A engenharia de tecidos é uma área de pesquisa que busca alternativas para a regeneração de tecidos danificados. Uma subárea dessa nova ciência faz uso das células-tronco, capazes de participar do processo de regeneração tecidual. As células são cultivadas em suportes porosos chamados de biomateriais ou scaffolds. As células-tronco mesenquimais de polpa de dente são células multipotentes e fáceis de serem obtidas. A policaprolactona, polímero atóxico, tem sido vastamente utilizada na fabricação de scaffolds devido as suas propriedades mecânicas, adequadas para a regeneração de alguns tecidos. A cultura dinâmica em biorreatores vem sendo utilizada pela sua capacidade de mimetizar as condições do ambiente natural das células-tronco no organismo humano. O objetivo do presente trabalho foi definir um protocolo de cultivo de células-tronco mesenquimais da polpa de dente em scaffolds de policaprolactona em multicamada, utilizando biorreatores de perfusão. Os biorreatores foram projetados com base em informações presentes na literatura. Os scaffolds de policaprolactona foram produzidos por electrospinning e apresentaram um diâmetro de 15 mm, aproximadamente 300 μm de espessura e diâmetro médio das fibras de 0,98 ± 0,24 μm. O cultivo estático, para fins de comparação, foi realizado em triplicata. Para o cultivo dinâmico, analisaram-se 3 diferentes vazões: 0,1 mL.min-1, 0,08 mL.min-1 e 0,05 mL.min-1. Nos dias 1, 3 e 7 de cultivo celular as análises de viabilidade celular foram realizadas pelo ensaio de WST-8, a citotoxicidade pela dosagem de LDH e de adesão celular pela histologia.. As células cultivadas nos scaffolds do topo da estrutura em camadas apresentaram maior viabilidade celular para as vazões de 0,1 e 0,08 mL.min-1 entre os dias 1 e 7 do cultivo celular. O cultivo dinâmico realizado na vazão de 0,05 mL.min-1 obteve um número baixo de células viáveis em todos os dias de análise. No cultivo estático, a viabilidade celular aumentou do primeiro ao sétimo dia de cultivo, mas permaneceu abaixo da maior vazão testada. As análises de histologia confirmaram a presença de células nas superfícies dos scaffolds. Os níveis maiores de citotoxicidade foram encontrados para o cultivo dinâmico, principalmente para a vazão de 0,1 mL.min-1, bem como foi observada maior viabilidade celular para essa vazão. Ainda é necessário repetir ambos os cultivos para confirmar os resultados e validar o protocolo definido para os experimentos. No entanto, no cultivo dinâmico, a vazão de 0,1 mL.min-1 proporcionou resultados promissores para o sistema de perfusão utilizado, podendo comprovar as vantagens do cultivo dinâmico de células-tronco para a medicina regenerativa. / Tissue engineering is an area of research which seeks for alternatives for the regeneration of damaged tissue. A sub-area of this new science uses stem cells, which are able to participate in the process of tissue regeneration. The cells are cultivated in porous supports called biomaterials or scaffolds. The mesenchymal stem cells from teeth pulp are multipotent cells and are easily obtained. Polycaprolactone, an atoxic polymer, has been extensively used in the production of scaffolds, due to its mechanical properties which are suitable for the regeneration of certain types of tissue. The dynamic cultivation using bioreactors is currently used because of its capacity to immitate the conditions of the natural environment of stem cells in the human organism. The aim of the present study has been to define a protocol for the cultivation of mensechymal stem cells from teeth pulp in multi-layered polycaprolactone scaffolds in perfusion bioreactors. The bioreactors were designed based on current information available in the literature. The polycaprolactone scaffolds were produced by electrospinning and had a diameter of 15 mm, with approximately 300 μm thickness and an average fiber diameter of 0.98 ± 0.24 μm. The static cultivation for use as a comparison was carried out in triplicate. Three different flow rates were analyzed for cellular cultivation: 0.1 mL.min-1, 0.08 mL.min-1 and 0.05 mL.min-1. The cellular viability analyses were carried out by WST-8 assay, cytotoxicity through an LDH dosage and cellular adhesion by histology on days 1, 3 and 7 of the cellular cultivation. The cultivated cells on the scaffolds, which were above the structure in layers, presented greater cellular viability when the flow rates 0.1 and 0.08 mL.min-1 were used, between days 1 and 7 of the cellular cultivation. When the flow rate of 0.05 mL.min-1 was used in the dynamic cultivation, the lowest number of viable cells in all the days of the analyses was observed. In the static cultivation, cellular viability increased between the first and seventh day of cultivation but remained lower than the dynamic cultivation with the higher flow rate tested. The histological analyses confirmed the presence of cells on the surface of the scaffolds. Both the greatest levels of cytotoxicity and cellular viability were observed in the dynamic cultivation, mainly with the flow rate of 0.1 mL.min-1. It is necessary to repeat both the cultivations to confirm the results and to test the efficiency of the protocol defined by the experiments. However, in the dynamic cultivation, the flow rate of 0.1 mL.min-1 presented promising results for the perfusion system of this study, showing the advantages of the dynamic cultivation of stem cells for regenerative medicine.

O crescimento de células em arcabouços tridimensionais porosos tem se tornado progressivamente ativo na engenharia de tecidos. Os arcabouços guiam o crescimento celular, sintetizam uma matriz extracelular e outras moléculas biológicas, e facilitam a formação de tecidos e órgãos funcionais. Um cimento deste tipo pode ser preparado misturando um sal de fosfato de cálcio com uma solução aquosa para que se forme uma pasta que possa reagir à temperatura corporal dando lugar a um precipitado que contenha hidroxiapatita (Ca10(PO4)6(OH)2). A similaridade química e morfológica entre este biomaterial e a parte mineral dos tecidos ósseos permite a osteocondução, sendo o cimento substituído por tecido ósseo novo com o tempo e com a vantagem de não desencadear processos inflamatórios e de corpo estranho, com eventual expulsão do material implantado. O objetivo do presente trabalho foi a obtenção e caracterização de suportes tridimensionais para a engenharia de tecido, com o uso de matérias-primas nacionais, por meio da utilização de microesferas de parafina como corpos geradores de poros. As microesferas foram produzidas por suspensão em solução aquosa de poli (álcool vinílico) (PVA) e sulfato de sódio (Na2SO4). Foram analisadas as fases presentes no cimento sintetizado e após a reação de cura do mesmo, a variação do tamanho de partícula e da resistência mecânica com o tempo de moagem. Foi analisada a porosidade dos suportes e a forma de extração da parafina daqueles que a utilizaram na sua formação. O tamanho de poro dos suportes gerados com a variação da quantidade de fase líquida ficou aquém do tamanho considerado ideal para o crescimento de tecido ósseo. A porosidade dos arcabouços fabricados com esferas de parafina foi observada por microscopia eletrônica de varredura (MEV), e seu comportamento foi analisado a partir de ensaios in vitro em solução SBF (simulated body fluid) e em cultura de células. A utilização de esferas de parafina permitiu a formação de poros com tamanho tal que possibilitam potencialmente o crescimento tecidual e celular. / The growth of cells in three-dimensional porous scaffolds has been extensively studied for use in tissue engineering. They guide grow of cells, synthesize extra cellular matrix and other biological molecules, and facilitate the formation of functional tissues and organs. Bone cements has been developed for biomedical applications for a decade approximately. This kind of cement can be prepared mixing a calcium phosphate salt with aqueous solution forming a paste that can react at body temperature generating a hydroxyapatite precipitated (Ca10(PO4)6(OH)2). The chemical and morphological similarity between the cement composition and the mineral part of the bones allows osteoconduction in the tissue with replacement of cement by new bone formed with the advantage to not unchain inflammatory processes and of strange body. The objective of this work was the use of the α-TCP cement for making these scaffolds, through the variation of the amount of liquid phase in the cement and of the use of paraffin spheres as pore source. These spheres were produced by suspension in water solution of poly (vinyl alcohol) and sodium sulphate (Na2SO4). The phases had been analyzed in the synthesized cement and after the reaction of cure of cement, beyond variation of the particle size and the resistance mechanics with the milling time. It was analyzed the porosity of the scaffolds and the extraction of the paraffin in that supports. The pore size of the supports generated with the variation of the amount of liquid phase was on this side of the size considered ideal for the bone tissue growth. The porosity of scaffolds manufactured with paraffin spheres was observed by Scanning Electron Microscopy (SEM), and its behavior was analyzed from test in vitro in SBF solution (simulated body fluid). The use of paraffin spheres allowed the formation of pores size able to permit tissue growth.

Orientadores: Antonio Celso Fonseca de Arruda, Cecília Amélia de Carvalho Zavaglia / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-16T09:25:08Z (GMT). No. of bitstreams: 1 Cardoso_GuineaBrasilCamargo_M.pdf: 3592565 bytes, checksum: f97086f3c3d7b2f1c44e717c77abe11f (MD5) Previous issue date: 2010 / Resumo: A engenharia tecidual, que tem como objetivo a reconstrução de tecidos de animais, enfrenta um desafio: a construção de suportes. Uma das propriedades relevantes desse eventual material é a sua taxa de absorção, que deve ser inversamente proporcional à taxa de crescimento tecidual. Nesse sentido, a poli ( -caprolactona) (PCL), que é um polímero biocompatível e bioreabsorvível, mostra-se ser um material promissor para reconstruções ósseas, já que possui uma taxa de reabsorção lenta. Esse polímero, por não ser capaz de atrair células osteoblásticas, deve ter a adição de cerâmicas bioativas com o propósito de fortalecer a estrutura do suporte. Adicionalmente, deve atuar como um condutor para a proliferação de células ósseas. O objetivo deste trabalho foi desenvolver suportes tridimensionais de poli ( -caprolactona) com fibras ou whiskers de hidroxiapatita. A estrutura deve ser porosa de modo a permitir a vascularização dessa área e consequentemente a osteocondução. Foram sintetizados reagentes primários tais como monetita e carbonato de cálcio, para a sinterização de fosfato tricálcico ( -TCP), que ao ser hidrolisado resultou em whiskers de hidroxiapatita. As fibras de hidroxiapatita foram obtidas pelo método de molten salt utilizando a hidroxiapatita precipitada. As cerâmicas foram caracterizadas por análises de difração de raios X (DRX), fluorescência de raios X (FRX) e microscopia eletrônica de varredura (MEV). Os suportes passaram por testes mecânicos de compressão e testes in vitro, além de MEV e DRX. Os resultados indicaram que os suportes poliméricos com adição de whiskers de hidroxiapatita apresentaram-se mais resistentes à compressão. A presença de whiskers, diferentemente das fibras, caracterizou-se como reforço na matriz polimérica. Os resultados do teste de imersão em fluído corpóreo mostraram que os suportes com whiskers de hidroxiapatita, diferentemente das fibras, resultaram em deposição de apatita em suas superfícies / Abstract: Tissue engineering, which aim is the reconstruction of animal tissues, faces a challenge: the building supports. One of the relevant properties of any material is its rate of absorption, which should be inversely proportional to the rate of tissue growth. Accordingly, to the poly ( -caprolactone) (PCL), which is a biocompatible polymer and bioresorbable shows to be a promising material for bone reconstruction since it has a slow rate of resorption. This polymer, for not being able to attract osteoblastic cells, should have the addition of bioactive ceramics in order to strengthen the supporting structure. Additionally, it should acts as an inducer for the proliferation of bone cells. The objective of this study was to develop three-dimensional scaffolds of poly ( -caprolactone) with fibers and whiskers of hydroxyapatite. The structure should be porous to allow vascularization of this area and consequently the osteoinduction. Were synthesized reagents such as monetita and calcium carbonate, for sintering of tricalcium phosphate ( -TCP), which when hydrolyzed results in hydroxyapatite whiskers. The fibers of hydroxyapatite were obtained by the method molten salt using precipitated hydroxyapatite. The ceramics were characterized by analysis of X-ray diffraction (XRD), X-ray fluorescence (XRF) and scanning electron microscopy (SEM). The scaffolds were tested by mechanical compression and in vitro tests, in addition to SEM and XRD. The results indicated that the scaffolds with the addition of hydroxyapatite whiskers were more resistant to compression. The presence of whiskers, unlike fiber, characterized as reinforcement in polymer matrix. The test results of immersion in body fluid showed that the scaffolds with hydroxyapatite whiskers, unlike fiber, aimed to the deposition of apatite on their surfaces / Mestrado / Materiais e Processos de Fabricação / Mestre 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.

No description available.

O objetivo do presente estudo foi sintetizar e caracterizar arcabouços tridimensionais de vidros bioativos contendo nióbio e avaliar a influência destes materiais no comportamento de células pré-osteoblasticas in vitro. A produção dos arcabouços foi realizada pelo método sol-gel a partir da mistura de precursores da matriz e modificadores minerais. Foram produzidos vidros contendo Nióbio (BAG-Nb) e vidros sem adição deste componente (BAG). A adição do nióbio foi feita por meio de NbCl5. Após a formação do sol foram adicionados um surfactante e um catalisador da condensação para que fosse possível produzir um gel poroso que, com o processo de queima deu origem aos arcabouços. A caracterização da estrutura química foi realizada por difração de raio-x (DRX) e espectroscopia Raman. A morfologia dos materiais foi avaliada por microscopia de varredura (MEV) e microtomografia computadorizada de raios-x (MicroCT). Células pré-osteoblasticas MC3T3-E1 foram cultivadas para avaliação da influência dos materiais na sua proliferação, mineralização e expressão gênica. Para este fim foram utilizados os testes da Sulforonamida B, a coloração por Vermelho de Alizarina e o teste de Reação em Cadeia da Polimerase (PCR), respectivamente. A caracterização do material demonstrou a presença de estruturas cristalinas nos vidros produzidos. O Nióbio foi encontrado no grupo BAG-Nb em sua forma de óxido disperso pela matriz do vidro de acordo com os resultados de DRX e Raman. Quanto à sua estrutura, a porosidade superficial e macroposidade, o tamanho dos poros e a interconectividade entre os poros mostraram-se favoráveis para o crescimento de tecido em ambos os grupos produzidos. O nióbio foi encontrado na estrutura do vidro na sua forma de óxido (Nb2O5). A incorporação de Nb2O5 ao vidro não interferiu na proliferação celular no entanto, os materiais contendo Nb2O5 promoveram maior aumento na mineralização das células pré-osteoblasticas após 7 e 21 dias de cultivo, indicando maior diferenciação celular. Estes resultados demonstram que a incorporação de Nióbio resultou em materiais com composição química e macroestrutura adequadas, induzindo maior e mais rápida taxa de diferenciação celular na cultura de células in vitro. / The aim of this study was to synthesize and characterized sol-gel derived bioactive glasses scaffolds containing Niobium and evaluate its influence in pre-osteoblastic cell behaviour. Sol-gel route was used to produce porous scaffolds by foaming method. Matrix precursors and mineral modifiers were used to produce the sol. Scaffolds were produced in two distinct compositions. One group containing Niobium (BAG-Nb) and one group without this component (BAG) were produced. NbCl5 was used as Nb precursor. After sol mixture a surfactant and catalyst for condensation was added under stirring to produce a porous gel structure. Heating treatment was applied to produce porous scaffolds. Chemical characterization was performed with X-ray diffraction (XRD) and Raman spectroscopy. To evaluate morphology, Scanning Electron Microscopy (SEM) and Microcomputed thomography (μCT) were used. MC3T3-E1 pre-osteoblastic cells were used in cell culture analysis of cell proliferation, cell mineralization and gene expression. For these analyses, SulphoronamideB, Alizarin S Red and quantitative Polymerase Chain Reraction (qPCR) were used, respectively. Niobium was found scattered in glass matrix in its oxide form (Nb2O5) according to Raman and XRD results. These results showed Nb2O5 did not bond to glass matrix. Scaffolds superficial and macro porosity, pore size and connectivity were found favourable for growth of tissue. Cell proliferation was not influenced by the addition of Nb2O5, however scaffolds containing Nb2O5 induced increased mineralization after 7 and 21 days in preosteoblastic cell cultures. This result indicate increased cell differentiation for glasses containing Nb2O5. The development of bioactive glass scaffolds containing Niobium resulted in material with suitable chemical properties and microstructure with increased and faster mineralization in cellular studies showing potential of Nb2O5 containing bioactive glasses for tissue engineering applications.

Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is an effective approach to overcome the shortcomings of delivery of growth factors in protein form. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold carrying polyethylenimine (PEI)-plasmid DNA (pDNA) complexes. Two different pDNA were used: pDNA encoding platelet derived growth factor-B (PDGF-B) and pDNA encoding vascular endothelial growth factor (VEGF). The complexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and then characterized for size, surface charge, as well as in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the PEI-pPDGF-B complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system, using PEI-pPDGF-B and PEI-pVEGF complexes, was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. A different biomaterial, chitosan, loaded with copper was also evaluated in vivo. The complexes were ∼100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. High magnification scanning electron microscopy imaging demonstrated the recruitment and attachment of BMSCs into the collagen scaffold containing PEI-pPDGF-B complexes. Confocal microscopy revealed significant proliferation of BMSCs on PEI-pPDGF-B complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the PEI-pPDGF-B complex-activated collagen scaffolds following 4 weeks of implantation when compared to the other treatment groups. Together these findings suggest that non-viral PDGF-B gene-activated collagen scaffolds effectively promote bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Diploma thesis is focused on development of method for preparation individualized bioscaffolds prepared by milling of HA foam prepared by gel casting method and consolidated via epoxy curing reaction. Diploma thesis continues in bachelor thesis Modern processing methods of porous bioceramics. Literature search is filled in the newest knowledge from the field of bioactivity enhancement of scaffolds. Experimental part of diploma thesis described development of system for individualized scaffold manufacturing from HA suspension preparation, preparation of ceramic blocks used as material for machining to final blocks machining. Pressure strength of foam were evaluated, too. Chosen samples were coated with calcium polyphosphate coating for biological response enhancement. Coated samples were given for in-vivo test to partner university in Beijing under the terms of project FP7-NMP-2013-EU-China: "Bioscaffolds"

Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for applications in tissue engineering.

Conventionally, adherent cells are cultured in vitro using flat 2D cell culture trays. However the 2D cell culture method is tedious, unreliable and does not replicate the complexity of the 3D dynamic environment of native tissue. Nowadays 3D scaffolds can be used to culture cells. However a number of challenges still exist, including the need for destructive enzymes to release confluent cells. Poly(Nisopropylacrylamide) (PNIPAAm), a temperature responsive polymer, has revolutionised the cell culture fraternity by providing a non-invasive means of harvesting adherent cells, whereby confluent cells can be spontaneously released by simply cooling the cell culture medium and without requiring enzymes. While PNIPAAm monolayer cell culturing is a promising tool for engineering cell sheets, the current technology is largely limited to the use of flat 2D substrates, which lacks structural and organisational cues for cells. The aim of this project was to develop a 3D PNIPAAm scaffold which could be used efficiently for non-invasive 3D culture of adherent cells. This project was divided into three phases: Phase 1 (preliminary phase) involved development and characterisation of cross-linked PNIPAAm hydrogels; Phase 2 involved development and characterisation of PNIPAAm grafted 3D non-woven scaffolds, while Phase 3 focused on showing proof of concept for non-invasive temperature-induced cell culture from the 3D PNIPAAm grafted scaffolds. In Phase 1, PNIPAAm was cross-linked with N,N’-methylene-bis-acrylamide (MBA) using solution free-radical polymerisation to form P(PNIPAAm-co-MBA) hydrogels. A broad cross-link density (i.e. 1.1 - 9.1 Mol% MBA) was investigated, and the effect of using mixed solvents as the co-polymerisation medium. The P(PNIPAAm-co-MBA) gels proved unsuitable as a robust cell culture matrix, due to poor porosity, slow swelling/deswelling and poor mechanical properties. Subsequently, in Phase 2, polypropylene (PP), polyethylene terephthalate (PET), and nylon fibers were processed into highly porous non-woven fabric (NWF) scaffolds using a needle-punching technology. The NWF scaffolds were grafted with PNIPAAm using oxyfluorination-assisted graft polymerisation (OAGP). The OAGP method involved a 2 step process whereby the NWF was first fluorinated (direct fluorination or oxyfluorination) to introduce new functional groups on the fibre surface. The functionalised NWF scaffolds were then graft-polymerised with NIPAAm in an aqueous medium using ammonium persulphate as the initiator. Following oxyfluorination, new functional groups were detected on the surface of the NWF scaffolds, which included C-OH; C=O; CH2-CHF, and CHF-CHF. PP and nylon were both easily modified by oxyfluorination, while PET displayed very little changes to its surface groups. Improved wetting and swelling in water was observed for the oxyfluorinated polymers compared to pure NWF scaffolds. PP NWF showed the highest graft yield followed by nylon and then PET. PNIPAAm graft yield on the PP NWF was ~24 ±6 μg/cm2 on grafted pre-oxyfluorinated NWF when APS was used; which was found to be significantly higher compared to when pre-oxyfluorinated NWF was used without initiator (9 ±6 μg/cm2, p= 1.7x10-7); or when grafting was on pure PP with APS (2 ±0.3 μg/cm2, p = 8.4x10-12). This corresponded to an average PNIPAAm layer thickness of ~220 ±54 nm; 92 ± 60 nm; and 19 ± 3 nm respectively. Scanning electron microscopy (SEM) revealed a rough surface morphology and confinement of the PNIPAAm graft layer to the surface of the fibers when oxyfluorinated NWF scaffolds were used, however when pure NWF scaffolds were used during grafting, homopolymerisation was observed as a loosely bound layer on the NWF surface. The OAGP method did not affect the crystalline phase of bulk PP as was determined by X-ray diffraction (XRD), however, twin-melting thermal peaks were detected from DSC for the oxyfluorinated PP and PP-g-PNIPAAm NWF which possibly indicated crystal defects. Contact angle studies and microcalorimetric DSC showed that the PP-g-PNIPAAm NWF scaffolds exhibited thermoresponsive behaviour. Using the 2,2-Diphenyl-1-1-picrylhydrazyl (DPPH) radical method and electron-spin resonance (ESR), peroxides, as well as trapped long-lived peroxy radicals were identified on the surface of the oxyfluorinated PP NWF, which are believed to be instrumental in initiating graft polymerisation from the NWF. A free radical mechanism which is diffusion controlled was proposed for the OAGP method with initiation via peroxy radicals (RO•), as well as SO4•- and OH• radicals, whereby the latter result from decomposition of APS. In Phase 3 of this study, proof-of-concept is demonstrated for use of the PNIPAAm grafted NWF scaffolds in non-invasive culture of hepatocytes. Studies demonstrated that hepatocyte cells attached onto the 3D PNIPAAm scaffolds and remained viable in culture over long periods. The cells were released spontaneously and non-destructively as 3D multi-cellular constructs by simply cooling the cell culture medium from 37°C to 20°C, without requiring destructive enzymes. The PP-g- PNIPAAm NWF scaffolds performed the best in 3D cell culture. Additionally the CSIR is developing a thermo responsive 3D (T3D) cell culturing device, whereby the 3D thermo responsive NWF scaffolds are used in the bioreactor for cell culture. Temperature-induced cell release was also verified from the 3D Thermo responsive scaffolds in the bioreactor. This technology could lead to significant advances in improving the reliability of the in vitro cell culture model. Please cite as follows: Chetty, AS 2012, Thermoresponsive 3D scaffolds for non-invasive cell culture, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06112013-151344/ > D13/4/713/ag / Thesis (PhD)--University of Pretoria, 2012. / Chemical Engineering / unrestricted