Multiphoton Fabrication of Fibronectin-Functionalized Protein Micropatterns: Stiffness-Induced Maturation of Cell–Matrix Adhesions in Human Mesenchymal Stem Cells

Abstract

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.

Precision 3D‐Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics

“…Furthermore, these cells displayed higher migratory activity compared to uncoated controls (Movie S3 and Figure S1, Supporting Information). In agreement with previous studies, we conclude that coating with [13] or an integrated printing of ECM [29] proteins are imminent for the successful colonization of cells in bioprinted 3D scaffolds.…”

“…In bottom-up print mode, evaporation can easily be prevented by using a resin cover, such as a silicon lid or a microfluidic device, that contains the resin drop. [29] For dip-in mode, we 3D printed a flexible water reservoir with conventional stereolithography that seals around the objective and the 35 mm cell culture dish. The design allows for objective translations while also maintaining humidity around the resin drop during printing.…”

“…Beginning with bovine serum albumin (BSA) and rose bengal (RB), various photoactivatable protein-based resins have been introduced to TPS [28] to print scaffolds for subsequent colonization with cells (Table S1, Supporting Information). [29][30][31] Other commonly used fluorescent photo-initiators, such as methylene blue, eosin Y or rhodamine B have also been used in combination with BSA. [32,33] While fluorescent cell scaffolds might be beneficial for specific applications, the intensity of residual photoinitiators such as RB within printed structures evokes high fluorescent background signals in the range from green to red often exceeding the signal of fluorescently labeled cells within scaffolds.…”

“…[32,33] While fluorescent cell scaffolds might be beneficial for specific applications, the intensity of residual photoinitiators such as RB within printed structures evokes high fluorescent background signals in the range from green to red often exceeding the signal of fluorescently labeled cells within scaffolds. [29,34] Furthermore, BSA is not native to the ECM and is therefore not ideal for cell cultivation. Hence, several groups have explored ECM proteins for their compatibility to a broad range of bioprinting methods, [35,36] including gelatin methacryloyl derivatives for TPS.…”

Immobilization of Active Substances in Food Using Self‐Organized Patterned Porous Film via Breath Figure Approach

“…Micropattern of bioactive substances such as proteins, [1] enzyme, [2] cells, [3] lectins, [4] nanoparticles [5] have received wide interests over recent years due to their appealing applications in many areas like food industry catalysis, [6] biological detection (such as a multitude of ELISA‐like biosensors, [7] and high‐throughput detection of proteins [8] ), film materials, [9] nutrition delivery system [10] and biomedicine areas [11] . Of the mostly immobilization of bioactive substances on solid surface, micropatterning of food protein and immobilization of enzymes has attracted great interest.…”