Optimization of Cryomill Settings to Create Extrudable Decellularized Extracellular Matrix for Bioink Applications

As the field of tissue engineering continues to revolutionize treatment strategies for a variety of conditions, 3D bioprinting platforms have driven advancements in recapitulating physiologically relevant characteristics of the native tissue microenvironment to support the regeneration of various tissues. Similarly, advancements in bioink formulations have allowed for greater biocompatibility, especially via the incorporation of decellularized extracellular matrix (dECM) to preserve the native and biochemically complex composition of the ECM. Decellularized ECM grafts derived from porcine small intestinal submucosa (SIS) are commercially available and have been widely used to promote healing in multiple clinical settings including orthopedics, gastrointestinal surgery, and cardiovascular surgery. This ECM’s biodegradability and favorable composition of fibrous proteins, glycosaminoglycans, and other bioactive molecules make it a promising candidate for use as a bioink component to promote wound healing. <br/><br/>One strategy for incorporating a porcine SIS-derived graft into a bioink formulation involves processing the graft into a particulate with a sufficiently small particle size distribution to allow for controlled extrusion through a bioprinter’s nozzle. This micronization may be effectively performed using a cryomill, as cooling with liquid nitrogen allows the intact graft to be embrittled for efficient milling into a fine particulate. Unsurprisingly, variations in cryomilling parameters impact the final particle size and, furthermore, the extrudability of resuspended porcine SIS particulate. In this study, various milling parameters were assessed for generating porcine SIS-derived particulate capable of passing through a 25-gauge needle upon resuspension with saline. Parameters of precooling time, milling time, grinding ball size, and grinding ball number were varied across multiple cryomill cycles to generate particulates and maximize the flow of solids through a 25-gauge needle. This study underscores the importance of optimizing cryomill cycle parameters to achieve particle size distributions appropriate for 3D bioprinting skin grafts from decellularized ECM.