Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease

International audience

The Effect of Architecture and Shear Stress on Endothelialization of 3D Printed Vascular Networks

Despite significant progress in the field of tissue engineering within the last decade, a number of unsolved problems still remain. One of the most relevant issues is the lack of proper vascularization that limits the size of engineered tissues to smaller than clinically relevant dimensions. In particular, the growth of engineered tissue in vitro within bioreactors is plagued with this challenge. Specifically, the tubular perfusion system bioreactor has been used for large scale bone constructs; however these engineered constructs lack inherent vasculature and quickly develop a hypoxic core, where no nutrient exchange can occur, thus leading to cell death. Through the use of 3D printed vascular templates in conjunction with a tubular perfusion system bioreactor, we attempt to create an endothelial cell monolayer on 3D scaffolds that could potentially serve as the foundation of inherent vasculature within these engineered bone grafts.

Silanisation de polysaccharides en milieu liquide ionique et réactivité d’un organoalkoxysilane vis-à-vis de nucléophiles simples : vers des hydrogels injectables pour l’ingénierie tissulaire du cartilage

Tissue engineering is a real challenge for the treatment of cartilage pathologies. In this field, biomimetic hydrogels based on natural polymers are among the most commonly used matrices. A hydrogel made of silanized hydroxypropylmethylcellulose (HPMC-Si) is especially promising because it can be injected in cartilaginous lesions by minimally invasive surgery. However, the current synthesis of HPMC-Si is limited by the insolubility of hydroxypropylmethylcellulose (HPMC). This thesis work was focused on finding new synthesis conditions for the design of HPMC-Si hydrogel. In order to obtain a complete solubilization of HPMC and to improve its functionalization by the (3-glycidyloxypropyl) trimethoxysilane (GPTMS), the use of ionic liquids (IL), which are excellent solvents for polysaccharides, was undertaken. The beginning of this study was first devoted to the selection of an IL and then to the development of new reaction conditions. With these new conditions, higher silicon rates were obtained for HPMC modified in ionic liquid medium, however no hydrogel could be formed. The second part was therefore devoted to the synthesis of GPTMS 13C. Indeed, thanks to this radiolabeling, a structural characterization by 13C NMR of the HPMC-Si could be achieved. Finally, the reactivity in organic solvents of three organosilanes, including the GPTMS, was investigated toward nucleophiles representing the common functions found in natural polymers (e.g. -NH2, -OH, -SH). The results of this thesis have provided insights into the GPTMS reactivity in organic medium and thus paves the way to new conditions for the silanization of polysaccharides.

Estudo das propriedades biocompatíveis de arcabouços poliméricos derivados de óleos vegetais para aplicação na engenharia de tecidos

Dissertação (Mestrado em Tecnologia Nuclear)

Développement de matériaux à base de protéines extraites du byssus de la moule bleue Mytilus edulis

Le byssus est un amas de fibres que les moules produisent afin de s’ancrer aux surfaces immergées sous l’eau. Ces fibres sont pourvues de propriétés mécaniques impressionnantes combinant rigidité, élasticité et ténacité élevées. De plus, elles possèdent un comportement d’auto-guérison de leurs propriétés mécaniques en fonction du temps lorsque la contrainte initialement appliquée est retirée. Les propriétés mécaniques de ces fibres sont le résultat de l’agencement hiérarchique de protéines de type copolymère blocs riches en collagène et de la présence de métaux formant des liens sacrificiels réversibles avec certains acides aminés comme les DOPA et les histidines. Bien que cette fibre soit très intéressante pour la production de matériaux grâce à son contenu élevé en collagène potentiellement biocompatible, cette ressource naturelle est traitée comme un déchet par les mytiliculteurs. L’objectif de cette thèse était de valoriser cette fibre en extrayant les protéines pour générer une nouvelle classe de matériaux biomimétiques. Un hydrolysat de protéines de byssus (BPH) riche en acides aminés chargés, i.e. ~30 % mol, et permettant de former des films a pu être généré. Lorsque solubilisé à pH 10.5, le BPH forme un hydrogel contenant des structures en triple hélice de collagène et des feuillets β anti-parallèles intra- et inter-moléculaires. Suite à l’évaporation de l’eau, le film de BPH résultant est insoluble en milieu aqueux à cause des structures secondaires très stables agissant comme points de réticulation effectifs. Les propriétés mécaniques des films de BPH sont modulables en fonction du pH. Au point isoélectrique (pI = 4.5), les interactions électrostatiques entre les charges opposées agissent comme points de réticulation et augmentent la rigidité des films et leur contrainte à la rupture sans affecter la déformation à la rupture. À pH plus élevé ou plus bas que le pI, les performances mécaniques des films sont plus faibles à cause de la répulsion entre les groupements fonctionnels de même charge qui interagissent plutôt avec les molécules d’eau et causent le gonflement de la matrice protéique des films. Le BPH contenant un nombre élevé d’acides aminés chargés et réactifs, nous avons pu réticuler les films de manière covalente à l’aide d’EDC ou de glutaraldéhyde. Les propriétés mécaniques des films sont modulables en fonction de la concentration d’EDC utilisée lors de la réticulation ou en employant du glutaraldéhyde comme agent réticulant. Les films sont à la fois plus rigides et plus forts avec un degré de réticulation élevé, mais perdent leur extensibilité à mesure que les segments libres de s’étirer lors d’une traction deviennent entravés par les points de réticulation. La réticulation augmente également la résistance à la dégradation enzymatique par la collagénase, les films les plus fortement réticulés lui étant pratiquement insensibles. La spectroscopie infrarouge montre enfin que la réticulation entraîne une transition de feuillets β anti-parallèles inter-moléculaires vers des structures de type hélices de collagène/PPII hydratées. Des liens sacrificiels ont été formés dans les films de BPH par traitement au pI et/ou avec différents métaux, i.e. Na+, Ca2+, Fe3+, afin de moduler les propriétés mécaniques statiques et d’évaluer le rôle de ces traitements sur le comportement d’auto-guérison lors de tests mécaniques cycliques avec différents temps de repos. Plus la valence des ions métalliques ajoutés augmente, plus les propriétés mécaniques statiques affichent un module, une contrainte à la rupture et une ténacité élevés sans toutefois affecter la déformation à la rupture, confirmant la formation de liens sacrificiels. Les tests mécaniques cycliques montrent que les traitements au pI ou avec Ca2+ créent des liens sacrificiels ioniques réversibles qui mènent à un processus d’auto-guérison des performances mécaniques dépendant du pH. L’ajout de Fe3+ à différentes concentrations module les performances mécaniques sur un plus large intervalle et la nature plus covalente de son interaction avec les acides aminés permet d’atteindre des valeurs nettement plus élevées que les autres traitements étudiés. Le Fe3+ permet aussi la formation de liens sacrificiels réversibles menant à l’auto-guérison des propriétés mécaniques. Les spectroscopies Raman et infrarouge confirment que le fer crée des liaisons avec plusieurs acides aminés, dont les histidines et les DOPA. Les résultats dans leur ensemble démontrent que les films de BPH sont des hydrogels biomimétiques du byssus qui peuvent être traités ou réticulés de différentes façons afin de moduler leurs performances mécaniques. Ils pourraient ainsi servir de matrices pour des applications potentielles dans le domaine pharmaceutique ou en ingénierie tissulaire.

Evaluation of the genotoxicity of cellulose nanofibers

Background: Agricultural products and by products provide the primary materials for a variety of technological applications in diverse industrial sectors. Agro-industrial wastes, such as cotton and curaua fibers, are used to prepare nanofibers for use in thermoplastic films, where they are combined with polymeric matrices, and in biomedical applications such as tissue engineering, amongst other applications. The development of products containing nanofibers offers a promising alternative for the use of agricultural products, adding value to the chains of production. However, the emergence of new nanotechnological products demands that their risks to human health and the environment be evaluated. This has resulted in the creation of the new area of nanotoxicology, which addresses the toxicological aspects of these materials.Purpose and methods: Contributing to these developments, the present work involved a genotoxicological study of different nanofibers, employing chromosomal aberration and comet assays, as well as cytogenetic and molecular analyses, to obtain preliminary information concerning nanofiber safety. The methodology consisted of exposure of Allium cepa roots, and animal cell cultures (lymphocytes and fibroblasts), to different types of nanofibers. Negative controls, without nanofibers present in the medium, were used for comparison.Results: The nanofibers induced different responses according to the cell type used. In plant cells, the most genotoxic nanofibers were those derived from green, white, and brown cotton, and curaua, while genotoxicity in animal cells was observed using nanofibers from brown cotton and curaua. An important finding was that ruby cotton nanofibers did not cause any significant DNA breaks in the cell types employed.Conclusion: This work demonstrates the feasibility of determining the genotoxic potential of nanofibers derived from plant cellulose to obtain information vital both for the future usage of these materials in agribusiness and for an understanding of their environmental impacts.

Transplantation of artificial gelatin-co-bletillastriata gelatin/Salvia miltiorrhiza Corium promotes dermal repair in rats

Purpose: To evaluate the growth of the composite corium (constructed with fibroblast cells and gelatinco- Bletillastriata gelatin/Salvia miltiorrhiza materials) on rats. Methods: The composite artificial corium was constructed by culturing fibroblast cells in gelatin-co- Bletillastriata gelatin/Salvia miltiorrhiza materials. Full-thickness area of skin was excised from the mice and subsequently, the composite corium was transplanted on the wound. Thereafter, the growth difference of the composite artificial corium and natural corium were compared. In addition, real-time fluorogenic reverse transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine vascular endothelial growth factor (VEGF) expression at gene and protein levels. Results: The composite artificial corium showed significant repair promoting effect on the skin, and the structure of the repaired skin was similar to that of natural corium. Interestingly, PCR and western blot results showed that the expressions of VEGF were higher in composite artificial corium than in natural corium on days 3 and 7 post-transplantation. Conclusion: The composite artificial corium has some clinical prospects for use in the treatment of wounds on large areas of skin.

Development of hydroxyapatite-based nanomaterials to enhance biological response of osteoblast cells for clinical application

Purpose: To develop a novel chitosan/gelatin-hydroxyapatite (CGHaP) microspheres for evaluating the biological response of pre-osteoblast cells. Methods: The microsphere was prepared by water-in-oil emulsion method. Cell proliferation was studied using AlamarBlue colorimetric assay and DAPI staining while alkaline phosphatase assay was carried out by colorimetric assay method. Chitosan microspheres as well as chitosan-hydroxyapatite microspheres was prepared and tested for biological response from MC3T3-E1 cell line. Results: The results showed that CGHaP promotes MC3T3-E1 cell proliferation and spread on the surface of microspheres. The cells were clustered with more actin filaments and well-linked with neighbouring cells or adjacent cells when cultured in CGHaP microspheres whereas fewer cells were spread on chitosan (CH) microspheres. CGHaP microspheres significantly (p < 0.05) promoted cell attachment, proliferation and extracellular matrix mineralization. CGHaP microspheres presented significantly (p < 0.02) higher calcium deposition (0.5 ng) than CH microspheres (0.28 ng). Specifically, CGHaP microspheres exhibited high ALP activity (8 units; 2-fold) compared to CH with 3 units, after 7 days of incubation. The results suggest that CGHaP possesses a great ability to facilitate bone ingrowth formation and possibility of good osteointegration in vivo. Conclusion: The nanomaterial enhances the proliferation of pre-osteoblast cells in tissue engineering microspheres. The outcome of this study may have a major impact on the development of novel nanomaterials for bone tissue engineering.

Positive biocompatibility of nanocrystalline glass-like carbon thin films

The interest in carbon nanomaterials with high transparency and electrical conductivity has grown within the last decade in view of a wide variety of applications, including biocompatible sensors, diagnostic devices and bioelectronic implants. The aim of this work is to test the biocompatibility of particular nanometer-thin nanocrystalline glass-like carbon films (NGLC), a disordered structure of graphene flakes joined by carbon matrix (Romero et al., 2016). We used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryo cells (Son et al., 1999). Some cells were cultured on top of NGLC films (5, 20 and 80 nm) and other with NGLC nanoflakes (approx. 5-10 mm2) in increasing concentrations: 1, 5, 10, 20 and 50 μg/ml, during 24 h, 3 days and 7 days. Cells growing in normal conditions were defined under culture with DMEM supplemented with 10% FCS, Glucose (0,6%), penicillin-streptomycin (50U/ml) and L-glutamine (2mM) at 5%CO2 humidified atmosphere. Nanoflakes were resuspended in DMEM at the stock concentration (2 g/l). The experiments were conducted in 96 well plates (Corning) using 2500 cells per well. For MTT analysis, the manufacturer recommendations were followed (Roche, MTT kit assay): a positive control with a 10% Triton X-100 treatments (15 minutes) and a negative control without neither Triton X-100 nor NGLC. As apoptosis/necrosis assay we used LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Invitrogen). In a separate experiment, cells were cultured on top of the NGLC films for 7 days. Primary antibodies: anti-synaptophysin (SYP, clone SY38, Chemicon) and goat anti-GIRK2 (G-protein-regulated inward-rectifier potassium channel 2 protein) (Abcom) following protocol for immunofluorescence. WB for proteins detection performed with a polyclonal anti-rabbit proliferating cell nuclear antigen (PCNA). Results demonstrated the biocompatibility with different concentration of NGLC varying the degree of survival from a low concentration (1 mg/ml) in the first 24 h to high concentrations (20-50 g/ml) after 7 days as it is corroborated by the PCNA analysis. Cells cultured on top of the film showed after 7 days axonal-like alignment and edge orientation as well as net-like images. Neuronal functionality was demonstrated to a certain extent through the analysis of coexistence between SYP and GIRK2. In conclusion, this nanomaterial could offer a powerful platform for biomedical applications such as neural tissue engineering

Multifunctional Nanoparticles in Cancer: in vitro Characterization, in vivo Distribution

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (pIn vivomouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p

Développement d’un dispositif d’extrusion tridimensionnelle de sucre vitrifié pour la production de réseaux fluidiques complexes par moulage rapide

L’objectif du vaste projet de recherche dans lequel s’inscrit ce mémoire est de guérir le diabète de type 1 en fabriquant un pancréas bioartificiel vascularisé contenant des cellules bêta (i.e. les cellules sécrétant l’insuline). Ce dispositif permettrait de rendre aux personnes atteintes par le diabète de type 1 la capacité de sécréter par elles-mêmes de l’insuline et de réguler leur glycémie. La vascularisation est actuellement un enjeu de taille dans le domaine du génie tissulaire. La plupart des tissus incorporant des cellules générées par le génie tissulaire sont actuellement fortement limités en épaisseur faute d’être vascularisés adéquatement. Pour les tissus dont l’épaisseur dépasse 400 μm, la vascularisation est nécessaire à la survie de la plupart des cellules qui autrement souffriraient d’hypoxie, les empêchant ainsi d’accomplir leurs fonctions [1]. Ce mémoire présente le développement et la mise en service d’un dispositif d’extrusion tridimensionnelle de sucre vitrifié pour la vascularisation d’un pancréas bioartificiel. Ce dispositif a été développé au laboratoire de recherche sur les procédés d’impression 3D ainsi qu’au bureau de design du département de génie mécanique de l’Université Laval. Grâce à cette technique d’impression 3D novatrice et à la caractérisation du procédé, il est maintenant possible de produire rapidement et avec précision des structures temporaires en sucre vitrifié pour la fabrication de réseaux vasculaires tridimensionnels complexes. Les structures temporaires peuvent, après leur production, être utilisées pour réaliser le moulage rapide de constructions vascularisées avec des matériaux tels que du polydiméthylsiloxane (PDMS) ou des hydrogels chargés de cellules biologiques. De par la nature du matériel utilisé, les moules temporaires peuvent être facilement et rapidement dissous dans une solution aqueuse et laisser place à un réseau de canaux creux sans créer de rejets toxiques, ce qui représente un avantage majeur dans un contexte de bio-ingénierie.

Evaluación de desenlace: trasplante meniscal versus segunda meniscectomía

Introducción: El incremento de pacientes sintomáticos de rodilla y la osteoartrosis en jóvenes con limitadas posibilidades terapéuticas después de una meniscectomía, genera la búsqueda de alternativas terapéuticas. A pesar que es poco utilizado en Colombia, el trasplante meniscal es una propuesta para el manejo sintomático. Según cifras norteamericanas, se practican entre 700.000 a 1.500.000 artroscopias de rodilla anualmente, el 50% termina en meniscectomía y de este un 40% persisten sintomáticos. Métodos: Estudio de cohorte retrospectivo, con el objetivo de evaluar dolor (Escala Visual Análoga-EVA) y funcionalidad (Escala de Tegner y Lysholm) en los pacientes a quienes se les realizó trasplante meniscal o meniscectomía por segunda vez, entre los años 2007 a 2015. Resultados: A partir de los 6 meses la EVA mostró una tendencia a la mejoría en el grupo de trasplante meniscal, pasando de Moderado a Leve (p: <0.000). La Escala de Tegner y Lysholm cambió de Pobre a Bueno en el grupo de segunda meniscectomía (p= 0.008) y de Bueno a Excelente en el grupo trasplantado (p=0.225). La calificación promedio de la EVA en el grupo de trasplante presentó mejoría (p=<0.000), a diferencia del grupo de segunda meniscectomía (p=0.591). La escala de Tegner y Lysholm, mostró significancia estadística con tendencia a la mejoría en el grupo de segunda meniscectomía. Discusión: Los resultados muestran que con trasplante meniscal hay mejoría del dolor y la funcionalidad versus un segunda meniscectomía. Para fortalecer la evidencia de este tratamiento son necesarios estudios prospectivos complementarios.

Applied biomaterials from sustainable sources

If we look back in time at the history of humanity, we can state that our generation is living an era of outstanding efficiency and progress because of globalization and global competition, even if this is resulting in the rapid depletion of energy sources and raw materials. The environmental impact of non-biodegradable plastic wastes is of increasing global concern: nowadays, imagining a world without synthetic plastics seems impossible, though their large-scale production and their extensive use have only spread since the end of the World War II. In recent years, the demand for sustainable materials has increased significantly and, with a view to circular economy, research has also focused on the enhancement and subsequent reuse of waste materials produced by industrial processing, intensive farming and the agricultural sector. Plastic polymers have been the most practical and economical solution for decades due to their low cost, prompt availability and excellent optical, mechanical and barrier properties. Biodegradable polymers could replace them in many applications, thus reducing the problems of traditional plastics disposability and the dependence on petroleum. Natural biopolymers are in fact characterized by a high biocompatibility and biodegradability and have already prompted research in the field of regenerative medicine. During my PhD, my goal was to use natural polymers from sustainable sources as raw materials to produce biomaterials, which are materials designed to interface with biological systems to evaluate, support or replace any tissue, organ, or function of the body. I focused on the use of the most abundant biopolymers in nature to produce biomaterials in the form of films, scaffolds and cements. After a complete characterization, the materials were proposed for suitable applications in different fields, from tissue engineering to cosmetics and food packaging. Some of the obtained results were published on international scientific and peer-reviewed journals.

Nuovo copolimero statistico a base di poli(butilene succinato) biocompatibile e bioriassorbibile da utilizzare nell'ingegneria tissutale del miocardio

Nonostante il forte calo della mortalità per malattie cardiovascolari, tali disfunzioni rappresentano ancora la prima causa di morte in Europa. L’unico vero trattamento è il trapianto di cuore che però presenta diverse complicanze, tra cui la scarsa disponibilità di organi e il rigetto da parte dell’organismo curato. Per superare questi problemi, la ricerca si sta focalizzando sullo studio di nuovi polimeri biocompatibili e biodegradabili, per la realizzazione di strutture porose tridimensionali in grado di supportare la crescita e l’adesione cellulare. Tra i polimeri sintetici sperimentati per questa applicazione, il poli(butilene succinato) (PBS) rappresenta un ottimo candidato. Nonostante i promettenti risultati già ottenuti dal punto di vista di biodegradabilità e biocompatibilità, il PBS presenta però proprietà meccaniche poco adatte all’impiego qui descritto, proprio perché l’applicazione miocardica richiede particolari caratteristiche di modulo di Young (E) e un ritorno elastico comparabile con quello del miocardio naturale. Nella presente Tesi è stato sintetizzato e caratterizzato un nuovo copolimero statistico a base di PBS che presenta proprietà meccaniche funzionali all’MTE (Miocardial Tissue Engineering). In particolare, è stato inserito all’interno della catena polimerica, il neopentil glicole, che ha portato a un aumento della stabilità termica, proprietà di particolare interesse in fase di lavorazione del materiale, e una diminuzione del grado di cristallinità. La ridotta capacità a cristallizzare del copoliestere ha un effetto diretto sulle proprietà funzionali, tra le altre, sulla risposta meccanica e sulla velocità di degradazione idrolitica in ambiente fisiologico. In particolare, i risultati ottenuti hanno evidenziato come la copolimerizzazione abbia determinato una maggiore plasticità del materiale finale insieme a una maggiore velocità di degradazione idrolitica, entrambi spiegabili sulla base del ridotto grado di cristallinità.

Utilizzo di sistemi bioreattori per l’ingegnerizzazione di sostituti del tessuto cartilagineo

La mancanza di vascolarizzazione dei costrutti tissutali cartilaginei ingegnerizzati rende opportuno e necessario l’impiego di bioreattori che permettano di indurre un flusso nel terreno di coltura favorendo il trasporto di massa e quindi lo sviluppo del metabolismo e del differenziamento cellulare. I bioreattori intendono replicare gli stimoli fisici fisiologici, nello specifico condrogenici, e a regolare la formazione della matrice extracellulare tramite meccanotrasduzione, il fenomeno biologico che traduce le sollecitazioni meccaniche applicate alle cellule in segnali biochimici che suscitano risposte adattative. In questo elaborato sono riportati i risultati di un recente lavoro - pubblicato da J. Hallas, A. J. Janvier, K. F. Hoettges & J. R. Henstock e intitolato “Pneumatic piston hydrostatic bioreactor for cartilage tissue engineering – che propone la realizzazione di un bioreattore idrostatico a pistone pneumatico, realizzato con componenti facilmente acquisibili a basso costo in commercio. Il bioreattore è collegato a una camera di coltura tramite un connettore pneumatico e un tubo per l’aria in polipropilene. La camera di coltura è realizzata in acido polilattico (PLA) tramite stampante 3D. Il dispositivo è in grado di applicare a una coltura cellulare tridimensionale una pressione idrostatica intermittente con ampiezza compresa tra 0 e 400 kPa e frequenza massima di 3,5 Hz. Condrociti provenienti dalla cartilagine di un’articolazione di ginocchio sono stati coltivati all’interno della camera di coltura del bioreattore dove sono stati sottoposti a una pressione di picco di 300 kPa per 3 ore al giorno per un totale di 5 giorni. Al termine della coltura si è ottenuto un aumento dell’attività metabolica cellulare del 21% e un aumento significativo del contenuto di glicosamminoglicani nell’ECM.