Abstract
The encapsulation of cells within hydrogel-based matrices has emerged over the past decades as an attractive approach for engineering tissue substitutes to repair, regenerate and replace damaged tissue and organs. The success of this strategy depends on the formulation of cell-instructive hydrogels through incorporation of different types of cues that steer cell differentiation and that steer the tissue formation within these matrices. These cues can be defined as biological cues (i.e. bioactive molecules in the form of proteins, peptides and/or nanoparticles) and physical cues (i.e. through adjusting hydrogel characteristics such as pore size, porosity and stiffness). This Thesis focusses specifically on the incorporation of physical cues within tissue substitutes in a way that mimics the spatial and temporal arrangement of physical cues in native tissues. Physical cues can be characterised at the microscale through the hydrogel’s physicochemical properties and the macroscale through construct architecture. While several strategies exist around the spatiotemporal incorporation of physical cues at the micro- and macroscale, current strategies have not yet achieved spatiotemporal biomimicry to formulate adequate tissue substitutes.
Therefore, the overall aim of this Thesis was to develop strategies that enable or improve spatiotemporal control over the microscale physicochemical properties and the macroscale architecture in cell-laden hydrogels for tissue engineering purposes. These strategies revolved around the use of light-activated biomaterials that can be photopolymerized to form hydrogels. To enable photopolymerization, hydrogel precursor solutions were formulated that contained gelatin-based macromers and a photoinitiating system based on co-initiators ruthenium (Ru) and sodium persulfate (SPS).
The ability to control physicochemical properties of allyl-modified gelatin (Gel-AGE) hydrogels through harnessing the macromolecular chemistry was investigated first. A thiol-persulfate redox reaction was identified that facilitated partial crosslinking of the Gel-AGE macromers. This reaction was successfully exploited in the development of a dual-step crosslinking method that allowed extrusion-based 3D printing (3DP) of otherwise unprintable low-viscous bioinks: 1) primary (partial) crosslinking in absence of light to alter the bioink's rheological profile for print fidelity, and 2) subsequent secondary post-printing crosslinking for shape maintenance. The extended ability to process otherwise unprintable low-viscous bioinks through 3DP allowed for the spatial control over the macroscale architecture of cell-permissive hydrogels that will be of particular interest in soft tissue applications.
In addition to the macromolecular chemistry of the hydrogel precursors, photoinitiator chemistry and the corresponding crosslinking conditions play a role in defining the physicochemical properties of hydrogels. The relationship between the macromolecular and photoinitiator chemistry is however largely unknown and was thus investigated. It was observed that the physicochemical properties of hydrogels are dependent on an interplay between several variables, namely macromolecular chemistry, photoinitiator chemistry, irradiation wavelength, hydrogel thickness and the presence of obstacles in the path of light (e.g. skin in transdermal photopolymerization). This clearly demonstrates the need to validate the formulation of novel light-activated biomaterials against these variables with respect to optimizing hydrogel formulations for desired tissue outcomes.
A novel technological platform was developed that enabled macroscale shape and size control over hydrogel architecture, whilst also allowing the fabrication of spatially varying physicochemical properties within these hydrogels. This platform was based around the controllable diffusion of SPS across a liquid-gel interface to form a SPS gradient within Gel-AGE hydrogels that in turn resulted in the formation of a stiffness gradient upon photopolymerization. Through the use of this platform, the behaviour of encapsulated cells in response to stiffness gradients within the Gel-AGE hydrogels could be successfully characterised.
Another technological platform was developed that uniquely controlled not only spatial, but also temporal variation in the macroscale architecture of cell-laden hydrogels through delayed introduction of microchannels within these “bulk” hydrogels. This strategy was made possible by the development of a sacrificial gelatin ink with tailorable delayed dissolution through exploiting the unique ability of the Ru/SPS co-initiating system to initiate crosslinking of non-chemically modified gelatin through their native tyrosines. Notably, the timing of introduction of microchannels within cell-laden bulk hydrogels had significant effects on cell behaviour and tissue formation in osteogenic and endothelial cultures, thereby underlining the relevance of this novel platform.
Collectively, the work demonstrated in this Thesis comprise several strategies that improved spatiotemporal control over the microscale physicochemical properties and the macroscale architecture in cell-laden hydrogels by harnessing macromolecular and photoinitiator chemistry in hydrogel precursor solutions. These outcomes aid in taking the next steps towards the fabrication of functional tissue substitutes.