Dissertations / Theses on the topic 'Bio-scaffold'

La ricerca riportata nel presente elaborato è stata focalizzata sull'emergente tecnologia degli stent coronarici riassorbibili (BRS, bioresorbable scaffold), con l obiettivo di generare dati a medio e lungo termine che potessero contribuire a chiarire il comportamento di tale tecnologia nei diversi contesti clinici, a ottimizzarne l uso e a definire gli ostacoli alla sua applicazione ordinaria, per poter definire strategie mirate di sviluppo e miglioramento dell intera tecnologia dei BRS. Per tale scopo, il presente progetto consta di diversi studi che possono essere raggruppati in: - valutazione dei risultati clinici a medio termine associati all impianto del BRS Absorb; - analisi di sottogruppo da registri monocentrici e multicentrici del BRS Absorb; - registro con survey multicentrica mirata a valutare nella pratica clinica quotidiana i criteri di selezione dei pazienti da trattare con i BRS; - documenti di consenso mirati a definire la tecnica di impianto ottimale e la selezione dei pazienti e delle lesioni potenzialmente candidate all'impianto del BRS Absorb; - analisi mediante tecniche di imaging intravascolare mirate a valutare le caratteristiche dell interazione tra lo scaffold e la parete coronarica, che possono traslare in outcome clinici; - valutazione dei risultati clinici a lungo termine associati all impianto del BRS Absorb. Le caratteristiche e i risultati di tali studi sono stati riportati nel presente elaborato finale.

In questo lavoro di tesi è stato progettato e caratterizzato uno scaffold 3D di cheratina innovativo tramite un approccio bio-ingegneristico integrato che unisce anche lo stimolo bio-meccanico generato da un campo elettromagnetico pulsato (PEMF). Lo scaffold è stato preparato mediante la fibrillazione di fibre di lana (cheratina) sfruttando i componenti istologici che le compongono (fibrille o cellule corticali), al fine di ottenere una struttura adatta alla rigenerazione ossea. E’ stato quindi progettato uno scaffold di cheratina (spugna di fibrille di lana) con micro e macro-porosità interconnesse di dimensione controllata, al fine di ospitare le cellule, favorendone l’adesione e guidando opportunamente la formazione di nuovo tessuto. Crosslinks aggiuntivi impartiti alle catene cheratiniche hanno permesso di ottenere uno scaffold con eccellente stabilità in acqua nonostante l’elevato rigonfiamento, resilienza alla compressione e stabilità alla degradazione. La cheratina contiene sequenze di adesione cellulare che facilitano la crescita delle cellule. Infatti, cellule SAOS-2 coltivate sulle spugne di fibrille di lana in condizioni proliferative (PM) e osteoinduttive (OM), hanno mostrato rispettivamente una crescita e differenziamento ottimali. Il differenziamento, in termini di aumento della mineralizzazione e deposizione di proteine della matrice è stimolato dall’applicazione del PEMF. Lo stimolo bio-meccanico velocizza il processo di differenziamento in condizioni osteoinduttive, mostrando una perfetta sinergia tra gli stimoli biochimici e meccanici nell’accelerazione del processo differenziativo. La valutazione della crescita di cellule staminali da midollo osseo su scaffold di cheratina 2D e 3D (film di fibrille di lana e idrogeli di cheratina) ha mostrato la loro efficacia nel supportare le cellule staminali; in particolare, i sistemi 3D, grazie al loro diverso tempo di degradazione, possono funzionare da cell-delivery system o da impalcatura a lungo termine. L’elevato tempo di degradazione mostrato dalla spugna di fibrille di lana suggerisce che questo scaffold possa essere promettente come supporto a lungo termine per la formazione ossea in vivo.
Novel keratin-based 3D scaffold for bone tissue engineering have been produced, characterized and tested, applying bio-mechanical stimuli generated by a pulsed electromagnetic field (PEMF). Controlled-size, interconnected porosity, tailored to match the natural bone tissue features, has been designed for cell guesting, proliferation and guided tissue formation, exploiting the natural histological structure of the wool fibers. Additional crosslinking of the keratin chains allowed obtaining excellent water stability and significant swelling due to the synergic contribution of hydrophilicity and porosity, associated to increased compression resilience and ageing resistance. Keratin contains cellular-binding motifs for cell attachment found in the native extra-cellular matrix which facilitate better growth, providing proliferation signals and minimising apoptotic cell death. Viability and consistent proliferation were observed for SAOS-2 human osteoblast cells cultured both in proliferative (PM) and osteogenic (OM) media, highlighted by PEMF application, especially in osteogenic conditions, with increased mineralization and higher ECM proteins deposition. PEMF stimulated an earlier differentiation in osteogenic conditions, showing a perfect synergy between biochemical and mechanical stimuli in acceleration of the differentiation process. Evaluation of the attachment and growth of human bone marrow mesenchymal cells on different 2D and 3D keratin-based scaffolds, made with wool fibril films, sponges and hydrogels, showed that keratin-based materials are an effective support for stem cell growth. In particular, 3D systems gave the best results and, thanks to the different ageing time, they can be suitable as cell delivery system or for long-term scaffolding. The longer degradation rate suggests that wool fibril sponges can be promising candidates for long-term support of bone formation in vivo.

PURPOSE. Conventional 2D culture systems do not consider the importance of tissue architecture that is particularly relevant since tissue microenvironment deeply contribute to determine the outcome of anti-cancer treatments. In this study we aimed to set up and use an in vitro 3D models for Hodgkin lymphoma (HL) to evaluate the activity of specific ADAM10 inhibitors LT4 and MN8, alone or in combination with the anti-CD30 ADC brentuximab-vedotin (Bre-Ved). METHODS. Three different 3D culture models were set up: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells), LN-derived de-cellularized matrices and collagen sponges repopulated with both LN-MSC and HL cells. RESULTS AND DISCUSSION. In these 3D systems LT4 and MN8 reduced the size of mixed spheroids and intracellular ATP content. In addition, sCD30 and TNFα shedding was limited by LT4 and MN8 that not only interfered with HL cell growth, but also enhanced the anti-lymphoma effect of Bre-Ved. This effect was evident at low and ineffective doses of Bre-Ved as well, indicating a possible synergistic scheme to potentiate ADC-based lymphoma therapy. CONCLUSIONS. Both direct and combined anti-lymphoma effect of ADAM10 inhibitors with Bre-Ved can be studied in in vitro 3D model recapitulating features of LN microenvironment and leading to ADC effects improvement. For this reason, scaffolds may represent a new promising tool to reproduce LN architecture and useful for the study of pharmacological response.

The peripheral nerve injuries represent 2.8% of the total annual trauma. More than 360.000 people at year in the U.S.A. and more than 300.000 in Europe undergo this type of lesion, which often can lead to a condition of permanent disability (Ciardelli and Chiono, 2005). The main cause is represented by motor vehicle and sports accidents, nevertheless knives, glass, metal lacerations and bone fractures cover as much as 30% of the total cases. Even the same surgery, especially orthopedic on upper limb, it is often due to nerve injury, as they are traction maneuvers that are performed in childbirth, which cover a rate of 0.12% in all lesions (Ichiara et al , 2008). Although surgery is a frequent medical practice in most of the cases, tissue engineering, based on knowledge and used of engineering materials, growth factors and cells (both staminal or somatic cells) is becoming more and more like a viable, and sometimes, only possibility of repair the peripheral nerve lesions (Geuna et al, 2007). Based on these considerations it has been designed, built, developed and tested a composed tubular scaffold in polyvinyl alcohol (PVA) from chemical, physical and biological point of view. The PVA is a synthetic polymer, water-soluble, biocompatible, durable, inexpensive and hardly degradable. In order to facilitate a better kinetic of degradation of the material it has been chemically modified, by a oxidative reaction. This same reaction has been shown, moreover, to promote the release of neurotrophic factors, including TAT-CNTF, whose activity is known to be essential in the various stages of the peripheral nerves regeneration process. The work course, from the material oxidation to the final product (through the freeze thawing technique) has developed a tubular polymer scaffold which is a patented industrial invention (No: VI2013A000019, class: A61K) deposited in “Camera di Commercio Industria, Artigianato e Agricoltura” of Vicenza, Italy.
Le lesioni dei nervi periferici rappresentano il 2,8 % dei traumi totali annui. Più di 360.000 persone all’anno negli USA e più di 300.000 in Europa vanno incontro a questo genere di lesione, che spesso può portare ad una condizione di disabilità permanente (Ciardelli e Chiono, 2005). La causa principale è rappresentata dagli incidenti automobilistici e pratica sportiva; ciò nonostante lacerazioni da coltelli, vetri, metalli e fratture ossee ricoprono il 30 % della casistica. Anche la stessa chirurgia, specialmente quella ortopedica degli arti superiori, è spesso causa di lesione nervosa, come lo sono anche le manovre di trazione che vengono effettuate durante il parto, che ricoprono il 0,12 % della totalità delle lesioni (Ichiara et al, 2008). Sebbene nella maggior parte dei casi si ricorra alla chirurgia, l’ingegneria tissutale, basata su conoscenze di ingegneria dei materiali, fattori di crescita proteici e cellule (sia staminale che somatiche) si sta affermando sempre più come una valida, ed a volte, unica possibilità di riparazione delle lesioni nervose periferiche (Geuna et al,2007). Basandosi su queste considerazioni è stato ideato, costruito, sviluppato e testato, da un punto di vista chimico, fisico e biologico uno scaffold tubulare di polivinilalcol (PVA). Il PVA è un polimero sintetico, idrosolubile, biocompatibile, resistente, poco costoso e difficilmente degradabile. Al fine di favorire una migliore cinetica di degradazione, il materiale è stato modificato chimicamente, mediante una reazione di tipo ossidativo. Questa stessa reazione si è dimostrata, inoltre, idonea a favorire il rilascio di fattori neurotrofici, tra cui TAT-CNTF, la cui attività è fondamentale nelle varie tappe del processo rigenerativo dei nervi periferici. Il percorso di lavoro svolto, dalla ossidazione del materiale fino al prodotto finito (attraverso la tecnica di freese thawing) ha prodotto uno scaffold polimerico tubulare coperto da brevetto come invenzione industriale ((No: VI2013A000019, classe: A61K) depositato presso “Camera di Commercio Industria, Artigianato e Agricoltura” di Vicenza.

Nuovi biomateriali impiantabili con sistema di rilascio di farmaci anti-tumorali stanno emergendo come terapia alternativa per la riparazione del tessuto osseo e il trattamento del cancro osseo, compreso l'osteosarcoma (OS). Biomateriali composti da idrossiapatite nanostrutturata, con stronzio (CD-HA2%Sr) funzionalizzati con i farmaci metotrexato (CD-HA2%Sr-MTX) e doxorubicina (CD-HA2%Sr-DOX), potrebbero rappresentare una nuova strategia terapeutica per la rigenerazione ossea e il trattamento dell’OS. L’OS è una neoplasia maligna dell’osso che colpisce pazienti pediatrici. Nonostante gli svantaggi, la linea terapeutica attuale consiste in chirurgia associata a chemioterapia con alte dosi di metotrexato e doxorubicina. Per la prima volta in questo studio, l’efficacia dei suddetti biomateriali è stata testata in vitro. Gli obiettivi dello studio erano la valutazione delle (1)citocompatibilità e (2)osteoinduttività del biomateriale CD-HA2%Sr con l’impiego di cellule staminali mesenchimali da tessuto adiposo umano (hASCs) e (3)dell’effetto citotossico dei biomateriali funzionalizzati CD-HA2%Sr-MTX e CD-HA2%Sr-DOX su cellule tumorali di OS. La citocompatibilità e osteoinduttività del biomateriale CD-HA2%Sr sono state analizzate nelle hASCs cresciute a contatto con i biomateriali fino a 14 giorni. La citocompatibilità è stata investigata tramite i saggi di vitalità cellulare Alamar Blue e Live/Dead, morfologia del citoscheletro e PCR Array per l’analisi dei geni coinvolti nella deposizione della matrice extracellulare. L’osteoinduttività è stata valutata tramite PCR Array per l’analisi dei geni coinvolti nel processo di osteogenesi, test ELISA specifico per l’osteocalcina (OCN) e l’analisi della deposizione di fosfati di calcio. L’attività citotossica dei biomateriali CD-HA2%Sr-MTX (45μg/mL) e CD-HA2%Sr-DOX (5μg/mL) è stata valutata utilizzando cellule umane di osteosarcoma ingegnerizzate con la proteina verde fluorescente (SAOS-eGFP) cresciute a contatto con i biomateriali fino a 7 giorni. L’effetto citotossico dei farmaci rilasciati è stato analizzato mediante la valutazione del numero di cellule e la misurazione dell’intensità della fluorescenza emessa dalle cellule cresciute a contatto con i biomateriali. Inoltre, la struttura dei biomateriale CD-HA2%Sr a contatto con hASCs e SAOS-eGFP, e CD-HA2%Sr-MTX e CD-HA2%Sr-DOX a contatto con SAOS-eGFP sono state analizzate con il microscopio elettronico a scansione (SEM) e confocale (CM). La proliferazione delle hASCs a contatto con il biomateriale, l’architettura del citoscheletro ben organizzata e l’up-regolazione dei geni coinvolti nella deposizione della matrice extracellulare come integrine, caderine, collagene e metalloproteinasi hanno dimostrato che il biomateriale CD-HA2%Sr presenta citocompatibilità in vitro. L’aumentata espressione di OCN e mineralizzazione della matrice nelle colture di hASCs, insieme all’up-regolazione dei geni coinvolti nell’osteogenesi, hanno confermato l’osteoinduttività in vitro del biomateriale CD-HA2%Sr. D’altra parte, i risultati delle analisi Alamar Blue, SEM e CM, insieme alla misurazione della fluorescenza, hanno mostrato una diminuzione del numero di cellule SAOS-eGFP a contatto con i biomateriali funzionalizzati, CD-HA2%Sr-MTX e CD-HA2%Sr-DOX, rispetto a quello non funzionalizzato, CD-HA2%Sr, confermando la loro proprietà citotossica nei confronti delle cellule tumorali di OS. I risultati in vitro hanno dimostrato le proprietà di citocompatibilità e osteoinduttività del biomateriale CD-HA2%Sr e che i biomateriali funzionalizzati, CD-HA2%Sr-MTX e CD-HA2%Sr-DOX, potrebbero rappresentare un buon sistema di rilascio dei farmaci metotrexato e doxorubicina per la cura dell’OS. Poiché questi biomateriali possiedono buone proprietà osteoinduttive, potrebbero rappresentare una nuova strategia terapeutica per la rigenerazione dell’osso.
Introduction: New implantable drug-delivery scaffolds, which combine bone substitutes and anti-cancer molecules, are emerging as an alternative therapy for bone tissue repair and treatment of bone cancer, including osteosarcoma (OS). Innovative ceramic scaffolds composed of strontium-substituted nanostructured calcium-deficient hydroxyapatite (CD-HA 2%Sr) with drugs methotrexate (CD-HA 2%Sr-MTX) and doxorubicin (CD-HA 2%Sr-DOX) may represent an innovative delivery system for a novel therapeutic strategy, both for bone regeneration and OS treatment. OS is an aggressive malignant neoplasm of the bone, which mainly affects pediatric and young adult patients. Despite the several disadvantages, treatments consist of surgery and chemotherapy using high-dose methotrexate and doxorubicin drugs. In this study, for the first time, these innovative biomaterials were tested in vitro for their efficacy. Aims. The aims of this study were to evaluate on human adipose-derived mesenchymal stem cells (hASCs) (i) the cytocompatibility and (ii) the osteoinductivity of CD-HA 2%Sr scaffolds and (iii) to assess the cytotoxic effect of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX drug-delivery scaffolds on OS cells proliferation. Material and methods. The cytocompatibility and osteoinductivity properties of CD-HA 2%Sr were assessed in hASCs grown on the scaffold, up to day 14. Cytocompatibility was investigated using Alamar Blue and Live/Dead assays, cytoskeleton morphology and human extracellular matrix PCR Array, whereas osteoinductivity was evaluated using human osteogenesis PCR Array, ELISA test specific for osteocalcin (OCN) and mineral matrix deposition analysis. The anti-OS cell proliferation activity of CD-HA 2%Sr-MTX (45μg/mL) and CD-HA 2%Sr-DOX (5μg/mL) was assessed employing the fluorescent engineered human osteosarcoma cell line SAOS-eGFP grown on biomaterials, up to day 7. The effects of released drugs were evaluated in terms of cell numbers and fluorescence intensity rate reductions in SAOS-eGFP cells grown on scaffolds. In addition, the structure of CD-HA 2%Sr scaffolds with both hASCs and SAOS-eGFP cells, and the structure of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds with SAOS-eGFP cells were analysed by scanning electron (SEM) and confocal microscopes (CM). Results. The increasing number of hASCs, the well-organised cytoskeleton architecture alongside the up-regulation of extracellular matrix genes including integrins, cadherins, collagens and MMPs suggested that CD-HA 2%Sr scaffold owns in vitro cytocompatibility. In hASC cultures, the increased OCN protein expression and matrix mineralization, alongside the up-regulation of genes involved in skeletal development, demonstrated CD-HA 2%Sr scaffold displays in vitro osteoinductivity. In addition, decreasing cell numbers, SEM and CM analyses, alongside fluorescence intensity measurement indicated that CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds displayed a cell-killing effect on SAOS-eGFP cells compared to CD-HA 2%Sr, thus validating the in vitro anti-proliferative properties of these scaffolds. Conclusion. Overall, these in vitro results demonstrated the cytocompatibility and osteoinductivity properties of CD-HA 2%Sr scaffold. In addition, experimental data with functionalized CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds indicate that these innovative biomaterials could represent a good delivery system for methotrexate and doxorubicin, which are anti-tumour drugs, for OS therapy. At the same time, since these innovative scaffolds, employed in vitro as with drug-delivery system, own a good osteoinductive properties too, they could be used as a novel therapeutic strategy for bone tissue regeneration.

Principal purpose of regenerative medicine for the cardiac tissue is to find the best way to inoculate stem cells in a specific myocardial area damage, improving their homing, integration and survival. To achieve this goal, the field of biomaterials is important to bypass this obstacles, modulating the environment for implanted cells and enhance CSC function in the heart. Biomaterials can mimic or include naturally occurring extracellular matrix and instruct stem cell function in different ways: promoting angiogenesis, enhancing stem cell engraftment and differentiation, and accelerating electromechanical integration of transplanted cells. The aim of this thesis was to assess whether the properties of three-dimensional polymer matrices in synthetic biomaterial such as polylactic acid and in natural origin as silk fibroin, if and how influence differentiative process of stem cells cardiac c-kit +. Another point considered was been the evaluation of expression of cardiac markers and sarcomeric proteins of cells isolated, inoculated in different types of scaffold and maintained in colture for 21gg in vitro and analyzed in RT-PCR and Real-time quantitative RT–PCR analysis. Also it was analyzed the immunogenicity of the scaffold when implanted in the dorsal subcutaneous region of nude mice, nude rats and SCID mice in order a possible use in vivo in the cardiac regeneration. These experiments showed a myocardial-like differentiation, in which the CSCs acquired a muscle-like shape, with the formation of initial intercalated disks, and a striated-like myofilament organization. In results shown below highlights evidence of an higher degree of differentiation using 3D scaffold for CSCs c-Kit+ that can be induced to differentiate definitely into cardiomyocytes thanks to three-dimensional culture of the scaffold, where is possible an environment similar to a cardiac niche in vivo.

Fabrication of scaffold is the key for bone tissue engineering, which is commonly regarded as the most potential route for repairing bone defects. Previously, porous ceramic scaffolds were fabricated through a variety of traditional methods, like moulding and casting, but most of them cannot produce customised tissue-engineered scaffolds. Therefore, 3D printing methods are gaining more attention and are currently being explored and developed to make scaffolds with acceptable biocompatibility. With the considerable development of bone tissue engineering, the bioactivity of scaffolds is becoming increasingly demanded, which leads to new methods and techniques to produce highly biomimetic bone scaffolds. In this study, a new fabrication process to optimise the structures of scaffolds was developed, and intensive researches were performed on the porous scaffolds to confirm their advantages in biological performance. Specifically, by combination of motor assisted extrusion deposition and gas-foaming (graphite as the porogen) technique, hierarchically porous scaffolds with improved microstructures, i.e. multi-scaled pores from nanometre to millimetre (nm-μm-mm), was successfully developed. In this thesis, the optimal content of porogen for scaffolds was studied in terms of compressive strength and in-rod porosities. The most concerned physicochemical properties of scaffolds were carefully examined and the results revealed that such scaffolds exhibit excellent physicochemical properties owing to hierarchically porous structures. Due to additional in-rod micropores and increased specific surface area, along with better hydrophilicity, hierarchically porous scaffolds exerted complete superiority in biological activity, including promoting cellular proliferation of osteoblasts, adhesion and spreading status, as well as the ability to induce cellular differentiation.

Secondo l'Organizzazione Mondiale della Sanità (OMS), le malattie della cornea sono la quarta causa di perdita della vista a livello globale. C'è una necessità universale sia per la cura oculare che per il ripristino dell'acuità oculare e nei decenni futuri la previsione rimane critica. Le malattie degli occhi colpiscono almeno 2,2 miliardi di persone in tutto il mondo, sia per problemi visivi che per cecità totale. Tra questi, più di 1 miliardo rappresenta una disabilità visiva che deve ancora essere affrontata o che si sarebbe potuta evitare. La cornea è l'interfaccia trasparente tra l'occhio e l'ambiente esterno e, a causa della sua posizione esterna, potrebbe essere danneggiata da lesioni e infezioni. In presenza di un danno stromale significativo, una delle tecniche più utilizzate per ripristinare l'acuità visiva è il trapianto di cornea allogenico (la procedura di cheratoplastica); in cui per il trapianto viene utilizzata una cornea di donatore umano come innesto. La cornea può essere sostituita nella sua totalità (cheratoplastica perforante) o in parte (cheratoplastica lamellare). Questa procedura dovrebbe essere superata a causa della carenza di donatori e delle complicazioni legate al rigetto immunologico, al glaucoma e alla cheratite microbica, che ancora limitano il successo. Pertanto, una vasta gamma di biomateriali viene sempre più presa in considerazione come sostituto della procedura di cheratoplastica. L'intento è quello di trovare substrati naturali e biocompatibili per la ricostruzione corneale. Pertanto, è importante considerare la funzione e l'anatomia della cornea umana come precursori per la valutazione di una varietà di diversi biomateriali per la rigenerazione corneale. Dunque, lo scopo di questo progetto è quello di ricostruire una cornea completamente autologa, seminando cellule corneali umane primarie su scaffold biocompatibili. Grazie alla nostra precedente conoscenza ed esperienza sulla coltivazione e caratterizzazione di cellule staminali limbari, abbiamo innanzitutto analizzato il corretto attaccamento, colonizzazione e crescita dei cheratinociti limbari umani su diversi scaffold biocompatibili e trasparenti. inoltre, poiché è importante che i nostri biomateriali abbiano proprietà specifiche utili per il trapianto clinico, abbiamo analizzato: l'origine naturale, la biocompatibilità e la biodegradabilità. Oltretutto, un biomateriale appropriato dovrebbe avere una forza biomeccanica significativa e una trasparenza molto elevata. Pertanto, abbiamo esplorato queste caratteristiche per selezionare il miglior candidato per la ricostruzione corneale. Infine, abbiamo ottimizzato anche le procedure per la standardizzazione. Infatti, abbiamo migliorato la realizzazione del nostro costrutto, controllando la semina cellulare e selezionando le caratteristiche appropriate degli scaffold naturali analizzati; al fine di utilizzare, in futuro, questi scaffold per test farmacologici in vitro o per scopi clinici.
According to the World Health Organization (WHO), diseases of the cornea are the fourth-largest cause of vision-loss globally. There is a universal need for both eye care and for restoring eye acuity and in future decades the projection remains critical. Eye diseases affect at least 2.2 billion people globally, both for vision impairment and blindness. Between those, more than 1 billion is a vision impairment that still needs to be addressed or that could have been intercepted. The cornea is the transparent interface between the eye and the external environment and due to its external location, the cornea could be damaged by injuries and infections. In the presence of significant stromal damage, one of the most used techniques to restore visual acuity is the allogeneic corneal transplantation (the keratoplasty procedure); in which for the transplantation is used a human donor cornea as a graft. The cornea can be replaced in its entirety (penetrating keratoplasty) or in part (lamellar keratoplasty). This procedure should be overcome due to the donor shortage and complications related to immunological rejection, glaucoma, and microbial keratitis, which still limit success. Therefore, a range of biomaterials is being evaluated as substitutes for the keratoplasty procedures. The intent is to find natural and biocompatible substrates for corneal reconstruction. Thus, it is important to consider the function and the anatomy of the human cornea as a forerunner for the evaluation of a variety of different biomaterials for corneal regeneration. The aim of this project is to reconstruct a fully autologous cornea, seeding primary human corneal cells on biocompatible scaffolds. Thanks to our previous knowledge and expertise on limbal stem cells cultivation and characterization, we firstly analyzed the correct attachment, colonization, and growth of human limbal keratinocytes on different biocompatible, transparent scaffolds. Since it is mandatory for our biomaterials to have specific properties useful for clinical transplantation, we analyzed: the natural origin, the biocompatibility, and the biodegradability. An appropriate biomaterial should have significant biomechanical strength and very high transparency. Thus, we explored these characteristics to select the best candidate for corneal reconstruction. The procedures for standardization were also optimized. Indeed, we have improved the manufacturing by controlling the cell seeding and selecting the appropriate characteristics of the selected natural scaffolds for the future use of the constructs for in vitro drug testing or for clinical purposes.

Carbon nanotubes (CNTs) are attractive candidates for the development of scaffolds for neural regeneration thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. This thesis work concerns the development of a freestanding nanocomposite scaffold composed of multi-walled CNTs in a poly-L-lactic (PLLA) matrix that combines the conductive, mechanical and topographical features of CNTs with the biocompatibility of PLLA. Such CNT-PLLA scaffold resulted to support growth and differentiation of neuronal SH-SY5Y cells better than PLLA alone. In order to mimic guidance cues from the neural environment, biomimetic peptides were designed to reproduce regulatory motifs from L1CAM and LINGO1 proteins, that are involved in neurite outgrowth control. Both peptides - which neither alter cell proliferation nor induce cell death - could specifically and positively modulate neuronal differentiation when either used to coat well bottoms or added to the culture medium (with highest efficiency at 1 uM concentration). Furthermore, cell differentiation resulted to be synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation. Then, the CNT-PLLA matrix was electrospun into fibres of submicrometric size in order to better mimic the neural environment, i.e. neuronal processes and collagenous components of the extracellular matrix. These scaffolds were shown to be biocompatible and to promote the formation of new neurites that extend along the scaffold fibres. Since cells are influenced by the scaffold topography, the orientation of the scaffold fibres opens up the perspective to promote a polarized neurite outgrowth. Moreover, the neuritogenic properties of the scaffolds are further enhanced when LINGO1 derivative peptide is added to culture medium; this represents a good starting point for developing next generation scaffolds upon peptide functionalization. Moreover, human circulating multipotent cells (hCMCs) were grown onto the scaffolds and treated with peptides in order to asses if this autologous and accessible source of stem cells is capable of neuronal differentiation thanks to the scaffold and peptide characteristics. The CNT-PLLA scaffolds and its respective electrospun version resulted to be suitable for hCMCs adhesion and growth, showing a very good level of biocompatibility, and the hCMCs growing onto the scaffolds showed typical features of cells from the neuronal lineage, such as long neuritic protrusions that are tipped with fan-shaped structures resembling growth cones. Moreover, soon after cell seeding, the scaffolds were shown to promote the upregulation of markers typical of the neuronal lineage.The biomimetic peptides were also shown to influence cell morphology and to upregulate neuronal markers. These results suggest that hCMCs can acquire neuronal commitment thanks to scaffold/peptide properties per se, i.e. even in the absence of those typical growth factors that are normally used to promote the neuronal differentiation of stem cells. Further improvements in the scaffold geometry and composition, functionalization with peptides and culture conditions are necessary to achieve the complete neuronal differentiation of cells and to control the neuron subtype obtained, but our system resulted to be a good starting point for setting up implantable scaffolds for autologous neuronal differentiation. Future functional assessment of synaptic transmission and electrophysiological properties of cells onto the scaffolds will be of great interest. Moreover, coupling such scaffolds with electrical stimulation (which is readily achievable using CNT based materials) can boost further analyses aimed at studying neuronal differentiation and has great potential in nerve injury repair as well as neuron prosthesis.
I nanotubi di carbonio (CNTs) sono i candidati ideali per lo sviluppo di supporti volti a promuovere la rigenerazione neurale grazie alla loro abilità di condurre gli stimoli elettrici e alla loro nanotopografia in grado di mimare l'ambiente neurale. Questo lavoro riguarda lo sviluppo di supporti nanocompositi costituiti da CNTs dispersi in una matrice di acido polilattico (PLLA) e quindi in grado di combinare le caratteristiche nanotopografiche e di conduttività dei CNTs con la biocompatibilità del PLLA. Tali supporti, sono risultati essere in grado di supportare la crescita e il differenziamento delle cellule neuronali SH-SY5Y in modo migliore rispetto al solo PLLA. Al fine di mimare gli stimoli guida dell'ambiente neurale, sono stati sintetizzati anche dei peptidi biomimetici ricavati da specifici motivi regolativi delle proteine L1CAM e LINGO1, le quali sono coinvolte nel controllo dell'accrescimento neuritico. Entrambi i peptidi non hanno dimostrato effetti negativi sulla vitalità e la proliferazione cellulare, promuovendo invece il differenziamento neuronale in modo sequenza specifico e con i maggiori effetti quando utilizzati a concentrazione 1 uM. Inoltre, quando usati in combinazione, supporti e peptidi sono in grado di agire in modo sinergico e di aumentare ulteriormente il differenziamento cellulare. Successivamente, al fine mimare al meglio l'ambiente neurale, la matrice CNT-PLLA è stata elettrospinnata in fibre di dimensione submicrometrica con lo scopo di rappresentare i processi neuronali e la componente collagenosa della matrice extracellulare. Tali supporti si sono rivelati essere biocompatibili e in grado di promuovere la formazione di nuovi neuriti che si allungano seguendo l'orientamento delle fibre del supporto. Dal momento che le cellule sono influenzate dalla topografia del supporto, l'allineamento delle fibre suggerisce la possibilità di poter ottenere una crescita neuritica polarizzata. Inoltre, le proprietà neuritogeniche del supporto aumentano quando il peptide derivato da LINGO1 viene aggiunto al terreno di coltura; questi risultati rappresentano un buon punto di partenza per sviluppare supporti più avanzati a seguito della funzionalizzazione con tale peptide. In aggiunta, cellule circolanti multipotenti umane (hCMCs) sono state coltivate sui supporti e trattate con i peptidi al fine di determinare se tale fonte di cellule staminali autologa ed accessibile sia capace di differenziazione neuronale grazie soltanto alle caratteristiche dei supporti e dei peptidi. I supporti CNT-PLLA e la rispettiva versione elettrospinnata sono risultati essere adatti all'adesione e alla crescita delle hCMCs, mostrando buoni livelli di biocompatibilità; inoltre, le hCMCs coltivate sui supporti hanno mostrato caratteristiche tipiche delle cellule neuronali come lunghe protrusioni neuritiche terminanti con strutture a forma di ventaglio simili ai coni di crescita. I supporti inoltre promuovono l'espressione di marcatori tipici del lignaggio neuronale. Anche i peptidi si sono rivelati essere in grado di influenzare la morfologia cellulare e di upregolare marcatori neuronali. Questi risultati suggeriscono che le hCMCs sono capaci di acquisire un commitment neuronale solo grazie alle caratteristiche dei supporti e dei peptidi e senza l'ausilio dei fattori di crescita che sono tradizionalmente usati per promuovere il differenziamento neuronale di cellule staminali. Sono necessari ulteriori studi riguardanti la composizione e geometria dei supporti, funzionalizzazione con i peptidi e condizioni di coltura per acquisire una completa differenziazione neuronale e controllare il tipo neuronale ottenuto; ma tale sistema sembra essere un buon punto di partenza per progettare supporti trapiantabili per promuovere la rigenerazione neurale. Sarebbe interessante poter valutare la trasmissione sinaptica e le proprietà fisiologiche delle cellule cresciute sui supporti così come utilizzare tali supporti per stimolare elettricamente le cellule e valutare un eventuale miglioramento nel differenziamento.

Abstract Introduction The demand for organ transplantation has rapidly increased during the past decades, thus requiring the development of a new interdisciplinary field, aimed at supplying this demand. Branching from regenerative medicine, the combination of elements usually applied separately for same or other purposes, was named Tissue Engineering (TE). Precisely, the components combined in TE constitute the so-called TE triad, are i) cells (derived from cell therapy), ii) scaffolding material (derived from material science) and iii) molecular signals (mainly derived from molecular biology and drug research). Among the organs and tissue that experience damages such that requiring implantation/transplantation, skeletal muscle represents no exception. As result of this, several approaches developed to efficiently repair the most common defects requiring surgery: Volumetric muscle loss (VML), abdominal wall defects (AWD) and defects of the diaphragm, namely traumatic diaphragmatic hernias or congenital diaphragmatic hernias (CDHs). While the first necessitates a substitute large in volume, the other two need a thinner but, according to defect size, wider surface. Thus far, between the materials chosen for repair of such defects, mostly synthetic materials were used, but decellularized tissue are rapidly covering the gap, likely to surpass them in the next future. Reason of this potential is the close resemblance to the organ or tissue of origin, while possessing features able to endpoint to constructive and functional remodeling. While decellularization-derivates of several organs and tissue were already attained, some were even tested in clinic and further moved to large-scale production (i.e. Surgisis®). However, only recently the proposal of developing a decellularized scaffold from diaphragm muscle for autologous repair purposes was reprised, and in general, was never considered in mouse. Methods Diaphragms (mouse and rabbit) were decellularized by a cyclical exposure to the sequence deionised water, sodium deoxycholate, DNase. Scaffolds were characterized in order to evaluate decellularization protocol efficiency, by the means of cell removal (mouse and rabbit), maintenance of microarchitecture (mouse and rabbit), ECM components (mouse), mechanical properties (mouse). Next, the interaction host-scaffold was tested in vivo, both in healthy and atrophic mice. Subsequent, a further characterization by disclosing the angiogenetic properties, both potential, taking advantage of the well-known CAM assay and subcutaneous transplant, as well as revealing angiogenesis-related protein content whithin the scaffold, and actual, by performing an orthotropic transplant in comparison with a synthetic material. To draw nearer the clinical validation, the characterized decellularized diaphragm (DD) were thus transplanted in the first surgically created model of CDH, again compared to the most commonly used material for the repair of this defect, polytetrafluoroethylene (PTFE), with the introduction recently of an allotransplant control. Lastly, as completion of a TE approach, it was developed a method to combine the cellular component, which was then applied to both mouse and rabbit derived scaffold. Results The adaption of a previously published detergent-enzymatic protocol aiming at obtaining a decellularized scaffold from diaphragm, resulted successful in with both mouse and rabbit sources, as cell removal while preserving microarchitecture and ECM components was attained. Moreover, it was confirmed that mechanical properties and growth factors were preserved in the mouse-derived scaffold. Upon implantation of the latter, angiogenesis and myogenic activation, while modulating the immune response were observed. In the healthy environment, the effect was transient, whereas implantation in a mouse model of atrophy led to long-term beneficial effects. The testing of mouse scaffold in a surgical model of CDH proved the previously seen results, as, compared to PTFE, yielded better outcomes, such as no recurrence, amelioration of diaphragm excursion and, differently from before, to a sustained myogenic response through time points analysed. The method used to re-populate both mouse and acellular muscles , after being developed, thus far resulted in a successful cell delivery, while the scaffold supported cell proliferation, survival and differentiation. Conclusions Muscle scaffold from mice and rabbit can be successfully obtained by decellularizing the diaphragm muscle via detergent enzymatic protocol. These scaffolds were proven to have several attractive properties both in vitro and in vivo, derived from their close resemblance to the tissue of origin. Precisely, it was demonstrated that while in vitro can sustain cell survival, proliferation and differentiation, in vivo positively interact with the recipient environment, driving a constructive response. Hence, scaling up of this type of construct is likely to be happening in the next future.
Riassunto Introduzione Negli ultimi decenni, la lista di attesa dei pazienti che necessitano trapianto di organi o un intervento chirurgico mirato alla sostituzione di grandi porzioni di tessuto, è andata aumentando. Al contrario però, la disponibilità di donatori, sia vivi che cadaverici, non ha seguito lo stesso andamento. Come conseguenza, nonostante i tentativi di supplire a queste necessità, molti ancora muoiono in attesa di essere curati. La medicina rigenerativa, ovvero la combinazione di diversi elementi dell’ingegneria tissutale, nasce proprio per rispondere alla ancora pressante domanda di organi e tessuti trapiantabili. Precisamente, un approccio di medicina rigenerativa si avvale di tre componenti principali: i) le cellule (dalla terapia cellulare), ii) il supporto o impalcatura per favorirne la crescita (derivato dalla scienza dei materiali, ad esempio prostetici, comunemente chiamato scaffold o mesh) e iii) segnali molecolari (principalmente dalla biologia molecolare e della farmaceutica). Tra gli organi e tessuti che sperimentano danni tali da richiedere l'impianto/trapianto, vi è anche il muscolo scheletrico. Di conseguenza, anche per questo sono stati sviluppati diversi approcci mirati a riparare efficacemente i più comuni difetti sia pediatrici sia adulti che richiedono un intervento chirurgico: A) la perdita muscolare di un grande volume di muscolo (causato da incidenti o dalla necessità di rimuovere tumori muscolari), B) difetti della parete addominale e C) difetti del diaframma, soprattutto congeniti. A seconda del tipo di difetto, il sostituto da sviluppare dovrà avere peculiari proprietà, oltre a quelle basilari necessarie per essere considerato un approccio di ingegneria tissutale completo. Infatti, mentre A necessita di un sostituto in grado di ripristinare un grande volume, B e C richiedono un costrutto sottile ma, a seconda delle dimensioni del difetto, di una più o meno ampia superficie. Finora, per la riparazione di tali difetti per lo più sono stati utilizzati più frequentemente scaffold/mesh sintetici, ma quelli di derivazione naturale, ed in particolare ottenuti dalla decellularizzazione di un tessuto nella sua completezza, stanno rapidamente recuperando strada, ed è probabile che diverranno il metodo di elezione nel prossimo futuro. La ragione di questo potenziale è la stretta somiglianza con l'organo o il tessuto di origine, pur possedendo caratteristiche in grado di culminare in un rimodellamento costruttivo e funzionale. Inoltre, mentre la decellularizzazione è stata ottenuta anche in organi più complessi, i prodotti derivati da tessuti più semplici sono stati anche già testati in clinica e per alcuni si è persino giunti alla commercializzazione su larga scala (ad esempio il Surgisis®). In questo lavoro si è partiti dal considerare la patologia dell’ernia diaframmatica, ad oggi riparata chirurgicamente tramite chiusura primaria con tessuto autologo, se il difetto lo permette, oppure ancora con mesh prostetici di natura sintetica. Tuttavia, gli svantaggi di questi tipi di materiali in termini di rigidità ed inerzia, rendono conto dell’attuale ricerca di una valida alternativa. Si è proposto quindi di derivare un costrutto muscolare partendo dalla decellularizzazione del muscolo diaframmatico utilizzabile come toppa tissutale per riparare il danno dell’ernia sia senza cellule sia con nuove cellule donatrici. Il modello studiato è stato, per la prima volta, quello murino. Metodi Diaframmi (topo e coniglio) sono stati decellularizati tramite esposizione ciclica alla sequenza: acqua deionizzata, sodio desossicolato, DNasi. Gli scaffold ottenuti sono stati caratterizzati per valutare l'efficienza del protocollo in termini di rimozione delle cellule (topo e coniglio), preservazione della micro architettura (topo e coniglio), componenti della matrice extracellulare (topo), proprietà meccaniche (topo). Successivamente, l'interazione ospite-scaffold (in topo) è stata testata in vivo, sia in topi sani che atrofici. Un ulteriore caratterizzazione è stata attuata approfondendo le proprietà angiogeniche, sia potenziali, sfruttando il noto test CAM, il trapianto sottocutaneo e rivelando il contenuto di proteine coinvolte nella angiogenesi ancora presenti dopo la decellularizzazione, sia effettive, eseguendo un impianto ortotopico, utilizzando come confronto un materiale sintetico. Per avvicinare la validazione alla pratica clinica, il diaframma decellularizato di topo precedentemente caratterizzato è stato quindi utilizzato per il riparo nel primo modello di ernia diaframmatica chirurgicamente indotta in topo. Il paragone è stato fatto anche in questo caso con il materiale più comunemente usato per la riparazione di questo difetto in clinica. Inoltre, più recentemente è stato introdotto un controllo rappresentato dall’allotrapianto. Infine, a completamento di un approccio di ingegneria tissuatle, è stato sviluppato un metodo per combinare la componente cellulare, poi applicato a entrambi gli scaffold derivati da topo e coniglio. Risultati L'adattamento di un protocollo detergente enzimatico precedentemente pubblicato, al fine di ottenere uno scaffold decellularizato da diaframma, si è rivelato efficiente sia utilizzando il topo che il coniglio come fonti, ottenendo così da entrami la rimozione delle cellule preservando la somiglianza della struttura con il tessuto di origine. Inoltre, è stato confermato, solo in topo, che le proprietà meccaniche e i fattori di crescita sono stati conservati dopo la decellularizzazione. L'impianto dello scaffold di topo, ha provocato sia angiogenesi che l'attivazione dei precursori muscolari, modulando al contempo la risposta immunitaria. Nell'ambiente sano, l'effetto è stato transitorio, mentre l'impianto in un modello murino di atrofia ha portato ad effetti benefici a lungo termine. La sperimentazione nel modello chirurgico di ernia ha confermato i risultati precedentemente osservati ed in aggiunta, rispetto al materiale sintetico, ha mostrato migliori risultati, come l'assenza di erniazione recidiva, il miglioramento dell’escursione diaframmatica e, diversamente da prima, una più sostenuta risposta miogenica nel tempo. Il metodo utilizzato per ripopolare entrambi i diaframmi acellulari di topo e muscoli, dopo essere stato sviluppato, ha finora portato a risulstati positivi, mentre lo scaffold ha dimostrato di poter supportare la proliferazione, la sopravvivenza e la differenziazione delle cellule. Conclusioni Scaffold derivati dal muscolo di diaframma possono essere conseguiti con successo sia da topi che conigli, utilizzando un protocollo di tipo detergente-enzimatico. Tali, hanno dimostrato di possedere diverse proprietà interessanti sia in vitro che in vivo, derivate dalla loro stretta somiglianza con il tessuto di origine. Precisamente, è stato dimostrato che, mentre in vitro possono sostenere la sopravvivenza, la proliferazione e la differenziazione cellulare, in vivo sono in grado di interagire positivamente con l'ambiente ricevente, guidando una risposta costruttiva. Quindi, è molto probabile che ulteriori passi vengano fatti nel prossimo futuro, fino a raggiungere infine l’applicazione in clinica.

Previous works showed that muscle substitutes composed by acellular matrix and myoblasts may represent a promising approach for the treatment of diseases characterized by congenital absence or loss of large areas of skeletal muscle tissue. Here, the regeneration process occurring in vivo within the prosthetic material has been studied verifying the expression, as mRNA and protein, of some skeletal muscle and nerve tissue markers during three month after reconstructive surgery. The experimental evidences indicate that a progressive muscle remodelling within the implants exists and it is not completed after three months. Indeed, the simultaneous presence of markers belonging to both early and late differentiative stages indicates the presence of activated progenitors towards the myogenic line and cellular elements of advanced stages. Moreover, the involving of circulating precursors, probably of marrow origin, in the regeneration process it is suggested by the following observations: i) persistence of poorly differentiated cells at 3 months after surgery, ii) important cell migration from vessels without inflammatory phenomena, and iii) implanted myoblasts, that are cells already committed toward a specific fate, presumably form multinucleated elements in a short time. Finally, there is a rapid appearence of both vascular network and the nervous component. Although these promising results, in view of medical application a problem arise when the function of muscle satellite cells is impaired or their number inside the muscle fibers is low. To overcome this problem, this research has been addressed to the identification of alternative cell sources. For this reason, it has been evaluated both in vitro and in vivo the myogenic potential of mesenchymal cells (MSCs) obtained from umbilical cord blood (UCB). There is the possibility to withdraw the umbilical cord blood at the birth time and to store it in tissue banks, and having an autologous MSC reserve to use for congenital muscular diseases. Evidence for myogenic differentiation was found when MSCs were seeded on Matrigel® coated plates and cultured with myogenic media. After 4 days Myf-5, detected by immunofluorescence, was expressed by 13% of MSCs increasing to 30% at 8 days. We used quantitative PCR for human MyoD and Myogenin mRNAs to further demonstrate myogenic differentiation of MSCs. To evaluate the regenerative capacity of MSCs we used a skeletal muscle chemical injury model (bupivacaine hydrochloride), resulting in necrosis of muscle fibers in Lewis rat Tibialis Anterior. Undifferentiated green fluorescent protein (GFP)-labeled MSCs were injected into the injured muscle, without immunosuppression. The cells engrafted after 1 week and stained positive for Myf-5 and MyoD. After 2 weeks, we noted striations in fibers containing UCB-MSCs suggesting an organization of cytoskeletal proteins into sarcomeres. The skeletal muscle appeared intact by histological analysis, without signs of significant immunologic response, and the presence of MSCs was detected by immunostaining with a monoclonal antibody against human-Nuclei. In addition, these cells also expressed myosin and sarcomeric tropomyosin, a motor-protein and an actin-binding filament protein, respectively. At both time points we observed fibers with centrally located nuclei, indicating regenerated fibers. Finally, the number of GFP-positive fibers/total fibers counted in several fields was 40% for both time points and the area covered by these fibers was 20% of all fibers at 7 days increasing to 26% at 14 days, indicating a maturation of these myofibers. Our in vitro and in vivo data indicate that human MSCs are able to differentiate towards the myogenic lineage and may be efficiently incorporated into injured skeletal muscle, supporting the muscle regenerative process.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell cultures, animal models and clinical research. In this thesis, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural proteins (COL1A1, COL1A2, and COL3A1) and secreted proteins such as DEFA3. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy colon mucosa, caused by direct effect of DEFA3. In addition, we demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation, compared with recellularized healthy mucosa and 2D conventional culture model. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings.
I modelli tumorali tridimensionali (3D) si stanno affacciando sul panorama scientifico con l’obiettivo primario di superare le limitazioni di colture cellulari convenzionali (2D) e modelli animali negli approcci di ricerca clinica. In questa tesi di dottorato, si descrive un innovativo approccio di ingegneria tissutale applicata alla ricerca oncologica mediante il quale, partendo da una biopsia tissutale decellularizzata, si genera un modello organo-tipico 3D bioattivo. Questo modello 3D, ricapitola, in vitro, l’ambiente ultra-strutturale del tessuto nativo come dimostrato da indagini istologiche, immunoistochimiche, di immunofluorescenza e di microscopia elettronica a scansione. L’analisi del proteoma e del secretoma mediante spettrometria di massa ha confermato una differente composizione stromale tra la mucosa colica sana decellularizzata e quella della controparte tumorale (CRC) in termini di proteine strutturali (Collagene 1A1, Collagene 1A2, Collagene 3A1) e di proteine secrete, come la Defensina alfa 3. Abbiamo dimostrato che le nostre matrici 3D mantengono le loro proprietà biologiche dopo il processo di decellularizzazione: mediante la CAM, abbiamo osservato un decremento del potenziale angiogenico della matrice decellularizzata di CRC comparata con la mucosa colica sana, causata da un effetto diretto della Defensina alfa 3. Inoltre, abbiamo dimostrato che dopo 5 giorni di ricellularizzazione con cellule HT-29 (linea stabilizzata di cancro del colon), le matrici tumorali 3D (comparate con le rispettive mucose coliche sane ed il metodo di coltura 2D) hanno indotto una sovra-espressione di IL-8, una chemochina a valle del pathway della Defensina alfa 3, che gioca un ruolo molto importante nella crescita e proliferazione tumorale. In conclusione, avendo dimostrato la capacità dei delle nostre matrici acellulari 3D di mucosa colica sana e CRC di mimare gli stimoli ultra-strutturali e biologici dei rispettivi tessuti nativi, crediamo che questo approccio possa essere un efficace strumento per migliorare il livello delle ricerche precliniche e nei test di screening di farmaci.

Systemic pathologies such as diabetes and Sjögren’s syndrome, medications, and radiation therapy often affect salivary gland morphology and functionality, giving rise to changes in quantity and composition of saliva and to numerous oral complications that severely compromise the patient’s life quality. The aim of this thesis, articulated into two sections, was to evaluate: 1) if salivary gland morphology and functionality are affected by type 2 diabetes mellitus even when there are not evident signs of oral injuries; 2) the possibility to use a native scaffold derived from human salivary glands as a substrate in which salivary cells can be host to restore damaged salivary glands. The first part of the investigations was carried out with surgical samples of salivary glands obtained from subjects, half diabetics and half non-diabetics, all without evident oral diseases. The samples were processed for light and electron microscopy and random images were subjected to morphometrical evaluation. The calculations revealed diabetes-related alterations such as acinar swelling and remarkable changes in serous cells, where secretory granules appeared enlarged but reduced in their total number. On the other hand, an increased number of granules anchored to the apical membrane was found, as well as an altered number of apical vesicles and microvilli (both involved in the mechanisms of membrane recycle). Taken together these findings suggest the occurrence of difficulties in the last step of exocytosis, and demonstrate that the structures involved in the secretory process are altered by diabetes per se. Other samples were analyzed by means of immunogold staining method to reveal the ultrastructural localization of melatonin and its receptors MT1 and MT2, until now never reported. Both melatonin and receptors were reactive in the secretory granules, in cytoplasmic vesicles and on cell surfaces, suggesting that their interaction could allow melatonin storage within specific cell compartments. Melatonin staining when performed on diabetic major salivary glands highlighted changes in the labeling intensity related with the diabetic condition. 4 The second part is focused on the characterization of a native salivary gland scaffold in view of its use in gland reconstruction. As well as diabetes, radiation therapy, Sjögren’s syndrome, and several medications also give rise to salivary gland degeneration and xerostomia, but the therapies commonly used are not satisfactory so far. An emerging alternative approach to ameliorate the life quality of xerostomic patients is the regeneration of salivary parenchyma. A “Native Human Submandibular Gland Scaffold” (nHSMGS) was isolated from healthy submandibular glands, and was analyzed by light and electron microscopy and by histochemistry. Morphological examinations showed a fiber arrangement similar to that of the intact glands, while histochemistry revealed the presence of collagen type I, III, and IV. Then, a human salivary gland cell line and human fibroblasts were seeded and cultured on the scaffold in order to verify its reliability as autologous substrate for cells expansion. Results showed that a high cell percentage was proliferating after 4 days of culture, and that most cells were still alive after 8 days. All these data encourage the use of the nHSMGS as autologous scaffold in which expand salivary cells for the restoring of damaged salivary glands.

The aim of the first part of this research project was to improve the knowledge about the existence and location of adult stem cells in the adrenal gland. Although some experiments might suggest that undifferentiated cells derived from the external periphery of cortical zone, the origin of the regenerating cells remains ambiguous, and there are not currently known surface markers for defining these cells. Two distinct cellular populations, from the capsular zone and from the inner part of the adrenal gland, were isolated and investigated in vitro in order to study their phenotype, their proliferative potential and plasticity. The second research project aims to design scaffolds for bone tissue engineering. The development of novel scaffolds for bone tissue engineering is very complex, because ideal support for cellular colonization would possess the same structural and functional characteristics of ECM. In this work, the creation of hybrid scaffolds, mixing a very well known biocompatible synthetic polymer, poly(ε-caprolactones), with different self-assembling peptides, is presented. The microfibrous structure of the scaffold is assured by the electrospinning process, whereas the nanofibrous structure is produces by the self-assembling process of peptides. We prepared six different scaffolds adding six different peptides to poly(ε-caprolactones). These peptides were synthesized by solid phase strategy, and one sequence was prepared condensing a RGD motif to a self-assembling sequence. The characterization of the different scaffolds was carried out through SEM morphological analysis, FT-IR spectroscopy and contact angle measurements. All prepared scaffold exhibit interwoven nanofibers comparable to the ECM ones. FTIR investigations showed that self-assembling peptides incorporated in the PCL nanofibers retain the -sheet conformation, and that an incubation with saline buffered solution can increase the percentage of this structure in the RGD containing peptide. The enrichment with peptides improves the wettability of the polymer scaffold adding an important requirement for better cellular adhesion. The biological assay showed that the presence of self-assembling peptides into the scaffold increases cellular adhesion, the calcium amount and the gene expression of some proteins important for osteoblast.
Premessa Il lavoro di ricerca svolto nel triennio di dottorato si è focalizzato su due progetti. Il primo ha riguardato l’individuazione di cellule staminali nel surrene di ratto, al fine di poterle isolare e caratterizzare. Nel secondo progetto ci si è occupati della progettazione e realizzazione di matrici biomimetiche nanofibrose per la rigenerazione del tessuto osseo. RIASSUNTO Il primo progetto di ricerca ha cercato di chiarire le conoscenze attuali riguardo l’esistenza di cellule staminali adulte residenti nel tessuto surrenale. Sebbene alcune evidenze sperimentali suggeriscano l’esistenza di tali cellule nella parte esterna della corticale surrenale, al riguardo non c’è ancora una teoria riconosciuta in modo unanime: non è stata identificata la loro zona d’origine, né tantomeno sono stati individuati dei marker caratteristici che permettano di isolarle. L’individuazione di tali cellule potrebbe trovare applicazione nella cura di malattie surrenali, ad esempio nell’ipocorticosurrenalismo permetterebbe di evitare la terapia ormonale a vita e quindi, rappresenterebbe la terapia d’elezione. Il progetto si è quindi prefisso come obiettivi di identificare e isolare una popolazione di cellule staminali all’interno della ghiandola surrenale di ratto. Tramite analisi immunoistochimica e di immunofluorescenza sono state ricercate: a) zone cellulari BrdU+ (ratti trattati alla nascita con BrdU), a ciclo cellulare lento; b) marker di staminalità CD105, CD90 e c-kit. Dai surreni di ratto sono state estratte due sottopopolazioni di cellule, capsulari e della parte interna: queste sono state coltivate in vitro, osservate e immunoseparate per CD105 e CD90. Infine, è stata testata la capacità differenziativa delle colture sia in senso osteogenico che adipogenico. Nel secondo progetto sono stati ideati e prodotti scaffold da impiegare nell’ambito dell’ingegneria del tessuto osseo. La progettazione di uno scaffold per l’ingegneria tessutale risulta essere alquanto complessa visto che un supporto ideale per la colonizzazione cellulare dovrebbe possedere le caratteristiche, strutturali e funzionali, della matrice extracellulare. Lo scaffold ideato ha natura ibrida essendo composto di un polimero di sintesi biodegradabile quale il poli(ε-caprolattone) e di peptidi auto-assemblanti. La struttura fibrosa dello scaffold su scala micrometrica è stata assicurata mediante un processo di elettrofilatura. D’altro canto, è noto che i peptidi auto-assemblanti formano spontaneamente matrici tridimensionali con fibre nanometriche estremamente gradite a differenti tipi di cellule, incluse quelle del tessuto osseo. Sono stati ottenuti sei differenti tipi di scaffold utilizzando sei diversi peptidi, ottenuti per sintesi su fase solida, tra i quali una sequenza auto-assemblante coniugata ad un motivo adesivo RGD. Le matrici sono state estesamente caratterizzate mediante analisi di spettroscopia elettronica a scansione, spettroscopia all’infrarosso in trasformata di Fourier e valutazione dell’angolo di contatto. Le matrici risultano essere composte da un intreccio di fibre di dimensioni comparabili a quelle della matrice extracellulare; inoltre, è stato possibile confermare che la struttura β-sheet, che è alla base dell’auto-aggregazione, è presente nelle matrici e viene incrementata, nel caso del peptide auto-assemblante con motivo RGD, dal pre-trattamento con soluzione salina. E’ stato inoltre dimostrato come l’arricchimento con piccole percentuali (5%) di peptidi dello scaffold in poli(ε-caprolattone) produca un grado di bagnabilità notevolmente superiore e quindi crei i presupposti per una maggior colonizzazione della matrice da parte delle cellule. I saggi biologici, eseguiti su tutti gli scaffold, hanno permesso di dimostrare che la presenza di sequenze auto-assemblanti incrementa in modo significativo l’adesione cellulare, la produzione di calcio e l’espressione di geni che codificano per proteine importanti per gli osteoblasti.

The chemical industry is ever more focused on the development of sustainable and selective procedures for the synthesis of high-added value fine chemicals, in order to meet the demand in society for environment-friendly chemical processes. The metal-catalysed carbene and nitrene transfer reactions have proved to be an excellent strategy to achieve the goal, due to their ability to form C-C, C-N and C heteroatom bonds with high stereo- and regioselectivities. In addition, the good choice of the carbene and nitrene precursors can increase the reaction sustainability and the atom efficiency, as when diazo compounds and azides were used as starting materials thanks to the formation of molecular nitrogen as the only by product of the reaction. Among all the metal catalysed carbene and nitrene transfer reactions, the formation of three-membered ring compounds, such as cyclopropanes and aziridines, is a very attractive topic due to their use as building blocks in organic chemistry and their biological and/or pharmaceutical activity. Considering the catalytic activity of metal porphyrin complexes in promoting cyclopropanation and aziridination reactions of alkenes, the following PhD thesis aims to develop a new class of hybrid catalysts which are able to mediate carbene and nitrene transfer reactions under mild experimental conditions. In order to confer chiral recognition properties and modulate the catalyst properties, the conjugation of metal porphyrin complexes with bio scaffolds such as carbohydrates, amino acids, cellulose and β-lactoglobulins was studied. Carbohydrates are natural chiral, polyhydroxylated compounds which play critical roles in several biological processes, including the energy storage, cellular communications and recognitions, and the immune system maintenance. Glycoporphyrins, derived from the conjugation of porphyrin rings with sugar units, are efficient photosensitizers extensively used in photodynamic therapy (PDT) and they are desirable ligands for organometallic synthesis. To assess how different glycosylated porphyrin ligands can influence the catalytic activity of corresponding ruthenium and iron complexes, different meso glycosyl-conjugated porphyrins were synthesised. The number and position of carbohydrate units on the porphyrin skeleton play an important role in determining the ligand chemo-physical properties. Mono and tetra-glycosylated porphyrins were synthesized and the corresponding iron and ruthenium complexes were tested as catalysts of cyclopropanation and aziridination reactions. In the model cyclopropanation reaction between α-methylstyrene and EDA, Fe(III)(porphyrin)OMe complexes showed higher activity then Ru(II)(porphyrin)CO derivatives and obtained data indicated the strong dependence of the reaction diastereoselectivity on the ligand structure. Among all the iron complexes which were tested as cyclopropanation catalysts, Fe(15)OMe complex was chosen to study the reaction scope by testing different styrenes in view of the good compromise between the synthetic accessibility of synthesize it and reaction productivity (yields and diastereoselectivities). All the desired cyclopropanes were obtained in very good yields (up to 80%) and trans diastereoselectivities (up to 98:2). The obtained iron and ruthenium glycoporphyrin complexes were also tested in the model aziridination reaction between α-methylstyrene and 3,5-bis-(trifluoromethyl)phenylazide. Unfortunately, Fe(III)(porphyrin)OMe complexes didn’t catalyse the aziridination reaction but the desired product was obtained in a very good yield (83%) in the presence of Ru(15)CO. The latter catalyst was used to study the reactivity of α-methylstyrene towards different aryl azides and the obtained data indicated the ability of Ru(15)CO to promote the aziridination of azides showing different electronic and steric nature. Low yields were obtained in the presence of coordinating methoxy groups which can be responsible for the catalyst deactivation. Obtained results showed the applicability of Fe(15)OMe and Ru(15)CO as catalysts for the synthesis of three-membered ring compounds and the amphiphilic nature of ligand 15 can be suitable, after deprotection of the saccharide units, to perform catalytic reactions in biphasic systems. The second biomolecules class that has been studied in this thesis consists of aminoacidic residues. In order to synthesise new bio-inspired catalysts, the conjugation of α2β2 USCl porphyrin with the DAP aminoacid was studied. The desired product can be formed as a mixture of two different regioisomers. A similar ligand was obtained by reacting USCl porphyrin with the methyl ester of DAP aminoacid. The presence of different porphyrin derivatives was revealed by the 1H NMR analysis where several tetrapyrrolic NH signals, as well as different β pyrrole proton signals, were detected at very high fields indicating the presence of different conformations of the ligand skeleton. The 50B regioisomer of the desired bis-strapped porphyrin was isolated in 10% yield and characterized by NMR spectroscopy. The new ligand was used to synthesise the corresponding iron(III) complex, which was tested to promote the cyclopropanation reaction. In the optimised conditions, the Fe(50B)OMe complex was very active in promoting the reaction between α-methylstyrene and EDA, forming the desired cyclopropane in 99% yield and a trans/cis ratio of 90:10. Considering the very good result obtained in the model reaction, the catalytic performance of Fe(50B)OMe was tested in the presence of different substituted alkenes. Very good results were achieved in terms of yields (up to 99%) and disteroselectivities (up to 99:1). Under the optimized catalytic conditions, the desired cyclopropanes were formed in quantitative yields; lower yields were observed by using 0.001 mol % of Fe(50B)OMe due to the low conversion of the starting diazo compound. Obtained data showed the applicability of Fe(50B)OMe as an efficient catalyst for carbene transfer reactions. In addition, the presence of carboxylic groups on the porphyrin strap makes the synthesised hybrid ligand suitable for the conjugation with long water soluble chains, like PEG, in order to perform catalytic reaction in biphasic systems. Besides the conjugation of porphyrin ligands with small bio-scaffolds, like sugars and aminoacids, new mono-substituted porphyrins were designed and synthesized for their conjugation with cellulose and β-lactoglobuline proteins. In order to develop metal porphyrin complexes supported on cellulose, ligands 71 and 72, with amino groups onto the para position of one porphyrin ring phenyl group, were synthesized. Ligand 73 was synthesized with a long aliphatic chain that can be accommodated inside the tridimensional structure of β-lactoglobulin protein by hydrophobic interactions. Preliminary studies were performed for the conjugation of porphyrin 71 with a cellulose derivative functionalised with aldehyde groups. The conjugated material was obtained by a reductive amination reaction and then reacted with FeBr2 affording the corresponding iron(III) complex. Preliminary tests as cyclopropanation catalyst revealed the ability of the cellulose-based iron catalyst to promote the reaction between α-methylstyrene and EDA, forming the desired cyclopropane in 15% yield and a trans/cis ratio of 97:3. The promising result suggested the potential use as cyclopropanation catalyst of the conjugated heterogeneous species. Considering that metal porphyrin complexes show high activity in promoting carbene and nitrene transfer reactions, the obtained data represent a starting point to develop a new generation of catalysts which can be employed for the synthesis of several fine chemicals through sustainable synthetic methodologies. For developing sustainable and selective procedures for the synthesis of high-added value fine chemicals, a part of the PhD thesis was devoted to the study of the CO2 cycloaddition to three membered ring compounds in the presence of metal porphyrin complexes. Ruthenium bis-imido porphyrins were able to catalyse the regioselective cycloaddition of CO2 to aziridines to form oxoazolidinones, as reported in a previous PhD work. The catalytic performances of Ru(TPP)(NAr)2 (Ar = 3,5(CF3)2C6H3) (77) were also tested in the CO2 cycloaddition to epoxides. Very good results were obtained in the synthesis of different cyclic carbonates and the formation of the ruthenium species, Ru(TPP)(NAr)(ArNCOO-TBA+) (93), suggested the fundamental role of imido-axial ligand in activating CO2. The results, achieved in the CO2 cycloaddition of both epoxides and aziridines, suggested a mechanism in which the interaction of CO2 with the nitrogen imido atom is the first step of the reaction. This hypothesis is supported by the very high electron density on the nitrogen atom due to the linkage with ruthenium, as indicated by a previous DFT study. Future kinetics and DFT studies of the reaction between bis-imido complexes and CO2 could be useful to shed some light on the reaction mechanism. Considering the high activity of ruthenium porphyrin complexes to promote alkene aziridination reactions, the obtained data are the starting point to develop a catalytic system in which the same catalyst can be able to promote both the synthesis of aziridines and their transformation into oxazolidinones in a two-steps one-pot reaction which affords added value compounds in an efficient and sustainable way.

L'ingegneria tissutale è un campo multidisciplinare in rapida crescita che si avvale delle scienze fisiche, ingegneristiche e della vita per sviluppare ed ottenere cellule funzionali, tessuti ed organi per riparare, sostituire o migliorare le funzioni biologiche perse a causa di anomalie congenite, traumi, malattie o invecchiamento. Nell’ambito della ricostruzione della cartilagine articolare sono stati fatti notevoli passi in avanti ma la soluzione per il ripristino completo del tessuto sembra ancora essere lontana. Nella prima parte di questo lavoro è stata valutata la capacità di uno scaffold a base di gelatina di indirizzare le ADSCs verso un differenziamento in senso condrogenico. Successivamente, con lo scopo di migliorare il grado differenziamento e diminuire i costi associati all’utilizzo di fattori differenziativi, l’attenzione è stata posta sullo sviluppo di un biomateriale a base di alginato e ioni cobalto in modo da mimare e sfruttare le caratteristiche fisiche della cartilagine piuttosto che il suo intorno chimico. In ultimo, è stato sviluppato un sistema low cost per la produzione di chip microfluidici sfruttabili per la realizzazione di sistemi micrometrici per incapsulazione cellulare.
Tissue engineering is an interdisciplinary and multidisciplinary field that aims at the developmentof biological substitutes that restore, mantain, or improve tissue function. Concerning the articular cartilage many improvments were made, but the complete tissue restoration approach still lacking. In the first part of this work, it was evaluated the ability of a gelatin scaffold to promote the condrogenic differentiation of ADSCs. Successively, in order to obtain a low cost sistem, a based alginate/Cobalt scaffold was designed with the aim to take advantage of the physical features of the cartilage tissue. Finally, it was developted a cost effective method to produce microfluidic chips with the aim to obtain micro-systems for cell encapsulation.

L'ingegneria tissutale è un campo multidisciplinare in rapida crescita che si avvale delle scienze fisiche, ingegneristiche e della vita per sviluppare ed ottenere cellule funzionali, tessuti ed organi per riparare, sostituire o migliorare le funzioni biologiche perse a causa di anomalie congenite, traumi, malattie o invecchiamento. Nell’ambito della ricostruzione della cartilagine articolare sono stati fatti notevoli passi in avanti ma la soluzione per il ripristino completo del tessuto sembra ancora essere lontana. Nella prima parte di questo lavoro è stata valutata la capacità di uno scaffold a base di gelatina di indirizzare le ADSCs verso un differenziamento in senso condrogenico. Successivamente, con lo scopo di migliorare il grado differenziamento e diminuire i costi associati all’utilizzo di fattori differenziativi, l’attenzione è stata posta sullo sviluppo di un biomateriale a base di alginato e ioni cobalto in modo da mimare e sfruttare le caratteristiche fisiche della cartilagine piuttosto che il suo intorno chimico. In ultimo, è stato sviluppato un sistema low cost per la produzione di chip microfluidici sfruttabili per la realizzazione di sistemi micrometrici per incapsulazione cellulare.
Tissue engineering is an interdisciplinary and multidisciplinary field that aims at the developmentof biological substitutes that restore, mantain, or improve tissue function. Concerning the articular cartilage many improvments were made, but the complete tissue restoration approach still lacking. In the first part of this work, it was evaluated the ability of a gelatin scaffold to promote the condrogenic differentiation of ADSCs. Successively, in order to obtain a low cost sistem, a based alginate/Cobalt scaffold was designed with the aim to take advantage of the physical features of the cartilage tissue. Finally, it was developted a cost effective method to produce microfluidic chips with the aim to obtain micro-systems for cell encapsulation.

Tissue engineering identifies procedures aimed to regenerate tissues of the body involving the seeding of cells on an appropriate support (scaffold), followed by their cultivation in appropriate bioreactors to form a new tissue to be transplanted into the patient. The scaffold, made of biodegradable or bioabsorbable material has a transitory but very important role: it must provide a physical support for cells adhesion and growth but, at the end of the process, it must be completely absorbed, leaving a newly formed tissue. Ideally, the cells necessary for scaffold seeding and colonization could be provided by the patient so that the new engineered tissue, once implanted, is devoid of immune reaction of rejection. In a longer term perspective, stem cell could be used for seeding and induced to differentiate in vitro in a particular tissue phenotype (bone, cartilage, muscle, etc.). In this thesis, attention was mainly focused on some techniques used to study structural scaffolds suitable for cell adhesion and growth. It is not superfluous to point out the fundamental importance that the material and structure of the scaffold have for the success of support function and cell growth. In particular, two specific techniques were used for analysis: scanning electron microscopy (SEM) and computed microtomography (micro-CT). Both structural and morphological analysis techniques were applied to the analysis of polymers PLLA and PCL. In the introduction the techniques have been well described, with a particular emphasis on the SEM technique that allowed analysis of polymer surfaces before and after cells implant. In the section Materials and Methods we have extensively described the techniques adopted for PLLA and PCL scaffolds preparation. The PLLA constructs are ideally addressed for use as vascular substitutes, especially if plasticized with triethyl citrate. Instead, the PCL scaffolds were prepared using an innovative technique called gel-forming and are intended for use as a substitute of bone. SEM observations of the numerous vascular PLLA scaffolds prepared show that the average porosity increases in agreement with the amount of added plasticizer. HUVEC cell adhesion to PLLA scaffolds, as evaluated by SEM observation, seems to depend mainly on the porosity, increasing in a certain range with it. PLLA scaffolds post-conditioning with fibronectin, a typical extracellular matrix protein appointed to cell adhesion, usually improves cells adhesion. PCL scaffolds assembling by gel-forming has enabled us to produce a solid matrix with uniform microporosity of interconnected pores, as demonstrated by SEM observations, fuchsin perfusion and Micro-CT analysis. The technique of PCL gel-forming allowed to realize bone substitutes of any shape and size, but a slight contraction of the final scaffold must be compensated by a 10% mold oversize. The gel-forming technique allows to incorporate various additives during the assembling, in particular a number of scaffolds were prepared in PCL/sucrose, PCL/HAp and, finally, PCL/sucrose/HAp and studied by SEM and Micro-TAC. Macropores were created in the matrix of microporous PCL after water removal of sucrose particle inclusions. A right degree of porosity facilitates cells colonization of the scaffold. The addition of hydroxyapatite increases the mechanical strength of the PCL scaffolds, but if excessive, may make them more fragile. It should also improve bone cells (osteoblasts) adhesion and growth.
Con il termine ingegneria tissutale vengono identificate le procedure indirizzate alla rigenerazione di tessuti del corpo che prevedono la semina di cellule su un supporto opportuno (scaffold) seguita dalla loro coltivazione in appositi bioreattori fino ad ottenere un nuovo tessuto da trapiantare nel paziente. Lo scaffold, realizzato in materiali biodegradabili o bioassorbibili ha quindi un ruolo transitorio ma molto importante: deve infatti fornire un supporto fisico per l’adesione e la crescita cellulare ma, alla fine del processo, deve essere completamente riassorbito lasciando il posto ad un tessuto neoformato. Idealmente le cellule necessarie alla semina e alla colonizzazione dello scaffold potrebbero essere fornite dal paziente in modo che il neotessuto formato, una volta impiantato, sia privo di reazione immunitaria di rigetto. In una prospettiva temporale più lunga, potrebbero essere utilizzate per la semina cellule di tipo staminale successivamente indotte a differenziarsi in vitro in un particolare fenotipo tissutale (osso, cartilagine, muscolo, etc.). In questo lavoro di Tesi, l’attenzione è stata focalizzata soprattutto su alcune tecniche di indagine strutturale applicate allo studio di scaffolds per ingegneria tissutale adatti alla adesione e alla crescita cellulare. Non è superfluo evidenziare, infatti, l’importanza fondamentale che il materiale e la struttura dello scaffold hanno al fine di arrivare al successo delle funzioni di supporto e di crescita cellulare. In particolare, sono state utilizzate due specifiche tecniche di analisi: la microscopia a scansione elettronica (SEM) e la microtomografia computerizzata (Micro-TAC). Le due tecniche di indagine strutturale e morfologica sono state applicate all’analisi di polimeri del tipo PLLA e PCL. Nell’introduzione sono state descritte le tecniche sopracitate, ponendo particolare enfasi alla tecnica SEM che ha permesso di analizzare le superfici polimeriche sia prima che dopo impianto di colture cellulari. Nella sezione dedicata ai materiali e metodi sono state descritte ampiamente le tecniche di preparazione degli scaffold in PLLA e in PCL. I costrutti in PLLA sono idealmente indirizzati per un utilizzo come sostituti vascolari, specialmente se elasticizzati con trietilcitrato. Invece gli scaffolds in PCL sono stati preparati utilizzando una tecnica innovativa detta gel-forming e sono indirizzati verso un utilizzo quali sostituti di osso. Le osservazioni al SEM dei numerosi scaffolds vascolari in PLLA evidenziano come la porosità media dei preparati aumenti in accordo con l’aumento della quantità di plastificante. L’adesione delle cellule HUVEC agli scaffolds realizzati in PLLA, sempre valutata mediante osservazione al SEM, sembra dipendere soprattutto dalla porosità, aumentando in un certo intervallo con essa. Il condizionamento post-assemblaggio degli scaffolds in PLLA con la fibronectina, una tipica proteina della matrice extracellulare deputata alla adesione cellulare, migliora generalmente l’adesione. L’assemblamento degli scaffolds in PCL mediante gel-forming ha permesso di produrre una matrice solida con microporosità uniforme di pori interconnessi, come dimostrato dall’osservazione al SEM, dal saggio di perfusione con fuscina e, infine, dall’analisi con Micro-TAC. La tecnica di gel-forming con PCL permette di riprodurre forma e dimensioni di qualsiasi osso, ma bisogna correggere la leggera contrazione dello scaffold finale predisponendo uno stampo sovradimensionato del 10% circa. La tecnica gel-forming permette di inglobare vari additivi durante la fase di assemblamento, in particolare sono stati preparati numerosi scaffolds in PCL/saccarosio, PCL/idrossiapatite e, infine, PCL/saccarosio/idrossiapatite che sono stati studiati con SEM e mediante Micro-TAC. Il saccarosio, di opportuna granulometria, realizza, dopo rimozione con acqua, dei macropori nella matrice microporosa del PCL che facilitano potenzialmente la colonizzazione da parte delle cellule dello scaffold. L’aggiunta di idrossiapatite aumenta la resistenza meccanica degli scaffolds in PCL ma, se eccessiva, può renderli più fragili; essa dovrebbe anche migliorare l’adesione e la crescita di cellule del tessuto osseo, in particolare degli osteoblasti. La crescita di cellule sulle varie tipologie di scaffolds in PCL, prodotte in questo lavoro, non è stata esaminata sperimentalmente.

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

Escafoldes em alumina foram fabricados e em suas superfícies impregnou-se biovidro e hidroxiapatita; realizou-se análise das propriedades mecânica e de interação célula-escafolde in vitro. Estruturas porosas denominadas escafoldes são utilizadas como suportes para crescimento de tecidos, devem apresentar poros abertos interconectados, com morfologia, distribuição e quantidade de poros que confiram resistência mecânica e induzam o crescimento ósseo. Os escafoldes simulam a matriz extracelular e são a chave para a engenharia de tecidos que está conceituada na cultura prévia de células com proteínas morfogenéticas, oferecendo suporte para o crescimento celular na formação do tecido maduro. Neste trabalho desenvolveu-se técnica de manufatura onde foram conformados escafoldes como corpos-de-prova em alumina, em hidroxiapatita e em alumina infiltrada com biovidro e hidroxiapatita. Os escafoldes foram submetidos a ensaios mecânicos de compressão e sofreram análise de interação com células in vitro. A morfologia e a concentração da porosidade dos escafoldes foram analisadas por microscopia de varredura eletrônica e apresentaram porosidade volumétrica de aproximadamente 70% e diâmetro médio de poros em torno de 190 µm. Observou-se interação das células mais vigorosas e com pronunciada mitose nos escafoldes infiltrados relativamente aos escafoldes de alumina e hidroxiapatita. Os resultados indicaram resistência mecânica para os corpos infiltrados de 43,27 MPa, valor inferior ao observado nos escafolde de alumina 52,27 MPa e muito superior aos de hidroxiapatita 0,28 MPa. Conclui-se que os escafoldes de alumina infiltrados com biovidro e hidroxiapatita apresentaram uma combinação promissora nas características mecânicas e biológicas in vitro com viabilidade econômica.
Alumina scaffolds were manufactured and surface impregnated with bio-glass and hydroxyapatite; the mechanical properties and the in vitro bone-cell and scaffold interaction were analyzed. Porous matrices are usually denominated as scaffolds in tissue engineering and they are used as supports for the tissue growing; they may have open and interconnected pores, with known porous geometry and distribution and with good mechanical strength and be able to induce the tissue cells growing. Scaffolds can work as extra cell matrices, mimic the desired tissue and are considered as the key for the tissue engineering, offering support for the cellular growing in the formation of mature tissue. In this work, manufacture techniques were developed where scaffolds were conformed in alumina, in hydroxyapatite and in alumina infiltrated with bio-glass and hydroxyapatite, as test bodies. The scaffolds were submitted to mechanical compression tests and to the interaction with bone cells in vitro. The morphology and the concentration of the scaffold porosity were analyzed by scanning electronic microscopy (SEM) and they presented porosity concentration near 70,0 vol% and medium diameter of pores around 190,0 µm. The cells interaction strongest and more vigorous bone cell interaction with pronounced mitosis was observed in the alumina scaffolds infiltrated with bio-glass and hydroxyapatite when compared with the alumina scaffolds and hydroxyapatite scaffolds. The results obtained shown lower values of the mechanical strength for the infiltrated scaffolds (43,27 MPa), higher values for non infiltrated alumina scaffold (52,27 MPa) and very low values for the hydroxyapatite scaffolds (0,28 MPa). As observed, final results shown that alumina scaffolds infiltrated with bio-glass and hydroxyapatite presented a promising combination in the mechanical and biological in vitro characteristics with economic viability.

A library of eight bifunctional 2,5-diketopiperazine (DKP) scaffolds, which are formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP1 - DKP7) or glutamic acid (DKP8) was designed and synthesized. The scaffolds differ for the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogens. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD-peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins alphaVbeta3 and alphaVbeta5, which are involved in tumor angiogenesis. Nanomolar values were obtained for the RGD-peptidomimetics derived from trans DKP scaffolds (DKP2 - DKP8). Conformational studies of the cyclic RGD-peptidomimetics by 1H-NMR experiments (VT-NMR and NOESY) in aqueous solution and Monte Carlo – Stochastic Dynamics (MC-SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cbeta(Arg)-Cbeta(Asp) average distance ≥ 8.8 A]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin alphaVbeta3 complexed with the cyclic pentapeptide Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex. Since alphaV integrins, which can be internalized by cells, are involved in tumor angiogenesis and are overexpressed on the surface of cancer cells, integrin ligands can be usefully employed as tumor homing peptidomimetics for site-directed delivery of cytotoxic drugs. A small library of integrin ligand - Paclitaxel conjugates 90-93 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the Paclitaxel-RGD constructs 90-93 inhibited biotinylated vitronectin binding to the purified alphaVbeta3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of Paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin alphaVbeta3, making them attractive to be tested in in vivo models. Cyclo[DKP-f3-RGD]-PTX 91 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 91 exhibited a superior activity than Paclitaxel, despite the lower (ca. half) molar dosage used.

The relatively new and still growing field of 3D-printing has opened up the possibilities to manufacture patient-specific medical devices with high geometrical accuracy in a precise and quick manner. Additionally, biocompatible materials are a demand for all medical applications while biodegradability is of importance when developing scaffolds for tissue growth for instance. With respect to this, this project consisted of developing biocompatible and bioresorbable polymer blend and composite filaments, for fused deposition modeling (FDM) printing. Poly(lactic acid) (PLA) and polycaprolactone (PCL) were used as supporting polymer matrix while hydroxyapatite (HA), a calcium phosphate with similar chemical composition to the mineral phase of human bone, was added to the composites to enhance the biological activity. PLA and PCL content was varied between 90–70 wt% and 10-30 wt%, respectively, while the HA content was 15 wt% in all composites. All materials were characterized in terms of mechanical properties, thermal stability, chemical composition and morphology. An accelerated degradation study of the materials was also executed in order to investigate the degradation behavior as well as the impact of the degradation on the above mentioned properties. The results showed that all processed materials exhibited higher mechanical properties compared to the human trabecular bone, even after degradation with a mass loss of around 30% for the polymer blends and 60% for the composites. It was also apparent that the mineral accelerated the polymer degradation significantly, which can be advantageous for injuries with faster healing time, requiring only support for a shorter time period.

碩士
龍華科技大學
工程技術研究所
99
The process parameters and raw materials of the current commercial rapid prototyping machines are limited, thus the bone scaffold model is not easily to fabrication. In order to improve these disadvantages, we use a self-developed rapid prototyping machine to produce bone model. In this work the hydroxyapatite, silica sol, and sodium tripolyphosphate suspending agent were mixed as raw materials. By adjusting the suitable process parameter, the bio-ceramic green parts could be formed, and then the green parts were sintered at 1200℃, 1300℃, 1400℃, respectively. The microstructure, shrinkage, density, porosity, surface roughness, compression strength, and bending strength were analyzed. Finally, the cell growth of the bone scaffold model were investigated through the osteoblast MG63 cells culture. The results showed that a optimum green part could be obtained when the sintering temperature at 1200℃, which causes the compression strength increasing significantly. In addition, a surface roughness of Ra=12.04μm could be also obtained, which is suitable for the growth of the osteoblast MG63. Above this temperature will decrease mechanical strength due to cracks expand, while the surface roughness was increase. After infiltration by the polyethylene glycol solution, the surface roughness was significantly decreased, and the mechanical strength due to cracks and holes have been filled so there are a substantial increase.

碩士
國立臺灣大學
機械工程學研究所
102
Abstract Two-Photon absorption Polymerization (TPP) can be used for micro-structures’ production, which allows accurate manufacturing of complicated three-dimensional structures at the micron or sub-micron levels. It has become well developed enough to offer a reliable fabrication method for micro electro-mechanical systems. Previous researches on TPP were mainly concentrated on the development of photosensitive resins, control of fabrication laser beams, capability of the fabrication of complicated structures and possible new applications (ex. Micro-fluidic chips and bio-scaffolds) of TPP. On the other hand, as to the mechanical properties of the micro-structures, few researches had been done. However, to design a practical machine component with reasonable structural integrity, reliability and functional operability, the mechanical properties must be known. For now, mechanical properties for the resins rely much on the data given by the factory which produces the resins. However, there are researches indicate that after the TPP process, the mechanical properties of the resins might change a lot. Because TPP fabrication has its limitation on the size of the structure, which makes the conventional mechanical testing methods impossible to be applied, and the lacking of the researches about the mechanical properties, the objective of this research is to measure the Young’s Modulus of the structure after the TPP process, and to understand the relationships between Young’s Modulus and some parameters during the production. Moreover, recently TPP is also used for manufacturing the bio-scaffold for the cell culture, but the researches on the mechanical properties of the bio-scaffold are also not enough. So the other half of this research is trying to understand and analyze some mechanical behavior and parameters of the bio-scaffold by Finite Element Method (FEM), and hope to give some ideas to later the production of the bio-scaffold. Keywords: Two-Photon absorption Polymerization (TPP), mechanical property(ies), Young’s Modulus, (bio-)scaffold

碩士
國立虎尾科技大學
機械與機電工程研究所
97
The purpose of this study is to develop an air pressure-aided deposition system for fabricating scaffold made of synthesized PCL-PEG-PCL copolymers and to validate the biocompatibility of fabricated scaffolds. The development of air pressure-aided deposition system is presented. The synthesis process of PCL-PEG-PCL copolymer is briefly introduced. Scaffolds with different mean pore sizes are fabricated using developed system and seed the fibroblast cells for culturing and validating its biocompatibility using MTT assay. It is apparent that the air pressure-aided deposition system is suitable for fabricating micro-porous cellular scaffold, especially for thermal-sensitive copolymers. In addition, through experimental results, it shows that molecular weight of 50,000 can be stable deposited the molten form through heating nozzle at air pressure of 0.3 MPa and no crack occurs after it solidify. The scaffold with mean pore size of 339*396 µm is suitable for fibroblast binding and ingrowth. The synthesized copolymers are non-toxicity, biocompatibility and could be used for biomedical application. In addition, the synthesized PCL-PEG-PCL copolymers are hydrophobic biomaterials and result in the fibroblast cells hard to attach on the surface of scaffolds. Consequently, the air pressure-aided deposition system is successfully proposed to construct 3D tissue scaffolds. Synthesized PCL-PEG-PCL copolymers is verified the biocompatibility and successfully fabricated of tissue scaffold with different mean pore sizes.

碩士
國立中央大學
機械工程學系
106
Recent developments in tissue-engineering techniques allow physicians to treat a range of previously untreatable conditions. In the development of such techniques, scaffolds with a controllable pore size and porosity have been manufactured to investigate cell interaction effects such as cell proliferation and differentiation. In this study, we describe the fabrication of scaffolds using a low-temperature deposition manufacturing system that we developed. This systems uses technology that enables the manufacture of three-dimensional microstructures and required the combination of several technologies, including motion card control (NI-motion card, National Instruments), movement system (Linmot system), low-temperature system, material dispesing system (Ultimus IV, NORDSON). In the other hand, this research will be researched the material (PLA) deposition process by this low-temperature deposition manufacturing and try to use our method such as: the trapezoidal move, the segment extrusion and the extrusion with variable moving velocities to analysis and improve the quality of the uniform width strands of scaffold and specially the over-accumulation of material located in the corner.

碩士
臺北醫學大學
生醫材料暨工程研究所
97
This experiment is based on the technique of bio-electrospinning to develop a combinative structure of electrospinning. According to the best model of nerve regeneration guidance conduits, we can use simple and specific technique of bioelectrospinnig, as meanwhile combining the applications of specific coaxial bi-component spinneret and rotating collector and combine these equipment and technique, the multi-hollow fiber systematic materials can be fabricated. Add nerve growth factor which can improve nerve cell regenerated, and evaluate the functional ability on nerve reparation. The multi-tubes can provide multiple surface area in order to make more cells attached. And the hollow tubes can efficiently induce the axon growing toward the right direction to prove quality and rate of reparation. This study used biodegradable materials including PLA(poly-lactic acid ), PVP(Polyvinyl pyrrolidone), PEG(Polyethylene glycol)、PEO (polyethylene oxide) to be the matrix of nerve guidance conduit., and successfully fabricated the hollow fiber by coaxial head. When the diameter of fiber were under 50 μm and the collective rotating rate was 15.6 m/s, we can get the high oriented fibric membrane. Add PC-12 cells to bio-electrospinning technique, the cells were successfully induced into inner conduit of PLLA by electrospinning. And add NGF into tubes, after observation on fluorescein-transfections PC-12 cells by fluorescent microscope, we can find that cells attach the tube wall, and successfully induced axons growth on the oriented tube wall. According these experiments, we can prove that it is easily to fabricate multi-functional nerve guidance conduits which were made of fibric membrane including PC-12 cells and high oriented arrangement via bio-electrospinning which combined coaxial head and rotating orientated collecting method.

碩士
國立中興大學
食品暨應用生物科技學系所
102
Chitosan is the product of deacetylated chitin, it is biodegradable and biocompatible. In addition, the degradation product of chitosan could be absorbed. Therefore, chitosan is widely used in skin tissue engineering currently. But chitosan scaffold showed lower water-uptake and dissolution rate under neutral environment, if we could find an alternative that can partially replace chitosan to improve the properties of bioscaffold, we might make the application of bioscaffold more widely. Therefore, in this study, we fabricated the bioscaffolds by mixing chitosan (C) and locust bean gum (L) or konjac gum (K) under a ratio of 1:1, 2:1 or 4:1 ratio, then, crosslinked the mixed polysaccharide with glutaraldehyde (GTA), genipin (GP), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC)+N-hydroxysuccinimide (NHS) or dimethyl 3-3, dithio bis’ propionimidate (DTBP) respectively, and taking the bioscaffold without crosslinking treatments as the control group. Characteristics of mixed polysaccharide bioscaffolds with different mixing ratio and crosslinking treatment were then investigated. For the locust bean gum/chitosan mixing system (LC), speaking to the effect of polysaccharides mixing ratio, mean pore diameter (except for GP group), water uptake and dissolution of scaffolds (except for control group and EDAC+NHS group) generally increased with increasing the proportion of locust bean gum. In contrast, crosslinking density (except GP group) and compressive stress decreased when the proportion of locust bean gum increased. Results of scanning electron microscopy revealed that the pore size of scaffolds were between 77~103 μm, and the porosity were above 85%. Relaxation test showed that relaxation time (λ) of scaffolds increased with increasing the amount of locust bean gum for groups crosslinked with GTA, EDAC+NHS and the control group, implying these scaffolds have lower elasticity. On the other hand, mixing ratio of polysaccharides had no significant influence on the elasticity of scaffolds crosslinked with GP and DTBP. Speaking to the effect of crosslinking agents, crosslinking agent did not play a significant role in terms of porosity and cell viability of scaffolds. Besides, water-uptake and dissolution of scaffolds from low to high were control groupcontrol group>GP group>EDAC+NHS group>GTA group. GTA-crosslinked scaffolds showed significant cell toxicity, and cell grew on the scaffold crosslinked with other agents had higher cell viability. For the konjac gum/chitosan mixing system (KC), speaking to the effect of polysaccharides mixing ratio, mean pore diameter, water uptake and dissolution of scaffolds (except for EDAC+NHS group) generally increased with increasing proportion of konjac gum. In contrast, crosslinking density and compressive stress decreased when the proportion of konjac gum increasesd. Results of scanning electron microscopy revealed that the pore size of scaffolds were between 78~114 μm, and the porosity were above 92%. Relaxation test showed that relaxation time (λ) of scaffolds increased with increasing the amount of konjac gum for the control group, implying that the viscoelasitc solid behavior (or higher elasticity) of scaffolds. In addition, mixing ratio of polysaccharides had no significant effect on the elasticity of scaffolds crosslinked with GTA, GP and DTBP group. In contrast, when the proportion of chitosan was increased, the scaffold crosslinked with EDAC+NHS had higher elasticity. On the other hand, mixing ration of polysaccharides did not play a significant role in terms of porosity and cell viability. Speaking to the effect of crosslinking agents, crosslinking agent did not play a significant role in terms of porosity. In addition, water-uptake and dissolution of scaffold from low to high were control groupcontrol group>GP group>EDAC+NHS group>GTA group. GTA-crosslinked scaffold showed significant cell toxicity, and cell grew on the scaffold crosslinked with other agents had higher cell viability. Based on the results, scaffolds crosslinked with GTA are not suitable due to its cell toxicity. In contrast, scaffolds crosslinked with DTBP had the highest cell viability, implying this crosslinking agent had the lowest cell toxicity. However, DTBP were more expensive than other crosslinking agents. Based on the performance and cost considerations, GP seemed to be a good candidate as a crosslinking agent, not only due to the fact that it is more economical, but also comparable cell viability and the better water-uptake and lower rate of dissolution as compared to the control group. We suggested that the scaffolds made of LC or KC under a ratio of 1:1 and 2:1 might be worthy to be applied and explored in the future tissue engineering.

碩士
國立臺灣科技大學
醫學工程研究所
98
This study investigated the induction of human adipose tissue-derived stem cells (hASCs) into chondrocytes, which made a possible source for cartilage tissue engineering in clinical therapy. The hASCs were cultured on three different biocompatible biomaterial groups including gelatin/collagen I: PCL (GCI), gelatin/collagen II:PCL (GCII) and gelatin:PCL (GP). Considering the high degradability of natural biomaterials, the addition of PCL for formulation of scaffolds is necessary to resist absorption in vitro. In addition, we characterized the attachment, growth and differentiation abilities of hASCs in biomaterials by SEM, fluorescence microscopy and cell growth curve in vitro. The immunofluorescence data showed that the hASCs were differentiated with the increasing time on biocomposite membrane groups. For investigation of cartilage differentiation, alcian blue staining and dimethylmethylene blue (DMMB) assay were used for both quality and quantity evaluation of glycosaminoglycans. The results showed that the groups of gelatin and GCII contained more glycosaminoglycans than GCI. For further distinguishing distinct collagen types produced by chondrocytes based on different biocomposite membranes, sirius red staining was used and type I collagen showed the absorbance at 540 nm and type II collagen at 350nm with UV-visble spectrophotometer. We demonstrated that the chondrocytes release mainly type I collagen on all biocomposite membranes. In detecting the long term expression of calcium accumulation in differentiated chondrocytes using von kossa stain, chondral calcification represented after induction of chondrocyte differentiation on biocomposite membranes for 30 days

La medicina rigenerativa è oggi uno tra i campi più interessante della biotecnologia, in grado di combinare diversi aspetti della medicina della biologia cellulare e molecolare dei biomateriali e dell'ingegneria dei tessuti, finalizzata a rigenerare, riparare o sostituire tessuti. La linea di ricerca proposta, sulla base di studi che analizzano vari aspetti della rigenerazione ossea in campo medico è rivolta al miglioramento delle tecniche già utilizzate in questo ambito e si propone di realizzare un sistema integrato standardizzato. Il substrato scelto per l'analisi è costituito da osso equino in blocchi di cui è stata analizzata con osservazioni S.E.M. la superficie dopo modellazione mediante strumentazione rotativa o vibrante, la possibilità di sterilizzazione raggiungendo SAL 10–6, valutando possibili modifiche macro e microstrutturali ed infine il possibile inserimento del materiale in un workflow digitale che partendo dalla rilevazione radiologica di un difetto osseo, passa alla modellazione di un modello tridimensionale computerizzato, che porti alla produzione di un blocco mediante tecnica sottrattiva, customizzato in relazione al difetto rilevato.