Stimuli-responsive biopolymers have attracted significant attention over the last decades as platforms for tissue engineering and regenerative medicine (TERM). In addition to their similarity with the native extracellular environment, stimuli-responsive biopolymers are also capable of responding to environmental changes in a tunable and determined way, supporting the development of innovative solutions. Among them, thermo-responsive biopolymers are the most studied for TERM purposes, since temperature can be regarded as the most available stimulus in the human body. In this thesis work, methylcellulose (MC) is presented as a noteworthy cellulose derivative for the preparation of hydrogels with thermo-responsive behavior. Specifically, the transition of MC hydrogels from the sol, hydrophilic state, to the gel, hydrophobic state, makes it interesting for different applications. A thorough literature review revealed that the main application areas of MC hydrogels are related to i) in situ gelling systems, ii) 3D (bio)printing, and iii) cell sheet engineering (CSE). In this regard, an optimal design of MC hydrogels was here disclosed as the fundamental stage to correctly tune their properties (e.g., thermal, mechanical, biological), first and foremost the lower critical solution temperature (LCST). For the scope of this thesis, MC hydrogels (8 % (w/v) in 50 mM Na2SO4) were optimally formulated to exhibit a LCST around 37 °C, in order to exploit physiological temperature as a trigger to activate their thermal transition. A new tool for the LCST quantification was then developed. On this topic, in situ Raman Spectroscopy was disclosed for the first time as an innovative technique for the precise identification of the LCST of MC hydrogels. In situ Raman Spectroscopy offered the distinctive advantage of being non-destructive compared to other well-established techniques (e.g., rheology, DSC) for LCST identification, also providing supplementary insights about the chemical make-up of MC during the thermal transition. Since MC hydrogels usually show reduced water stability and low mechanical properties, chemical crosslinking is required to enable the use of MC hydrogels for different TERM applications. A citric acid (CA)-based method for MC hydrogels crosslinking was thus designed and developed. Following a design of experiment (DOE) approach, three crosslinking parameters (CA concentration, crosslinking time, crosslinking temperature) were studied. Through this approach, three optimized crosslinked MC hydrogels (MC-L, MC-M, MC-H) were obtained and characterized. Particular consideration was given to the preservation of MC thermo-responsive character after crosslinking. In this regard, MC-M samples were shown to preserve their responsive behavior, while displaying superior properties (i.e., water stability and rheological) compared to MC control. Higher crosslinking (i.e., MC-H) was instead demonstrated to impair MC thermo-responsiveness. The optimized crosslinked MC hydrogels were then tested as substrates for CSE. First, CA was disclosed as a non-cytotoxic crosslinking agent for MC. Confluent cell sheets (CSs) were harvested from crosslinked MC hydrogels (MC-L, MC-M) without the need for any proteolytic enzyme (e.g., trypsin), simply by lowering the external temperature below the LCST. The harvested CSs displayed adhesive and proliferative activity after their transfer to a new culture surface, indicating their high regenerative potential. MC-H surfaces did not allow the detachment of any CSs, due to the loss of their thermo-responsive character caused by over-crosslinking. In conclusion, this thesis describes, for the first time, the use of chemically crosslinked MC hydrogels for CSE. The crosslinking approach and the tool developed for LCST assessment (i.e., in situ Raman Spectroscopy) synergistically contributed to the development of crosslinked MC hydrogels with superior properties (compared to pristine MC) and preserved thermo-responsiveness. Crosslinked MC hydrogels have been demonstrated as suitable substrates for CSE, but lend themselves well for a wide range of innovative applications. For instance, these systems could be used in the field of soft robotics or as smart sensors. Furthermore, such systems could be used as innovative thermo-responsive platforms for the development of smart water harvesting systems.
I biopolimeri responsivi hanno attratto molta attenzione, negli ultimi anni, come piattaforme per l’ingegneria dei tessuti e la medicina rigenerativa (TERM). Oltre alla loro somiglianza con l'ambiente extracellulare nativo, i biopolimeri responsivi sono anche in grado di rispondere ai cambiamenti ambientali in modo controllabile e definito, supportando lo sviluppo di soluzioni innovative. Tra questi, i biopolimeri termo-responsivi sono i più studiati in ambito TERM, poiché la temperatura può essere considerata lo stimolo più disponibile nel corpo umano. In questo lavoro di tesi, la metilcellulosa (MC) viene presentata come un derivato della cellulosa interessante per la preparazione di idrogeli con comportamento termo-responsivo. In particolare, la transizione degli idrogeli di MC dallo stato sol, idrofilo, allo stato gel, idrofobo lo rende interessante per diverse applicazioni. Da una attenta analisi di letteratura è emerso che le principali aree di applicazione degli idrogeli di MC sono: i) in situ gelling systems, ii) 3D (bio)printing, e iii) cell sheet engineering (CSE). A tal riguardo, un design ottimale degli idrogel di MC viene qui presentato come fase fondamentale per regolare le loro proprietà (e.g., termiche, meccaniche, biologiche), prima fra tutte la lower critical solution temperature (LCST). Per questa tesi, gli idrogeli di MC (8% (p/v) in Na2SO4 50 mM) sono stati formulati in modo ottimale per avere una LCST di circa 37 ° C, al fine di sfruttare la temperatura fisiologica come trigger per attivare la loro transizione termica. È stato quindi sviluppato un nuovo strumento per la quantificazione della LCST. Su questo argomento, la spettroscopia Raman in situ è stata utilizzata per la prima volta come una tecnica innovativa per l'identificazione della LCST degli idrogeli di MC. La spettroscopia Raman in situ ha offerto il vantaggio di essere non distruttiva rispetto ad altre tecniche tradizionali (e.g., reologia, DSC) per l'identificazione della LCST, fornendo anche informazioni integrative sulla struttura chimica della MC durante la transizione termica. Poiché gli idrogeli di MC mostrano in genere una ridotta stabilità in ambiente acquoso e basse proprietà meccaniche, è necessaria la loro reticolazione chimica per consentirne l'uso in diverse applicazioni in ambito TERM. È stato quindi sviluppato un metodo di reticolazione degli idrogeli di MC basato sull'acido citrico (CA). Seguendo un approccio di tipo DOE (Design of Experiment), sono state studiati tre parametri di reticolazione (concentrazione di CA, tempo di reticolazione, temperatura di reticolazione). Utilizzando questo approccio, sono stati ottenuti e caratterizzati tre idrogeli di MC reticolati ottimizzati (MC-L, MC-M, MC-H). Particolare considerazione è stata data alla conservazione del carattere termo-responsivo della MC dopo la reticolazione. A questo proposito, è stato dimostrato che i campioni MC-M preservano il loro comportamento termo-responsivo, pur mostrando proprietà superiori (ovvero stabilità in ambiente acquoso e reologiche) rispetto al controllo di MC. È stato invece dimostrato che una maggiore reticolazione (MC-H) compromette la termo-responsività della MC. Gli idrogeli di MC reticolati ottimizzati sono stati quindi testati come substrati per CSE. In primo luogo, il CA si è dimostrato un agente reticolante per la MC non citotossico. Foglietti cellulari (CSs) confluenti sono stati distaccati dagli idrogeli di MC reticolati (MC-L, MC-M) senza la necessità di alcun enzima proteolitico (e.g., tripsina), ma semplicemente abbassando la temperatura esterna al di sotto della LCST. I CSs distaccati hanno mostrato attività adesiva e proliferativa dopo il loro trasferimento su una nuova superficie di coltura, indice del loro elevato potenziale rigenerativo. Gli idrogeli MC-H non hanno consentito il distacco di alcun CS, a causa della perdita del loro carattere termo-responsivo causato dall'eccessiva reticolazione. In conclusione, questa tesi descrive, per la prima volta, l'uso di idrogeli di MC reticolati chimicamente per CSE. L'approccio di reticolazione e lo strumento sviluppato per la valutazione della LCST (spettroscopia Raman in situ) contribuiscono sinergicamente alla messa a punto di idrogeli di MC reticolati aventi proprietà superiori (rispetto al controllo di sola MC) e preservata termo-responsività. Gli idrogeli di MC reticolati sono stati presentati come substrati utili per CSE, ma ben si prestano per un’ampia gamma di applicazioni innovative. Ad esempio, tali sistemi potrebbero essere utilizzati nell’ambito della soft robotics o come sensori intelligenti. Inoltre, questi sistemi potrebbero essere utilizzati come piattaforme termo-responsive innovative per lo sviluppo di sistemi intelligenti di water harvesting.
Chemically crosslinked methylcellulose hydrogel platforms: from design to smart applications
Bonetti, Lorenzo
2020/2021
Abstract
Stimuli-responsive biopolymers have attracted significant attention over the last decades as platforms for tissue engineering and regenerative medicine (TERM). In addition to their similarity with the native extracellular environment, stimuli-responsive biopolymers are also capable of responding to environmental changes in a tunable and determined way, supporting the development of innovative solutions. Among them, thermo-responsive biopolymers are the most studied for TERM purposes, since temperature can be regarded as the most available stimulus in the human body. In this thesis work, methylcellulose (MC) is presented as a noteworthy cellulose derivative for the preparation of hydrogels with thermo-responsive behavior. Specifically, the transition of MC hydrogels from the sol, hydrophilic state, to the gel, hydrophobic state, makes it interesting for different applications. A thorough literature review revealed that the main application areas of MC hydrogels are related to i) in situ gelling systems, ii) 3D (bio)printing, and iii) cell sheet engineering (CSE). In this regard, an optimal design of MC hydrogels was here disclosed as the fundamental stage to correctly tune their properties (e.g., thermal, mechanical, biological), first and foremost the lower critical solution temperature (LCST). For the scope of this thesis, MC hydrogels (8 % (w/v) in 50 mM Na2SO4) were optimally formulated to exhibit a LCST around 37 °C, in order to exploit physiological temperature as a trigger to activate their thermal transition. A new tool for the LCST quantification was then developed. On this topic, in situ Raman Spectroscopy was disclosed for the first time as an innovative technique for the precise identification of the LCST of MC hydrogels. In situ Raman Spectroscopy offered the distinctive advantage of being non-destructive compared to other well-established techniques (e.g., rheology, DSC) for LCST identification, also providing supplementary insights about the chemical make-up of MC during the thermal transition. Since MC hydrogels usually show reduced water stability and low mechanical properties, chemical crosslinking is required to enable the use of MC hydrogels for different TERM applications. A citric acid (CA)-based method for MC hydrogels crosslinking was thus designed and developed. Following a design of experiment (DOE) approach, three crosslinking parameters (CA concentration, crosslinking time, crosslinking temperature) were studied. Through this approach, three optimized crosslinked MC hydrogels (MC-L, MC-M, MC-H) were obtained and characterized. Particular consideration was given to the preservation of MC thermo-responsive character after crosslinking. In this regard, MC-M samples were shown to preserve their responsive behavior, while displaying superior properties (i.e., water stability and rheological) compared to MC control. Higher crosslinking (i.e., MC-H) was instead demonstrated to impair MC thermo-responsiveness. The optimized crosslinked MC hydrogels were then tested as substrates for CSE. First, CA was disclosed as a non-cytotoxic crosslinking agent for MC. Confluent cell sheets (CSs) were harvested from crosslinked MC hydrogels (MC-L, MC-M) without the need for any proteolytic enzyme (e.g., trypsin), simply by lowering the external temperature below the LCST. The harvested CSs displayed adhesive and proliferative activity after their transfer to a new culture surface, indicating their high regenerative potential. MC-H surfaces did not allow the detachment of any CSs, due to the loss of their thermo-responsive character caused by over-crosslinking. In conclusion, this thesis describes, for the first time, the use of chemically crosslinked MC hydrogels for CSE. The crosslinking approach and the tool developed for LCST assessment (i.e., in situ Raman Spectroscopy) synergistically contributed to the development of crosslinked MC hydrogels with superior properties (compared to pristine MC) and preserved thermo-responsiveness. Crosslinked MC hydrogels have been demonstrated as suitable substrates for CSE, but lend themselves well for a wide range of innovative applications. For instance, these systems could be used in the field of soft robotics or as smart sensors. Furthermore, such systems could be used as innovative thermo-responsive platforms for the development of smart water harvesting systems.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/177103