Significance As is known, biological science research focuses on revealing the essence of life and studying the mystery of growth, development, disease, and aging. To understand all of these, we must begin with understanding the cell behavior. Cell proliferation, differentiation, migration, movement, and other behaviors are closely related to life processes. Thus, understanding the mechanism of these behaviors is of great significance.

The surface microstructure of the material can influence the biological behavior of cells, such as inducing cell growth,proliferation and migration, and promoting specific cell functions. While hydrogels are widely used in tissue engineering and regenerative medicine, drug delivery, in vitro cell culture and other fields due to their good biocompatibility and similar composition to extracellular matrix. Therefore, hydrogel micronanometer patterned surfaces and scaffolds prepared by using micro-nano manufacturing technology can provide a more simulated in vivo development environment for the growth of cells and tissues in vitro, which are of great significance for the exploration of the interaction mechanism between cells and cells, between cells and the surrounding environment.

Femtosecond laser two-photon polymerization (TPP) is a recently developed micro-nano manufacturing method that can realize the controlled preparation of two-dimensional and three-dimensional (2D/3D) micro-nano structures with high precision morphology. The micro-nano structure of hydrogels prepared by this technology, with both the biocompatibility of hydrogels and the mechanical clues, can more accurately simulate the microenvironment of cell growth in vivo, which has attracted more and more attention in the field of tissue engineering. It is worth mentioning that the resolution of the micro-nano structure fabricated by TPP depends largely on the initiator efficiency. A series of efforts has been made to improve the efficiency of photoinitiators, and significant achievements have been made. However, because most of the traditional initiator molecules are soluble in organic solvents, cytotoxicity originating from organic solvent residue in the micro-nano structure will occur. Therefore, it is particularly important to design and prepare bionontoxic water-soluble two-photon initiators.

In the past few years, many advances have been achieved in the preparation of hydrogel micro-nano structures by TPP and its application in tissue engineering. However, there are still many challenges in biosafety initiator design and in vitro cell culture experiments of hydrogel micro-nano structures. Hence, it is essential to summarize the existing relevant researchs and understand the problems in this field in a more comprehensive way, which has guiding significance for the future development direction and implementation methods of this field.

Progress In this study, the basic principle of femtosecond laser TPP (Fig.1) and the design and synthesis of a two-photon initiator (Fig.2) are briefly introduced. The research progress of initiators for TPP of hydrogels is mainly introduced, including expanding the length of conjugation system, introducing strong donor/electron acceptor group, adding a coinitiator system to increase the two-photon absorption cross-section, introducing free radical quenching group to reduce the fluorescence quantum yield, and decreasing the cytotoxicity of microstructures by increasing the water solubility of the initiator. Perry et al. designed a series of D-A-π-D-type organic molecules that increase the two-photon absorption cross-section by expanding the conjugate bridge (Fig.3). Belfield et al. synthesized fluorene substituents with different electron-donating and electron-absorbing abilities, which had large transition dipole moment and strong two-photon absorption efficiency, providing new materials for imaging and other two-photon related applications. Xing et al. synthesized new C_2v symmetric anthraquinone derivatives by Wittig reaction. These compounds exhibit stronger intramolecular charge transfer bands and lower fluorescence quantum yield (Fig.5). Considering the cytotoxicity of organic solvent residues, the study of water-soluble two-photon initiators has become a hotspot. Bazan's group synthesized a series of organic and water-soluble diphenyl TPA initiators containing dialkyl amino donors by introducing alkyl halide terminal units. On the basis of the principle of host and guest chemistry, the hydrophobic photosensitizer is coated with cyclodextrin and cucurbit urea 7 with large hydrophobic inner cavity size and good water solubility, which is also a simple and environmentally friendly preparation method of a water-soluble two-photon initiator. In our group, Zheng et al. have proposed designing and synthesizing a series of carbazole-based ionic two-photon initiators and further improving its two-photon absorption property by using host-guest chemistry. By varying the chemical microenvironment during polymerization, the TPP of precise configuration in an aqueous phase can be realized, which is important to avoid the cytotoxicity usually caused by conventional two-photon initiators (Fig.7). These studies are the scientific basis of the fabrication of 3D biocompatible hydrogels' micro-nano structures and are the necessary conditions for better simulating the biological microenvironment in vitro. Then, the micro-nano structure of hydrogel made using TPP and the application of these structures in tissue engineering are introduced. Furthermore, the existing problems and the future development trend in the application of biocompatible hydrogel microstructures were summarized and prospected.

Conclusion and Prospect In past decades, conventional TPP initiators have made significant progress. Researchers have made much effort to develop two-photon initiators with high initiation efficiency and low polymerization threshold. A series of water-soluble two-photon photoinitiators without cytotoxicity have been designed, considering biosafety, to improve the biocompatibility of the 3D microstructure of hydrogels while ensuring the two-photon absorption characteristics. At present, although the research and development of water-soluble two-photon photoinitiators have been achieved, there are still some shortcomings, such as understanding the polymerization mechanism and low initiation efficiency, which still need to be further studied. Also, the size of the current TPP hydrogel microstructure is small (nanometer level), unable to meet the needs of a large amount of cell culture and tissue growth in vitro, so the rapid preparation of large hydrogel microstructure with TPP will be an important research focus in the future.