Bone has the capacity to regenerate itself, however for challenging defects such as non-uniform factures, repair can be problematic. A similar challenge is presented in the repair of osteoporotic bone. Osteoporotic bone becomes increasingly porous and brittle and the risk of fracture is greatly increased. A drug mimic, poly(vinyl phosphonic acid – co – acrylic acid)(PVPA), has been incorporated into FDA approved poly(ε-caprolactone)(PCL), and aims to mimic the action of bisphosphonates to reduce the activity of osteoclasts. The PVPA polymer contains pendant phosphonic acid groups which are hypothesised to mimic the P-C-P backbone found in bisphosphonates. The PCL/PVPA scaffold has been found to have sufficient mechanical strength in order to be used as a bone void filler as well as providing a hydrophilic surface for superior cell attachment. The substrate has been found to significantly enhance the deposition of collagen, alkaline phosphatase activity and the expression of osteocalcin. Alizarin red staining revealed an increase in the rate of mineralisation in the presence of the drug mimic. The PCL/PVPA substrates have been suggested to induce osteoblast cells from a proliferative phase to a mineralisation stage. This is believed to be due to the presence of phosphorous within the scaffold which could lead to the critical concentration required for the initiation of mineralisation being reached more rapidly and effectively. The PVPA polymer has been found to mimic the action of bisphosphonates by inducing osteoclast apoptosis in vitro, and its actions of osteoclast apoptosis are comparable to that of Alendronate, a commonly administered bisphosphonate. A critical size defect model has demonstrated that the PVPA polymer has the ability to heal critical size defects; the healing potential was two fold greater than the control PCL substrate. Initial in vivo studies using a subcutaneous model demonstrated an improvement in mineralisation in the presence of PVPA. Untreated PCL/PVPA substrates displayed a high level of branched blood vessel formation, essential for healthy bone formation. However PVPA samples pre-treated with VEGF, hindered blood vessel formation and the infiltration of cells. This suggests that the PVPA alone is capable of inducing neovascularisation without the addition of VEGF. The findings suggest that the PCL/PVPA system could be used to treat challenging bone defects such as non-unions and osteoporotic fractures.

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

L’objectif de cette thèse était de développer de nouveaux biomatériaux nanofibreux architecturés (2D ou 3D) grâce à la méthode d’électrospinning puis d’étudier l’influence de ces structures nanofibreuses sur le comportement des cellules osseuses. L’électrospinning est une technique qui permet d’obtenir des nanofibres en projetant sous l’action d’un champ électrique intense une solution de polymère sur un collecteur. Les nanofibres sont alors généralement disposées aléatoirement sous forme de mats (ou scaffolds). Ces scaffolds trouvent des applications en ingénierie tissulaire grâce à leur structure mimant la matrice extracellulaire des tissus vivants. Toutefois, il a été montré que lorsque le collecteur est micro-structuré, il est alors possible de contrôler l’organisation des fibres lors de leur dépôt grâce à la perturbation locale du champ électrique au voisinage de la surface du collecteur. Ces collecteurs architecturés jouent ainsi le rôle de « templates » électrostatiques. Dans un premier temps, nous avons développé des scaffolds 2D nanofibreux monocomposants en forme de nids d’abeilles grâce à l’utilisation d’un collecteur micro-structuré en nids d’abeilles lors du procédé d’électrospinning. Ces scaffolds ont été développés à partir de deux biopolyesters le poly(ε-caprolactone) (PCL) ou le poly(lactic acid) (PLA). Nous avons prouvé que la morphologie des nanofibres de PCL (distribution bimodale du diamètre des fibres) conduisait à un scaffold présentant un relief beaucoup plus marqué alors qu’avec les fibres de PLA, qui présentent une distribution monomodale du diamètre des fibres, les scaffolds obtenus sont beaucoup plus plats. Nous avons montré qu’il est possible de contrôler l’organisation spatiale de cellules osseuses de type MG-63, des ostéoblastes, en jouant sur le relief et l’architecture du scaffold. Puis, nous avons démontré qu’en couplant la micro-structuration des nanofibres de PCL (par l’utilisation d’un collecteur en nid d’abeilles lors du procédé d’électrospinning) avec les propriétés d’auto-assemblage du PCL, nous pouvions élaborer de nouveaux scaffolds nanofibreux 3D ayant la particularité de présenter des pores de tailles contrôlées ainsi qu’un gradient de porosité dans l’épaisseur du scaffold. Puis nous nous sommes intéressés à l’élaboration de membranes composites micro-structurées 2D et 3D. En couplant le procédé d’électrospinning avec le procédé d’électrospraying sur des collecteur micro-structurés, nous avons démontré que nous pouvions déposer de manière contrôlée les particules spécialement sur les murs des nids d’abeilles grâce notamment à la présence d’une très fine couche de fibres électrospinnées au préalable sur le collecteur. Cette fine couche de nanofibres joue le rôle de « template électrostatique » pour le dépôt des particules. Nous avons ensuite appliqué cette technique pour développer des membranes composites nanofibreuses bicouches à base de nanofibres de PCL et de microparticules d’hydroxyapatite (HA). Ces membranes composées de 21 microarchitectures différentes (barres, plots, hexagones, labyrinthe) ont ensuite été intégrées dans des mini plaques de culture cellulaire, formant ainsi un nouveau type de biopuce, appelés biochips, qui permettent pour le screening des microarchitectures nanofibreuses. Enfin, en combinant simultanément l’électrospinning de nanofibres et l’électrospraying de particules sur des collecteur micro-structurés en nid d’abeilles, des scaffolds composites 3D présentant des pores cylindriques de tailles contrôlées ont été élaborés. / The aim of this thesis was to develop new architectured nanofibrous biomaterials (2D or 3D) using the electrospinning method and to study the influence of these nanofibrous structures on bone cells behaviors. Electrospinning is a technique allowing the production of nanofibers by projecting, under the action of a strong electric field, a polymer solution on a collector. The nanofibers are generally randomly deposited and form mats or scaffolds. These scaffolds are interesting for tissue engineering applications because of their structure mimicking the extracellular matrix of living tissues. However, it has been shown that when the collector is microstructured, it is possible to control the organization of the fibers during their deposition through the local perturbation of the electric field at the vicinity of the surface of the collector. These micropatterned collectors act as "electrostatic templates". First, 2D honeycomb nanofibrous scaffolds were elaborated using micropatterned honeycomb collectors during the electrospinning process. These scaffolds were made either with poly(ε-caprolactone) (PCL) or poly(lactic acid) (PLA). We showed that the morphology of the PCL nanofibers (bimodal distribution of the fiber diameter) led to a scaffold with a strong relief. Despite, with PLA fibers which presented a monomodal distribution of the fiber diameter, the obtained scaffolds were much flatter. It was possible to control the spatial organization of bone-like cells MG-63 (osteoblasts), playing on the relief and the architecture of the scaffold. Subsequently, 3D materials were elaborated using micropatterned collectors in order to open new paths for the development of filling materials for bone regeneration. Microstructuration of PCL nanofibers (by the use of micropatterned honeycomb collector during the electrospinning process) coupled with the self-assembling properties of the PCL lead to the development of new 3D nanofibrous scaffolds, with controlled pore size and porosity gradient in the thickness of the scaffold. Afterwards, micropatterned composite 2D and 3D membranes were elaborated. By coupling the process of electrospinning with the process of electrospraying on micropatterned collector, we demonstrated that we can deposit the particles in a controlled way, especially on the walls of honeycomb patterns thanks to the presence of a thin fiber layer first deposited on the collector. This thin nanofiber layer plays the role of an "electrostatic template" for the particles deposition. Thereafter, this technique was applied to develop bilayers composite nanofibrous membranes containing PCL nanofibers and hydroxyapatite (HA) microparticles. These membranes consisted of 21 different microarchitectures (bars, blocks, hexagons, maze) were then incorporated into a small cell culture plate, thereby forming a new type of biochip for the screening of nanofibrous architectures. Indeed, these biochips allowed the screening of nanofibrous microarchitectures to identify the most relevant for bone regeneration. It turned out that the HA hexagonal structures (with an average diameter of 300 microns) and circular HA structures (with an average diameter of 150 microns) are the structures that enhance the most the mineralization process of bone cells. Finally, by combining simultaneously electrospinning nanofibers and electrospraying particles on micropatterned honeycomb collector, 3D composite scaffolds were elaborated. It was possible to control the size of cylindrical pores of these 3D composite from tens to hundreds of microns by changing the size of the honeycomb patterns of the collector.

Les objectifs de cette thèse concernent la caractérisation des polymères à l’état fondu via la rhéologie non linéaire dans les modes de cisaillement ou en élongationnel et les procédés faisant intervenir de fortes élongations tel que l’électrospinning en voie solvant et en voie fondue. Pour atteindre le premier objectif, nous nous sommes concentrés sur la caractérisation des polymères fondus enchevêtrés dans les régimes viscoélastiques linéaires et non linéaires. L'influence de la masse moléculaire, Mw et de sa distribution MWD, la présence de longues chaînes branchées (LCB) ou encore l'addition de nanoparticules dans la matrice de polymère à l'état fondu ont été étudiées en utilisant des techniques rhéologiques en cisaillement et en élongationnel. Dans le cas des écoulements de cisaillement oscillatoires à grandes amplitudes (LAOS), nous avons proposé de nouveaux paramètres mécaniques qui ont permis de définir les relations structure-propriétés des différents systèmes étudiés. / The goals of this thesis are the characterization of polymer melts using mainly non-linear shear and extensional rheological techniques. The fabrication of scaffolds with excellent physical and mechanical properties using solution electrospinning technology for tissue engineering applications and the development of melt electrospinning equipment to facilitate the fabrication of solvent free scaffolds. To achieve the first goal, we focused on the characterization of entangled polymer melts in the linear and nonlinear viscoelastic regimes. The influence of molecular weight, Mw, molecular weight distribution (MWD), long-chain branching (LCB) and addition of particles to the polymer matrix on polymer melt properties were investigated using shear and extensional rheological techniques. The resulting structure-property relationships were established using newly introduced mechanical parameters under large amplitude oscillatory shear (LAOS) flow.

The goals of this thesis are the characterization of polymer melts using mainly non-linear shear and extensional rheological techniques. The fabrication of scaffolds with excellent physical and mechanical properties using solution electrospinning technology for tissue engineering applications and the development of melt electrospinning equipment to facilitate the fabrication of solvent free scaffolds. To achieve the first goal, we focused on the characterization of entangled polymer melts in the linear and nonlinear viscoelastic regimes. The influence of molecular weight, Mw, molecular weight distribution (MWD), long-chain branching (LCB) and addition of particles to the polymer matrix on polymer melt properties were investigated using shear and extensional rheological techniques. The resulting structure-property relationships were established using newly introduced mechanical parameters under large amplitude oscillatory shear (LAOS) flow.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Materials based on polyhydroxybutyrate (PHB), chitosan (CHI) and fibroin (SF) are biocompatible and attractive for applications in bone tissue engineering. In this work, 3D scaffolds of PHB/CHI and PHB/CHI/SF in different proportions was prepared, characterized and evaluated the in vitro behavior: group I: PHB/CHI (50:50 wt.%), group II: PHB/CHI/SF (50:45:5 wt.%) and group III: PHB/CHI/SF (50:35:15 wt.%). The scaffolds were produced by the lyophilization method of the components mixtures. The physical-chemical characterization of the scaffolds was performed by X-ray diffraction, infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. A highly porous nature was revealed by SEM analysis of the scaffolds. The FTIR analysis revealed that the constituents used in the preparation of the scaffolds interacted chemically with each other. Thermal analysis showed that fibroin increases the thermal stability of the scaffolds. The viability and cell proliferation were assessed by the MTT method and the cytotoxicity results showed that all scaffolds are non- cytotoxic. In addition, the scaffolds stimulated cell proliferation and are promising for tissue engineering applications. / Materiais à base de polihidroxibutirato (PHB), quitosana (QUI) e fibroína (SF) são biocompatíveis e atrativos para aplicações na engenharia tecidual óssea. Neste trabalho foi preparado, caracterizado e avaliado o comportamento in vitro de arcabouços 3D de PHB/QUI/SF em diferentes proporções: grupo I: PHB/QUI (50:50 % em massa), grupo II-PHB/QUI/SF (50:45:5 % em massa) e grupo III- PHB/QUI/SF (50: 35:15 % em massa). Os arcabouços foram produzidos pelo método da liofilização das misturas dos componentes dos três grupos. A caracterização físico-química dos arcabouços foi realizada por difração de raios X, espectroscopia no infravermelho, microscopia eletrônica de varredura e análise termogravimétrica. A análise de MEV mostrou que os arcabouços 3D apresentam uma boa porosidade. Por FTIR observou- se que os componentes utilizados na preparação dos arcabouços interagiram quimicamente entre si. As análises térmicas indicam que a fibroína aumenta a estabilidade térmica dos arcabouços. A viabilidade e a proliferação celular foram avaliadas pelo método do MTT e os resultados de citotoxicidade mostraram que ambos os arcabouços não são citotóxicos. Além disso, os arcabouços estimularam a proliferação celular, sendo promissores para aplicações em engenharia tecidual.

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