Monitoring of cell-induced deformations in biocompatible hydrogels via 3D multiphoton grafting

Abstract

Recent openings in regenerative medicine turned the site of tissue engineering science towards the new approaches of healing the damaged tissues: not replace, but repair and regenerate became the directional vectors. In this light, old artificial constructions gave way to the principally new `vital-avital¿ compounds, which combine synthetic material and live cells, embedded in this material. Such compounds not only mimic the natural tissue in the best way, they also enable and accelerate a regeneration process in tissues and allow one to avoid the immune response of a host. For successful application of these systems, it is important to enable monitoring of live cells inside of material. In this work live MC3T3 preosteoblast cells and their interactions with a 2D hydrogel substrate were monitored via fluorescent grid, produced by multiphoton grafting (MPG) on the surface of the substrate. A proposed model should allow one to see cell-induced deformations in hydrogel layer, via deformations of a grid. Hydrogel substrates were produced from mixtures of biocompatible, enzymatically degradable and photosensitive chemically modified gelatin (GelMOD) with photoinitiators (PIs). Photorheology was utilized for cross-linking of initial reaction mixtures and also for determination of an optimal type and concentration of PI - factors, which influence on mixture reactivity and mechanical properties of produced hydrogels. Two PIs were compared in this work: a commercially used IG2959 and Li-TPO-L, concerning their cytotoxicity, reactivity and influence on stiffness of produced substrates. Produced hydrogel was then decorated with fluorescent molecules in a particular pattern of a square grid, which could be visible if observed with laser scanning microscopy (LSM). The main benefit of MPG process, rising from a group of technologies, utilizing multi-photon absorption (MPA) chemistry, over commonly used stereolithografic techniques is possibility to create precise computer-designed patterns with high resolution. For our studies water-soluble grafting agents (GAs) were preferential, therefore, a commonly used insoluble in water BAC-M was compared with a new water-soluble GA. Toxicity studies were performed in order to determine cell-friendly concentration of a new grafting agent (GA). Finally, processing window for MPG of selected hydrogel substrates was also determined. Presented system should not only enable an in vivo monitoring of cell-induced deformations in 2D substrates. Ability to change and control matrix stiffness, which is one of key factors, influencing differentiation process of stem cells, makes it potentially applicable for studying the influence of environment on differentiation process of stem cells, which is a distant motivation of our work. Later, the same method can be applied for 3D matrices, which better represent a natural cell environment ¿ extracellular matrix (ECM). Better understanding of the mechanisms and processes, taking place in the cell, more precise study of cell-cell and cell-environment interactions becomes possible, if this method succeeds.