Bovine serum albumin in dh20

Electric field stimulation was used in order to assess the functional capacity (force generation) of tissue engineered constructs. Constructs were washed twice in PBS, and one end of the construct removed from the supporting mould pin. The free end of the construct was then attached to the force transducer (403A Aurora force transducer, Aurora Scientific, CA) using the eyelet present in the construct. The construct was positioned to ensure its length was equal to that before removal from the pin and covered (3mL) with Krebs-Ringer-HEPES buffer solution (KRH; 10mM HEPES, 138 mM NaCl, 4.7mM KCl, 1.25mM CaCl2, 1.25mM MgSO4, 5mM Glucose, 0.05% Bovine Serum Albumin in dH20, Sigma, UK).
Aluminium wire electrodes, separated by 10mm, were positioned parallel either side of the construct to Instruments, UK) connected to a custom-built amplifier. Maximal twitch force was determined using a single 3.6V/mm, 1ms impulse and maximal tetanic force was measured using a 1 second pulse train at 100Hz at 3.6V/mm, generated using LabVIEW 2012 software (National Instruments). Twitch and tetanus data were derived from 3 contractions per construct, and a minimum of 2 constructs per time point per biological repeat. Data was acquired using a Powerlab system (ver. 8/35) and associated software (Labchart 8, AD Instruments, UK).