Two-Photon Techniques in Tissue Engineering

Abstract

Purpose

NIR radiation in the range of about 800 nm is less absorbed by biological tissues and is suited for triggering photonic effects using femtosecond pulsed Ti: Sa lasers. Especially in the life sciences, two-photon techniques are gaining greater importance. We introduce two laser applications for tissue engineering: the autofluorescent visualization of cells seeded on 3D scaffolds after two-photon excitation; and the manufacturing of 3D-structured hydrogel-like scaffolds by triggering free-radical polymerization processes within polymerizable precursors.

Methods

Primary bovine chondrocytes were cultivated on collagen I/III scaffolds using a flow chamber system coupled with a two-photon laser scanning microscope (2PLSM). During the incubation the cell population was hydrostatically stimulated. The selective visualization of unlabeled cells and scaffolds was achieved by spectral autofluorescence imaging. To gain some insight into scaffold-mediated effects on cell growth and cell differentiation, hydrogel-like scaffolds with well defined 3D structures were generated by two-photon polymerization (2PP) using methacrylated urethane and polyethyleneglycol diacrylate.

Results

We were able to show that spectral autofluorescence imaging provides spatially resolved data for the non-invasive online control of the tissue engineering process as well as the quantification of cell distribution within the scaffold. The fabrication of 3D 2PP scaffolds made from hydrogel-forming monomers and their effect on cell attachment and cell growth were also shown.

Conclusions

Two-photon techniques provide powerful tools for both the non-invasive online visualization of 3D cell-scaffold constructs and the structuring of 3D cultivation environments. The application of these techniques is also suitable for integration into micro-systems technology (e.g. BioMEMS, Cells-on-Chip, Lab-on-a Chip).