With physicochemical properties similar to extracellular matrices, hydrogels have good mechanical properties, self-healing ability, and responsiveness, which can be used to construct micro-nano biomimetic structures for tissue regeneration, and provide micron-scale surface morphology to regulate cell behavior, such as cell adhesion, migration, or the release of survival proliferation differentiation factors. Therefore, hydrogels are widely used in tissue engineering and drug delivery. However, the preparation of high-precision three-dimensional (3D) arbitrary biocompatible hydrogel scaffolds is challenging. In order to adapt to the future development of the biomedical field, it is urgent to develop new hydrogel materials with fine 3D geometries.
Recently, Zheng Meiling, a researcher at the Organic Nanophotonics Laboratory of the Biomimetic Intelligent Interface Science Center of the Institute of Physical and Chemical Technology, Chinese Academy of Sciences, published a report entitled 22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a in ACS Applied Materials & Interfaces Hyaluronic Acid Vinyl Ester Hydrogel's research. This study proposes a new strategy for the regulation of single cells with true 3D high-precision arbitrary designable topology.
The researchers used femtosecond laser two-photon polymerization technology, vinyl ester hyaluronic acid (HAVE) hydrogel as monomer material, P2CK as a high-efficiency water-soluble two-photon initiator, dithiothreitol (DTT) as thiol-ene click chemical crosslinker and PBS buffer solution to prepare HAVE precursors, through formula optimization and laser focus regulation has made important progress in hydrogel structural resolution, that is, the maximum resolution is 22 nm. A 3D microscaffold of a hydrogel comparable to the size of the cells was prepared and the biocompatibility of the material and structure was verified, indicating that the HAVE hydrogel cell scaffold can be further used to study cell migration and manipulation behaviors.
The team carried out formulation optimization experiments to screen HAVE precursor formulations with good solubility, easy processing and good polymerization performance by changing the mass ratio of monomers and initiators and controlling the thiol-ene functional group ratio.
At resolutions of tens of nanometers, the position of voxels relative to the substrate is a non-negligible influencing factor. In order to further improve the structural resolution, the team adjusted the relative position of the focal point and substrate according to laser focus voxel theory to obtain a higher resolution linear structure. As shown in Figure 2, the high-power laser focal spot is bright and the voxel volume is large, and it is not easy to obtain the best focus position, while the low-power laser focus spot is weaker, the voxel volume is smaller, and it is easier to obtain the best focus position, and a higher resolution line structure is obtained based on this method.
Through the above formulation optimization and focus control, the researchers carried out the resolution study of the HAVE precursor C formulation. At a scanning speed of 6 μm/s, the quality of the linear structure is significantly improved (Figure 3a), and the structure is intact and dense. A resolution of 22 nm was achieved using the HAVE precursor C formulation (Figure 3c).
Furthermore, the two-photon polymerization of 3D hydrogel microstructure was carried out on the HAVE precursor formulation, and the Young's modulus of the 3D cell scaffold was measured by atomic force microscopy, and the average value of 94 kPa was close to the mechanical properties of in vivo tissues. Biocompatibility tests were carried out on the water-soluble initiator P2CK and 3D cell scaffolds in the formulation, which verified that the material and structure had good biocompatibility.
In summary, the team comprehensively studied the two-photon polymerization performance of HAVE hydrogel photoresist, obtained a characteristic linewidth of 22 nm by optimizing the formulation of the photoresist precursor and adjusting the focal position, and verified the biocompatibility of the material and the 3D hydrogel cell scaffold. The proposed scheme in this study is expected to create complex biocompatible 3D hydrogel structures and explore its potential applications in the fields of personalized microenvironment regulation, tissue engineering, biomedicine and biomimetic science.
The above results are an extension of the team's previous series of biomimetic hydrogel work. The research work has been supported by the National Key R&D Program "Nanotechnology", the National Natural Science Foundation of China, and the International Partnership Program of the Chinese Academy of Sciences.
Figure 1.3D Schematic diagram of the preparation of a hydrogel
Table 1 Formulation optimization and performance comparison of A-E series HAVE precursors
Figure 2.Effects of voxel morphology and relative substrate position on the structural resolution of polymerization lines with high power changes (a) and low power changes (b).
Figure 3. Study on two-photon polymerization performance of HAVE precursor C formulation
Figure 4. SEM comparison chart of 3D cell scaffold structure prepared by A and C formulations and confocal fluorescence microscopy image of co-cultured L929 cells on a hydrogel scaffold
Source: Institute of Physical and Chemical Technology, Chinese Academy of Sciences
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