Protein-Derived Jammed Microgels as Inks for Extrusion 3D Printing of Bioactive Scaffolds

Extrusion 3D printing has emerged as a promising technique in tissue engineering, enabling the creation of personalized scaffolds for individual patients. Typically, natural hydrogels are utilized as inks due to their resemblance to the extracellular matrix (ECM), as well as their biodegradability and biocompatibility, making them ideal for tissue engineering applications. One such biomaterial is human platelet lysates (PL) derived from whole blood, which contains a rich assortment of growth factors, cytokines, and proteins that can stimulate cell proliferation and differentiation, and tissue regeneration. However, the use of PL as ink for printing scaffolds is limited by its liquid nature even at high concentrations, preventing the formation of a solid and stable filament upon deposition.<br/>To address this challenge, granular hydrogels of methacrylated PL (PLMA) were developed through chemical coupling. These granular hydrogels consist of jammed microgels that exhibit viscoelastic solid properties at low levels of strain while flowing under high levels of strain. This unique characteristic enables filament formation and facilitates their application as injectable biomaterials. Additionally, a granular ink of methacrylated bovine serum albumin (BSAMA) was prepared for comparison, leveraging the high albumin content of PL. The microgels were generated by extruding bulk hydrogels through a series of nozzles with progressively higher gauges (18G, 21G, and 23G), followed by centrifugation to remove excess liquid phase and obtain granular hydrogels. Both materials displayed irregular microgels and a wide size distribution. Moreover, the process resulted in albumin microgels with a larger average size, leading to increased porosity compared to the PLMA hydrogel.<br/>Rheological analysis revealed that both inks exhibited shear-thinning, yield-strain, and self-healing properties—essential characteristics for materials used in 3D printing. Additionally, a time sweep test utilizing UV light demonstrated a rapid increase in the storage modulus of the hydrogels due to photo-crosslinking, indicating rapid stabilization of the printed scaffolds. The study of filament formation using different nozzles allowed an evaluation of the printing fidelity of BSAMA and PLMA inks. In all cases, the filament diameter closely matched the internal diameter of the needles, and thus the smallest nozzle tested (22G) was selected for printing the scaffolds. Due to its larger microgels, the printing of the BSAMA presented some clogging, requiring higher pressure for constant extrusion compared to the PLMA ink. Nevertheless, stable scaffolds with varying geometries and layer numbers were successfully printed, confirming the efficacy of this strategy in producing protein inks with the necessary printing properties.<br/>Finally, the bioactivity of the granular hydrogels was assessed by seeding human adipose-derived stem cells (hASCs) for a period of 7 days. Metabolic activity increased at each time point, being consistently higher in PLMA hydrogels. This can be attributed to cell proliferation in the spaces between the microgels, with cells adhering around them from the first day, as indicated by the Live/Dead assay. Furthermore, only a small number of dead cells were detected, indicating the non-cytotoxic nature and biocompatibility of the inks. The DAPI/Phalloidin assay confirmed a higher cell count in the PLMA scaffolds from day 1, highlighting the superior bioactivity of this material. In summary, bioactive granular hydrogels of protein sources with microporosity were successfully created, demonstrating their potential for 3D printing stable scaffolds with excellent resolution for use in tissue engineering applications.<br/>[1] Qazi, T. H., et al. (2022). ACS Biomater. Sci. Eng. 8, 1427<br/>[2] Santos, S. C., et al. (2018). Adv. Healthc. Mater. 7, 1800849