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Gomiero, Chiara (2017) Novel regenerative medicine approaches with the use of adult mesenchymal stem cells: in vitro and in vivo experimental procedures. [Tesi di dottorato]

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Abstract (inglese)

Tendon injuries are often associated with skeletal muscle lesions that can originate from a variety of events, including direct trauma, tendon and muscle lacerations and contusions, indirect insults and degenerative diseases as muscular dystrophies. Currently, a complete cure for musculoskeletal diseases is not present and the restitutio ad integrum is difficult to obtain.
In the last decade, adult MSCs gained general attention in both human and veterinary medicine and the understanding of MSC function is improved promoting the application of cell therapy and the development of powerful cell-derived therapeutics for regenerative medicine.
The first part of this research focused on the reprogramming of stromal cells derived from equine and sheep mesenchymal tissue towards tenogenic and myogenic fate in vitro using new non-viral transfection system.
1) Equine MSCs isolated from peripheral blood (PB-MSCs) can develop the tenogenic pathway using four specific growth factors such as TGFβ3 (transforming growth factor β3), EGF2 (epidermal growth factor 2), bFGF2 (fibroblast growth factor 2) and IGF1 (insulin-like growth factor 1) in presence or without Low Level Laser Technology (LLLT).
2) PB-MSCs were induced to differentiate towards myogenic fate using the complex TAT-MyoD in presence of a conditioned medium obtained from co-culturing PB-MSCs with C2C12 without a direct contact.
3) A novel surface-active maghemite nanoparticles (SAMNs) were tested as vectors for eukaryotic cell transfection of coding gene in PB-MSCs without the application of external magnetic fields.
The full characterization of these three techniques was achieved using molecular and immunohistochemistry analysis.
Real-time PCR (rt-PCR) was performed to study the expression level of the typical tenogenic genes markers Early Growth Response Protein-1 (EGR1), Tenascin C (TNC) and Decorin (DCN) to discover the best combination of GFs in presence or without LLLT.
To evaluate the myoblasts differentiation, rt-PCR analysis was executed to study Myf5 and Myogenin gene expression while immunofluorescence experiments was performed to estimate MyoD, Myf5 and Myogenin protein expression.
The cytotoxicity effects of SAMNs nanoparticles was observed with XTT cell proliferation assay and to evaluate SAMNs efficacy as vector for pDNA coding GFP, an immunofluorescence analysis was performed.

The second topic of this research project was on skin regeneration studied in vivo. Skin is a soft tissue and covers the entire surface area of body. It is a self-repairing, self-renewing organ that forms an important barrier from the outer environment to the inner environment. Therefore, damage to the skin leads to debilitating wounds that is an impairment of the anatomical structure and function of the skin. In the two papers of the second section, the capability of adult equine and ovine MSCs to regenerate skin injuries has been studied.
1) Wounds were induced in the gluteus region of six horses and treated with autologous epithelial stem cells (EpSCs), allogeneic EpSCs, vehicle treatment or untreated control.
2) Sheep allogeneic PB-MSCs were utilized to treat experimental lesions on the back of six sheep. This project is part of a large scheme where conventional treatments (Manuka Honey, Connettivina and Acemannane) were compared to innovative cures (MSCs and gas-ionized plasma). In this thesis, only the data about skin regeneration with PB-MSCs was reported.
In the first work of the second section, rt-PCR was performed on tissue biopsies collected after one and five weeks of treatment and IFN-y, IL-6, VEGF, EGF, IGF-1 and epidermal keratin (eKER) were analyzed to study cellular immune response, neovascularization and the epidermal keratinization.
In the second paper, clinical analysis have been performed to analyze the healing time, the presence, the color and the nature of exudate, the aspect of gauze, the hydration of the wound, the percentage of re-epithelization and contraction of the lesions. Tissue biopsies were collected after 15 and 42 days of treatments to conduct molecular analysis, histological and immunohistochemical staining.
Molecular analysis were performed to study the expression level of genes such as Collagen 1α1 (Col1α1) and Keratin of hair (hKER). Dermal and subcutaneous inflammation, granulation tissue and skin adnexa were evaluated using histological analysis while the expression of MHCII, von Willebrand factor (vWF) and a cellular proliferation marker (KI67) were estimated with immunohistochemical staining.

Abstract (italiano)

Nell’ultima decade, le cellule staminali mesenchimali adulte (CSM) sono state considerate una cura innovativa per la medicina umana e veterinaria. Questo progetto di ricerca supporta l’efficacia delle cellule staminali nella rigenerazione dei tessuti muscolo-scheletrici e cutanei. In particolare è stata analizzata la loro potenzialità in vitro nel differenziare in tenociti e mioblasti e la loro capacità, in vivo, nel riparare danni alla cute.
Le lesioni tendinee e muscolari sono frequenti e altamente debilitanti, possono originare da diversi eventi come traumi, lacerazioni, contusioni e malattie degenerative (distrofie muscolari). Attualmente una cura efficace non è ancora nota per cui risulta molto difficile riacquisire la restitutio ad integrum del tessuto lesionato.
La prima parte di questa tesi di dottorato si è focalizzata sulla riprogrammazione in vitro di cellule stromali isolate da tessuti mesenchimali di cavalli e pecore verso la via tenogenica e miogenica usando nuove metodologie di trasfezione senza l’uso di vettori virali.
1) CSM isolate da sangue periferico di cavallo sono state indotte a differenziare verso la via tenogenica in presenza di quattro fattori di crescita come il TGFβ3 (fattore di crescita trasformante-3), EGF2 (fattore di crescita dell’epidermide-2), bFGF2 (fattore di crescita dei fibroblasti-2) e IGF-1 (fattore di crescita insulino-1) combinati tra loro in presenza o in assenza della Tecnologia Low Level Laser (LLLT).
2) CSM isolate da sangue periferico di cavallo sono state indotte a differenziare verso la via miogenica usando il complesso TAT-MyoD in presenza di un terreno di crescita condizionato ottenuto dalla co-coltura tra CSM e le cellule C2C12 senza il loro diretto contatto.
3) Nanoparticelle di maghemite (SAMNs) sono state testate come vettori di trasfezione in CSM isolate da sangue periferico di cavallo senza l’impiego di campi magnetici esterni.
La caratterizzazione di queste tre tecniche è stata effettuata usando analisi molecolari ed immunoistochimiche.
Per individuare la miglior combinazione di fattori di crescita nel differenziamento verso la linea tenogenica, in presenza o assenza della tecnologia LLLT, sono state effettuate real-time PCR (rt-PCR) analizzando i livelli di espressione genica dei markers tenogenici Early Growth Response Protein-1 (EGR1), Tenascina C (TNC) e Decorina (DCN).
Per valutare il differenziamento delle CSM verso la miogenesi, sono state effettuate analisi con rt-PCR valutando l’espressione genica di Myf5 e Miogenina, mentre saggi di immunofluorescenza sono stati eseguiti per stimare l’espressione delle proteine MyoD, Myf5 e Miogenina nelle cellule staminali mesenchimali adulte.
Infine, saggi di immunofluorescenza sono stati effettuati per valutare l’efficienza di trasfezione delle SAMNs mentre gli effetti citotossici delle nanoparticelle sono stati osservati con il saggio di proliferazione cellulare XTT.

La seconda sezione di questa tesi di dottorato si è focalizzata sulla rigenerazione della cute in vivo. La cute è un organo che ricopre l’intera superficie del corpo e possiede la capacità di auto-riparazione e di auto-rinnovo formando un’importante barriera tra l’ambiente esterno ed interno. Danni alla pelle possono creare ferite debilitanti che intaccano la struttura anatomica e la funzione della cute stessa.
Nei due lavori presenti nella seconda parte di questa tesi, è stata studiata la capacità delle CSM isolate da sangue periferico di cavallo e di pecora di rigenerare lesioni cutanee sperimentali.
1) Lesioni cutanee sono state indotte in corrispondenza dei muscoli dei glutei di sei cavalli e trattate con CSM epiteliali autologhe (Ep-MSCs), allogeniche, soluzione salina o non trattate.
2) CSM allogeniche isolate da sangue periferico di pecora sono state utilizzate per trattare lesioni effettuate sul dorso di sei pecore. Questo progetto rientra in uno studio molto più ampio dove trattamenti convenzionali come Miele di Manuka, Connettivina e Acemannano, sono stati comparati a trattamenti innovativi come le CSM e il gas ionizzato plasma. In questa tesi di dottorato, è stato riportato solo l’articolo inerente la rigenerazione della cute utilizzando CSM allogeniche.
Il primo lavoro di questa sezione riporta i risultati ottenuti con analisi molecolari (rt-PCR) su tessuto cutaneo bioptico equino dopo una e cinque settimane di trattamento con Ep-MSCs. I livelli di espressione dei geni Interferone-y (IFN-y), Interleuchina-6 (IL-6), fattore di crescita dell’endotelio vascolare (VEGF), fattore di crescita dell’epidermide-2 (EGF2), fattore di crescita insulino-1 (IGF-1) e cheratina dell’epidermide (eKER) sono stati studiati per analizzare la risposta immunitaria, la neo vascolarizzazione e la cheratinizzazione epidermica.
Nel secondo lavoro, sono state effettuate analisi cliniche per analizzare il tempo di guarigione, la presenza, il colore e la natura dell’essudato, l’aspetto della garza, l’idratazione della ferita, la percentuale di ri-epitelizzaione e di contrazione delle lesioni. Il tessuto bioptico prelevato dopo 15 e 42 giorni di trattamento è stato utilizzato per analisi molecolari, istologiche ed immunoistochimiche. Mediante le analisi molecolari sono stati valutati i livelli di espressione dei geni Collagene 1α1 (Col1α1) e cheratina del pelo (hKER). L’infiammazione dermica e sottocutanea, il tessuto di granulazione immaturo e maturo e gli annessi cutanei sono stati valutati mediante analisi istologiche mentre il complesso maggiore di istocompatibilità (MHCII), il fattore di von Willebrand e il marker di proliferazione cellulare KI67 sono stati studiati con saggi di immunoistochimica.

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Tipo di EPrint:Tesi di dottorato
Relatore:Patruno, Marco Vincenzo
Correlatore:Martinello, Tiziana
Dottorato (corsi e scuole):Ciclo 29 > Corsi 29 > SCIENZE VETERINARIE
Data di deposito della tesi:30 Gennaio 2017
Anno di Pubblicazione:26 Gennaio 2017
Parole chiave (italiano / inglese):Tenogenesi, fattori di crescita, LLLT, Miogenesi, complesso TAT-MyoD, Nanoparticelle di maghemite (SAMNs), lesioni cutanee, guarigione delle ferite, pecore, cavalli, cellule staminali mesenchimali adulte, medicina rigenerativa Tenogenesis, growth factors, Low level laser technology (LLLT), myogenesis, TAT-MyoD complex, maghemite nanoparticles (SAMNs), skin lesions, wound healing, sheep, horses, adult mesenchymal stem cells (MSCs), regenerative medicine.
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/01 Anatomia degli animali domestici
Struttura di riferimento:Dipartimenti > Dipartimento di Biomedicina Comparata ed Alimentazione
Codice ID:10088
Depositato il:03 Nov 2017 10:58
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