Polysorbate 20

Heparin microparticles were fabricated as previously described (37 (link),38 (link)). Briefly, EDC/Sulfo-NHS chemistry was used to substitute methacrylamide groups on heparin. Heparin methacrylamide was then dissolved in phosphate-buffered saline (PBS) and mixed with equimolar amounts of the free radical initiators, ammonium persulfate (Sigma Aldrich) and tetramethylethylenediamine (TEMED, Sigma Aldrich). A water-in-oil emulsion was then formed by adding the heparin solution dropwise into 60mL of corn oil and 1mL of polysorbate 20 (Promega) and then homogenized on ice for 5 min at 3000rpm (Polytron PT3100 Homogenizer, Kinematica). Free radical polymerization and thermal cross-linking of the methacrylamide groups was carried out by immersing the emulsion in a 55˚C water bath under constant stirring and nitrogen purging for 30 minutes. The HMPs were collected following centrifugation for 10 minutes at 3000rpm and subsequently washed in acetone, deionized water several times, and 70% ethanol for sterilization. HMPs were lyophilized and stored at 4˚C until ready for incorporation into the alginate constructs. HMPs were characterized following fabrication and confirmed to retain their functionality with evaluation of growth factor binding and release kinetics (38 (link)).

Lyophilized GMA was fully dissolved in water (10% w/v) at 37°C. Sixty mL of corn oil (Mazola) was heated to 37°C prior to the addition of 1 mL of polysorbate 20 (Promega, Fitchberg, WI) and homogenized at 1500 rpm (Polytron PT-3100 homogenizer) for 3 minutes. The 10% GMA solution was mixed with 3 μL of 0.3 M ammonium persulfate (APS) (Bio-Rad, Hercules, CA) and added dropwise to the corn oil phase. The oil and water emulsions were homogenized for 5 minutes at 1800 rpm for 15% and 50% GMA, and 1500 rpm for 90% GMA (to obtain MPs of similar size as 15% and 50% GMA MPs), and placed on a hotplate set to 45°C with agitation via stir bar. N₂ gas was bubbled through the emulsion for 20 minutes to purge oxygen. Under constant stirring, the hotplate was increased to 100°C to initiate the thermal cross-linking reaction and allowed to proceed for 40 minutes. The particle/corn oil mixture was centrifuged at 2500 rpm at 4°C to harvest the microparticles, and excess corn oil was removed with four successive washes with deionized water. In order to visualize the hydrogel microparticles, fluorescent labeling was performed by incubation with Alexa Fluor ® succimidyl ester 594 (Invitrogen, Carlsbad, CA) in 0.1 M sodium bicarbonate buffer followed by four deionized water washes. All particles were stored at 4°C in dH₂O until further use.

HMPs were fabricated as previously described [32 (link)] and as schematized in Figure 1A. Briefly, 55.6 mg of heparin methacrylamide, 18 mM ammonium persulfate (APS; Sigma Aldrich), and 18 mM tetramethylethylenediamine (TEMED; Sigma Aldrich) were dissolved in phosphate buffered saline (PBS; Corning Mediatech, Manassas, VA). 500 μL of heparin solution was homogenized for 5 minutes at 3000 rpm into a mixture of 60 mL of corn oil (Mazola) and 1 mL of polysorbate 20 (Promega, Madison, WI) using a Polytron PT3100 homogenizer (Kinematica, Lucerne, Switzerland). The emulsion was immersed in a 55°C water bath, and the free radical cross-linking reaction proceeded under constant stirring and nitrogen purging for 30 minutes. The solution was centrifuged for 10 minutes at 3000 rpm, and the resulting HMP pellet was washed once in acetone and several times in ddH₂O, prior to being disinfected in 70% ethanol for 30 minutes. The HMPs were lyophilized (Labconco, Kansas City, MO) and stored at 4°C until use.