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Paganin, Piergiorgio (2011) REALIZZAZIONE DI SCAFFOLDS TRIDIMENSIONALI IN VITRO
PER LA RIGENERAZIONE OSSEA.
[Tesi di dottorato]

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Abstract (inglese)

Bone regeneration in tissue engineering is proposed for the treatment of various orthopedic problems due to various causes. Since the last decade, tissue engineering to provide a viable alternative to compensate the therapeutic and methodological limitations that have the organ transplant (autograft or allograft) and surgical reconstruction. Due to the fast development on biomaterial technologies, to repair orthopedic defects scaffolds made by biocompatible and bioresorbable polymers and composite materials were developed for providing temporary support of damaged bone and sustain tissue regeneration. A key component in tissue engineering for bone regeneration is the scaffold that serves as a template for cell interactions and formation of bone extracellular matrix and to provide structural support for the new tissue. The scaffolds for bone regeneration should meet certain criteria, including mechanical properties similar to tissue repair, biocompatibility, biodegradability and possess a degree of remodelling. For a biomaterial used in bone regeneration is also important porosity, which promote the migration and proliferation of cells and the vascular tissue being formed. Furthermore, the porosity improves the interconnection between the natural system and bone, and shall ensure a high mechanical stability at this critical interface.
The polycaprolactone (PCL)-α is a linear polyester, semi-crystalline approved by the Food and Drug Administration (FDA) for devices to be implanted in the human body. It degrades through hydrolytic scission of ester bond at physiologic pH. Because of its biocompatibility properties, degradation rate and mechanical strength, it has been investigated as biomaterial for medical-drug delivery device, and load bearing bone tissue engineering applications. The aim of the present study was to obtain PCL scaffolds by means of phase separation technique using different porogen agents in order to achieve three-dimensional porous matrices with defined porosity, mechanical properties and microstructure which allow the osteoblastic adhesion, growth and differentiation.
In this work, we were carried out several scaffolds of PCL with various porogen (PEG, sucrose, fructose, spheres and threads of calcium alginate), also to improve the mechanical properties and biocompatibility, but with no effect on the porosity, we added hydroxyapatite and a natural bone powder.
The scanning electron microscopy analysis showed that the porosity of the scaffolds made of calcium alginate ratio (w/w porogen/polymer 200/100) (PCL-AT2) had indices morphometric (porosity, interconnection, distribution and pore size) very similar to natural trabecular bone as well as PCL-AT2 with the addition of hydroxyapatite (PCL-AT2-HA) or bone powder (PCL-AT2-BP).
In parallel, the microcomputed tomography (microCT) analysis confirmed the presence of interconnected void spaces suitable to allow the formation of a biological environment for cellular adhesion, growth and differentiation.
Moreover the mechanical test showed that all scaffolds have a maximum deformation around 3 Kg. The low load resistance, in all matrices in comparison to natural bone, is due of the lack of mineral component (hydroxyapatite), which provide a hard-wearing material. On the other hand, the elastic recovery of PCL-AT2-HA, PCL-AT2-BP was higher than that PCL-WP, PCL-AT2, because the HA and BP make more rigid the polymer and less elastic, that is more like natural bone.
The viability assay (MTS assay) performed on two cell types (MC3T3-E1 cells and mesenchymal stem cells from bone marrow of rabbit) shows that all three types of scaffolds, as described above, have a microenvironment suitable for cell growth compared to the polymer without the addition of porogen PCL-WP we use as a reference. In fact, PCL-WP has a porosity of about 21% while the other matrices between 72% -85%. These date indicates, that greater porosity have a positive influence on adhesion and cell growth because to allow a better transport of nutrients and oxygen.
Scanning electron microscope analysis revealed that after 24 h of seeding the cells were adhered to the scaffolds and appeared flattened showing an irregular and elongated form. A 7-14 days can be seen that the cell populations have created a complete monolayer and started to ECM deposition
The activity of alkaline phosphatase and the mineralized matrix deposition PCL-AT2-HA e PCL-AT2-BP was higher than that PCL-WP e PCL-AT2, this date means that the presence of factors such as hydroxyapatite and bone powder create the optimum conditions for osteogenic differentiation.
The RT-PCR analysis performed on MSCs seeded onto PCL confirmed the osteogenic differentiation as has been observe by the expression of typical markers, in addition MSC have a similar profile of alkaline phosphatase activity and deposition of calcium salts, where however, it was observed that the MSC on PCL-AT2-HA under basal conditions, express a significant difference (p<0,05) of production of calcium compared with other scaffolds, this date was confirmed at 14 and 21 days.
In conclusion, we have identified a polymer suitable for the construction of a scaffold for bone regeneration, and identified a reproducible method, easy to implement and cheap, development of scaffolds with trabecular bone morphometric indexes similar nature with the use of calcium alginate as porogen which allow adhesion, proliferation and differentiation of preosteoblast cells line and mesenchymal stem cells. The addition of HA and bone powder to polymer scaffolds, does not affect on total porosity but provides greater rigidity and also improves the osteogenic potential of the matrices. Thanks to the characteristics of the method of production, it was possible to form scaffolds as a phalanx of human with the possibility to create a customized bone prosthesis.

Abstract (italiano)

La rigenerazione ossea è proposta in ingegneria tessutale per il trattamento di diversi problemi ortopedici dovuti a molteplici cause. A partire dall’ultima decade, l’ingegneria tessutale provvede a fornire una valida alternativa per ovviare alle limitazioni terapeutiche e metodologiche che presentano il trapianto d’organo (autologo o eterelogo) e la ricostruzione chirurgica. Grazie al rapido miglioramento delle conoscenze dei biomateriali, si è potuto creare degli scaffold polimerici, biocompatibili e biodegradabili i quali possono garantire un sostegno temporaneo all’osso danneggiato e favorire la rigenerazione di nuovo tessuto. La componente chiave dell’ingegneria tessutale ossea è lo scaffold, che serve da stampo per l’interazione con le cellule e la formazione della matrice extracellulare dell’osso, inoltre provvede a fornire un supporto per il nuovo tessuto in via di formazione.
Lo scaffold per la rigenerazione ossea deve rispondere a ben determinati criteri, come proprietà meccaniche simili al tessuto da riparare, biocompatibiltà e biodegradabilità, e deve possedere un certo grado di rimodellamento. Per un biomateriale da utilizzare nella rigenerazione ossea inoltre è importante la porosità, la quale garantisce la migrazione e la proliferazione delle cellule e la vascolarizzazione del tessuto in via di formazione. Inoltre la porosità migliora l’interconnessione tra l’impianto e l’osso naturale, e provvede a garantire una grande stabilità meccanica in questa interfaccia critica.
Il policaprolattone (PCL) è un α-poliestere lineare , semicristallino approvato dalle Food and Drug Administration (FDA) per dispositivi da essere impiantati nel corpo umano. Esso viene degradato attraverso scissione idrolitica del legame estereo a pH fisiologico. Grazie alle sue proprietà di biocompatibilità, tasso di degradazione e di resistenza meccanica, è stato studiato come biomateriale per drug delivery e applicazione in ingegneria tessutale dell’osso. Lo scopo di questo lavoro è quello di ottenere uno scaffold di PCL realizzato attraverso la tecnica dell’inversione di fase usando un porogeno di dimensioni definite, in modo da ottenere una matrice con porosità, proprietà meccaniche e microstruttura tali da permettere l’adesione osteoblastica, la crescita e la differenziazione.
In questo lavoro sono state realizzate diversi scaffolds di PCL con vari porogeni (PEG, saccarsosio, fruttosio, sfere e fili di alginato di calcio), inoltre per migliorare le proprietà meccaniche e la biocompatibilità, ma non influendo sulla porosità, alle matrici polimeriche sono state aggiunte idrossiapatite e una polvere d’osso naturale. Gli scaffold sono stati caratterizzati attraverso microscopia ottica a scansione e con microtomografia computerizzata (MicroCT).
La microscopia elettronica a scansione e l’analisi della porosità hanno indicato che gli scaffolds realizzati con alginato di calcio (rapporto w/w rispetto al polimero 200/100) (PCL-AT2) presentano indici morfometrici (porosità, interconnessione, distribuzione e dimensione pori) molto simili a quella dell’osso trabecolare naturale come anche PCL-AT2 con l’aggiunta di idrossiaptite (PCL-AT2-HA) o polvere d’osso (PCL-AT2-BP). In parallelo l’analisi (MicroCT) ha confermato che gli scaffolds preparati con i fili d’alginato di calcio presentano spazi vuoti interconnessi tra loro, che permettono la formazione di un ambiente biologico adatto all’adesione, crescita e differenziamento cellulare. Inoltre le prove meccaniche hanno evidenziato che tutte le matrici hanno una massima deformazione attorno ai 3 Kg. La bassa resistenza al carico in tutte le matrici, rispetto all’osso naturale, è riconducibile alla scarsa presenza della componente minerale (idrossiapatite) la quale permette di avere un materiale più resistente. Le matrici PCL-WP, PCL-AT2 presentano un ricovero elastico abbastanza elevato, mentre si è riscontrato un valore basso per PCL-AT2-HA, PCL-AT2-BP, questo perché HA e BP rendono il polimero più rigido e meno elastico, cioè più simile all’osso naturale.
Il saggio di vitalità (MTS Assay) eseguito su due tipologie cellulari (MC3T3-E1 e cellule mesenchimali staminali da midollo osseo di coniglio) mostra che tutti e tre i tipi di scaffolds, sopra indicati, presentano un microambiente adatto alla crescita cellulare rispetto al polimero senza l’aggiunta di porogeno PCL-WP da noi usato come riferimento. Infatti PCL-WP presenta una porosità di circa 21% mentre gli altri tra 72%-85%. Questo dato indica come una maggiore porosità influenzi positivamente l’adesione e la crescita cellulare in quanto garantisce migliore trasporto di nutrienti e di ossigeno. Inoltre avendo uno scaffold poroso, questo permette la migrazione delle cellule anche all’interno dello scaffold, colonizzando completamente la matrice, cosa non permessa dalla matrice PCL-WP in quanto più compatto.
L’analisi al microscopio a scansione ha evidenziato come le cellule dopo appena 24 h dalla semina siano completamente adese alla matrice e presentano una tipica forma allungata. A tempi più lunghi, 7-14 giorni si nota che le popolazioni cellulari hanno realizzato un monostrato completo, con difficoltà nel evidenziare i contorni cellulari di una singola cellula e con un inizio di deposito di matrice extracellulare.
I saggi per la valutazione dell’attività osteogenica in cellule MC3T3-E1 ha evidenziato come nei polimeri PCL-AT2-HA e PCL-AT2-BP ci sia una maggiore espressione di fosfatasi alcalina e una maggiore calcificazione della matrice rispetto a PCL-WP e PCL-AT2 questo dato significa che la presenza di fattori come idrossiapatite e polvere d’osso creano delle condizioni maggiormente favorevoli alle popolazioni celllulari affinché esse si differenzino in cellule dell’osso.
L’analisi di RT-PCR eseguita su cellule MSC seminate su PCL ha confermato l’avvenuto differenziamento in senso osteogenico, in quanto è stato osservato l’espressione di marcatori tipici come osteocalcina, RUNX-2, osteopontina, collagene I. In aggiunta anche in MSC si è riscontrato un profilo simile per quanto riguarda la produzione di fosfatasi alcalina e depositi di sali calcio, dove però si è osservato che le cellule MSC su PCL-AT2-HA mantenute in condizioni basali di coltura esprimano un significativa differenza (p<0,05) nella produzione di calcio rispetto a quelle seminate sugli altri scaffolds. dato è confermato anche a 14 e 21 giorni.
In conclusione possiamo dire di avere individuato un polimero adatto per la realizzazione di uno scaffold per la rigenerazione ossea, e individuato una metodica riproducibile, facile da eseguire ed economica, ottenendo scaffolds con indici morfometrici simili all’osso trabecolare naturale con utilizzo di alginato di calcio come porogeno, e capace di permettere l’adesione, la proliferazione e il differenziamento di preosteoblasti di linea e cellule mesenchimali staminali. L’aggiunta di HA e polvere d’osso agli scaffolds polimerici, non influisce sulla porosità ma garantisce maggiore rigidità, inoltre ottimizzano la potenzialità osteogenica delle matrici. Grazie alle caratteristiche della metodica di produzione, è stato possibile realizzare scaffolds con una forma di falange umana dimostrando che il metodo permette la possibilità di creare una protesi ossea su misura.

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Tipo di EPrint:Tesi di dottorato
Relatore:Grandi , Claudio
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:22 Gennaio 2011
Parole chiave (italiano / inglese):bone regeneration, scaffold porosity, polycaprolactone, calcium alginate threads, scaffold pore size, mesenchymal stem cells
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/09 Farmaceutico tecnologico applicativo
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:3355
Depositato il:01 Ago 2011 09:37
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