In this work, I investigated the influence of different surfaces on stem cell proliferation and osteogenetic differentiation. Surface properties of biomaterials are important factors that influence cell fate such as cell adhesion, viability, proliferation and differentiation. Herein, mesenchymal stem cells (MSCs) were cultured on composite electrospun nanofibrous membranes with varied surface wettability for designed periods and cell morphologies, proliferation and viability were characterized via analysis methods such as Infrared attenuated total reflectance Spectroscopy (IR-ATR), scanning electron microscopy (SEM) and MTT cell proliferation assay. The expression of genes associated with osteogenesis, including bone sialoprotein (BSP), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) were measured by real-time RT-PCR on different time points. Through western blot analysis, ERK1/2 pathway was found to be responsible for the differentiation of MSCs on nanofibrous membranes with different wettability.

A huge discrepancy between the number of patients on the waiting list for organ transplants and the actual available donors has led to search for alternative approaches to substitute compromised or missing tissues and organs. Tissue engineering is a promising alternative to organ transplantation with the aim to fabricate functional organs through the use of biological or biocompatible scaffolds. Nanogels made from self-assembling ultrashort peptides are promising biomaterials for a variety of biomedical applications. Our group at KAUST is interested in the development of novel synthetic peptide-based biomaterials that combine the advantages of both natural and synthetic hydrogels for various applications. In this study, we have investigated two compounds of a novel class of rationally designed ultrashort peptides, Ac-IVFK-NH2 (Ac-Ile-Val-Phe-Lys-NH2) and Ac-IVZK-NH2 (Ac-Ile-Val-Cha-Lys-NH2). These compounds have an innate tendency to self-assemble into nanofibrous hydrogels which can be used as 3D scaffolds, for example for the fabrication of 3D skin grafts for wound healing. We have evaluated the efficacy of the peptide scaffolds in treating full-thickness wounds in minipigs. Additionally, we assessed the ability of these scaffolds in supporting skeletal muscle tissue proliferation and differentiation. We found that our innovative nanogels supported a substantial increase in human dermal fibroblast and myoblast growth and cells viability, and supported myoblast differentiation. Also, microscopic observation of the direct contact of keratinocytes and fibroblasts revealed enhancement in keratinocytes proliferation. In addition, we demonstrated the ability of human umbilical vein endothelial cells to form tube like structure within peptide nanogels using immunofluorescence staining. Moreover, we successfully produced artificial 3D vascularized skin substitutes using these peptide scaffolds. We selected these peptide nanogels and were able to produce in situ silver nanoparticles within the nanogels, solely through UV irradiation, with no reducing agent present. We then assessed the efficacy of the silver nanoparticle-containing peptide nanogels on minipigs with full-thickness excision wounds. The application of the peptide nanogels on full thickness minipig wounds demonstrated that the scaffolds were biocompatible and did not trigger wound inflammation, and thus safe for topical application. The effect of nanogels, both with and without the addition of the silver nanoparticles, revealed that the scaffold itself has a high potential to act as an antibacterial agent. Interestingly, the effect of the peptide nanogels on wound closure was comparable to that of standard care hydrogels. Furthermore, we have demonstrated that both peptides can act as printable bioinks which opens up the possibility of 3D bioprinting of different cell types in the future. We believe that the described results represent an advancement in the context of engineering skin and skeletal muscle tissue, thereby providing the opportunity to rebuild missing, failing, or damaged parts.

Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.

Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.

Master of Science / Department of Architectural Engineering and Construction Science / Julia A. Keen / Nanofibers are classified as fibers less than 1 micrometer in diameter. These fibers can be layered to form nanofibrous membranes, and these membranes offer great potential in the filtration industry. The membranes' smaller fiber diameters and pore sizes permit such filters to filter out more and smaller particulate. Additionally, antimicrobial agents can be incorporated into the membrane to inhibit fungal and bacterial growth on the membrane’s surface. This report evaluates nanofibrous membranes with antimicrobial agents and their potential in two specific locations: cleanrooms and protective environment rooms, where bacterial and fungal growth would have a detrimental effect on the process or occupant of the space.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Polymeric nanofibrous membranes that act as surfaces offer two compelling properties; they have extremely large surface areas that can be modified and they can offer multiple reactive sites depending on which polymer they were electrospun from. Combining nanofibrous surfaces such as these with photochromic dyes can give remarkable, light responsive materials. In this study, a terpolymer was electrospun and crosslinked (to impart insolubility to the material) to produce a nanofibrous mat that contained surface secondary-hydroxyl units and benzyl chloride units, from which monomers could grow using surface-initiated atom transfer polymerization (SI-ATRP). The surface was further fluorinated though the secondary-hydroxyl moieties to produce a hydrophobic crosslinked nanofibrous surface. n-Butyl acrylate and a photochromic spiropyran dye were copolymerized from the surface using SIATRP, in order to produce a surface that exhibited reversible adhesion towards a water droplet using ultraviolet and visible light as a stimulus to change between the two states. This in principle would allow the droplet to roll off and stick to the surface respectively. Although the surface displayed a colour change when switched, proving that the SI-ATRP had taken place, the droplet of water remained attached to the surface in both states. / AFRIKAANSE OPSOMMING: Polimeriese nanovesel membrane wat dien as oppervlaktes bied twee aantreklike eienskappe; hulle het baie groot oppervlakareas wat gemodifiseer kan word en hulle bied veelvuldige reaktiewe punte, afhangende van die polimeer waarvan hulle ge-elektrospin is. Deur sulke nanovesel oppervlaktes met fotochromiese kleurstowwe te kombineer, kan uitstekende lig reagerende materiale verkry word. In hierdie studie is ‘n terpolimeer ge-elektrospin en daarna gekruisbind (om die materiaal onoplosbaar te maak) om ‘n nanovesel web te kry wat oppervlak sekondêre-hidroksiel en benzielchloried eenhede bevat. Monomere kan dan vanaf hierdie eenhede groei deur middel van oppervlak-geïnisieerde atoomoordrag polimerisasie (OI-AOPR). Die oppervlak was verder gefluorineer via die sekondêre-hidroksiel eenhede om ‘n hidrofobiese, gekruisbinde nanovesel oppervlak te vorm. n-Butielakrilaat en ‘n fotochromiese spiropiraan kleurstof is gekopolimeriseer vanaf die oppervlak deur middel van OI-AORP om sodoende ‘n oppervlak te skep wat omkeerbare adhesie van ‘n water druppel gee as ultraviolet en sigbare lig as stimulus gebruik word om tussen die twee toestande te wissel. In beginsel sal die water druppel vanaf die oppervlak kan afrol én daaraan vassit, afhangende van die toestand van die oppervlak. Alhoewel die oppervlak van kleur verander het met die wisseling tussen die twee toestande, wat as bewys dien dat OI-AORP wel plaasgevind het, het die druppel water bly vassit aan die oppervlak in beide toestande.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapes are lost annually due to spoilage by Botrytis cinerea. Botrytis cinerea is currently controlled using fungicides and SO2 fumigation in storage rooms but with limited success. Although these techniques have been used for decades they have been associated with numerous of disadvantages. Fungicides are restricted from being used during storage due to legislation and toxicity of the fungicides present on the surface of the berries. SO2 fumigation does not kill the dormant infections present inside the grape tissue and if the SO2 concentration is too high the berries are damaged. During this study two different polymer nanofibrous platforms was synthesized in an attempt to prevent the rotting effect of B. cinerea. The first polymer was modified to yield a polymer with a positively charged quaternized nitrogen moiety, which was subsequently reacted with sodium metabisulfite through an ion exchange process. The modified polymer was electrospun into nanofibrous mats for the benefit of the nanofibers’ high available surface area. A further functionalization was done to increase the quantity of sodium metabisulfite on the surface of the polymer nanofibers. Sodium metabisulfite salt released SO2 gas upon reaction with water vapour present in the atmosphere which resulted in the inhibition of conidial germination of B. cinerea. The second polymer was synthesized and then electrospun into polymer nanofibrous mats followed by chemical modification of the electrospun polymer nanofibers. This post-electrospun modification resulted in the covalent attachment of a fungicide-derivative to the surface of the polymer nanofibers. The fungicide-derivative showed excellent inhibition of B. cinerea mycelium growth. Anti-fungal studies were conducted using the two modified polymer nanofibrous mats against B. cinerea to evaluate these nanofibrous surfaces as B. cinerea inhibiting membranes. Results indicated that B. cinerea conidial germination and mycelium growth were successfully inhibited. / AFRIKAANSE OPSOMMING: Druiwe bederf jaarliks weens die verrottende effek van die fungi Botrytis cinerea. Botrytis cinerea word tans beheer deur gebruik te maak van swamdoders en swaweldioksied (SO2) besproeiings in stoorkamers, maar met beperkte sukses. Alhoewel hierdie tegnieke al vir dekades in gebruik is, word dit geassosieer met verskeie probleme. Die gebruik van swamdoders word in stoorkamers verbied, weens die moontlike skadelike inname daarvan deur die mens aangesien die swamdoders op die druiwe se oppervlakte teenwoordig is. SO2-gasbesproeiings maak nie dormante infeksies binne-in die druiwe self dood nie en indien SO2 -konsentrasies te hoog is kan dit die druiwe beskadig. Tydens hierdie studie is twee verskillende polimeer-nanoveselplatforms gesintetiseer met die doel om die verrottende effek van B. cinerea te voorkom. Die eerste polimeer is aangepas met ’n primêre amienverbinding met die doel dat dit deur ‘n opvolgende modifikasiereaksie verander om sodoende ’n polimeer met ‘n kwatenêre ammoniumgroep te verkry. Die doel van die kwatenêre eienskap is dat ioniese uitruiling plaasvind tussen die anioon van die polimeer en die natruimmetabisulfiet-anioon van die sout. Die voorbereide kopolimeer is geëlektrospin in nanoveselmatte deur middel van die enkelnaald-en-enkelbal-elektrospintegniek om SMI-qC12 nanovesels te lewer wat gefunksionaliseer is. Die nanoveselmatte is na die elektrospinproses verder aangepas om die hoeveelheid natruimmetabisulfiet op die oppervlak van die nanovesels te verhoog. Die natruimmetabisulfiet stel SO2-gas vry sodra dit in aanraking kom met waterdamp in die lug wat ’n beperkende effek op spoorontkieming van B. cinerea tot gevolg het. Die tweede polimeer is voor en na die elektrospinproses gefunksionaliseer met gesintetiseerde organiese verbindings (swamdoder). Die aanpassing van die polimeer met die organiese verbindings het plaasgevind op ‘n kovalente wyse om te verhoed dat die organiese verbinding vrygelaat word tydens gebruik. Die organiese verbindings het effektiewe beperking van miselium-groei getoon. Anti-swamstudies is uitgevoer tussen die twee gefunksionaliseerde polimeer platforms en B. cinerea om die oppervlaktes van die gefunksionaliseerde polimeer/nanovesels te evalueer as B. cinerea beperkende platforms. Resultate het aangetoon dat spoorontkieming en miselium groei van B. cinerea suksesvol beperk is.

Dans le travail de thèse, nous avons étudié le processus de croissance et l’évolution des propriétés structurales de l’alumine ultra-poreuse (UPA) monolithique pendant le traitement thermique dans le domaine des températures entre 20 et 1600 °C. Un modèle théorique a été proposé permettant de décrire et prédire sa structure. La formation de mullite à partir d’alumine imprégnée par la silice a été également étudiée. Les premières mesures conduites sur les propriétés diélectriques des UPA dans le domaine de fréquences GHz-THz montrent des possibilités d’application dans le domaine de l’optique réfractrice. L’installation expérimentale pour l'élaboration des monolithes ultra poreux est décrite. L’étude de la cinétique de croissance à partir d’aluminium technique, ultra pur et monocristallin a permis d’analyser l’influence des différentes impuretés présentes. Les matériaux ont été caractérisés par les méthodes DRX, MET/MEB, ICP/OES, TGA et PL. Les UPA modifiées chimiquement par imprégnation de TMES et TEOS en phase vapeur ont également été analysées. L’étude en fonction de la température des différents matériaux a été menée sur la taille des fibreuse, la surface spécifique, la densité massique, ainsi que la quantité de l’eau structurelle et adsorbée, d’une part, et la structure cristalline d’autre part. L’utilisation des méthodes DRX et PL corrélées a permis d’expliquer une transformation de la phase α de l’UPA en mullite 2:1.Le modèle proposé distingue deux principaux régimes de modification thermique de la structure des UPA : transport de masse sur la surface d’une fibre ou de la masse globalement (frittage). Des énergies d’activation du transport massique et des constantes pre-exponentielles ont été obtenues pour les deux régimes. Les résultats obtenus permettent de conclure que les transformations morphologiques, de la composition chimique et de la phase cristalline sont d’origine commune. / In this PhD work we investigated growth process and evolution of structural properties of ultraporous alumina (UPA) monoliths during thermal treatment in the range between 20 and 1600 °C. A simple theoretical model was proposed permitting description and prediction of the material structure. A particular extension of this study concerns the mullite formation with an increase of the silica loading. Furthermore, first measurements of dielectric properties (refraction index and losses) in GHz-THz range of frequencies were performed, indicating this material to be potentially interesting for fabrication of the refraction optics.The experimental installation for UPA elaboration is described, and the analysis is presented of principal impurities and their influence on the growth kinetics using technical, high-purity and monocrystalline aluminum. The obtained materials were characterized by XRD, TEM, SEM, ICP/OES, TGA and PL methods. UPA modified with TMES and TEOS vapor impregnations were also obtained and analyzed. The fibril size, specific surface area, mass density and content of structural, adsorbed water and crystalline phase were measured for different UPA materials as a function of the annealing temperature. The correlated XRD and PL analyses was performed explaining the conversion of α phase UPA to 2:1 mullite, which fundamental band gap was set to 7.55 eV.The proposed model distinguished two principal regimes of thermal modifications: surface diffusional mass transport over a single fibril and bulk mass transport involving total material mass (sintering). The activation energies of the mass transport and pre-exponential constants (diffusion coefficient and free volume) in both regimes were obtained, providing a better understanding of the underlying physical processes in different UPA materials. Based on these results, we conclude about a common origin of morphological, chemical composition and phase transformations.

Orientador: Cecília Amélia de Carvalho Zavaglia / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-23T08:49:24Z (GMT). No. of bitstreams: 1 Brito_TalitaAlmeidaVidade_M.pdf: 2327615 bytes, checksum: 1370c441c447a5a8dae05303883562b5 (MD5) Previous issue date: 2013 / Resumo: Este trabalho apresenta a preparação e caracterização de nanofibras da blenda poli (L-ácido láctico) (PLLA)/poli (?-caprolactona) (PCL) através dos processos de eletrofiação e rotofiação destinada à produção de fibras visando futuras aplicações como suporte para a engenharia tecidual. As blendas foram preparadas através da dissolução do polímero em clorofórmio e clorofórmio mais acetona, resultando em uma solução de 6%. A eletrofiação é um processo relativamente simples e de baixo custo, que consiste na aceleração de uma solução polimérica, inicialmente contida em um capilar metálico, pela presença de um campo elétrico externo, para produzir fibras com diâmetro médio reduzido. Apesar da popularidade e da versatilidade, o processo de eletrofiação apresenta algumas desvantagens, tais como o uso de fonte de alta tensão, baixa taxa e longo tempo de produção das fibras. Com a necessidade de encontrar um método de produção de fibras para sanar eventuais obstáculos encontrados na eletrofiação, foi realizado um estudo com um novo processo: a rotofiação. O processo de rotofiação é um processo simples que forma fibras durante o jateamento da solução polimérica através de um orifício central utilizando alta velocidade de rotação e não utiliza campo elétrico de alta voltagem, como na eletrofiação. As fibras obtidas por meio dos dois processos foram analisadas e caracterizadas pelos seguintes métodos: microscopia eletrônica de varredura (MEV), análise termogravimétrica (TGA), calorimetria exploratória diferencial (DSC), e espectroscopia na região do infravermelho por transformada de fourier (FTIR). A análise das fibras por microscopia eletrônica de varredura (MEV) mostrou que é possível a formação de nanofibras da blenda PLLA/PCL através dos dois processos. Verificou-se que ocorreram diferenças significativas no diâmetro médio dos fios entre os processos, onde na rotofiação, os diâmetros foram maiores, os dois processos estudados permitiram a obtenção de fibras porosas, uma característica importante requerida na engenharia tecidual. Os resultados das análises térmicas indicaram o comportamento imiscível das blendas PLLA/PCL. Através da análise de FTIR foi possível demonstrar eliminação completa dos solventes durante o processamento e também a imiscibilidade dos polímeros / Abstract: This work presents the preparation and characterization of nanofibers of poly (L-lactic acid) (PLLA) / poly (?-caprolactone) (PCL) through electrospinning and rotary jet spinning processes for the production of fibers aiming future applications as scaffolds for tissue engineering. The blends were prepared using the two polymers cited above in chloroform and chloroform plus acetone, resulting in a solution of 6%. Electrospinning is relatively simple and low cost process, which consists in the acceleration of a polymer solution initially contained in a capillary, the presence of an external electric field to produce fibers with reduced average diameter reduced. Despite the popularity and versatility, the electrospinning process has some disadvantages such as the use of high voltage supply, low spinning rates and takes time for long fiber production. With the need to find a method of producing fibers to remedy any obstacles encountered in electrospinning, a study was conducted with a new process: rotary jet spinning. The process of rotary jet spinning is a simple process that forms fibers during the blasting of the polymer solution through a central hole using high-speed rotation and does not use high intensity electric fields, as in electrospinning. The fibers obtained by the two processes were analyzed and characterized by the following methods: scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and infrared spectroscopy fourier transform (FTIR). The fiber analysis by scanning electron microscopy (SEM) showed that it is possible to form nanofibers of PLLA / PCL through two processes. It was found that there were significant differences in the average diameter of the fibers between the processes, where higher diameters were observed in rotary jet spinning, the two processes studied allowed to obtain porous fibers, an important feature required in tissue engineering. The results of thermal analysis indicated that it was formed immiscible blends of PLLA / PCL. By FTIR analysis it was demonstrated complete elimination of the solvents during processing and also the immiscibility of the polymers / Mestrado / Materiais e Processos de Fabricação / Mestra em Engenharia Mecânica

The limited reparative capacity of the skin and the inadequacy of conventional treatments have necessitated the development of tissue engineered skin substitutes. Several substitutes, including Integra Dermal Regeneration Template, are finding increasingly widespread application in the treatment of acute and chronic wounds. To date, these substitutes are unable to fully recreate the functionality and aesthetics of skin prior to injury. This thesis applied an integrated approach combining solution preparation, material fabrication control and biological testing to investigate electrospun silk fibroin (SF) nano-microfibrous scaffolds as potential biomimetic skin substitutes. Further to this, the improvement of the existing Integra scaffold through the incorporation of hyaluronan (HA) was assessed. Through rheological analysis of regenerated SF solutions under shear and extensional deformation a concentration regime transition at 20 wt% SF was identified. Solutions with relaxation times under 0.001 seconds were found to be unsuitable for electrospinning. The incorporation of poly(ethylene oxide) (PEO) was found to significantly increase solution relaxation times and extensional viscosity, making them much more suitable for electrospinning. Solution viscoelastic properties were shown to directly influence electrospun fibre morphology, with increases in viscosity resulting in increases in fibre diameter under stable spinning conditions. The effects of electrospinning parameters on electrospun fibre morphologies were investigated using SF-PEO blended solutions. Increased electrical field, spinneret height and decreased flow rate were found to decrease fibre diameter. In vitro assessment of the attachment, spreading, proliferation, viability and gene expression of primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) was conducted. Both PHDFs and BM-MSCs attached and proliferated with greater rapidity on fibres of the smallest diameters (~250-300 nm) with proliferation decreasing as fibre size increased until fibre diameters reached ~1200 nm. Cells were observed to be spread, with multiple attachments between fibres in scaffolds composed of ~250-300 nm diameter fibres. Cells aligned themselves to single fibres in scaffolds composed of fibres greater than 1 micrometre. HA supplementation to Integra resulted in increased proliferation, viability and migration of PHDFs. In ex vivo cutaneous wound healing models, the invasion of Integra was enhanced when scaffolds were supplemented with HA, with increased matrix deposition observed. Optimal supplementation concentrations for in vitro and ex vivo increases in cell proliferation and migration were at 1.5 – 2 mg ml-1 HA. SF electrospun scaffolds facilitated epithelial migration in ex vivo artificial wounds, with the migratory epidermis more closely resembling the structures observed in vivo. Additional preliminary investigations into the efficacy of a paste-form of Integra, Integra Flowable Wound Matrix (IFWM) were performed ex vivo, with cell invasion comparable to the conventional scaffold format. The potential for the incorporation of viable PHDFs and BM-MSCs was also investigated and keratinocyte migration was enhanced in these scaffolds. The results in this thesis provide valuable optimisation information on the development of SF electrospun scaffolds for skin engineering. Additionally, the supplementation of Integra with HA may provide a simple and effective way to enhance the performance of the scaffold in vivo.