Abstract

In this dissertation, a report of my PhD research activity is provided. The activity was carried out in Biotech Research Center, part of the Industrial Engineering Department, of the University of Trento (Italy), under the supervision of Prof. Claudio Migliaresi and Dr. Devid Maniglio. Biofabrication, an approach to the bottom-up paradigm of tissue engineering, represents the research topic. This technology is defined as the production of complex biological constructs using cells, components of the ECM, biomolecules, and biomaterials that are assembled with different techniques in an engineered tissue fragment. The general aim of the work was to address some of the problems that currently limited the development and applicability of biofabrication. In particular, two issues were considered in the experimental part: the cryopreservation of cell-laden hydrogel constructs and the development of novel building blocks containing cells using alginate-based hydrogels. Alginate was the material of choice for investigation, as an accepted support for different tissue engineering applications that can sustain several modification and fabrication methods. In the first chapter, the concepts of bottom-up tissue engineering and biofabrication are introduced. The role and state of the art of hydrogels to manufacture cell-laden building blocks, the techniques for cell encapsulation and the commonly used fabrication strategies for biofabrication and bioprinting are reviewed together with their applications. Moreover, the limitations that currently restrict the applicability of hydrogel-based tissue engineering are discussed. In chapter two, the role of alginate hydrogels in tissue engineering and biofabrication is described. In particular, its chemical content, crosslinking behavior, manufacturing capacity, and applications are reviewed with emphasis on the possible modification of alginate hydrogels in order to enhance biocompatibility and functionality of encapsulated cells. The experimental part is described in the following chapters. Chapter three introduces the concept of cryopreservation and in particular the issues concerning the preservation of cell-laden building blocks. Subsequently, the impact of cryopreservation on the viability and functionality of cells encapsulated in alginate matrices is evaluated comparing different cryoprotective agents. The experimental methods for manufacturing and preserving cell-laden alginate fibers and for performing the biological and structural tests are reported. The results are presented, discussed and compared with the state of the art. In chapter four, a novel method for encapsulating cells within alginate-based hydrogel films with micrometer thickness is described. The procedure for immobilizing cells within hydrogel films with different composition is described, together with the performed biological assays aimed at selecting the best matrix composition. The results are reported and discussed, emphasizing the potential applications and future developments of the proposed method.