Background: In this paper we present generation and preliminary assessment of multiphase anisotropic tissue structures by microdroplet based hydrogel printing method. Current cell/tissue scaffolding methods present shortcomings due to the lack of control over the spatial and temporal control over cell seeding and extracellular matrix composition. Microdroplet based hydrogel bioprinting technology can be used to engineer complex tissue anisotropies with multiple phases by producing scaffolds with controlled micro-scale spatial heterogeneity in extracellular, cellular compositions and physical properties. Therefore, we hypothesized that the microdroplet-based hydrogel bioprinting approach developed in our laboratory will successfully facilitate engineering of the complex tissue anisotropies that are composed of multiple phases. In order to test this hypothesis we printed agarose hydrogel bioinks colored with red, green and blue (RGB) high molecular weight (35-38 kDa) fluorescent dyes and assessed the phase transitions via image processing to evaluate the anisotropy of the resulting multiphase structure by measuring the RGB color intensities.
Methods: Microdroplet generation process was performed with multiple ejectors in sterile laminar flow hood under controlled humidity. The inter-droplet distance was determined by the size of the droplets residing on the substrate. The prepared bio-inks (RGB colored hydrogels) were printed in a staggered configuration. The ejector was kept warm (37 degC) to minimize viscosity changes and premature gelation of the hydrogel. Printed staggered phases were gelled by incubation at 4 degC for 5 minutes. The diffusion and integration of the phases was assessed immediately after and 3 hours after printing by taking micographs and analyzing using ImageJ software. RGB color relative intensity values were used analytically to analyze the anisotropic gradient of the phases and phase transitions.
Results and Discussion: The printed multiphase hydrogel structure representing an anisotropic tissue unit displayed sharp RGB boundaries between the phases immediately after printing. These sharp boundaries disappeared and smooth transitions emerged within 3 hours. These results suggest that microdroplet based hydrogel printing technology can be used to create highly anisotropic structures with smooth boundaries mimicking the complex cellular and extracellular gradients in the natural tissues. Our long term goal is to develop effective bioprinting methodologies to engineer micro-scale anisotropic complex tissue structures with multiple phases, which can be incorporated into currently available biomaterials to face the challenges of incompatibility at tissue-biomaterial interfaces.