Tooth pathologies are widespread within the population and can lead to severe functionality impairments and tooth loss. Current therapeutic approaches consist in the replacement of the compromised tissues with inert substitutes, resulting in the reduction of teeth mechanical properties and frequent postoperative issues. Furthermore, tooth implants do not provide for the complete functional recovery and are prone to mechanical failure. Regenerative medicine strategies can help in overcoming these limitations by relying on the use of stem cells and environmental/pharmacological stimuli to generate functional tissues. Teeth are composed of a sophisticated combination of hard and soft tissues. The enamel is the hardest tissue covering the crown of the tooth. It is supported by dentine, an additional mineralised porous matrix that protects an inner core made of soft connective tissue providing for vascularisation and innervation: the dental pulp. Dental pulp hosts mesenchymal stem cells (DPSCs) which are multipotent and guarantee the homeostasis of dental tissues. In this regard, DPSCs properties are being studied for regeneration purposes but, even though encouraging results are being achieved, their application is still far from clinical practice. As a matter of fact, translational process for new therapeutic approaches is costly and risky: most of the experimental programs are suspended during human trials due to low drug efficacy and safety that failed to be predicted by preclinical studies. The development of more accurate in-vitro models is crucial to speed up translational process and better predict the outcome of clinical trials. In order to address these needs, increasing attention is being dedicated in the last years to Organ-on-chip (OoC) technology. OoCs are miniaturised culture systems intended for mimicking in-vivo microenvironment by controlling cell-cell and cell-matrix interactions as well as biochemical and mechanical cues that are crucial for the development and the remodelling of tissues. Recently these systems started to be exploited for dental biology purposes but, to date, a complete Tooth-on-a-chip model is still missing. The aim of this work is the design and the validation of an OoC to generate a 3D in-vitro model of dental pulp for drug discovery purposes. The device is intended for multiple cell lines co-culture, reproducing the physiological interaction between different cell types in the dental pulp. In particular, the OoC comprises different channels that are designed to host a variety of cells reproducing all the principal tooth components: hDPSCs, Human umbilical endothelial cells (HUVEC), Odontoblast-like cells differentiated from hDPSCs (pre-Ob) and embryonal mouse trigeminal ganglia (TGG). All cell types, except from TGG, have been cultured in a fibrin-based hydrogel. Furthermore, an additional compartment was engineered to include gel-embedded dentin microparticles in order to prevent pre-Ob from losing their phenotype and to induce them to deposit dentin matrix. The OoC has been technically validated by assessing the dimensions of the microscale features. Furthermore, computational simulations and FITC-dextran diffusion tests have been performed to assess correct diffusion of metabolites and hypothetical drug molecules from the medium channels and reservoirs to cells. The OoC has been biologically validated through live/dead assays, proliferation assays, Immunofluorescent stainings, RT-qPCR analysis. The designed device was proved to be a useful tool to develop a model of dental pulp comprising stem-cells niche, vascularisation, dentin-odontoblast interface and innervation and represents a starting point for dental microanatomy modelling to be further improved and refined. Once the model is fully optimised it will be useful to study dental tissue remodelling and developmental processes in order to develop novel strategies for tooth regeneration.
Le patologie orali sono largamente diffuse tra la popolazione e possono compromettere la funzionalità dei denti fino a provocare la loro caduta. Gli approcci terapeutici convenzionali consistono nella rimozione dei tessuti compromessi e nella loro sostituzione con surrogati inerti che modificano le proprietà meccaniche del dente e possono provocare problemi post-operatori. Inoltre, gli impianti dentali non garantiscono il completo recupero funzionale e sono soggetti a fallimento meccanico. Le strategie di medicina rigenerativa possono essere una soluzione per superare tali limiti e si basano sull’uso di cellule staminali e stimoli ambientali e farmacologici per generare tessuti funzionali. Il dente si compone di una combinazione unica di tessuti duri e molli. Lo smalto è il tessuto mineralizzato che ricopre la corona dentale; esso aderisce alla dentina, un’altra matrice porosa mineralizzata che protegge un nucleo di tessuto connettivo molle: la polpa dentale. La polpa dentale è altamente vascolarizzata ed innervata ed è sede di una popolazione di cellule staminali mesenchimali (dental pulp stem cells, DPSC) che provvedono all’omeostasi dei tessuti dentali. Le proprietà delle DPSC sono oggetto di studi che mirano allo sviluppo di strategie rigenerative; tuttavia, nonostante il raggiungimento di risultati incoraggianti, la loro applicazione clinica è ancora lontana nel tempo. Infatti, il processo traslazionale per lo sviluppo di nuovi approcci terapeutici è dispendioso e rischioso: gran parte dei programmi sperimentali sono arrestati durante gli studi sull’uomo a causa dei risultati di efficacia e sicurezza non previsti durante gli studi preclinici. Pertanto, risulta necessario lo sviluppo di nuovi modelli in-vitro che velocizzino la traslazione clinica e predìcano in modo più accurato i risultati degli studi preclinici. Per soddisfare tale necessità, negli ultimi anni sta acquisendo sempre più rilevanza la tecnologia degli Organ-on-Chip (OoC). Gli OoC sono sistemi miniaturizzati di coltura cellulare progettati per mimare le condizioni fisiologiche. Permettono il controllo delle interazioni cellulari e degli stimoli biochimici e meccanici alla base dello sviluppo e rimodellamento dei tessuti. Solo di recente questi sistemi sono stati adottati per studi di biologia orale e, ad oggi, un modello completo di dente su chip non è presente in letteratura. Lo scopo del presente lavoro è la progettazione e la validazione di un OoC in grado di generare un modello in-vitro tridimensionale della polpa dentale per lo studio di nuovi approcci rigenerativi. Il dispositivo è progettato per la co-coltura di molteplici linee cellulari in modo da riprodurre l’interazione tra le diverse cellule della polpa dentale. In particolare, esso comprende canali microfluidici differenti che alloggiano diversi fenotipi cellulari: hDPSC, cellule endoteliali (HUVECs), pre-Ob e TGG. Tutti i tipi cellulari, eccetto il TGG, sono stati coltivati in un hydrogel a base di fibrina. Inoltre, un canale aggiuntivo è stato progettato per includere particelle di dentina con lo scopo di indurre i pre-Ob a mantenere il loro fenotipo e produrre matrice extracellulare. L’OoC è stato validato attraverso la misura delle caratteristiche micrometriche. Inoltre, sono stati condotti simulazioni computazionali e test di diffusione con destrano fluorescente in modo da verificare la corretta diffusione di metaboliti ed ipotetiche molecole terapeutiche all’interno dell’ambiente di coltura. Successivamente sono state eseguite la validazione biologica e la definizione di protocolli per la riproduzione dei tessuti dentali in-vitro. L’OoC progettato si è dimostrato essere uno strumento utile per lo sviluppo di un modello di polpa comprendente la nicchia staminale, la vascolarizzazione, l’interfaccia dentina-odontoblasti e l’innervazione. Tale dispositivo rappresenta un punto di partenza per la modellizzazione della microanatomia dentale che necessita di essere ulteriormente migliorato. Una volta che il dispositivo sarà completamente ottimizzato e validato, potrà essere utile per lo studio dei fenomeni di sviluppo e rimodellamento finalizzato all’implementazione di nuove strategie rigenerative in ambito dentale.
Design and validation of a microfluidic platform for dental tissues emulation
Cordiale, Alessandro
2020/2021
Abstract
Tooth pathologies are widespread within the population and can lead to severe functionality impairments and tooth loss. Current therapeutic approaches consist in the replacement of the compromised tissues with inert substitutes, resulting in the reduction of teeth mechanical properties and frequent postoperative issues. Furthermore, tooth implants do not provide for the complete functional recovery and are prone to mechanical failure. Regenerative medicine strategies can help in overcoming these limitations by relying on the use of stem cells and environmental/pharmacological stimuli to generate functional tissues. Teeth are composed of a sophisticated combination of hard and soft tissues. The enamel is the hardest tissue covering the crown of the tooth. It is supported by dentine, an additional mineralised porous matrix that protects an inner core made of soft connective tissue providing for vascularisation and innervation: the dental pulp. Dental pulp hosts mesenchymal stem cells (DPSCs) which are multipotent and guarantee the homeostasis of dental tissues. In this regard, DPSCs properties are being studied for regeneration purposes but, even though encouraging results are being achieved, their application is still far from clinical practice. As a matter of fact, translational process for new therapeutic approaches is costly and risky: most of the experimental programs are suspended during human trials due to low drug efficacy and safety that failed to be predicted by preclinical studies. The development of more accurate in-vitro models is crucial to speed up translational process and better predict the outcome of clinical trials. In order to address these needs, increasing attention is being dedicated in the last years to Organ-on-chip (OoC) technology. OoCs are miniaturised culture systems intended for mimicking in-vivo microenvironment by controlling cell-cell and cell-matrix interactions as well as biochemical and mechanical cues that are crucial for the development and the remodelling of tissues. Recently these systems started to be exploited for dental biology purposes but, to date, a complete Tooth-on-a-chip model is still missing. The aim of this work is the design and the validation of an OoC to generate a 3D in-vitro model of dental pulp for drug discovery purposes. The device is intended for multiple cell lines co-culture, reproducing the physiological interaction between different cell types in the dental pulp. In particular, the OoC comprises different channels that are designed to host a variety of cells reproducing all the principal tooth components: hDPSCs, Human umbilical endothelial cells (HUVEC), Odontoblast-like cells differentiated from hDPSCs (pre-Ob) and embryonal mouse trigeminal ganglia (TGG). All cell types, except from TGG, have been cultured in a fibrin-based hydrogel. Furthermore, an additional compartment was engineered to include gel-embedded dentin microparticles in order to prevent pre-Ob from losing their phenotype and to induce them to deposit dentin matrix. The OoC has been technically validated by assessing the dimensions of the microscale features. Furthermore, computational simulations and FITC-dextran diffusion tests have been performed to assess correct diffusion of metabolites and hypothetical drug molecules from the medium channels and reservoirs to cells. The OoC has been biologically validated through live/dead assays, proliferation assays, Immunofluorescent stainings, RT-qPCR analysis. The designed device was proved to be a useful tool to develop a model of dental pulp comprising stem-cells niche, vascularisation, dentin-odontoblast interface and innervation and represents a starting point for dental microanatomy modelling to be further improved and refined. Once the model is fully optimised it will be useful to study dental tissue remodelling and developmental processes in order to develop novel strategies for tooth regeneration.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/179372