Scala, Stefania (2023) Design of a novel 3D bioprinted in vitro model for blood brain barrier. [Tesi di dottorato]

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The blood-brain barrier (BBB) is a crucial component of the central nervous system that protects the brain from harmful substances while allowing essential nutrients to pass through. Overcoming the BBB remains a crucial aspect for the delivery of drugs or therapeutics in neurological disease modelling. It is important to underline that in vivo models could provide experimental environments that closely mimic the complexity of human physiology, although no animal model can faithfully reproduce all the manifestations of human diseases. In this context, in vivo models must be interpreted as an approximation of human biology limited to particular regions or other features. The most important disadvantage of in vivo models is the translation of results towards human application. Furthermore, using an in vitro model it should be possible to closely reproduce the essential features of the human BBB in vivo. The main limit of 2D in vitro models is the lack of a life-like model of the neural tissue, because of the difficult human reparability. In this scenario, we propose an ideal in vitro BBB 3D model that should have: (i) 3D vessel-like structure design; (ii) multiple cell lines in co-culture; (iii) high reproducibility. It is possible to study how to have a co-culture by overcoming traditional 2D co-culture systems, manufacturing a 3D structure that is closer to the geometry of a blood vessel. Accordingly, a novel 3D BBB in vitro model has been provided adopting a bipartite vessel-like bioprinted scaffold using cell-laden sodium alginate hydrogels with two different cell lines, namely endothelial and neuronal cells. The optimization of both design and printing parameters has been carried out to achieve high-quality BBB models. The critical parameters that influence print quality and cell behaviour were also explored and tailored for achieving optimal results. Preliminary results suggested that cell-laden alginate hydrogels represent a valuable bioink for complex biological system modelling, also taking into account the possibility of adopting a bioreactor to study how the shear stress impacts on tight junction protein expression. This innovative in vitro BBB model could also be an interesting tool towards high-throughput drug screening. On one hand, to assess the functionality of BBB models a trans-endothelial electrical resistance (TEER) can be measured, which uses a current between two electrodes as a measure of BBB permeability and integrity. TEER measurement is a quantitative technique that has the great advantage of being able to be performed in real time by monitoring the various growth and differentiation phases of a cell culture. Many systems for TEER measurements are commercially available such as the EVOM2 and cellZscope systems but they are highly dependent on the geometry and positioning of electrodes, generally made of metallic materials, and used mostly in static 2D cellular environments. In order to have the implementations of TEER sensors in LB2 bioreactors of the company. Using conductive polymers, the design of an innovative prototype was identified as a result of a careful literature study. The principle behind the proposal is to obtain a holder with an integrated sensor inside the LB2 bioreactor to measure the transepithelial resistance of the cell membrane during its growth. The proposal involves the realisation of the holder with measuring islands and the modification of the chamber for its integration. This will be the state of art for future studies on a 3D prototypal bioreactors in which it will be possible to integrate a TEER measurement. On the other hand, bioinspired design, intended as the emulation and replication of the innate capacity of native tissues to respond to both physical and chemical signals, represents a key aspect in advancing functional tissue regeneration. This rapidly evolving concept drives the advancement of innovative hybrid biomaterial platforms able ability to dynamically and selectively respond to on-demand signals or physiological stimuli. Hydrogels and nanoparticles are highly prized among the existing range of biomaterials due to their exceptional versatility and ability to incorporate stimuli-responsive properties, making them ideal for a wide range of applications such as stem cell therapy, drug delivery, and biosensing. Particularly, hydrogels emerged as outstanding scaffolds for tissue regeneration and possess the unique ability to mimic essential characteristics of the native extracellular matrix (ECM). On the other hand, nanoparticles are uniquely suited as reservoirs for bioactive molecules, efficiently accommodating both hydrophilic and hydrophobic compounds. Based on this, the combination of these two distinct classes results in the creation of nanoparticle-hydrogel hybrids, which seamlessly integrate the beneficial attributes of both systems while mitigating their respective limitations. The integration of acoustic-responsive polymeric nanoparticles offers the possibility of localized and targeted release triggered by acoustic stimulation but also the acoustic waves could induce mechanical stress on the hydrogel matrix, leading to changes in the arrangement of polymer chains and subsequent modification of cell behaviour such as orientation, proliferation, and direction of cell migration. Furthermore, by engineering the composition and structure of these nanoparticles, such as their tuneable size, surface chemistry, and drug encapsulation capabilities, it is possible to modulate their acoustic properties to optimize their interaction with sound waves. Ultrasound as a stimulus for drug delivery and tissue engineering may be used readily as an available equipment and is minimally invasive. However, there may be limitations in how deep within the tissue the ultrasound can penetrate. In the light of that, in the third chapter it was optimized a synthesis protocol for Janus nanoparticles of PLA-PCL blend to verify in future studies if and how the external acoustic stimulation of the nanoparticle-hydrogel complexes can modulate the cell behaviour.

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