Ex vivo CAM assay

Fertilised White Leghorn chicken eggs were incubated at 37°C in a humidified atmosphere (at >60% relative humidity). After 3 days incubation, 2 to 3 mL of albumin was withdrawn, using a 21 gauge needle, through a small opening at the large blunt edge of the egg to minimise adhesion of the shell membrane with CAM. A square window of 1 cm² was opened in the egg shell and sealed with transparent adhesive tape to prevent dehydration. The eggs were returned for further incubation.

On embryonic day 8, the following implants were placed on top of the CAM:

(1) clusters of equivalent size of hMSCs cultured on CoO 0% and 2% microspheres for 7 days.

(2) equivalent volumes of CoO 0%, CoO 2% and Synth II microspheres embedded in 35 µL type I collagen gel (BD, Bioscience), produced according to manufacturer instruction.

After implantation, eggs window where sealed with transparent adhesive tape and returned to the incubator. Images were taken 7 days post implantation and samples of CAM containing the implant material were harvested and fixed in 4% PFA for 1 h at RT.