| Abstract [eng] |
Aim of this work was to form Young ‘s modulus micropatterns on polyacrylamide hydrogel and adapt these hydrogels for adhesion and mechanical stimulation of cells. Polyacrylamide formulation used in this work involved photoinitiators such as Irgacure 2959 or riboflavin, allowing for control of hydrogel crosslinking via light. Hydrogel was copolymerised with methacrylic acid (MA) or aminoethyl methacrylate hydrochloride (AM), allowing for various protocols of covalent attachment of extracellular matrix protein – fibronectin, to hydrogel surface. While forming stiffness patterns, hydrogel mixture was polymerised in 2-step approach. Firstly, hydrogel mixture was exposed to patterned UV light through inverted microscope with photomask. Secondly, sample was exposed to uniform UV light. Another method employed was stereolithography – 3D printing. When forming stiffness patterns with 3D printer, sample height was kept constant, and only the light pattern from printer was changing. When evaluating these patterns with scanning probe microscopy, it was observed that for PAM-MA hydrogels, higher Young’s modulus values are obtained from areas exposed to UV light in 1st polymerisation step, however these areas also exhibited higher topography. For PAM-AM hydrogels we observed inverse effect, with areas of higher Young’s modulus values forming lower. When measuring PAM-MA samples, height of line topography was estimated to be between 2-4 µm and Young’s modulus values of lines to be around 9 kPa, while gaps between them had 6 kPa. When trying to evaluate same composition samples with disk patterns, it was impossible to get high resolution QI images. 8 samples with over 60 QI measurements were taken, to establish that height of disk patterns exceeds measuring range, and could be over 10 µm in height, Young’s modulus values for these disks varied wildly, ranging from 10 kPa to 90 kPa, across samples. For PAM-AM samples with line patterns, line topography only reached up to 2µm, and Young’s modulus of gaps was 10% larger than modulus of lines and ranged somewhere between 3-4 kPa. Cells on prepared PAM-MA samples, to which the fibronectin protein was bound via the EDC reagent, grew rather poorly, few cells adhered to the sample after seeding, and even fewer survived the first 24 hours. The cells that survived during the experiment differentiated into various shapes, either on the topography or between them. On PAM and PAM-AM samples to which fibronectin was bound via the sulfo-SANPAH reagent, cells grew much better, many cells adhered to the samples, and these oriented themselves along the prepared stiffness patterns. Mechanical measurements of the cells performed using a scanning probe microscope revealed that the large cellular structures that formed differed significantly in their Young’s modulus values from the surrounding cells, and their Young’s modulus was more similar to that of cells growing on glass. The hydrogel platforms prepared during this work can be adapted for mechanobiological studies. The greatest advantage of the platform is the applied photopolymerization protocol, which allows for the simple preparation of platforms with various stiffness patterns and topographies. Cells exhibited different morphologies when growing on these platforms. |
