Elisa for human decorin

After loading, decorin microrods were divided into Protein Lo-Bind tubes (Eppendorf, Hamburg, Germany) consisting of 50,000 microrods/microcentrifuge tube, and then suspended in 1 mL of phosphate buffered saline (PBS) containing 0.05% Tween-20 (pH 7.4). Samples were placed onto a tube revolver (14 RPM) within an incubator (37°C). Decorin microrods were spun down at 8,000 RPM for 3 minutes and the entire supernatant was collected and replenished with 1 mL at 4, 8, 16, 24, 72, 96, 120, 144, 168, 240, 336, 432, 504, and 744 hours. Collected supernatants were immediately flash frozen and stored at − 80°C until further use. An ELISA for human decorin (Abcam, Cambridge, UK) was performed per the manufacturer’s instructions and hourly release amounts were calculated using an established standard curve using human decorin (Abcam, Cambridge, UK).

To passively load microrods with decorin, 750,000 microrods were concentrated in 1 mL of deionized (DI) water and then 333 μL of decorin (600 μg/mL) was added. The microrods were passively loaded via incubation with inversion over four days at 4°C. After incubation, the microrods were centrifuged at 15.000 × g for 10 minutes and the supernatant was removed. The microrods were then recentrifuged at 15.000 × g for 10 minutes once more to concentrate them and remove any residual solution. The microrods were then resuspended in saline to the desired concentration. Decorin loading was assessed via NanoOrange Protein Quantitation Kit (Thermo Fisher, Waltham, MA). Values were validated with an ELISA for human decorin (Abcam, Cambridge, UK) (data not shown). Decorin loading was determined by calculating the amount of free decorin in the supernatant post-centrifugation, and then subtracting that value from the initial amount of decorin added (200 μg), and then dividing that value by 750,000 to get the amount of decorin μg) per microrod.

After loading, decorin microrods were divided into Protein Lo-Bind tubes (Eppendorf 022431102, Hamburg, Germany) consisting of 50,000 microrods/microcentrifuge tube, and then suspended in 1 mL of phosphate buffered saline (PBS) containing 0.05% Tween-20 (pH 7.4). Samples were placed onto a tube revolver (14 RPM) within an incubator (37 °C). Decorin microrods were spun down at 8000 RPM for 3 min and the entire supernatant was collected and replenished with 1 mL at 4, 8, 16, 24, 72, 96, 120, 144, 168, 240, 336, 432, 504, and 744 h. Collected supernatants were immediately flash frozen and stored at −80 °C until further use. An ELISA for human decorin (Abcam) was performed per the manufacturer’s instructions and hourly release amounts were calculated using an established standard curve using human decorin (Abcam).