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

STUDIES OF FUNCTIONALIZED NANOPARTICLES FOR SMART SELF-ASSEMBLY AND AS CONTROLLED DRUG DELIVERY

This dissertation is related to the studies of functionalized nanoparticles for self-assembly and as controlled drug delivery system. The whole topic is composed of two parts. In the first part, the research was conducted to design and synthesize a new type of ionic peptide-functionalized copolymer conjugates for self-assembly into nanoparticle fibers and 3D scaffolds with the ability of multi-drug loading and governing the release rate of each drug for tissue engineering. The self-assembly study confirmed that such peptide-functionalized amphiphilic copolymers underwent different self-assembly behavior. The bigger nanoparticles were more easily assembled into nanoparticle fibers and 3D scaffolds with larger pore size, while the smaller nanoparticle underwent faster self-assembly to form more compact 3D scaffolds with smaller porosity but more stable structure. Controlled release studies confirmed the ability of governing simultaneous release of different model drugs with independent release rate from a same scaffold. Cytotoxicity tests showed that all synthesized peptides, copolymers and peptide-copolymer conjugates were biocompatible with SW-620 cell lines and NIH3T3 cell lines. This new type of self-assembled scaffolds combined the advantages of peptide nanofibers and versatile controlled release of polymeric nanoparticles to achieve simultaneous multi-drug loading and controlled release of each drug, uniform distribution and flexibility of hydrogel scaffolds. The investigations in second part were first to design and synthesize organic biocide-loaded nanoparticles for low-leaching wood preservation using a cost-effective one-pot method to synthesize amphiphilic chitosan-g-PMMA nanoparticles loading with ~25-28 wt.% of the fungicide tebuconazole with particle size of ~100 nm diameter by FESEM. FESEM analysis confirmed efficient penetration of nanoparticles throughout the treated wooden stake with dimension of 19 × 19 × 455 mm^3. Leaching studies showed that biocide introduced into sapwood via nanoparticles leached only ~9% compared with the amount leached from tebuconazole solution-treated control, while soil jar tests showed that the nanoparticle-treated wood blocks were effectively protected from biological decay tested against G. trabeum, a brown rot fungus. Copper oxide nanoparticles with and without polymer stabilizers were also investigated to use as inorganic wood preservatives to clarify the factor affecting copper leaching from treated wood. Copper oxide nanoparticles with uniform diameters of ~10 nm and ~50 nm were prepared, and the leachates from southern pine sapwood treated with these nanoparticles were analyzed. It was found by TEM and EDS analysis that significant numbers of nanoparticles leached from the treated wood. The 50 nm nanoparticles leached slightly less than a soluble copper salt control, but 10 nm nanoparticles leached substantially more than the control. The effect of polymer stabilizers on nanoparticle leaching was also investigated. Results showed that polymer stabilizers increased leaching. The trends showed that nanoparticle size was a major factor in copper leaching.

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

Movement effects on the flow physics and nutrient delivery in engineered valvular tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-:τ: explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-:τ: environments.^

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.

To Evaluate the Synergistic Effect of Different Crosslinking Strategies on Collagen Derivatives to Develop Funcional Biomimetic Scaffolds

The preliminary objective of this work was to study how the effect of different crosslinking methodologies can functionally modify various characteristics of biological macromolecules relevant for scaffold development in bone tissue engineering. The research study was classified and studied in three different phases: (i) different crosslinking strategies in gelatin functionalization, (ii) ribose mediated crosslinking in collagen-hydroxyapatite scaffold (iii) different crosslinking mechanisms in functional modification of bone-like scaffold. The obtained results were highly positive in all the three investigated studies. Though the core aim of this research was to explore the available crosslinking strategies in different biological macromolecules, the present study generated significant findings, largely contributing to provide optimum solutions in understanding how the crosslinking density can fine-tune the overall performance of a scaffold, relevant for its functioning in vivo. In particular, this study demonstrated that different crosslinkers at different conditions (pH and temperature) can modify the functional properties of the scaffolds differently, therefore this optimization strategies on these crosslinkers as obtained from this study results will help material scientists in the design and development of bioactive hybrid biomaterials for hard tissue regeneration.

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.

Nuovo copoliestere alifatico del PBS da fonte rinnovabile biocompatibile e bioriassorbibile per impiego nell’ Ingegneria dei tessuti molli

L’enorme progresso nel campo della biologia cellulare ha consentito lo sviluppo di tecnologie per la ricostruzione in vitro di tessuti, definendo una nuova branca di scienze biomediche: l’ingegneria dei tessuti. Tra le sue numerose applicazioni, la riparazione del tessuto cardiaco infartuato rappresenta un’importante obiettivo. 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 proprietà meccaniche poco adatte a questo impiego: l’applicazione miocardica richiede particolari caratteristiche di modulo di Young (E) e un ritorno elastico comparabile a quello del miocardio. Al fine di conferire al PBS proprietà meccaniche funzionali all’MTE (Miocardial Tissue Engineering), in questa Tesi è stato sintetizzato e caratterizzato un nuovo copolimero statistico a base di PBS contenente subunità Pripol 1009, un diacido prodotto dalla Croda, biobased e biodegradabile. Sono stati preparati film attraverso pressofusione e scaffold tramite elettrofilatura. Oltre alla caratterizzazione molecolare, volta a determinare il peso molecolare, la struttura e la composizione, film e scaffold sono stati sottoposti anche ad analisi termica, diffrattometrica, meccanica e a studi di degradazione idrolitica in condizioni fisiologiche. I risultati ottenuti hanno evidenziato che l’inserimento di segmenti Pripol all’interno della catena polimerica ha portato, oltre che a un incremento della stabilità termo-ossidativa, anche a un importante miglioramento delle proprietà meccaniche: il materiale sintetizzato, sia sotto forma di film che di scaffold, possiede le caratteristiche di elastomero termoplastico che lo rendono adatto ad applicazioni nell’ingegneria tissutale. Da ultimo, rispetto al PBS, il copolimero statistico mostra una maggiore velocità di degradazione in condizioni fisiologiche.

Electrospun scaffolds for regeneration of musculoskeletal interface tissues

L’apparato muscolo scheletrico è composto da strutture muscolari, articolari e ossee. Tali tessuti sono molto diversi tra loro e hanno proprietà meccaniche estremamente variabili, pertanto presentano una transizione graduale in corrispondenza della loro giunzione, onde evitare l’insorgere di concentrazioni di tensione. L’evoluzione ha portato alla formazione di particolari interfacce che permettono la corretta trasmissione dei carichi distribuendo le tensioni su una superficie più ampia in corrispondenza della giunzione. Le interfacce che vanno a inserirsi nell’osso vengono definite entesi e in particolare, in questa review, analizzeremo il caso di quelle tra tendini/legamenti e osso. In questo lavoro ci siamo anche concentrati sulla giunzione miotendinea, ovvero tra muscolo e tendine. Sono numerose le lesioni che riguardano muscoli, ossa, tendini o legamenti e molto spesso l’infortunio avviene a livello della giunzione. Quando ciò accade vi sono diverse strade, ciascuna con i suoi vantaggi e svantaggi: sutura, autograft, allograft o xenograft. Oltre a queste soluzioni si è fatta gradualmente più spazio la possibilità di realizzare degli scaffold che vadano temporaneamente a sostituire la parte danneggiata e a promuovere la sua rigenerazione, degradandosi man mano. L’elettrofilatura (Elettrospinning) è un processo produttivo che negli ultimi decenni si è affermato come tecnica per la fabbricazione di questi scaffold, fino a diventare uno tra i principali processi utilizzati dai ricercatori in questo campo. Questa tecnica infatti permette di realizzare scaffold di nanofibre porose utilizzando polimeri biodegradabili e soprattutto biocompatibili. Lo scopo della review è proprio quello di scoprire tutti i lavori e gli studi che utilizzano l’elettrofilatura per realizzare degli scaffold per interfacce, delineando così lo stato dell’arte sui progressi fatti e sulle varie tecniche utilizzate.

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.