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International Journal of Bioprinting 4D heterojunction shape reconfiguration by two-photon polymerization

Figure 1. The schematic flowchart begins with facile preparation of homogeneous responsive precursors (mixed PEG-DA, NIPAM, MB, and TAIC in
polyethylene glycol), followed by a biomimetic femtosecond laser direct writing method scanning at a pre-designed path to generate high-fidelity function-
controllable monolayer nanostructured interactive hydrogels (MNIHs), subsequently, the residual precursor solution was rinsed off, and MNIHs were
loaded stimuli for high-kinematic shape reconfiguration

conductive background for scanning electron microscopy the composite MNIHs here. The equipped precise scale
(SEM) images or secondary-electron images. Besides the reflected the mass changes at a resolution of <0.1 μg. Solvents
Olympus digital microscopes, 3D micrographs of the inside fully-swelled MNIHs were gradually evaporated in
shape-reconfigured MNIHs were achieved by utilizing a TGA tests when the ambient temperature increased at a step
laser scanning confocal microscope (LSCM, VK-X1000, of 5°C/min at the range of 30°C – 60°C by infrared heating.
Keyence) to digitally reconstruct the sample profile at The relative mass loss was calculated at (m initial -m )/m initial
real
nanometer accuracy (shown in Supplementary File). to reflect the water retention of MNIHs. The dehydration
Geometric parameters were figured out by the Keyence speed of water was calculated by differential operation on
analysis software. mass loss along time.

2.3. Two-photon polymerization 2.5. Cell cytotoxicity tests
TPP was done by a femtosecond laser system on isolating The as-fabricated MNIH samples (one PC sample and one
optical platform to realize monolayer heterojunction hollow scaffold) were first disinfected and then added cell
hydrogel. The laser system contained Ti: sapphire culture medium placed in a non-dust room. The used cell
laser (density of optical power ranges from 2 to culture medium contained small ratios of amino acids,
20 mW/μm ). A 2D galvanometer cooperated with one glucose, vitamins, and trace elements for fibroblast cell’s
−2
longitudinal translation platform to realize 3D trajectories survival and reproduction. A fluorescence microscope
(Videoclip S1). A self-developed analysis software (Nikon Ti-U) reflected and summarized the cell activities
converted stereolithography file (STL) of models into over two weekends as experimental verification of
structure data and directed the movement of laser focus. cytocompatibility. The viability of fibroblasts was examined
The focused oil objective provided ultra-precise feature using the live-dead assay kit (Abcam, ab115347), and the
sizes down to 180 nm confined inside the laser focus voxel ratios of live/dead cells were calculated with Image J.
using the hydrogel-nature precursors.
2.6. Optical spectroscopic investigation
2.4. Thermogravimetric analysis (TGA)
To interpret the process of TPP-induced molecular
We conducted TGA to estimate the dynamic solvent linking, we conducted Fourier-transform infrared (FTIR)
retention of MNIHs affected by temperature. The TGA spectroscopic observation on MNIHs and precursor
analyzer (Q500, TA Instruments) adjusted the ambient material compositions. The FTIR spectra were read by
temperature inside the chamber at the vacuum degree of Nicolet ™ nexus670 to reflect the molecule structural
100 torr. The pure PEG-DA hydrogel was compared with transfer. Another optical spectrograph (SCT-320,

Volume 9 Issue 3 (2023) 16 https://doi.org/10.18063/ijb.678