microsystems, Wetzlar, Germany). For semiquantitative collagen analysis, picrosirius red stain was eluted from the constructs using 0.2 M NaOH/methanol (1:1, v/v) for 30 min under shaking. A 100 μl of this solution per well of a 96-well plate (Greiner, Bio-One, Alphen aan den Rijn, Netherlands) was used to determine the absorbance at 490 nm, with a microplate reader (BioRad Laboratories Inc., Veenendaal, Netherlands). Constructs were weighed after 24 h of air drying at room temperature. Data were normalized to the weight of the dried construct and expressed as absorbance/g. Constructs were assayed in triplicate.

Dissections were performed under a Nikon SMZ‐10 stereo microscope (Nikon) in wax‐poured 35 × 15 mm Petri dishes (Fisher Scientific). Before RNA isolation, the tissues were dissected and separated into 1.5 ml Eppendorf tubes containing 250 μl of RNAlater stabilization solution (Thermo Fisher Scientific), and held at 4°C for 24 h, after which the RNAlater solution was decanted following the manufacturer protocol. Subsequently, the tissues were stored at −80°C until RNA isolation. The whole‐body samples of respective life stages were cut into ca. 0.25–0.50 cm‐sized pieces with dissection scissors and then processed in RNAlater using the same protocol described above for various tissues. To isolate protein from whole bodies or tissues, homogenization was conducted in PBS (pH 7.4 and 0.1 M) using a glass mortar and Teflon pestle. Homogenates were then centrifuged for 15 min at 15,000g and 4°C to obtain nuclear and mitochondrial fraction pellets and supernatant containing the cytosolic protein fraction. Only the cytosolic protein fractions were used for further analyses.