VALIDAZIONE DI UN IDROGEL A BASE DI SETA COME MODELLO TRIDIMENSIONALE DI MIDOLLO OSSEO PER IL SUPPORTO E LO STUDIO DELLA MEGACARIOPOIESI

Bone marrow is located in the trabecular cavities of flat and long bones. Within it hematopoietic processes take place, which are responsible for the formation, starting from the hematopoietic stem cell, of all blood cells. The structure of the organ, with a dense and non-uniform vascular network, presents two main niches: the endosteal niche and the vascular niche. The first is located close to the bone and is characterized by a stiffer matrix. The second is located near the sinusoids, and the matrix shows more elastic mechanical properties. Stromal cells contribute to the creation and functionality of this environment directly through the secretion of cytokines and growth factors, and indirectly, through deposition and remodeling of the extracellular matrix due to matrix metalloprotease production. Its localization, which makes its study difficult, and the challenges associated with adequately recreating in two-dimensional systems the high structural complexity just described, highlight the strong need for the development of three-dimensional ex vivo bone marrow models. Research in this area has produced various types of systems over the years, but they have problems to fully recapitulate all the key aspects. In this doctoral thesis, I therefore validated the application of a silk fibroin–based hydrogel developed in Prof. Balduini’s laboratory as a three-dimensional bone marrow model. More specifically, I evaluated whether the scaffold was able to sustain the viability of human hematopoietic stem cells once directly embedded in the hydrogel and subsequently printed. Flow cytometry analysis of the cells recovered from the structure highlighted their degree of maturation, with the presence of the main lineage-specific differentiation clusters (CD) on the surface.Megakaryocyte functionality occurs through the formation of structures called proplatelets, which derive from cytoskeletal and cytoplasmic remodeling to form protrusions from which platelets originate. Image analysis using confocal microscopy as a tool to calculate parameters such as cell volume and sphericity allows us to better observe and evaluate these processes. In particular, it was observed that during differentiation into megakaryocytes, the cells experience an increase in volume; this increase is associated with a decrease in sphericity, especially during the phase of proplatelet formation. Comparison of the formulation with other commercially available showed that only the silk fibroin–containing hydrogel was able to support cell differentiation toward megakaryocytes. This difference is due to the fact that only our formulation has the mechanical and softness properties that fall within the physiological ranges of bone marrow. To validate the clinical translatability of the hydrogel, cells derived from patients affected by inherited thrombocytopenias were cultured in the hydrogel to evaluate whether the model was able to identify these diseases and be useful for their study. These pathologies are genetically determined, and present low platelet counts, which leads to bleeding risk and related complications; they are caused by defects that mainly involve megakaryocyte differentiation, cytoplasmic and cytoskeletal remodeling processes, or increased clearance of platelets from circulation. Culture of patient-derived cells within the three-dimensional scaffold made it possible to distinguish between cells from healthy donors and those from patients with thrombocytopenia with described mutations in MYH9 or ANKRD26. In particular, the healthy group showed normal differentiation with formation of proplatelets and functional platelets; the MYH9 group showed a reduction in proplatelets associated with the release of enlarged platelets; the ANKRD26 group, in conclusion, showed a defect limited to the reduction of proplatelet-forming cells. Finally, the data from my work demonstrated that the hydrogel, thanks to its composition, is an excellent bone marrow model.

Il midollo osseo è localizzato nelle cavità trabecolari delle ossa piatte e lunghe ed è la sede dei processi ematopoietici, responsabili della produzione di tutte le cellule del sangue a partire dalle cellule staminali ematopoietiche. La sua struttura, caratterizzata da una rete vascolare densa e disomogenea, presenta due principali nicchie: quella endostale, più vicina all’osso e con matrice rigida, e quella vascolare, situata presso i sinusoidi e con caratteristiche meccaniche più elastiche. Le cellule stromali regolano questo ambiente tramite la secrezione di fattori solubili e il rimodellamento della matrice extracellulare. La complessità strutturale del midollo e le difficoltà di riproduzione in sistemi bidimensionali rendono necessario lo sviluppo di modelli tridimensionali ex vivo. In questa tesi è stato validato un idrogel a base di fibroina di seta sviluppato nel laboratorio della prof. Balduini come modello tridimensionale di midollo osseo. È stata valutata la sua capacità di sostenere la vitalità delle cellule staminali ematopoietiche umane dopo semina e stampa. L’analisi citofluorimetrica delle cellule recuperate ha mostrato la presenza dei principali marker di differenziamento. La funzionalità dei megacariociti è legata alla formazione di strutture dette propiastrine, da cui originano le piastrine. L’analisi confocale ha evidenziato che durante la differenziazione il volume cellulare aumenta mentre la sfericità diminuisce, soprattutto nella fase di formazione delle propiastrine. Il confronto con altri idrogel commerciali ha mostrato che solo quello contenente fibroina di seta supporta efficacemente la differenziazione megacariocitaria, grazie a proprietà meccaniche compatibili con quelle fisiologiche del midollo. Per valutarne la trasferibilità clinica, cellule di pazienti con trombocitopenie ereditarie sono state coltivate nel modello. Le cellule sane, utilizzate come controllo, hanno mostrato una normale formazione di propiastrine ed il rilascio di piastrine funzionali; quelle con mutazioni MYH9 una riduzione dei propiastrine e piastrine aumentate di dimensione; quelle con mutazioni ANKRD26 una riduzione selettiva delle cellule formanti propiastrine con megacariociti più piccoli. I risultati dimostrano che l’idrogel rappresenta un valido modello tridimensionale di midollo osseo.