Dynamic and reversible double network hydrogels based on two types of crosslinking chemistry, dynamic covalent (DCvC) and photo-switchable covalent functional groups, were developed. First, the synthesis of new photo-switchable polymers were synthesized, using coumarin functionalization as strategy to make both synthetic and natural polymers light-responsive (objective 1). Furthermore, RGD peptide (biomolecule) was attached to dynamic network via DCvC cross-linking (objective 2) to bring adhesive sites for cells. The light responsiveness was corroborated via UV-Vis spectroscopy and photo-rheology for the polymer and double network hydrogels, respectively. After corroborating the changes in mechanical properties stimulated by light (365 nm), we decided to switch the order of experiments and left the printability as last step. These photo-responsive mechanical-instructive hydrogels were explored for modulating the cellular behavior (objective 3). Fibroblasts and stem cells were tested as cells which can respond to the stiffening observed in our gels. We found promising and interesting outcomes from cell response, which were deeply studied, dedicating extra time than expected. For this reason, the printability of these hydrogels is still in progress.
Regarding the milestones, the achievements for each milestone are explained in the following list:
M1. Proof-of-principle for printing dynamic and photo-switchable networks. It was proven the successful synthesis of photo-switchable networks. Natural and synthetic polymers were successfully functionalized with coumarin to give them light-responsive properties. The photo-responsiveness was corroborated via UV-Vis and NMR spectroscopy. Furthermore, the double network was also photo-responsive, which was corroborated via photo-rheology.
M2. Proof-of-concept for biologically instructive bioinks. Hydrogels showed mechanical changes by light irradiation. The stiffening at 365 nm was observed in both single and double network hydrogels. The magnitude of change depends on the intensity and time of irradiation. The softening of hydrogels were observed by irradiation at 254 nm, but this process was discarded for cell studies owing to the wavelenght. Cells (fibroblasts, hMSC) were shown to be able to sense that change via cell culture and immunoflourescence assays.
M3. Establishment of instructive constructs influencing macrophage polarization. Different cell lines (fibroblasts, hMSCs) were tested, and the range of mechanical changes for the studied formulations drove us to use fibroblasts as proof-of-concept. Immunostaining and qPCR were employed for exploring the cell response to the stiffening. Cell morphology, adhesion, and proliferation were evaluated. Macrophage polarization experiments were delayed, and will be executed in follow-up studies.
M4. Achieve my career development plan. I have grown professionally during my fellowship, improving both scientific and transferable skills. Hands-on training and courses. Supervising Master’s students, and teaching at UM. Participating in outreach activities. Attend national and international conferences.
I have disseminated the research outputs both in written form in conference proceedings and as oral presentations in seminars (Hasselt University, University of Erlangen-Nuremberg, Max-Planck Institute for Polymer Research) and conferences (TERMIS, ESB, IUPAC, Chains) to the scientific community. By all these scientific events, I had the opportunity to network with internationally leading experts with a variety of backgrounds such as medicine, physics, and engineering, among others. Detailed versions of the most significant results are in progress to be submitted to journals in the following months.
