Microgel-Embedded DLP Bioink for Printing Complex Tissue Structures

<b>Purpose: </b>Digital light processing (DLP) is a vat polymerization printing method that forms complex structures through a layer-by-layer process. The high resolution, fast printing time, and cell-friendly nature of the printing process has resulted in numerous biomedical applications for DLP. Monolithic polymer solutions are generally used to form DLP inks, leading to the formation of bulk hydrogel structures with limited tunability. By leveraging the heterogeneity of composite systems, DLP can be used to print structures that more accurately represent the behavior of natural tissues. In this study, we propose the use of embedded spherical microgels within a DLP photoink to form complex cell-laden tissue grafts. To achieve this, a DLP photoink will first be fabricated using gelatin-methacrylate (GelMA). Microfluidics will then be used to generate monodisperse GelMA microparticles with two different polymer concentrations. These particles will then be incorporated with the ink at varying volume ratios to assess effects on printability and mechanical properties of the resulting structures.<br/> <br/><b>Methods: </b>The DLP photoink was formed by mixing GelMA (12.5%), LAP photoinitiator, and tartrazine photoabsorber. Microgels were formed with a concentration of 8% or 12.5% GelMA and added to the ink at varying volume fractions from 5% to 75%. Rheological measurements were used to assess the pre-print viscosity and gelation time of different groups. Compression testing and nanoindentation were performed to further investigate how both the bulk and surface stiffness were affected by microgel inclusions. Using ANSYS, a computational simulation of compression was performed to assess the force distribution within printed constructs. To assess the feasibility of use as a complex tissue graft, multi-cell laden constructs will be formed using DLP. By encapsulating hMSCs within microgels and adding HUVECs to the ink precursor, anatomical structures with a heterogenous cell population will be formed. Alamar blue assays and staining cultured constructs for angiogenic markers will indicate the ability of the microgel-embedded system to promote blood vessel formation <i>in vitro</i>. The chick chorioallantoic membrane assay (CAM assay) will assess <i>in ovo</i> angiogenesis by quantifying the enhancement of blood vessel formation.<br/> <br/><b>Results: </b>Fluorescence imaging indicated control over microgel size using microfluidics, showing a uniform size of 120±15μm. Rheological tests showed that above 50%, microgel-embedded inks are too viscous for proper use as a DLP ink. The addition of microgels also reduces crosslinking density of the overall system, reducing the gelation time and overall stiffness of the structures. This trend was consistent through measurement of the storage modulus, bulk compressive modulus, and local Young’s modulus at the surface. Computational simulations showed that the embedded structures produced more representative non-uniform stresses in response to applied strain as compared to uniform stress in the bulk structure. Additionally, lower stresses were produced by microgels which speaks to their ability to preserve encapsulated cells via stress shielding. Cell encapsulation in addition to in vitro and in ovo assays on printed structures will highlight the ability of the composite system to integrate with host systems and promote angiogenesis.