Ultrafast Laser Micromachining to Form Capillary-Sized Networks within Bioprinted Constructs

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Bioprinting has emerged as a promising approach to generate functional tissues and organs, as well as tissue analogs for regenerative medicine, pharmacology, toxicology, and disease modeling. Bioprinting technologies generally involve the encapsulation of living cells within biocompatible bioinks (such as hydrogels). However, with current technologies, capillary-sized networks cannot be printed within bioprinted constructs. These capillary-sized networks are critical for maintaining viability of tissue constructs and, therefore, are at the center of establishing the widespread application of current technologies. Ultrafast pulsed lasers are uniquely capable of machining capillary-sized networks within bioprinted constructs, however the impact of ultrafast laser machining on biological constructs is not known. This award supports fundamental research on effects of ultrafast laser micromachining on cellular damage. Research results will be useful for the development of a novel hybrid process combining laser micromachining and stereolithography bioprinting to produce bioprinted constructs that contains capillary-sized networks.The research objective is to establish the relationship between ultrafast laser variables and cellular damage within cell-laden gelatin-based hydrogel biomaterials. To achieve this research objective, hydrogel constructs will be prepared by encapsulating mesenchymal progenitor cells within gelatin methacrylate hydrogel via ultraviolet crosslinking. Femtosecond laser multiphoton absorption process will be used to micromachine channels (about 10 microns in diameter) within the cell-laden hydrogel constructs. These constructs will have different cell densities (10,000 - 10,000,000 cells/mL), hydrogel concentration (7-18% w/v), and photoinitiator concentration (0.05-0.5% w/v). Experiments will be conducted under different ultrafast laser parameters: laser fluency from 0 to 25 J/cm^2, pulse energy from 0 to 100 nJ, and scanning speed from 0.1 to 10 mm/s. Cellular damage within hydrogel constructs will be evaluated by the following parameters: radial zones of cellular viability measured by confocal imaging of calcein AM-ethidium homodimer biomarkers, DNA damage measured using Comet assay, and function (ability to proliferate and produce extracellular matrix) measured using confocal imaging of EdU and Movat's fluorescent biomarkers.