Mimicking the dynamic and complex ECM: designing bioprinted immune-instructive hydrogels

Project Information

INKspired

Grant agreement ID: 101028471

DOI

10.3030/101028471

Project closed

EC signature date 17 March 2021

Start date 15 September 2021

End date 14 September 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 175 572,48

EU contribution

€ 175 572,48

Coordinated by

UNIVERSITEIT MAASTRICHT
Netherlands

Periodic Reporting for period 1 - INKspired (Mimicking the dynamic and complex ECM: designing bioprinted immune-instructive hydrogels)

Reporting period: 2021-09-15 to 2023-09-14

Summary of the context and overall objectives of the project

The global demand for organ transplantation has increasingly exceeds the supply of donors. Organ shortage is now the limiting factor in treating many patients with chronic organ failure and has led to record numbers of patients
on waiting lists. In Europe, over 150,000 patients have been registered on organ waiting lists in 2019, alone. Six new patients are added to this list every hour; the chronic lack of organs, tissues and cells has been attributed to over 18 patients deaths every day.
In this context, the overall aim of the INKspired project is to generate novel ECM-inspired hydrogels, which could further recapitulate the dynamicity and complex structure of native tissue. By double network strategy, I will combine two dynamic hydrogel networks to control the overall mechanical and biological properties of constructs. This biomaterial design bridges multiple disciplines, from chemical synthesis and material science to engineering and biology, for creating advanced solutions in medicine. My research background, the guidance of Prof. Moroni and Dr. Baker (with a vast experience in biofabrication and biomaterials synthesis, respectively) and the ideal international setting (CTR, MERLN, UM) were fundamental for driving the multidisciplinary aspects of this proposal. Thus, this prestigious Marie Curie fellowship has boosted my professional career plan, by improving my research and advance publishing and communication skills, expanding my research network, developing administrative skills in project management, empowering me to take leading positions in the field of tissue engineering worldwide.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The ECM plays a key role to regulate cell behavior through multiple functions such as mechanical and structural support, traction and movement, and the ability to remodel in response to a cell or external stimuli. Many research groups around the world have made significant progress in rationally designed systems, pursuing to closely mimic the responsiveness, instructiveness, and dynamic structure of the ECM. Several researchers have demonstrated that matrix stiffness and elasticity affects cell differentiation (mechanotransduction). The effect of stiffness and elasticity on cells have been studied on (or in) static hydrogel models. However, most of the process where mechanical cues impact on cells are dynamic, such as disease progression (e.g. cancer, fibrosis). Designing biomaterials that change their mechanical properties through an external stimulus at user-defined time point is still challenging.
In this project, we synthesized new photo-responsive and switchable polymers using coumarin chemistry as reversible cross-linkers. Both natural and synthetic polymers were functionalized successfully with the photo-chrome. Their photo-responsiveness was corroborated by spectroscopic techniques. Furthermore, we developed novel double network hydrogels that are able to change, and also switch, their mechanical properties. The double network design allowed for modulating the mechanical and photo-responsiveness properties. Changing the mechanical properties of the cell microenvironment during culture-directed changes in cell morphology, adhesion, and proliferation. Definitively, the mechanical-instructive photo-responsive hydrogels developed thanks to this granted project represent very promising platform to investigate dynamic mechanical cues. Furthermore, the outcomes of this project further impact on Good Health and Well-being (n°3, UN sustainable development goal), contributing to create biomaterials promising for addressing the global tissue/organ demand.

Mechanical-instructive photo-responsive hydrogels tune cell behavior