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Boldrin, Elisa (2011) Trapianto di cellule staminali in un modello animale di danno osseo da glucocorticoidi. [Tesi di dottorato]

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Abstract (inglese)

Bone, a specialized connective tissue, consists of cells and mineralized extracellular matrix. The main cell types of bone tissue are: the osteoblasts, the osteocytes and the osteoclasts. Osteoblasts produce extracellular matrix, osteoclasts are responsible of its resorption, hence bone physiology is a delicate balance between synthesis of new bone and resorption of the old one. Osteoporosis is a disease in which catabolic activity of osteoclasts overtakes anabolic activity of osteoblasts leading to increased bone resorption and progressive bone fragility.
Primary osteoporosis is a common disease among post-menopausal female population. Pathologies as diabetes mellitus, hyperparathyroidism and long-term treatment with glucocorticoids cause secondary osteoporosis. Glucocorticoid-induced osteoporosis is the most common type of secondary osteoporosis.
Glucocorticoid treatment is a well known method to induce osteoporosis in animal models, hence it could be an example of “translational model” in which injured bone could be repopulated by stem cells or progenitors in clinical trials.
The aim of this thesis is to investigate whether preosteoblasts could repopulate injured bone in an animal model treated with glucocorticoids. Preosteoblasts have been isolated from newborn calvariae of GFP mice.
In these cells, expression of the osteogenic marker Runx2 has been assessed by Real time PCR, while osteogenic potential has been analysed by cytochemistry assays to detect alkaline phosphatase and mineralized bone nodules (Alizarin Red and Von Kossa staining).
To realize the in vivo model, C57BL/6 three months aged mice have been divided into three groups [group I (n=4): mice not treated with drug and not infused with cells, group II (n=4): mice treated with drug and not infused with cells, group III (n=4): mice treated with drug and infused with cells]. Drug (methylprednisolone) has been administered for one month with a dose of 75 mg/Kg/week. In mice of group III, 5 x 105 GFP preosteoblasts, previously expanded in vitro, have been infused with injection into the tail vein. Mice have been sacrificed, tibial and femoral bones have been harvested, processed and analysed by istomorphometry and immunoistochemistry. Expression of Runx2, osteonectin (SPARC) and alkaline phosphatase (ALP) in these tissues has been detected with Real time PCR.
In vitro preosteoblasts produce alkaline phosphatase during early time in culture with normal medium, while the level decreases in differentiating conditions with medium containing ascorbic acid and β-glycerophosphate. Preosteoblasts maintained in differentiation medium for 30 days are positive to Alizarin and Von Kossa staining, hence they are able to produce mineralized extracellular matrix that is a feature of functional mature osteoblasts. Runx2 expression increases during differentiating conditions; in cells maintained in differentiation medium for 30 days there is an increase of 50% compared to cells maintained in normal medium (p<0.05).
In mice of group III an increased level of parameters concerning osteoid has been detected (O.Th, OS/BS, OV/BV) and an increased number of active osteoblasts (during synthetic activity) has been observed compared to group II. Between these two groups, significant variations of bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) have not been detected.
Microarchitecture parameters (Nd.N/TV, Nd/Tm) have not been affected. Similar results have been obtained from inderect microarchitecture parameters as Marrow Star Volume and Fractal Dimension.
Real time PCR analysis revealed a reduction in osteogenic gene expression in group II compared to group I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05).
In group III there is a recovery of expression of osteogenic markers (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%; p<0.01) compared to group II.
Immunoistochemistry is under investigation.
In our glucocorticoid-induced osteoporosis model we sacrificed mice only one week after infusion of cells, this preparatory investigation shows that our model induces the engraftment of preosteoblasts in injured bone. However, a longer time, at least of 1-2 months, is needed to investigate if preosteoblasts are able not only to graft onto host tissue, but also to proliferate in vivo and to differentiate in full mature and functional osteoblasts.

Abstract (italiano)

L’osso è un tessuto connettivo specializzato costituito da cellule e matrice extracellulare mineralizzata. Le cellule principali sono gli osteoblasti, gli osteociti e gli osteoclasti. Gli osteoblasti depongono la matrice ossea, mentre gli osteoclasti sono i responsabili del suo riassorbimento.
La fisiologia dell’osso è quindi il risultato di un delicato equilibrio tra deposizione di matrice ossea e suo riassorbimento.
Quando l’azione catabolica degli osteoclasti è maggiore rispetto a quella anabolica degli osteoblasti si produce una progressiva fragilità ossea che porta ad un quadro clinico osteoporotico.
L’osteoporosi primaria colpisce soprattutto la popolazione femminile dopo la menopausa. Molte patologie come il diabete mellito, l’iperparatiroidismo ed il trattamento a lungo termine con glucocorticoidi causano l’osteoporosi secondaria. L’osteoporosi indotta da glucocorticoidi è la più comune causa di osteoporosi secondaria.
Il trattamento con glucocorticoidi è un noto procedimento di induzione dell’osteoporosi in modelli animali e può dunque rappresentare un primo esempio di modello “traslazionale” potenzialmente applicabile in clinica per indurre un ripopolamento dell’osso con cellule staminali mesenchimali o precursori osteogenici.
Lo scopo di questo lavoro è stato pertanto valutare nel modello animale se sia possibile ripopolare l’osso danneggiato con preosteoblasti.
I crani di topi neonati transgenici (GFP) sono stati prelevati e messi in coltura per ottenere preosteoblasti. Nelle colture in vitro è stata valutata l’espressione del gene Runx2 con la tecnica di Real time PCR, mentre la capacità osteogenica è stata analizzata con colorazioni citochimiche per la fosfatasi alcalina e per la deposizione di matrice ossea mineralizzata (Alizarin Red e Von Kossa). Per la realizzazione del modello in vivo topi C57BL/6 maschi di 3 mesi sono stati divisi in 3 gruppi [gruppo I (n=4): topi non trattati con farmaco e non infusi con cellule; gruppo II (n=4): topi trattati con farmaco non infusi con cellule; gruppo III (n=4): topi trattati con farmaco ed infusi con cellule]. Il farmaco (metilprednisolone) è stato somministrato per un mese alla dose di 75 mg/Kg/settimana.
Negli animali appartenenti al gruppo III sono state infuse, attraverso iniezione nella vena della coda, 5 x 105 preosteoblasti GFP precedentemente espansi in vitro. I topi sono stati sacrificati, le tibie ed i femori sono stati prelevati e processati per l’analisi istomorfometrica e della microarchitettura ossea e per l’ immunoistochimica. In questi tessuti, l’espressione genica di Runx2, osteonectina (SPARC) e fosfatasi alcalina (ALP) è stata valutata tramite Real time PCR.
In vitro i preosteoblasti producono fosfatasi alcalina durate i primi giorni di coltura in medium non differenziante, mentre il livello decresce in condizioni differenzianti, cioè in medium contenente acido ascorbico e β-glicerofosfato. I preosteoblasti mantenuti in medium di differenziamento per 30 giorni sono positivi alle colorazioni Alizarin Red e Von Kossa, quindi sono in grado di produrre matrice ossea mineralizzata, caratteristica degli osteoblasti funzionali e maturi. L’espressione del gene Runx2 aumenta durante il differenziamento, si ha un aumento del 50% nelle cellule differenziate per 30 giorni rispetto alle cellule non differenziate (p<0.05).
L’inoculazione dei preosteoblasti nei topi del gruppo III ha evidenziato un aumento dei parametri statici di neoformazione ossea relativi all’osteoide (O.Th, OS/BS, OV/BV) ed un aumento del numero di osteoblasti attivi, cioè in corso di deposizione di osteoide, rispetto al gruppo II. Tra questi due gruppi non si sono osservate, invece, variazioni significative in termini di volume osseo (BV/TV), spessore trabecolare (Tb.Th) numero delle trabecole (Tb.N) e separazione fra esse (Tb.Sp).
Non sono state rilevate, inoltre, differenze dei parametri di microarchitettura (Nd.N/TV, Nd/Tm). Risultati simili sono emersi dalla valutazione dei parametri indiretti di microarchitettura (Marrow Star Volume e Fractal Dimension).
L’espressione genica ha dimostrato che nel gruppo II si ha una riduzione dell’espressione dei geni osteogenici rispetto al gruppo I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05).
Nel gruppo III si è avuto un recupero dell’espressione dei geni osteogenici (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%, p<0.01) rispetto al gruppo II. I campioni di tessuto per l’ immunoistochimica devono essere processati.
Nel nostro modello sperimentale di osteoporosi indotta da glucocorticoidi nel topo, abbiamo sacrificato gli animali solo una settimana dopo l’infusione delle cellule; questi dati preliminari dimostrano che il nostro modello induce l’engraftment dei preosteoblasti nell’osso danneggiato.
Tuttavia è richiesto un tempo di osservazione più lungo, di almeno 1-2 mesi per valutare se le cellule trapiantate siano in grado, non solo di integrarsi nel tessuto dell’ospite, ma anche di proliferare in vivo e di differenziare in osteoblasti maturi e funzionali.

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Tipo di EPrint:Tesi di dottorato
Relatore:Realdi, Giuseppe
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > SCIENZE MEDICHE, CLINICHE E SPERIMENTALI > FISIOPATOLOGIA CLINICA
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:27 Gennaio 2011
Parole chiave (italiano / inglese):Preosteoblasti, glucocorticoidi, osteoporosi, preosteoblasts, glucocorticoids, osteoporosis
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/09 Medicina interna
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Mediche e Chirurgiche
Codice ID:3518
Depositato il:13 Lug 2011 12:12
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