Magnetic polystyrene beads

Collagenase 1A (Sigma Aldrich) was adsorbed to magnetic polystyrene beads (4–5 μm in diameter, Spherotech, Lake Forest, IL), as previously described [30 (link)]. Briefly, a 25 μL aliquot of Spherotech bead solution was incubated in 1 mL of 2 mg/mL of collagenase for 2 hours at room temperature in a low retention 1.5 mL tube (Axygen, Pittsburgh, PA). Collagenase-coated beads were separated from solution via a magnet, the supernatant removed, and unbound collagenase was removed via 3 PBS washes. A BCA assay was used to determine the amount of beads necessary to obtain 1 μg collagenase. Beads with 1 μg of collagenase were added to 0.5 mg of each MP formulation in 1 mL PBS with 0.036 mM CaCl₂. The collagenase beads and MPs were rotated continuously at 37°C, and the collagenase coated-beads were replaced with freshly coated beads under sterile conditions every 48 hours. At specified time points, collagenase-coated magnetic beads were removed and the MP suspension was transferred to a new tube with 0.5 mL of 0.03% fluorescamine in DMSO. Increasing values, indicating the generation of new amino terminus groups by peptide bond cleavage by collagenase, were determined on a Biotek plate reader with an excitation of 390 nm and emission of 465 nm, and compared to non-degraded MPs and completely degraded MPs (50 μg collagenase for 48 hours).