par Siminska-Stanny, Julia
Président du jury Gilis, Dimitri
Promoteur Shavandi, Armin
Co-Promoteur Delporte, Christine
Publication Non publié, 2025-10-30
Thèse de doctorat
Résumé : Summary and Outline Vascularization remains one of the most critical and unresolved challenges in tissue engineering. Without afunctional vascular network, engineered tissues cannot sustain cell viability, while nutrient delivery, oxygentransport, and waste removal are severely limited. This constraint hinders the development of larger, morecomplex tissue constructs and restricts their integration and performance in vivo. Despite progress inbioengineering, replicating the intricate architecture and dynamic function of native vasculature remains achallenging task. Many current approaches struggle to achieve the spatial and temporal organizationnecessary for stable and perfusable networks, which are essential for tissue maturation and long-termfunction. Addressing vascularization is therefore a central objective in the field. It is a technical challenge anda biological imperative, as successful vascular integration determines whether an engineered tissue cansurvive and function after implantation. Developing strategies that promote and guide vascular formation iskey to advancing tissue engineering toward clinically effective solutions. In this thesis, some of the fundamental assumptions surrounding vascular integration in engineered tissuesare critically examined to explore how specific material properties and biological factors can influence andguide vascular formation in a controlled and effective manner. The experimental chapters of this thesispresent a systematic exploration of biomaterial formulation and advanced 3D printing strategies forfabricating perfusable, mechanically tunable, and biologically functional hydrogel scaffolds suitable for softtissue and vascular applications. The work is structured around five interconnected studies, each addressingspecific limitations in hydrogel scaffold fabrication: insufficient printability and shape fidelity, inadequatemechanical stability of vascular-like structures, slow fabrication times, and a limited understanding of howconstruct geometry influences cell behavior in 3D environments. By addressing these technical barriers, thethesis provides a framework for engineering hydrogel constructs that are structurally stable, rapidlyfabricated, and biologically relevant. Chapter 1 provides a comprehensive introduction to the central problem of vascularization in tissueengineering. It outlines the biological importance of functional vascular networks for delivering nutrients,transporting oxygen, and removing waste in engineered tissues. The chapter emphasizes the complexity ofmimicking native vascular architecture and function, framing vascularization as a technical limitation and abiological necessity. This literature review chapter presents the motivation and objectives of the thesis, layingthe groundwork for the development of material-based and biofabrication strategies to address these unmetneeds. Chapter 2 focuses on the development and optimization of bioinks for the 3D printing of soft tissue scaffolds. 15 • Part 1 introduces a dual-crosslinkable hydrogel ink composed of tyramine-modified hyaluronic acid(HA-Tyr) and gelatin methacrylate (GelMA), designed for extrusion-based printing. A Box-Behnkendesign is used to systematically optimize viscosity, shape fidelity, and mechanical stability by varyingpolymer ratios. The resulting ink exhibits shear-thinning behavior, high swelling capacity, andmechanical properties in the physiological range (~300 Pa), with robust structural fidelity during andafter extrusion. In vivo and CAM assays confirm the biocompatibility and host tissue integration ofthe scaffolds, supporting their potential use in soft tissue engineering. • Part 2 extends the use of HA-Tyr-based formulations toward coaxial extrusion printing of perfusablevascular structures in a single step. A composite ink combining PEG-Tyr, alginate, and methylcellulose(MC) is investigated to enhance flexibility, pressure resistance, and mechanical robustness whilemaintaining favorable flow behavior. PEG-Tyr provides enzymatically crosslinkable covalent bonds,significantly improving elasticity and burst pressure resistance (up to 283.3 mbar). These hollow,elastic scaffolds support cell attachment and neoangiogenesis, as confirmed in CAM and in vivoassays. However, the process remains time-consuming, and the constructs lack the long-termmechanical durability required for extended perfusion studies, motivating a transition to faster andmore robust fabrication strategies. Chapter 3 addresses the limitations identified in Chapter 2 by transitioning to Digital Light Processing (DLP)printing using a PEGDA/PVA-based resin to enhance mechanical strength and production scalability. A postfabricationmodification approach was introduced using borax and tannic acid (TA). Borax treatmentincreased compressive modulus by ~44% and toughness by ~53%, while TA confers antibacterial andantioxidant functionality via sustained release. DLP enables the simultaneous fabrication of multiple highresolutionconstructs, effectively overcoming the limitations of low throughput in coaxial extrusion. Theprinted tubular scaffolds demonstrate structural integrity across ~32 mechanical cycles and exhibitbiocompatibility in a larval in vivo model. However, they still limited cell adhesion and biological interaction,highlighting the need for more biofunctional materials – an issue that is addressed in the following chapter. Chapter 4 introduces volumetric 3D printing (Vol3DP) as an emerging additive manufacturing approach thatsubstantially shortens fabrication times and enables the creation of branched, hierarchical geometries usingbiocompatible materials such as gelatin. Unlike digital light processing (DLP), which requires filling an entireresin bath, Vol3DP operates with minimal starting material, making it more resource-efficient whileovercoming the extended production times and limited biocompatibility associated with conventional layerby-layer orextrusion techniques.• Part 1 presents a GelMA–PEGDA resin optimized for Vol3DP, which combines the cell-adhesive anddegradable properties of GelMA with the mechanical reinforcement of PEGDA. Volumetric printingenables rapid, single-step fabrication of perfusable vascular-mimetic scaffolds with embedded 16channels in under one minute. A custom-built perfusion chamber, developed in collaboration withthe industry partner (A4BEE), allows precise control of shear stress, flow rate, and flow modality(pulsatile vs. continuous). Simulations and experiments show that perfusion dynamics and masstransport behavior closely mimic physiological conditions, with diffusion through the hydrogel matrixas the dominant transport mechanism. • Part 2 leverages this Vol3DP platform to investigate how scaffold geometry affects cell behavior.Constructs with angular geometries of 60°, 90°, and 110°, seeded with human umbilical veinendothelial cells (HUVECs) and 143b osteosarcoma cells, display geometry-dependent spreading andCD31 expression, consistent with physiological mechanosensitivity. In contrast, 143b cancer cellsexhibit aggressive, geometry-independent proliferation and form dense agglomerates, reflectingtheir invasive phenotype. Label-free holographic microscopy enables long-term, non-invasivemonitoring of these cell behaviors, demonstrating the potential of the system for disease modelingand mechanobiological studies. Chapter 5 summarizes the stepwise progression achieved throughout the thesis in developing hydrogelscaffolds with enhanced printability, mechanical performance, and biological functionality. Beginning withthe development of a printable HA-Tyr/GelMA ink and the implementation of coaxial extrusion for vascularchannels (Chapter 2), the work advances to DLP printing, accompanied by post-processing treatments formechanical reinforcement and scalability (Chapter 3). It culminates in volumetric printing for rapid scaffoldfabrication and functional perfusion analysis (Chapter 4). The geometry-based study in Chapter 4 furtherdemonstrates how scaffold architecture influences the behaviors of endothelial and cancer cells,underscoring the biological significance of spatial design in tissue engineering. Together, the five chapters (Schematic 1) represent a continuous advancement in scaffold development,from optimizing printability and exploring vascularization strategies to enhancing mechanical reinforcementand enabling high-speed, high-resolution fabrication with real-time biological assessment. Each chapter builtupon the challenges of the previous one, with tailored methodological innovations addressing specificlimitations. Despite these advances, several challenges remain in the engineering of vascular scaffolds. These includeensuring long-term mechanical and biological stability under physiological flow, optimizing microscalegeometries for hierarchical vascular networks, and integrating dynamic vascular remodeling in vitro. Futurework will aim to incorporate multicellular co-cultures and extended perfusion models to further enhance thetranslational relevance of these systems, thereby enabling the development of more physiologicallyrepresentative vascularized tissues.

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