Preparation for transmission electron microscopy (TEM)

Another set of hMSC-seeded samples was prepared for TEM. The samples were washed with sodium cacodylate buffer and post-fixed post-fixation in 1% osmium tetraoxide for 1 hour and en bloc stained with 2% aqueous uranyl acetate before dehydrated in a graded ethanol series. Infiltration of Spurr’s resin was initiated with an ethanol/resin mixture in a 1:1 followed by 1:2 ratio. Finally the samples were embedded in resin with the orientation that the sectioning surface was perpendicular to the grating axis. The samples were embedded with 2 changes of resin and cured at 60 °C until hardened.

The embedded samples were trimmed and sectioned to a thickness of 90 nm using the ultratome cutter (Duke University School of Medicine Research Electron Microscopy Service, Durham, NC). The samples were sectioned perpendicular to the grating axis to obtain a cross-sectional view of the gratings and the aligned cells. After section, samples were imaged with a transmission electron microscope (CM-12, Philips/FEI, Hillsboro, OR, USA) at 80 kV. As the 5 mm wide samples were sectioned, a population of cells sectioned at the center and the edge would be observed. Cross-sectional images were taken both at the center and at the edge of cells. The nucleus was used as a reference as the size and height of the hMSC nuclei have been previously characterized¹⁸. Specifically, the cell height and width of the cells, whether the nucleus was observed, and the width and height of the nucleus were used as an indicator if the section was made at the center or at the edge.

To ensure that the integrity of hMSC and substrate grating samples remain intact after the sectioning process, the integrity of the intracellular structures in hMSCs were observed before accepting the samples for the subsequent TEM measurement (see Supporting Material).