• BDNF
• cholangiocytes
• graphene
• MSCs
• organoids
• regeneration
• scaffold
• tendon

Regenerative medicine is a rapidly evolving field that merges biology, medicine, and engineering. Regenerative medicine attempts to repair or replace damaged tissues and organs, offering transformative solutions to a wide spectrum of diseases and injuries. This interdisciplinary approach encompasses a wide range of strategies, including tissue engineering, stem cell therapy, and the use of growth factors or small molecules. In this thesis, different approaches were investigated.
In the first chapter, an innovative approach to regenerate the enthesis, which is a specialized tissue positioned between bone and tendon was explored in depth. Specifically, we recreated the enthesis structure, using a multi-material biomimetic scaffold able to support autologous Mesenchymal Stem Cells (MSCs) differentiation in both tenocyte and osteoblasts. The tendon and bone-like structure were composed of elettrospunned PLGA (Poly (lactic-co-glycolic acid)) and 3D-printed PCL (polycaprolactone) produced in collaboration with the Department of Engineering (University of Pisa). Their ability to support the growth and differentiation of mesenchymal stem cells (MSCs) was demonstrated. Additionally, a stretch stimulus applied to the multimaterial scaffold in a biodynamic reactor further increased the ability of MSCs to differentiate into tenocytes, but not osteoblasts. Overall, the results highlight the possible use of these scaffolds as an approach to enhance the tendon/bone interface engineering.
The second chapter focused on the generation of a bioengineered human-sized bile duct, combining both decellularized porcine scaffolds and human-derived organoids. All the experiments were performed during the candidate period abroad at the Cambridge Stem Cells Institute under the supervision of Prof. Fotios Sampaziotis. Cholangiocytes were obtained from bile ducts and gallbladder biopsy of patients under hepatic surgery. Cholangiocytes formed organoids in culture expressing the specific markers (such as EPCAM, SOX9, CK7, CK19). Pig common bile ducts were decellularized and repopulated with the human cholangiocytes obtaining a good viability and a regular distribution of the cells within the duct lumen. The cellularized ducts were successfully transplanted in pigs demonstrating its possible use for regenerative medicine.
In the third chapter, the properties of graphene derivatives, which are widely used in conduits for peripheral nerve regeneration, were investigated. Highly crystalline graphene and tungsten disulfide (WS2) were produced in collaboration with the group of Professor Camilla Coletti (Center for Nanotechnology Innovation CNI@NEST, Istituto Italiano di Tecnologia), and tested on non-neuronal cell types such as neutrophils and MSCs. Results evidenced that materials were able to trigger the neutrophil activation promoting neutrophil extracellular trap (NET) production, even if no degradation was evidenced. Of note, specific graphene substrates reduced MSC viability. Overall, the results confirm the possibility of regulating cell responses by varying graphene properties driving the selection of the most suitable graphene forms to produce nerve conduits.
The fourth chapter focuses on the development of novel BDNF-based therapeutic strategies for neurodegenerative disorders. The administration of neurotrophins as therapeutics is challenging. Thus, a small peptide mimicking the N-terminal region of BDNF (d-BDNF) was produced in collaboration with Prof. La Mendola (Dept. Pharmacy, University of Pisa) and tested on a neuron-like model to evaluate the ability to activate the BDNF pathway. It demonstrated to act as a TrkB agonist enhancing TrkB dimerization. Likewise, d-BDNF sustained neurite outgrowth, increasing the expression of differentiation (LAMC1 and NEFM) and polarization genes (MAPT and MAP2) confirming its neurotrophic activity. These results provide light on the neurotrophic effects of a new BDNF mimetic peptide and open the way for future research into the pharmacological basis of d-BDNF activity and the development of innovative BDNF-based treatment techniques.