Gamma 1 20

MSCs in 3D scaffolds at 1 day of culturing were fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, EMS, USA) for 2 hours, post-fixed with 1% osmium tetroxide (EMS), and rinsed with sterile DPBS followed by washing with DI water. Samples were snap-frozen at –80 °C and lyophilized by a freeze dryer (Gamma 1–20, Christ, Germany). The samples were then mounted on a strip and sputter-coated with gold (Ion Sputter JFC-1200, Jeol, Japan). All images were photographed under an ultra-high resolution SEM (JSM 7600 F, Jeol, Japan) at an accelerating voltage of 5 kV. At least three independent repeats were performed. Cells within a pore in the 3D scaffold were individually pseudocolored using Photoshop CC software (Adobe Systems Incorporated, USA).

The minced meat of the left MPS was freeze-dried in a lyophilization unit (Gamma 1-20, Christ^®, Osterode, Germany), pulverized in a mixer mill (MM 400, Retsch^®, Haan, Germany) and analyzed by gaschromatography (GC TRACE 1300, ThermoScientific^®, Dreieich, Germany; SP-2560 Column, Supelco, Bellefonte, USA; carrier gas: nitrogen) with a modified method according to Lepage and Roy [17 (link)]. In brief, a methanol-hexane-tridecanoic-acid mixture was utilized as standard. Subsequently, acetyl chloride was added and the sample was heated, followed by the addition of potassium chloride solution. The measurement was carried out after centrifugation with the superior hexane phase.