Improving functionality of living tissue constructs by expanding the applicability of melt electrowriting: Three-dimensional tissue regeneration guided by biochemical and structural cues using converged (bio)fabrication technologies

Abstract

The persistent societal challenge of organ donor shortage, driven by factors such as population growth, increased lifespan, and higher disease prevalence, leaves thousands awaiting life-saving transplants. Tissue Engineering and Regenerative Medicine (TERM) emerge as promising solutions, with 3D biofabrication technologies capable of creating functional tissues and preclinical testing models. These technologies, spanning soft tissue structures to musculoskeletal replacements, aim to address the critical shortage of organ donors.

Melt electrowriting (MEW), enable precise recreation of native tissue equivalents due to its high resolution and fine control over architectures. MEW, and other biofabrication technologies automate the generation of biologically functional products with structural organization using living cells, bioactive molecules, and biomaterials. Tailoring tissue architecture, porosity, and structural organization is crucial for directing cell differentiation toward specific target tissues. While offering fine control over cell alignment and differentiation, ensuring the durability of engineered constructs is vital. Hydrogels, commonly used in TERM, provide a suitable environment for cell growth but often lack mechanical strength. Techniques such as MEW reinforce hydrogels, addressing this challenge. Vascularization within thick tissue structures remains a hurdle, necessitating strategies like modifying scaffold porosity and incorporating growth factors.

To incorporate biochemical cues, atmospheric-pressure plasma jet (APPJ) functionalization was used to covalently immobilize transforming growth factor beta-1 (TGFβ1), improving hydrophilicity, and chondrogenic differentiation. Results indicated enhanced compressive modulus and glycosaminoglycan production compared to conventional TGFβ1 supply in-medium.

Structural cues were then explored to guide cell behavior using MEW. Advances in 3D fiber scaffold manufacturing were discussed for their potential in repairing cardiac tissues. Methacrylated hydroxyl-functionalized PCL was investigated to covalently bond MEW microfibers to methacrylated silk fibroin hydrogels, resulting in more durable constructs with improved mechanical strength for articular cartilage tissue engineering. The introduction of z-porosity into MEW box structure scaffolds using isomalt as a sacrificial material demonstrated minimal impact on structural integrity, offering a promising method for creating z-directional pores crucial for cellular interconnectivity and vascularization.

MEW and extrusion-based bioprinting were converged to create complex tissue patterns with native-inspired pre-vascular pathways. Optimal bioinks were identified for myocardial cellular arrangement and vascular cell connectivity. Preservation methods for tissue-engineered constructs were compared, highlighting hypothermic preservation as a promising approach for short-term preservation and transportation.

Building upon myocardial tissue engineering approaches explored in this thesis, steps were taken to generate a full-thickness transmural tissue engineered construct. Co-culturing cardiomyocytes with fibroblasts and optimizing culture substrates improved alignment and functionality, showcasing the potential for sophisticated in vitro cardiac models with native-like architecture.

In conclusion, this thesis explored MEW as a versatile technology, combining it with other fabrication strategies to achieve biochemical- and/or structural-guided tissue regeneration. By manipulating design and technologies, functional tissues for a broad range of target applications were generated, including contractile, muscular, myocardial tissue, and fibrous, dense articular cartilage tissue.