Tunable and cell-responsive 3D poly(ethylene glycol) microenvironments for the development of tissue models

The recapitulation of tissue organization and function in vitro is one of the main objectives of tissue engineering. Proper tissue function depends on the spatial assembly and interactions between key components such as cells, growth factors, signaling cues and extracellular matrix (ECM). The ECM is of utmost importance in the design of engineered tissues, since it not only provides support but also actively interacts with cells and growth factors to regulate tissue function. Tissue engineering has thus pushed towards the development of novel biocompatible and biodegradable materials capable of both supporting and interacting with cells. Such biomaterials are expected to allow the positioning of different cell types and the localization of biological factors, as well as having controlled adhesive properties and stiffness. Yet despite constant improvement on biomaterial design, technological advances and the increasing knowledge gained on tissue functions, the synthesis of artificial tissues and organs still remains far ahead. Current achievements in research have enabled the generation of engineered constructs with either controlled positioning of cells or patterning of growth factors. But the combination of both aspects into a reproducible and high-throughput strategy is still a task at hand. Another limitation is the current inability to recreate stable vascularization in tissue constructs. Vascularization is a compulsory aspect of engineering artificial tissues, since survival of any clinical-sized constructs depends on the availability of oxygen and nutrients normally provided by blood circulation in vivo. Such a shortcoming is directly linked to the necessity of establishing new strategies for the localized control of cellular function in 3D-arranged microenvironments, since vascularization is a complex and well-orchestrated phenomenon involving cell migration and differentiation under the control of specific guidance and signaling cues. To this end, a platform for the synthesis of tunable tissue constructs was developed in this thesis work. Taking advantage of the biocompatible and highly customizable enzymatically cross-linked poly(ethylene glycol) hydrogel platform, a strategy for the controlled incorporation of bioactive factors via peptide linkers was developed. The successful localized immobilization of growth factors and their ability to differentiate cells in a 3D-controlled manner was demonstrated by the osteogenic differentiation of mesenchymal cells by tethered bone morphogenic protein-2. Furthermore, using the same model, the release dynamics of the immobilized growth factors were investigated and a cell-dependant release strategy was devised by designing a proteolytically degradable peptide linker. The availability of immobilization strategies that are cell-responsive and that can mimic ECM properties is a desirable feature that can also contribute to the improvement of growth factor dose-response relationship. Additionally, selected factors modulating angiogenesis were evaluated for their potential role and use in the design of tissue constructs. Pro-angiogenic, anti-angiogenic effects and influence on cell migration were assessed in vitro and using the highly vascularized chicken chorioallantoic membrane. Preliminary experiments support the hypothesis that the selected growth factors could be used for establishing prevascular structures in engineered constructs.


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