Dependence of cell migration on covalently immobilized RGD gradients in PEGDA-Based hydrogels

Directionality and orientation of cell movement are critica1 in many physiological processes, including morphogenesis, immune response, and wound healing [l]. It is well known that, in these processes, ce11 response can be guided by gradients of various diffusible and non-diffusible chemical signals, such as cytokines, chemokines and extracellular matrix (ECM) components. Also in tissue engineering, where it is crucial to control cell behaviour to promote engineered tissue formation designing suitable "cell instructive" materials, able to guide cell response, is necessq. Several studies have demonstrated that gradients of covalently irnmobilized biochemical signals influence cell behaviour [2]. However, few works have elucidated the effect of these _P.dients on cell migration. This work aims at investigating the role of scaffolds with a controlled spatial distribution of adhesive signals in controlling ce11 migration. To this purpose. pol~(ethyleneg hcol) $-acrilate (PEGDAt.based hydrogel scaffolds. functionalized with gradients of covalentl>- irnmobilized RGD adhesib-e peptides were obtained by using photopolimerization reaction and a system based on a home-made gmdient maker. This system alloa-s to obtain a continuous linear RGD gradient by using a fluidic chamber having a channel network M-ith nvo inlets in order to combine hvo different precursor solutions (5% W/W PEGDA and 5% w/w PEGDA + RGD-PEG-acrylate in 10 mM HEPES buffer supplemented with a photoinitiator). Thanks to this technology, gradients with different RGD gradient slope (0.7 and 1 mM1cm) and PEGDA-based hydrogel with a uniform distribution of RGD [l .5 mM], as control, were obtained. The effect of RGD gradients on NIH3T3 fibroblast migration was studied by time-lapse experiments and the behaviour of each individua1 cell in time, compared to that of cells seeded on control hydrogel scaffolds, was qualitatively analyzed by an image analysis software. Furthermore, we developed an automated numerica1 tool that is able to provide quantitative data on the random (stochastic) and biased components of the ce11 motion, without the drawbacks of indirect measurements via non linear curve fitting of averaged quantities [3]. Our results suggest that cells recognize the RGD gradient and adhere to it assuming a stretched shape. Moreover, on RGD gradient, cells describe longer trajectories mainly aligned along gradient direction. As concern cell speed, we did not observe difference for random speed in al1 the samples. Conversely, bias speed, representing the drift of the cell population migrating on a signal gradient, results zero on control substrate, 0.04 pmlmin on 0.7 mM/cm gradient and 0.08 pmlmin 1 mMIcm gradient, indicating an evident effect of RGD gradient on cell migration.