Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. The focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture systems using fibroblasts as an in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here we investigate the niche factor(s) that influence(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning. First, the bovine serum albumin (BSA)-made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of cross-linked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.

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it is critical to be able to reconstitute the complex cell niche in native tissues in vitro. An ideal in vitro cell niche should feature spatially controlled heterogeneity and the free selection of niche factors for individual control. The availability of a cell niche where the components can be so finely controlled will provide information regarding how a particular cell niche factor regulates cellular fate processes and what combination of cell niche factors leads to optimal cellular activities. Ultimately, a complex cell niche with "programmable" niche factors can be fabricated to model the microenvironment of cells in native and pathological tissues. Previous studies show that human mesenchymal stem cells (hMSCs) are sensitive to the ECM composition [7] as well as the matrix compliance or mechanical force applied. [8] Numerous microfabrication technology platforms, for example, replica molding, [9] microcontact printing, [10] dippen nanolithography, [11] and micro-inkjet printing [12] have been developed to fabricate microstructures and micropatterns. However, they have several drawbacks such as time-consuming fabrication process and low fabrication resolution. Two-photon microfabrication [13] has several advantages such as sub-micrometer resolution, free-form 3D fabrication with spatial control ability. Moreover, the noncontact laser-based crosslinking makes it possible to functionalize the surface of extremely soft 3D microstructures at selected locations with specific densities without damaging their topological features. Previous studies demonstrated the ability of two-photon microfabrication to fabricate complex protein micropatterns with precisely controlled voxels, topological structures and porosity, [14] as well as mechanical properties. [15] The ECM niche is one of the most important components of cell niche, and thus plays a critical role in cell-matrix interactions. Nevertheless, micropatterning the ECM niche with precise control of the density, spatial location of multiple ECM components, and decoupling this niche with the mechanical properties of the protein microstructures, has not been demonstrated.Here, we demonstrate the critical capability of the multiphoton microprinting technology to micropattern the ECM niche with heterogeneity (Figure 1). The multiphoton 3D fabrication system including a femtosecond laser (Figure 1a) has been used to fabricate the underlying protein substrateReconstituting the biomimetic cell niche in vitro is important as it allows to determine how niche factors may affect cellular fate processes. Many biofabrication and micropatterning technologies are developed to achieve this goal. However, the critical outstanding challenges are the capabilities to spatially control the micropatterns and to decouple the different niche factors. Multiphoton microfabrication is an emerging technology with unique advantages. A multiphoton 3D microprinting platform has been previously established to create protein microstructures and micropatterns with sub-micrometer resolution and it...

A cell niche is a complex microenvironment that cells are exposed to in tissues. In article number https://doi.org/10.1002/adbi.201800053, Barbara Pui Chan and co‐workers use multiphoton micro‐printing technology to create protein micro‐patterns with independently and spatially controlled matrix and mechanical properties. Human mesenchymal stem cells cultured on a protein micro‐niche spread towards selective protein islands with different extracellular matrices. This platform contributes to highthroughput screening of cell niche factors for biomimetic scaffold design.