One of the most common clinical problem that affects athletes is tendinopathy. The main cause of this pathology is overuse and excessive load in tendons, which can develop in stress concentrations at the enthesis, i.e. the interface between bone and tendon. The degeneration of enthesis can also be caused by age, weight and genetic factors. The current gold standard treatments for tendon repair include transplantation of autografts and allografts. Autografts, due to their immunogenic properties remain the best option. Nevertheless, their limited availability and the necessity of a second site surgery represent important drawbacks. On the other hand, allografts are generally associated with lower incorporation to bone and can induce an immune response. Tendon injuries can affect also the attachment site of tendon to bone. The tendon/ligament to bone transition, called enthesis, is a highly specialized tissue with gradients in structure and extracellular matrix composition. The maintenance of this structure is essential for enthesis function. This structure is generally not regenerated after injury, leading to the formation of fibrotic tissue with lower mechanical properties compared to native tissue. Growth factors are proteins that regulate several cell functions including morphogenesis, proliferation, differentiation and tissue repair. Considering the multiphasic structure of the enthesis, the administration of several growth factors or the generation of concentration gradients may be beneficial for its regeneration. Though the effects of combinations of growth factor has been extensively examined in tendon/ligament and bone, few studies explored their role in enthesis regeneration. Growth factors from the transforming growth factor β (TGF-β) family are involved in musculoskeletal development and homeostasis. In particular as reported by Font Tellado et al., TGF-β2 is involved in mice fetal development and induces scleraxis (SCX) expression in mouse and chick embryos. SCX is necessary for tenogenesis since the early stage of embryo formation and it is expressed also in all mature tendon cells, making it the most specific marker for tendon and ligament differentiation. Another relevant member of the TGF-β family is growth and differentiation factor-5 (GDF5). GDF5 is involved in bone, cartilage, ligaments and tendons formation and plays a fundamental role during enthesis development. Engineering complex tissues, such as the enthesis, requires a combination of factors, and microfluidics could be a useful strategy for this aim. Microfluidics has several advantages compared to traditional in vitro cell culture system. At first, it offers a more accurate temporal and spatial control over fluid flow and cell microenvironment. Indeed, the micrometer scale in which microfluidic works is in the range of single cell size. In this thesis, we developed a platform to establish an in vitro model of enthesis, by using a microfluidic approach. In detail, a diffusion-based microfluidic device was generated in order to create gradients of growth factors, which could improve enthesis regeneration.
Una delle problematiche più diffuse che affligge gli atleti sono le tendinopatie. La causa principale di questa patologia è l’eccessivo carico nei tendini, che può svilupparsi in concentrazioni di stress a livello dell’entesi, l’interfaccia tra osso e tendine. La degenerazione dell’entesi può però anche essere causata dall’età, il peso e fattori genetici. Gli attuali trattamenti gold standard per il ripristino del tendono includono l’impianto di autografts e allografts. Gli autografts, grazie alle loro proprietà immunogeniche, rimangono l’opzione migliore. Ma la loro limitata disponibilità e la necessità di effettuare una seconda operazione rappresentano degli svantaggi importanti. Dall’altro lato, gli allografts sono generalmente associati a una minore integrazione con l’osso e possono indurre una risposta immunitaria. Le lesioni al tendine possono interessare anche il sito di inserzione tra tendine all’osso. La transizione del tendine/legamento verso l’osso, chiamata entesi, è un tessuto altamente specializzato che presenta una struttura a gradiente e una composizione simile alla matrice extracellulare. Questa struttura generalmente non si rigenera dopo una lesione, portando alla formazione di tessuto fibrotico con proprietà meccaniche ridotte rispetto al tessuto nativo. I fattori di crescita sono proteine che regolano diverse funzioni cellulari, come la morfogenesi, la proliferazione, il differenziamento e la rigenerazione tissutale. Considerando la struttura multifasica dell’entesi, la somministrazione o la generazione di concentrazioni di gradienti di diversi fattori di crescita possono essere di beneficio per la sua rigenerazione. Sebbene gli effetti della combinazione di più fattori di crescita sia stata ampiamente esaminata nei tendini/legamenti e nell’osso, pochi studi si sono focalizzati sulla rigenerazione dell’entesi. I fattori di crescita della famiglia del fattore di crescita trasformante-β (transforming growth factor-β, TGF-β) sono coinvolti nello sviluppo e nell’omeostasi del tessuto muscoloscheletrico. In particolare, come riportato da Font Tellado et al., il TGF- β2 è coinvolto nello sviluppo del feto nei topi e induce l’espressione di Scleraxis (SCX) negli embrioni di topo e di pollo. SCX è necessario per la tenogenesi sino dai primi stadi della formazione embrionale ed è inoltre espresso nelle cellule mature del tendine, rendendolo il marker più specifico per il differenziamento di tendine e legamento. Un altro membro importante della famiglia del TGF-β è il fattore di differenziazione della crescita-5 (growth and differentiation factor-5, GDF5). GDF5 è coinvolto nella formazione di osso, cartilagine, legamenti e tendini, e riveste un ruolo importante durante lo sviluppo dell’entesi. L’ingegnerizzazione di tessuti complessi come l’entesi richiede una combinazione di fattori, e la microfluidica può essere una tecnologia utile a questo scopo. La microfluidica ha diversi vantaggi rispetto ai sistemi tradizionali di coltura in vitro: offre un controllo spazio-temporale più accurato sui fluidi e sul micro-ambiente cellulare, inoltre la scala dimensionale su cui si basa questa tecnologia è nel range della singola cellula. In questo lavoro di tesi è stata sviluppata una piattaforma per definire un modello in vitro dell’entesi, usando un approccio microfluidico. Nel dettaglio, è stato realizzato un dispositivo microfluidico basato sulla diffusione per la generazione di fattori di crescita, con lo scopo di migliorare la rigenerazione dell’entesi.
Development of on-chip gradient generator for enthesis regeneration
BERNARDELLO, CLARA
2019/2020
Abstract
One of the most common clinical problem that affects athletes is tendinopathy. The main cause of this pathology is overuse and excessive load in tendons, which can develop in stress concentrations at the enthesis, i.e. the interface between bone and tendon. The degeneration of enthesis can also be caused by age, weight and genetic factors. The current gold standard treatments for tendon repair include transplantation of autografts and allografts. Autografts, due to their immunogenic properties remain the best option. Nevertheless, their limited availability and the necessity of a second site surgery represent important drawbacks. On the other hand, allografts are generally associated with lower incorporation to bone and can induce an immune response. Tendon injuries can affect also the attachment site of tendon to bone. The tendon/ligament to bone transition, called enthesis, is a highly specialized tissue with gradients in structure and extracellular matrix composition. The maintenance of this structure is essential for enthesis function. This structure is generally not regenerated after injury, leading to the formation of fibrotic tissue with lower mechanical properties compared to native tissue. Growth factors are proteins that regulate several cell functions including morphogenesis, proliferation, differentiation and tissue repair. Considering the multiphasic structure of the enthesis, the administration of several growth factors or the generation of concentration gradients may be beneficial for its regeneration. Though the effects of combinations of growth factor has been extensively examined in tendon/ligament and bone, few studies explored their role in enthesis regeneration. Growth factors from the transforming growth factor β (TGF-β) family are involved in musculoskeletal development and homeostasis. In particular as reported by Font Tellado et al., TGF-β2 is involved in mice fetal development and induces scleraxis (SCX) expression in mouse and chick embryos. SCX is necessary for tenogenesis since the early stage of embryo formation and it is expressed also in all mature tendon cells, making it the most specific marker for tendon and ligament differentiation. Another relevant member of the TGF-β family is growth and differentiation factor-5 (GDF5). GDF5 is involved in bone, cartilage, ligaments and tendons formation and plays a fundamental role during enthesis development. Engineering complex tissues, such as the enthesis, requires a combination of factors, and microfluidics could be a useful strategy for this aim. Microfluidics has several advantages compared to traditional in vitro cell culture system. At first, it offers a more accurate temporal and spatial control over fluid flow and cell microenvironment. Indeed, the micrometer scale in which microfluidic works is in the range of single cell size. In this thesis, we developed a platform to establish an in vitro model of enthesis, by using a microfluidic approach. In detail, a diffusion-based microfluidic device was generated in order to create gradients of growth factors, which could improve enthesis regeneration.File | Dimensione | Formato | |
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https://hdl.handle.net/10589/164844