Three-dimensional (3D) cell culture scaffolds promote 3D cell adhesion and growth, and are capable of directing cell fate. The physical impact provided by the scaffolds is crucial for revealing the behavior of the cells. In this study, 3D scaffolds with woodpile and honeycomb structures were fabricated using two-photon polymerization (TPP) technology. The effects of scaffold geometry and pore size on the proliferation, morphology and orientation of human mesenchymal stem cells (hMSCs) were investigated. To assess the efficacy of the scaffolds in promoting cell growth, scanning electron microscopy and laser scanning confocal imaging were utilized. The results showed that hMSCs cultured in 3D scaffolds exhibited higher proliferation rates compared to 2D planar surfaces. Notably, cells on the scaffold exhibited reduced nuclear area, roundness, and aspect ratio, highlighting the profound effect of the physical properties of the scaffold on cell morphology. In addition, hMSCs grown on the scaffolds displayed a unique growth pattern, aligned along specific directions to form an orderly arrangement. Furthermore, hMSCs grown on the woodpile scaffolds displayed elongated morphology, aligning along the columns and forming an orderly arrangement. While hMSCs on honeycomb structures exhibit triangular and crescent cellular shapes with a more random orientation. These TPP-fabricated 3D scaffolds are promising in facilitating the understanding of cell behavior and provide a versatile platform for exploring tissue engineering strategies, disease modeling, and pharmacological evaluation.
 

two photon polymerization,three-dimensional scaffolds,human mesenchymal stem cells,cellular morphology