Double sided carbon tape

Samples were fixed in 4% formaldehyde for 24 h and dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) for 10 min each and then treated with hexamethyldisilane (Sigma-Aldrich) overnight for further water extraction. Dehydrated samples were then mounted on aluminium stubs with double sided carbon tape (Agar Scientific, Essex, UK), and imaged with a tabletop scanning electron microscope (TM3030Plus, Hitachi Hightech, Tokyo, JP) at appropriate magnifications.

Joint surface replicas were cast using Crystal Clear 202 acrylic resin (Smooth-On, Bentley Advanced Materials, London, UK) according to manufacturer’s instructions (Fig. 2). Pre-warmed (37 °C) parts A and B (1:9 ratio) were mixed thoroughly and vacuum de-gassed. Resin was poured into joint surface molds by gravity flow using a fine pipette tip with the aid of a dissecting microscope (MZ9, Leica Microsystems, UK) and bubbles were removed from mold-resin interfaces. Casts were set for 16 h at 18–23 °C, post cured for 6 h at 65 °C and removed from molds after 7 days at room temperature, according to manufacturer’s instructions. Each cast was secured to a custom-produced 10 × 10 × 0.3 cm aluminum raft with double-sided carbon tape (Agar Scientific, Stansted, UK). Rafts of samples were coated with >20 nm carbon using a high-vacuum bench-top carbon coater (Agar Scientific).

Biomaterials were fixed in 4 % formaldehyde overnight and were dehydrated with increasing concentrations of ethanol (30 %, 50 %, 70 %, 90 %, 100 % and 100 % again) for 10 min each. The biomaterials were then treated with hexamethyldisilazane (Sigma-Aldrich, USA) overnight for further water extraction. Finally, they were mounted on aluminium stubs using double sided carbon tape (Agar Scientific, UK), and imaged using a tabletop scanning electron microscope (TM3030Plus, Hitachi Hightech, Japan) at various magnifications.