N isopropylacrylamide 99 nipam

Laponite crosslinked pNIPAM‐co‐DMAc (NPgel) hydrogel was prepared as previously described.45 Briefly a 10 mL exfoliated suspension of 0.1 g Laponite clay nanoparticles (25‐30 nm diameter, <1 nm thickness) (BYK Additives Ltd, Cheshire, UK) in 18 mΩ deionized H₂0 was prepared. To 10 mL exfoliated clay suspension, 0.773 g N‐isopropylacrylamide 99% (NIPAM) (Sigma, Poole, UK); 0.117 g N, N′‐dimethylacrylamide (DMAc) (Sigma, Gillingham, UK), and 0.01 g 2‐2′‐azobisisobutyronitrile (AIBN) (Sigma, Poole, UK) were added, mixed well, and strained through 5 to 8 μm pore filter paper, polymerization was performed at 80°C for 24 hours. Hydrogel suspension was cooled to 38°C to 39°C prior to cell incorporation.

An exfoliated suspension of Laponite^® clay nanoparticles (25–30 nm diameter, <1 nm thickness) (BYK Additives Ltd, Cheshire UK) was prepared by vigorous stirring of Laponite^® (0.1 g) in deionised H₂0 (18 MΩ) (9.0 mL) for 24 h. N-isopropylacrylamide 99% (NIPAM) (0.9 g) (Sigma, Poole, UK) and 2–2ʹ-azobisisobutyronitrile (AIBN) (9 mg) (Sigma, Poole, UK) were added to the suspension and stirred for 1 h. After passing the suspension through 5–8 μm pore filter paper, polymerisation was initiated by heating to 80° C and the reagents were allowed to react for either 4 h (L-pNIPAM-4h) or 24 h (L-pNIPAM-24h), to investigate the effect of reduced synthesis time on the biocompatibility of pre-set hydrogel constructs following subcutaneous implantation (see section 2.2.1). It was observed that after heating the monomeric suspension to 80° C, the transparent liquid transformed to a milky suspension. Following 4 (L-pNIPAM-4h) or 24 h (L-pNIPAM-24h) the hydrogel suspension was cooled to room temperature which resulted in a solidified hydrogel.