Two photon polymerization-derived micro 3D structures for application in regenerative medicine

Abstract

Patterned surfaces have been shown to be capable of influencing cellular behaviour. However, depending on the intended application their assembly may be a complex process. To overcome the limitations of common fabrication processes, a laser lithography technique based on the two-photon absorption effect, was used to construct several micro and nanostructures. Initial stages of the project were devoted to developing the technical skills and assessing the limitations of the two photon polymerization (TPP) fabrication process. It was additionally shown, the possibility of mass producing TPP constructs by replicating them, via soft lithography, in polystyrene (PS) and polylactic acid (PLA) imprints, without losing the original geometry. Following a series of optimizations and design alterations, two classes of patterned surfaces were fabricated, replicated in PS and seeded with a pre-osteoblastic stem cell lineage, MG-63, for 72 hours. The first class, was developed to explore the effect of micro (beams) and nano (dashes) structures within the same sample. The 72 hour timepoint analysis has shown a significant influence of the micrometric structures (beams), as opposed to the dashes, in inducing cell orientation and migration following a direction parallel to the beams as well as a tendency to form a tissue like conformation in the spaces between beams. The second class of patterns included two uniform cylinder based pillar topographies, with different heights (low: 5μm and high: 13μm) and a ramp-like structure with variable pillar height (3 to 15μm). Contrary to cells expanded in the smaller pillars, both the high and variable height patterns promoted an elongated shape, with cells orientated across the pillar spaces. The height gradient created by the variable pillars, suggested the existence of a height threshold capable of inducing morphological change, since only cells within the patterned region where pillar height is greater than 6μm, exhibit similar behaviour to the high pillars topography. As future work, it was also suggested that this fabrication technique may find wide application in single cell analysis and migration studies. Lastly, several custom PLA-based biopolymers were developed\optimized, for two photon fabrication. The best formulation was obtained for a 50/50 ratio of a PLA\NVP (N-vinylpyrrolidone) monomer mixture and 5%Wt. of a Benzophenone derivative photoinitiator.

Date of Award	26 Jan 2016
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Pamela Habibovic (Supervisor)