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High-throughput studies of cells mechanotransduction are usually performed using 2D biomaterials. However, cells-extracellular matrix interactions in the body occur in 3D environment. By using cells entrapped in hydrogels, it is not easy to isolate the mechanical effect from the chemical cues of biomaterials. We used a cytocompatible and non-expensive platform based in the patterning of wettable spots in superhydrophobic surfaces to deposit porous biomaterials in these regions. Freeze-dried alginate/chitosan scaffolds were used to create an array of mechanical properties and porosities. Adaptation of dynamic mechanic analysis equipment allowed performing on-chip single scaffold analysis. Micro-computed tomography allowed acquir- ing data for whole chips simultaneously. Results were validated using individual scaffolds and single-formulation chips. A sub-array with combined modulus/porosity properties was selected. Fibronectin (Fn) in different concentration was adsorbed in the scaffolds, and fibroblasts and osteoblast-like cells were seeded. The independent study of variables influence in cell response was performed by image-based methods. In the absence of Fn fibroblasts did not respond to mechani- cal properties in the chip range. Osteoblast-like cells showed higher cell adhesion in stiffer substrates. The adsorption of Fn was studied qualita- tively by image methods. Results related with Fn amount and scaffolds’ mechanical properties were analyzed for each cell type.

The development of optimized products in the tissue engineering (TE) field is a time and resource consuming process due to the unpredictable influence of the combination of variables such as biomaterials, cells and soluble factors. As multiple combinations can be considered highthroughput (HT) methods were suggested as a way to master complexity in TE. Usually, HT systems test cell-2D biomaterials interactions and, more recently, cell-3D hydrogels interactions. Using a chip consisting of superhydrophobic surfaces patterned with wettable regions we tested cells-hydrogels interactions in three-dimensional environment [1]. The versatility of the chip allowed its use for the first time on-chip combinatorial study of 3D miniaturized porous scaffolds. Arrays of biomaterials were dispensed and processed in situ as porous scaffolds with distinct composition, surface characteristics, porosity/pore size and mechanical properties. Those characteristics were assessed by adapting microcomputed tomography equipment and a dynamic mechanical analyzer. The interactions between cell types of two distinct origins – osteoblast-like and fibroblasts - and the scaffolds modified with distinct amounts of fibronectin were studied by image-based methods and validated by comparison with conventional destructive methods. Physical and biological on-chip results were coherent with conventional measures, and conclusions about the most favorable media for the growth of both cell types were taken. Growth factors (GF) proved to play an important role in TE approaches, mainly for determining cell fate in applications containing stem cells. We developed a chip based on wettability contrast with torus-shaped hydrophilic transparent regions disposed in an array matrix. Concentrically to these wettable regions a superhydrophobic circle was maintained, so the hydrogels could be processed as protein-loaded spheres with minimum protein loss [2] and fixed with an indentation. A combinatorial system of BSA-FITC – a commonly used GF model – encapsulated in alginate hydrogels was designed. The protein release from the hydrogels could be studied by image analysis, avoiding manipulation and protein loss. The results were compared with conventional protein release tests and similar tendencies were observed. We believe that the proposed innovative uses for the superhydrophobic chip and their upgrade in future applications may constitute a promising breakthrough in integrated technologies for the rapid development of TE systems.

Cells-biomaterials interactions are dependent on a wide range of fac- tors, namely substrate mechanical properties. Usually in the study of mechanotransduction phenomena 2D models are used and surface modification of the biomaterials is performed to suppress the effect of materials chemistry. However, 3D structures provide a more accurate mimicry of the physiological media. In high-throughput analysis cell encapsulation is commonly used, although in different stiffness hydro- gels the maintenance of chemical features is difficult to achieve. Wetta- ble spots were patterned in superhydrophobic surfaces in order to deposit polymeric precursors and generate combinatorial porous scaf- folds. On-chip dynamic mechanical analysis was performed, as well as simultaneous quantification of porosity of several scaffolds. After select- ing groups of scaffolds with a wide range of mechanical and morpho- logical properties, a new array was created, consisting of these scaffolds with fibronectin adsorbed through the whole solid structure. Protein adsorption was studied by image analysis. The adhesion of two different cell types was studied. A tendency for an increased metabolic activity/cell number rate could be observed for pre-osteoblast cells in high stiffness scaffolds, while an opposite tendency was verified for fi- broblasts. In both cell types, mainly for pre-osteblasts, a tendency for increased metabolic activity and cell number in the scaffolds in the presence of fibronectin was observed.