ECM-mimicking biomaterial research has driven numerous advances in tissue engineering in recent years, bringing novelties in polymer chemistry and cell-material interactions. New 3D biomaterial platforms can provide pivotal insights for bioengineering, allowing the investigation of cell fate, drug delivery, or novel translational applications in regenerative medicine. Recently, hydrogels have received a considerable interest as leading candidates for engineered tissue scaffolds due to their unique compositional and structural similarities to the natural extracellular matrix. This research topic aims to implement recent advances in the areas that focus on developing next-generation biomaterials and biotechnologies with controlled material composition, defined structural architectures, dynamic functionality, and biological complexity targeted toward various applications in regenerative engineering and translational medicine. In this thesis the functionalization of gelatin and chitosan with methylfuran is reported. The obtained polymers were combined with a maleimide PEG formulating GelChiDA, an enhanced 3D hybrid hydrogel platform. The click chemistry-based hydrogel is formed spontaneously by mixing reactive compounds, and occurs in mild conditions without further purifications, catalysts or UV irradiation. The starting materials and the resulting hybrid biomaterial were characterized in terms of physical-chemical properties. The proposed analytical strategy guarantees a global view of the complex biomaterials properties and can be applied to characterize all platforms of this nature. GelChiDA showed interesting rheological properties, including self-healing characteristics and promising preliminary biocompatibility tests. GelChiDa was formulized as bioink to be used with pneumatic 3D bioprinters, and the protocol was optimized by assessing the optimal timing to perform bioprinting at low pressure into grid-like structure, without affecting cell viability. Moreover GelChiDA was investigated as construct for in vitro tumor model spheroids, proving to be a stable platform for future pharmacological tests as pathological models. The ability to control the shape, porosity and surface morphology of scaffolds has created new opportunities to overcome various challenges in tissue engineering such as vascularization and complex architecture. In this context, a novel application of Ice Binding Proteins is proposed: the use of these proteins allows to tune the biomaterial according to the endmost need without affecting cellular vitality. Starting from GelChiDA construct, elastin, identified as a potential scaffold that renders extensibility and elasticity to tissues, was included as component to a new hybrid construct. The multimodal three component GELCHEL (GELatin, CHitosan, ELastin) hydrogel conceived and studied in our work, combines the various properties and advantages that each component provides and lends itself to future massive variety of biomedical applications. After chemical and physical deep characterization, GELCHEL hydrogel has been formulated and bioprinted with U87 cell line and cytotoxicity tests highlight great biocompatibility. Moreover, antibacterial property of chitosan in the construct is maintained in the network, giving a relevant advantage to the platform. Project perspective is to expand the Diels- Alder based hydrogel library to other materials and composition. The final aim is to create an extensive standardized library of formulations that includes all features that are required and could be used as a versatile material for a wide range of bioapplications. In the final chapter U87 spheroids were employed as tumor models to evaluate the inhibitory effect of sodium hyaluronate functionalized with matrix metalloproteinases (MMPs) inhibitor molecules. Spheroids were included into the functionalized platform and the specific inhibition of membrane metalloproteins was evaluated by immunofluorescent quantification.

Negli ultimi anni, la ricerca sui biomateriali in grado di imitare la matrice extracellulare ha portato numerosi progressi nel campo dell’ingegneria tissutale, così come nella chimica dei polimeri e della conoscenza dell’interazioni cellula-materiale. Le nuove piattaforme di biomateriali 3D possono fornire informazioni fondamentali per la bioingegneria, consentendo lo studio del destino cellulare, della somministrazione di farmaci e di nuove applicazioni traslazionali nella medicina rigenerativa. Recentemente, gli idrogel sono stati oggetto di notevole interesse: a causa delle loro somiglianze compositive e strutturali con la matrice extracellulare naturale, sono i candidati principali per lo sviluppo di scaffold tissutali ingegnerizzati. In questa tesi viene riportata la funzionalizzazione di gelatina e chitosano con metilfurano. I polimeri ottenuti sono stati combinati con un PEG maleimmidico per formulare GelChiDA, una nuova piattaforma ibrida di idrogel. La reazione di crosslinking avviene tramite reazione di click chemistry, si verifica in condizioni blande, non necessita di ulteriori purificazioni e avviene spontaneamente senza l'uso di catalizzatori o irradiazione UV. I materiali di partenza e il biomateriale ibrido risultante sono stati caratterizzati in termini di proprietà fisico-chimiche. La strategia analitica proposta garantisce una visione globale delle complesse proprietà dei biomateriali e può essere applicata per caratterizzare tutte le piattaforme di questa natura. GelChiDA ha mostrato interessanti proprietà reologiche, comprese capacità di autoriparazione, e promettenti test preliminari di biocompatibilità. GelChiDa è stato formulato come bioinchiostro e il protocollo è stato ottimizzato valutando la tempistica ottimale per eseguire la biostampa a bassa pressione in modo da non influire sulla vitalità cellulare. Inoltre, GelChiDA è stato impiegato per lo studio di modelli tumorali in vitro con sferoidi, rivelandosi una piattaforma stabile per futuri test farmacologici. La capacità di controllare la forma, la porosità e la morfologia superficiale degli scaffold ha creato nuove opportunità per superare le varie sfide nell'ingegneria tissutale. In questo contesto, viene proposta una nuova applicazione delle Ice Binding Proteins: l'uso di queste proteine consente di calibrare il biomateriale in base alle esigenze di applicazione senza compromettere la vitalità cellulare. Partendo dal costrutto GelChiDA, l'elastina è stata inclusa come componente di una nuova piattaforma. L'idrogel multimodale a tre componenti GELCHEL (GELatin, CHItosan, ELastin) concepito e studiato nel nostro lavoro, combina in un unico costrutto i vari vantaggi di ciascuna componente e si presta a future applicazioni biomediche. Dopo una profonda caratterizzazione chimica e fisica, GELCHEL idrogel è stato formulato e biostampato con la linea cellulare U87 e i test di citotossicità hanno evidenziato una grande biocompatibilità. Inoltre, la nota proprietà antibatterica del chitosano viene mantenuta nel costrutto, conferendo alla piattaforma un vantaggio rilevante. L'obbiettivo futuro del progetto è di espandere la libreria di idrogel ottenuti tramite reazione di Diels-Alder ad altri materiali e composizioni. Il fine è creare un'ampia libreria di formulazioni standardizzata che includa tutte le proprietà richieste e possa essere utilizzata come materiale versatile per un'ampia gamma di bioapplicazioni. Nel capitolo finale viene valutata la capacità inibitoria dello ialuronato di sodio funzionalizzato con piccole molecole di sintesi. Nello scaffold sono stati inclusi sferoidi come modelli tumorali di glioblastoma utilizzando la linea cellulare U87. L’inibizione specifica dell'espressione di metalloproteinasi di membrana è stata valutata e quantificata mediante saggio di immunofluorescenza, aprendo le porte a future applicazioni in campo biomedico.

(2022). Multifunctional Three-Dimensional Hydrogel Architectures for Biomedical Applications. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).

Multifunctional Three-Dimensional Hydrogel Architectures for Biomedical Applications

MAGLI, SOFIA
2022

Abstract

ECM-mimicking biomaterial research has driven numerous advances in tissue engineering in recent years, bringing novelties in polymer chemistry and cell-material interactions. New 3D biomaterial platforms can provide pivotal insights for bioengineering, allowing the investigation of cell fate, drug delivery, or novel translational applications in regenerative medicine. Recently, hydrogels have received a considerable interest as leading candidates for engineered tissue scaffolds due to their unique compositional and structural similarities to the natural extracellular matrix. This research topic aims to implement recent advances in the areas that focus on developing next-generation biomaterials and biotechnologies with controlled material composition, defined structural architectures, dynamic functionality, and biological complexity targeted toward various applications in regenerative engineering and translational medicine. In this thesis the functionalization of gelatin and chitosan with methylfuran is reported. The obtained polymers were combined with a maleimide PEG formulating GelChiDA, an enhanced 3D hybrid hydrogel platform. The click chemistry-based hydrogel is formed spontaneously by mixing reactive compounds, and occurs in mild conditions without further purifications, catalysts or UV irradiation. The starting materials and the resulting hybrid biomaterial were characterized in terms of physical-chemical properties. The proposed analytical strategy guarantees a global view of the complex biomaterials properties and can be applied to characterize all platforms of this nature. GelChiDA showed interesting rheological properties, including self-healing characteristics and promising preliminary biocompatibility tests. GelChiDa was formulized as bioink to be used with pneumatic 3D bioprinters, and the protocol was optimized by assessing the optimal timing to perform bioprinting at low pressure into grid-like structure, without affecting cell viability. Moreover GelChiDA was investigated as construct for in vitro tumor model spheroids, proving to be a stable platform for future pharmacological tests as pathological models. The ability to control the shape, porosity and surface morphology of scaffolds has created new opportunities to overcome various challenges in tissue engineering such as vascularization and complex architecture. In this context, a novel application of Ice Binding Proteins is proposed: the use of these proteins allows to tune the biomaterial according to the endmost need without affecting cellular vitality. Starting from GelChiDA construct, elastin, identified as a potential scaffold that renders extensibility and elasticity to tissues, was included as component to a new hybrid construct. The multimodal three component GELCHEL (GELatin, CHitosan, ELastin) hydrogel conceived and studied in our work, combines the various properties and advantages that each component provides and lends itself to future massive variety of biomedical applications. After chemical and physical deep characterization, GELCHEL hydrogel has been formulated and bioprinted with U87 cell line and cytotoxicity tests highlight great biocompatibility. Moreover, antibacterial property of chitosan in the construct is maintained in the network, giving a relevant advantage to the platform. Project perspective is to expand the Diels- Alder based hydrogel library to other materials and composition. The final aim is to create an extensive standardized library of formulations that includes all features that are required and could be used as a versatile material for a wide range of bioapplications. In the final chapter U87 spheroids were employed as tumor models to evaluate the inhibitory effect of sodium hyaluronate functionalized with matrix metalloproteinases (MMPs) inhibitor molecules. Spheroids were included into the functionalized platform and the specific inhibition of membrane metalloproteins was evaluated by immunofluorescent quantification.
RUSSO, LAURA
NICOTRA, FRANCESCO
Ingegneria Tissutale; Click Chemistry; Diels-Alder; Colture cellulari 3D; Biostampa 3D
Tissue engineering; Click Chemistry; Diels-Alder; 3D cell culture; Biostampa 3D
CHIM/06 - CHIMICA ORGANICA
English
28-gen-2022
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
34
2020/2021
open
(2022). Multifunctional Three-Dimensional Hydrogel Architectures for Biomedical Applications. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/365344
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