Sputter coater

For scanning electron microscopy (SEM), 100 μl of the OB-containing solution (10⁹ OBs/ml) were incubated with 300 μl of acetone at 25°C for 1 hour. The solution was loaded in a metallic stub, dried overnight at 37°C, coated with gold in a Sputter Coater (Balzers) for 3 min, and observed in a scanning electron microscope Jeol JSM 840 A at 10 kV. For transmission electron microscopy (TEM) pellets of purified granules were fixed in Karnovsky fixative (2.5% glutaraldehyde, 2% paraformaldehyde, in 0.1 M, cacodylate buffer, pH 7.2) for 2 h, post-fixed in 1% osmium tetroxide in the same buffer for 1 h and then stained en bloc with 0.5% aqueous uranyl acetate, dehydrated in acetone, and embedded in Spurr’s low viscosity embedding medium. The ultrathin sections were contrasted with 2% uranyl acetate and observed in a ZEISS TEM 109 at 80 kV.

Following MSC culture on micropatterned substrates, samples were incubated in BRB80 buffer (PIPES [80 mm], MgCl₂ [1 mm], EGTA [1 mm], pH 6.8 with KOH) + digitonin [0.001%] for 10 min. Fixation was performed for 2 h in PHEM buffer (HEPES [5 mm], PIPES [60 mm], EGTA [10 mm], MgCl2 [2 mm], pH 7 with KOH) supplemented with glutaraldehyde [2%] and tannic acid [0.1%]. Samples were subsequently incubated in Osmium tetroxide 0.1% for 30 min, dehydrated through 5 min washes in 10%, 20%, 40%, 60%, and 80% ethanol and washed twice for 5 min in 100% ethanol. Critical point drying was then performed using a Balzers CPD 030, followed by sputter coating with a 10 nm platinum layer using a 328 Cressington sputter coater. Samples were imaged using a Hitachi S5200 scanning electron microscope.

Surgically debrided tissue was fixed and processed for SEM imaging. First, fresh tissues were sectioned into approximately 5 mm³ pieces, fixed in 2.5% paraformaldehyde (PFA)/glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4; Electron Microscopy Sciences, Hatfield, PA) for 2 h at room temperature followed by a second fixation in 1% osmium tetroxide (Electron Microscopy Sciences) for 20 min. Subsequently, samples were rinsed with ddH₂O and serially dehydrated in a 50/75/80/90/100/100% ethanol series before critical-point drying in hexamethydisilazane (HMDS; Electron Microscopy Sciences). The desiccated samples were coated with gold using a sputter coater (Balzers; Schaumburg, IL, USA) and surface topography was examined by scanning electron microscope (S-4800; Hitachi, Troy, MI, USA, in the Biomedical Engineering and Physical Science Shared Resource, NIBIB, National Institutes of Health, Bethesda, MD, USA) at 5 kV with various magnifications.