Spinal Cord Injury (SCI) affects thousands of people all over the word every year, leading to a permanent paralysis, with a different degree of severity. The body is not able to spontaneously regenerate the damaged Spinal Cord, and in general Central Nervous System (CNS), for this reason it is necessary to find an effective therapy to regenerate the tissue independently to the extent of the injury. Different therapies were proved to have a positive effect, as cell therapies and injection of neurotrophic factors, but with a very low efficiency due to events that occur after trauma as inflammation, inhibitory environment, demyelization and glial scar formation. As a possible alternative therapy, synthetic biomaterials have shown a potential tool for the regeneration of the nervous tissue as it is engineered to mimic the native Extra Cellular Matrix (ECM). A further improvement of the success can be achieved combining cell therapy and biomaterials where cells create connections with the cells of the tissue and the biomaterial supports their survival and create a permissive environment. In the last few years, different technique were developed to obtain a 3D structures, also called 3D scaffolds, which better resemble the ECM of the tissue in which stem cells can differentiate in a way that is more similar to the biological tissue. In this way, when the stem cells encapsulated in a 3D scaffold are transplanted, it recreate a permissive environment in which the cells are able to produce neurotrophic factors and recreate neuronal connections. In our laboratory, we focus our attention on a particular type of biomaterial, specifically self-assembling peptides (SAPs). Our goal was to identify and characterize the best SAP through rheological analysis and in vitro 3D cell culture, using human neural stem cells (hNSC). The two sequences analyzed were tris(LDLK)3/(LDLK)3/Ac-KLP and tris(LDLK)3/(LDLK)3/Ac-KLP/SSL, which are a combination of peptide sequences that were previously demonstrate to positively influence β-sheets structures and neuronal differentiation. The two peptides were mixed with hNSC, sucrose and NaOH to create a 3D-structure called hNSC-HydroSAPs (hNSC-HSAPs). As first, the hNSC-HSAPs were prepared with a different pH with the addition or not of NaOH in order to determine if it can influence the viability and the differentiation of the cells. The changed pH demonstrated a positive effects and, starting from that, we focus on the optimal concentration of NaOH. Moreover, the order of components addition was evaluated: the sucrose and NaOH directly added to the peptides mix before the addition of hNSC and on the other hand the inverted order, characterized by the addition of peptides to the mixture of sucrose and NAOH. Results demonstrated that the inverted order forms a stable hNSC-HSAPs, which does not fragment. From those results, we were able to choose tris(LDLK)3/(LDLK)3/Ac-KLP/SSL as the best peptide and to set up the optimal conditions to study the highest number of hNSC inside the hNSC-HSAPs that shows an equilibrium between cell viability, cell differentiation and maturation. At the end, the hNSC-HSAP with the optional condition and with the highest number of cells was kept in culture for different weeks. The timing choices were 2, 4, 6, 8 and 10 weeks in order to follow the expression of the mature neuronal markers and avoid cell suffering. The hNSC-HSAP at 6 weeks shows the best results regarding the cell death/cell viability and the expression of mature neuronal markers, as GAP43, SMI31 and GABA; additionally we demonstrate that the cells at 6 weeks are able to produce an action potential. Additionally, hNSC at 6 weeks display a very complex network, that in the following weeks does not increase. Finally, the hNSC-HSAP maintained in culture for 6 weeks is a promising model that could be induce regeneration of nervous tissue when transplanted into Spinal Cord Injury animal model.
La lesione del midollo spinale colpisce migliaia di persone in tutto il mondo ogni anno, portando a una paralisi permanente, con un diverso grado di gravità. Il corpo non è in grado di rigenerare spontaneamente il tessuto danneggiato e, in generale, il sistema nervoso centrale (SNC), perciò è necessario trovare una terapia efficace per rigenerare il tessuto indipendentemente dall'entità della lesione. Diverse terapie hanno dimostrato di avere un effetto positivo, come le terapie cellulari e l'iniezione di fattori neurotrofici, ma con un'efficienza molto bassa a causa di eventi che si verificano dopo un trauma come infiammazione, ambiente inibitorio, demielinizzazione e formazione di cicatrici gliali. Come possibile terapia alternativa, i biomateriali sintetici hanno mostrato un potenziale strumento per la rigenerazione del tessuto nervoso poiché è stato progettato per imitare la matrice cellulare extra cellulare (ECM). Un ulteriore miglioramento del successo può essere ottenuto combinando terapia cellulare e biomateriali in cui le cellule creano connessioni con le cellule del tessuto e il biomateriale sostiene la loro sopravvivenza e crea un ambiente permissivo. Negli ultimi anni sono state sviluppate diverse tecniche per ottenere strutture 3D, chiamate anche scaffold 3D, che assomigliano meglio all'ECM del tessuto in cui le cellule staminali possono differenziarsi in un modo più simile al tessuto biologico. In questo modo, quando le cellule staminali incapsulate in un'impalcatura 3D vengono trapiantate, ricrea un ambiente permissivo in cui le cellule sono in grado di produrre fattori neurotrofici e ricreare connessioni neuronali. Nel nostro laboratorio focalizziamo la nostra attenzione su un particolare tipo di peptidi biomateriali, specificamente autoassemblanti (SAP). Il nostro obiettivo era identificare e caratterizzare il miglior SAP attraverso l'analisi reologica e la coltura cellulare in vitro 3D, utilizzando cellule staminali neurali umane (hNSC). Le due sequenze analizzate erano tris (LDLK) 3 / (LDLK) 3 / Ac-KLP e tris (LDLK) 3 / (LDLK) 3 / Ac-KLP / SSL, che sono una combinazione di sequenze peptidiche che in precedenza avevano dimostrato di positivamente influenza le strutture dei fogli β e la differenziazione neuronale. I due peptidi sono stati miscelati con hNSC, saccarosio e NaOH per creare una struttura 3D chiamata hNSC-HydroSAPs (hNSC-HSAPs). Come prima cosa, gli hNSC-HSAP sono stati preparati con un pH diverso con l'aggiunta o meno di NaOH al fine di determinare se può influenzare la vitalità e la differenziazione delle cellule. Il pH modificato ha dimostrato effetti positivi e, a partire da quello, ci concentriamo sulla concentrazione ottimale di NaOH. Inoltre, è stato valutato l'ordine di aggiunta di componenti: il saccarosio e il NaOH aggiunti direttamente alla miscela di peptidi prima dell'aggiunta di hNSC e dall'altra l'ordine invertito, caratterizzato dall'aggiunta di peptidi alla miscela di saccarosio e NAOH. I risultati hanno dimostrato che l'ordine invertito forma un hNSC-HSAP stabile, che non si frammenta. Da questi risultati, siamo stati in grado di scegliere tris (LDLK) 3 / (LDLK) 3 / Ac-KLP / SSL come il miglior peptide e impostare le condizioni ottimali per studiare il maggior numero di hNSC all'interno degli hNSC-HSAP che mostra un equilibrio tra vitalità cellulare, differenziazione cellulare e maturazione. Alla fine, hNSC-HSAP con la condizione opzionale e con il più alto numero di cellule è stato tenuto in coltura per diverse settimane. Le scelte temporali sono state 2, 4, 6, 8 e 10 settimane per seguire l'espressione dei marcatori neuronali maturi ed evitare la sofferenza cellulare. L'hNSC-HSAP a 6 settimane mostra i migliori risultati per quanto riguarda la vitalità e l'espressione di marcatori neuronali maturi, come GAP43, SMI31 e GABA; inoltre dimostriamo che le cellule a 6 settimane sono in grado di produrre un potenziale d'azione.
Progettazione e caratterizzazione in vitro di uno scaffold tridimensionale composto da peptidi auto-assemblanti multi-funzionalizzati e cellule staminali neuronali per lesioni al midollo spinale
ABD EL MALEK, MARINA
2016/2017
Abstract
Spinal Cord Injury (SCI) affects thousands of people all over the word every year, leading to a permanent paralysis, with a different degree of severity. The body is not able to spontaneously regenerate the damaged Spinal Cord, and in general Central Nervous System (CNS), for this reason it is necessary to find an effective therapy to regenerate the tissue independently to the extent of the injury. Different therapies were proved to have a positive effect, as cell therapies and injection of neurotrophic factors, but with a very low efficiency due to events that occur after trauma as inflammation, inhibitory environment, demyelization and glial scar formation. As a possible alternative therapy, synthetic biomaterials have shown a potential tool for the regeneration of the nervous tissue as it is engineered to mimic the native Extra Cellular Matrix (ECM). A further improvement of the success can be achieved combining cell therapy and biomaterials where cells create connections with the cells of the tissue and the biomaterial supports their survival and create a permissive environment. In the last few years, different technique were developed to obtain a 3D structures, also called 3D scaffolds, which better resemble the ECM of the tissue in which stem cells can differentiate in a way that is more similar to the biological tissue. In this way, when the stem cells encapsulated in a 3D scaffold are transplanted, it recreate a permissive environment in which the cells are able to produce neurotrophic factors and recreate neuronal connections. In our laboratory, we focus our attention on a particular type of biomaterial, specifically self-assembling peptides (SAPs). Our goal was to identify and characterize the best SAP through rheological analysis and in vitro 3D cell culture, using human neural stem cells (hNSC). The two sequences analyzed were tris(LDLK)3/(LDLK)3/Ac-KLP and tris(LDLK)3/(LDLK)3/Ac-KLP/SSL, which are a combination of peptide sequences that were previously demonstrate to positively influence β-sheets structures and neuronal differentiation. The two peptides were mixed with hNSC, sucrose and NaOH to create a 3D-structure called hNSC-HydroSAPs (hNSC-HSAPs). As first, the hNSC-HSAPs were prepared with a different pH with the addition or not of NaOH in order to determine if it can influence the viability and the differentiation of the cells. The changed pH demonstrated a positive effects and, starting from that, we focus on the optimal concentration of NaOH. Moreover, the order of components addition was evaluated: the sucrose and NaOH directly added to the peptides mix before the addition of hNSC and on the other hand the inverted order, characterized by the addition of peptides to the mixture of sucrose and NAOH. Results demonstrated that the inverted order forms a stable hNSC-HSAPs, which does not fragment. From those results, we were able to choose tris(LDLK)3/(LDLK)3/Ac-KLP/SSL as the best peptide and to set up the optimal conditions to study the highest number of hNSC inside the hNSC-HSAPs that shows an equilibrium between cell viability, cell differentiation and maturation. At the end, the hNSC-HSAP with the optional condition and with the highest number of cells was kept in culture for different weeks. The timing choices were 2, 4, 6, 8 and 10 weeks in order to follow the expression of the mature neuronal markers and avoid cell suffering. The hNSC-HSAP at 6 weeks shows the best results regarding the cell death/cell viability and the expression of mature neuronal markers, as GAP43, SMI31 and GABA; additionally we demonstrate that the cells at 6 weeks are able to produce an action potential. Additionally, hNSC at 6 weeks display a very complex network, that in the following weeks does not increase. Finally, the hNSC-HSAP maintained in culture for 6 weeks is a promising model that could be induce regeneration of nervous tissue when transplanted into Spinal Cord Injury animal model.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/24107