Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

Implanted combination devices comprising both biological as well as biomaterial components may trigger non-specific inflammatory responses against the biomaterial component as well as specific immune responses against the biological component. This specific immune response may be enhanced by the biomaterial, thereby implying a biomaterial-mediated adjuvant effect, or in contrast may be mitigated by the biomaterial. Since adjuvants function by triggering dendritic cell (DC) maturation, biomaterials may regulate DC responses and hence facilitate DC-orchestrated host responses. This research work has focused on examining DC responses to different model self-assembled monolayer (SAM) biomaterial chemistries, as an in vitro readout of the potential of these biomaterials to trigger DC maturation. The underlying hypothesis was that DCs recognize and respond to biomaterials either indirectly through the adsorbed protein layer, specifically through carbohydrate modifications of these proteins, or through carbohydrates inherent in the biomaterial chemistry, using PRRs to initiate an immune response. Towards this goal, DCs were derived from human peripheral blood mononuclear cells (PBMCs) by culture with DC differentiation cytokines and the culture systems were characterized as being composed of DCs as well as associated T and B lymphocytes. Culture of DCs on different SAM chemistries implied differential DC responses in terms of morphology, maturation marker expression and allostimulatory capacities as well as distinct underlying mechanisms responsible for these responses. Enzyme-linked lectin (ELLA) assays were used to characterize the profiles of carbohydrates associated with serum/plasma proteins adsorbed to different SAM chemistries. Differential profiles of DC carbohydrate ligands of CLRs were present on different chemistries. Furthermore, the profiles of human serum/plasma proteins adsorbed to and eluted from different SAM chemistries were assessed using immunoblot analysis. Finally, to observe the roles of carbohydrates in supporting DC maturation in the presence of a biomaterial, DCs were cultured in the presence of partially de-glycosylated FBS from which DC carbohydrate ligands were selectively removed. This research is significant towards the ultimate development of optimal design criteria for biomaterials for use in diverse tissue-engineering or vaccine development applications for which a wide spectrum of adjuvant effects are required.

Background Surface treatments and irregularities in the surfaces may affect the fracture of ceramics. The effects of various treatments on the surface texture of different types of ceramic cores/substructures was therefore qualitatively, quantitatively and numerically evaluated. Since fractures in ceramics are not fully understood, the fracture behavior in dental ceramic core/substructures was also studied using both established laboratory methods and newly developed numerical methods. Methods The surfaces of dental ceramic cores/substructures were studied qualitatively by means of a fluorescence penetrant method and scanning electron microscopy, quantitatively evaluated using a profilometer and also numerical simulation. In order to study fracture in zirconia-based fixed partial denture (FPD) frameworks, fractographic analysis in combination with fracture tests and newly developed two-dimensional (2D) and three-dimensional (3D) numerical modeling methods were used. In the numerical modeling methods, the heterogeneity within the materials was described by means of the Weibull distribution law. The Mohr–Coulomb failure criterion with tensile strength cut-off was used to judge whether the material was in an elastic or failed state. Results Manual grinding/polishing could smooth the surfaces on some of the types of dental ceramic cores/substructures studied. Using the fluorescence penetrant method, no cracks/flaws apart from milling grooves could be seen on the surfaces of machined zirconia-based frameworks. Numerical simulations demonstrated that surface grooves affect the fracture of the ceramic bars and the deeper the groove, the sooner the bar fractured. In the laboratory tests the fracture mechanism in the FPD frameworks was identified as tensile failure and irregularities on the ceramic surfaces could act as fracture initiation sites. The numerical modeling codes allowed a better understanding of the fracture mechanism than the laboratory tests; the stress distribution and the fracture process could be reproduced using the mathematical methods of mechanics. Furthermore, a strong correlation was found between the numerical and the laboratory results. Conclusion Based on the findings in the current thesis, smooth surfaces in areas of concentrated tensile stress would be preferable regarding the survival of ceramic restorations, however, the surfaces of only some of the ceramic cores/substructures could be significantly affected by manual polishing. The newly developed 3D method clearly showed the stress distribution and the fracture process in ceramic FPD frameworks, step by step, and seems to be an appropriate tool for use in the prediction of the fracture process in ceramic FPD frameworks.

A limitation to engineering viable thick tissues (greater than a few hundred microns in thickness) has been the lack of vascularisation and a vascular supply. A key element in engineering such tissues is the generation of a supporting scaffold with a defined and wellcharacterized architecture. To date relatively little attention has been paid to characterization. The objective of this research was to develop well-characterized structures which will inform the rational design of the next generation of engineered thick tissues. Specifically, this research aimed to test combinations of various culturing environments, cell mono- and co-cultures, and scaffold architectures; develop improved imaging techniques and structural/spatial analytical methods to characterise porous polymer scaffolds; and use various spatial and morphological measures to quantify the relationships between scaffold geometric structure and cell distribution. Isotropic and anisotropic pore scaffolds were manufactured and then processed with nondestructive and destructive imaging methods, and characterised using image analysis methods to measure geometric parameters such as the degree of anisotropy/isotropy, porosity, and fractal parameters of pore and strut networks. Cells were introduced into scaffolds using a range of seeding methods and cultured in static and hydrodynamic environments. Quantification of the spatial cell distribution in cell-seeded scaffolds was done with first-order spatial statistics and fractal analysis. Findings comparing various destructive and non-destructive imaging methods found that cryotape cryohistology was the most accurate method for processing bare polymer scaffolds and eliminated histological artefacts common to other techniques. It was found with the various image analysis methods, surface and internal scaffold geometric architectures were strongly isotropic for porogen-fused porogen-leached scaffolds and anisotropic for TIPS scaffolds. For both isotropic and anisotropic pore scaffolds, collagen hydrogel infusion and droplet methods gave the highest cell seeding efficiencies (at 100% efficiency). The key finding in this study was that first-order spatial statistics and fractal analysis of cell distribution revealed that the geometric structure of the scaffolds had the strongest effect on spatial cell infiltration and distribution compared to the influence of culture environment or mono- and co-culture. Isotropic pore scaffolds had a higher level of cell distribution. Further work with optimizing the growth environment parameters, and utilizing collagen-infused cell-seeded scaffolds, may assist in achieving better cell growth. The work presented therefore provides the analytical basis for the rational design of tissue engineering scaffolds.

This thesis focuses on surface science and bioengineering investigations, first for the development of an improved biomaterial for orthopaedic implant applications, and second, for the development of a biosensor device for biomedical diagnostics. A key component considered in this thesis was the covalent linkage of proteins to the material’s surface for retaining the protein’s immunological and biological activities and for generating a functional interface. Part 1 of this thesis investigated surface modification procedures for improving the bioactivity of titanium substrates. Titanium is first coated with a bioactive silica film grown by plasma enhanced chemical vapour deposition (PECVD), referred to as PECVD-Si-Ti. In previous studies, the bone-implant integration process was enhanced 1.6-fold for titanium implants coated with PECVD-Si films compared to uncoated titanium implants in vivo. However, in vitro studies carried out in this thesis showed that the growth of MG63 osteoblast-like cells was 7-fold higher on uncoated titanium compared to PECVD-Si coated titanium. Therefore, to improve cell growth on the surface and, by inference, the integration of PECVD-Si-Ti implants into bone tissue, the implant’s surface was functionalised with a mitogenic factor, insulin-like growth factor-1 (IGF-1). This was accomplished by modifying the PECVD-Si-Ti surface with an alkoxysilane, 3-isocyanatopropyl triethoxysilane (IPTES), and then by covalent bioconjugation of IGF-1 through isocyanate-amino chemistry. After 72 h of in vitro cell culture in serum-free medium, the growth of MG63 cells was enhanced 1.9-fold on IPTES functionalised PECVD-Si-Ti, which was loaded with covalently immobilised IGF-1 compared to IPTES functionalised PECVD-Si-Ti without IGF-1 (isocyanate reactive groups were quenched with ethanolamine hydrochloride). The attachment and adhesion of MG63 cells were also enhanced on PECVD-Si-Ti by the covalently immobilised IGF-1 in serum-free cell culture conditions. Therefore, the bioactivity of PECVD-Si-Ti was improved by covalently linking IGF-1 to the substrate surface through isocyanate-amino chemistry. Part 2 of this thesis involved the development of a new optical interferometric biosensor. The biosensor platform was constructed from electrochemically-prepared thin films of porous silicon that acted as a sensing matrix and transducer element. By reflective interferometry using white light, an enzyme-catalysed reaction was discovered (horseradish peroxidase (HRP) mediated oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB)), which led to an acceleration in the rate of porous silicon corrosion and represented the biosensor’s readout signal. We discovered that another substrate, which is also oxidised by HRP, OPD, produces an even more pronounced readout signal. The HRP-OPD system was used in an immunoassay for detecting human IgG from an Intragam solution. An important part in the design of the biosensor was the surface functionalisation approach where anti-human IgG, referred to as the capture antibody, is immobilised on the porous silicon surface. The readout signal (produced from the capture of human IgG) was enhanced 4-fold on the porous silicon biosensing platform functionalised with covalently linked anti-human IgG through isocyanate-amino chemistry compared to the porous silicon biosensing platform functionalised with adsorbed anti-human IgG. The optimised biosensor was used to detect IgG from a total human protein concentration of Intragam to a sensitivity of 100 ng/ml. In summary, isocyanate-amino bioconjugate chemistry was used to covalently link either IGF-1 to PECVD-Si-Ti for improving the biological activity of the orthopaedic implant and to covalently link IgG to porous silicon for developing a sensitive biosensor for the detection of proteins. This surface chemistry approach is very useful for biomaterial and biosensing applications.

A limitation to engineering viable thick tissues (greater than a few hundred microns in thickness) has been the lack of vascularisation and a vascular supply. A key element in engineering such tissues is the generation of a supporting scaffold with a defined and wellcharacterized architecture. To date relatively little attention has been paid to characterization. The objective of this research was to develop well-characterized structures which will inform the rational design of the next generation of engineered thick tissues. Specifically, this research aimed to test combinations of various culturing environments, cell mono- and co-cultures, and scaffold architectures; develop improved imaging techniques and structural/spatial analytical methods to characterise porous polymer scaffolds; and use various spatial and morphological measures to quantify the relationships between scaffold geometric structure and cell distribution. Isotropic and anisotropic pore scaffolds were manufactured and then processed with nondestructive and destructive imaging methods, and characterised using image analysis methods to measure geometric parameters such as the degree of anisotropy/isotropy, porosity, and fractal parameters of pore and strut networks. Cells were introduced into scaffolds using a range of seeding methods and cultured in static and hydrodynamic environments. Quantification of the spatial cell distribution in cell-seeded scaffolds was done with first-order spatial statistics and fractal analysis. Findings comparing various destructive and non-destructive imaging methods found that cryotape cryohistology was the most accurate method for processing bare polymer scaffolds and eliminated histological artefacts common to other techniques. It was found with the various image analysis methods, surface and internal scaffold geometric architectures were strongly isotropic for porogen-fused porogen-leached scaffolds and anisotropic for TIPS scaffolds. For both isotropic and anisotropic pore scaffolds, collagen hydrogel infusion and droplet methods gave the highest cell seeding efficiencies (at 100% efficiency). The key finding in this study was that first-order spatial statistics and fractal analysis of cell distribution revealed that the geometric structure of the scaffolds had the strongest effect on spatial cell infiltration and distribution compared to the influence of culture environment or mono- and co-culture. Isotropic pore scaffolds had a higher level of cell distribution. Further work with optimizing the growth environment parameters, and utilizing collagen-infused cell-seeded scaffolds, may assist in achieving better cell growth. The work presented therefore provides the analytical basis for the rational design of tissue engineering scaffolds.

Regensburg, Univ., Diss., 2003. / Erscheinungsjahr an der Haupttitelstelle: 2003.

O titânio comercialmente puro (Ti-Cp) e suas ligas destacam-se como biomateriais metálicos devido a sua biocompatibilidade sendo amplamente utilizados. Buscando aprimorar o sucesso clínico dos implantes de Ti-Cp em longo prazo, é necessário revestir a sua superfície, proporcionando uma eficiente ancoragem mecânica do implante com o tecido ósseo. Dentre os diversos revestimentos superficiais destacam-se os revestimentos híbridos orgânicos-inorgânicos à base de precursores alcóxidos de silício, obtidos via processo sol-gel. Para que ocorra uma satisfatória adesão do revestimento no substrato, precisa-se levar em consideração a natureza e a preparação da superfície metálica antes da aplicação deste revestimento. Na etapa inicial do trabalho, pré-tratamentos superficiais foram propostos previamente à aplicação dos revestimentos buscando aumentar a aderência entre o titânio e este revestimento híbrido. Utilizou-se três diferentes pré-tratamentos na superfície do Ti-Cp: o "piranha" (ácido sulfúrico e peróxido de hidrogênio), o "kroll" (ácido fluorídrico, ácido nítrico e água) e o "hidróxido de sódio". Em sequência, aplicou-se por processo de dip-coating sobre as superfícies tratadas, um revestimento híbrido produzido a partir dos precursores alcóxidos de silício tetraetoxisilano (TEOS) e metiltrietoxisilano (MTES), obtido pelo processo de sol-gel. Em uma segunda etapa, sobre a superfície do Ti-Cp com o pré-tratamento superficial que proporcionou uma maior aderência do revestimento híbrido ao titânio, aplicou-se por dip-coating, um revestimento híbrido à base de precursores alcóxidos de silício TEOS e MTES com adição de partículas de hidroxiapatita buscando aprimorar a diferenciação celular sobre o revestimento híbrido. O pré-tratamento com o hidróxido de sódio promove o melhor resultado dentre os pré-tratamentos, pois o revestimento híbrido aplicado posteriormente apresenta recobrimento regular e adesão ao substrato de Ti-Cp. Os resultados morfológicos por MEV-FEG mostraram um revestimento híbrido com boa dispersão da hidroxiapatita e um recobrimento regular e adesão ao substrato de Ti-Cp. Nos resultados biológicos Ti-Cp revestido com TEOS/MTES com a presença de partículas de hidroxiapatita obteve uma adesão celular semelhante ao Ti-Cp sem tratamento. Porém este mesmo revestimento não propiciou a proliferação e diferenciação celular. Os resultados indicaram que a combinação de fatores como a sua superfície hidrofóbica (91°) e a presença da hidroxiapatita no revestimento tornou a superfície desorganizada, acarretando em uma superfície com comportamento desfavorável para o desenvolvimento das células-tronco mesenquimais. / Commercially pure titanium (cp-Ti) and its alloys stand out among the metallic materials due to their biocompatibility being widely used in biomaterials. In order to improve the clinical success of cp-Ti implants in the long term, it is necessary to coat the surface, providing an efficient mechanical anchoring of the implant with the bone tissue. Among the various surface coatings are the hybrid coatings based on silicon alkoxide precursors, obtained by the sol-gel process, however, considering the nature and the preparation of the metal surface prior to the application of this coating. In the initial stage of the work, surface pre-treatments were proposed prior to the application of the coatings seeking to increase the adhesion between the titanium and this hybrid coating. Three different pretreatments were used on the cp-Ti surface: "piranha" (sulfuric acid and hydrogen peroxide), "kroll "(hydrofluoric acid, nitric acid and water) and "sodium hydroxide". Subsequently, a hybrid coating produced by the tetraethoxysilane silicon (TEOS) and methyltriethoxysilane (MTES) precursors obtained by the sol-gel process was applied by the dip-coating process onto the treated surfaces. In a second step, on the surface of the cp-Ti with the surface pretreatment that gave a greater adhesion of the hybrid coating to the titanium, dip-coating, a hybrid coating based on precursors silicon alkoxides TEOS and MTES with the addition of hydroxyapatite particles to enhance cell differentiation on the hybrid coating. Pretreatment with sodium hydroxide promotes the best result among the pre-treatments, since the hybrid coating applied afterwards presents regular coating and adhesion to the cp-Ti substrate. The morphological results by SEM-FEG showed a hybrid coating with good dispersion of the hydroxyapatite and a regular coating and adhesion to the cp-Ti substrate. In the biological results Ti-Cp coated with TEOS / MTES with the presence of hydroxyapatite particles obtained a cell adhesion similar to Ti-Cp without treatment. However, this same coating did not promote cell proliferation and differentiation. The results indicated that the combination of factors such as its hydrophobic surface (91°) and the presence of the hydroxyapatite encapsulated in the coating rendering the surface disorganized led to a surface with unfavorable behavior for the development of mesenchymal stem cells.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-11-13T17:34:42Z No. of bitstreams: 1 Tese_ODONTO_Luiz Guilherme Pinheiro Soares.pdf: 3345860 bytes, checksum: 283f739a64a95083e8cecd801d43636c (MD5) / Approved for entry into archive by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2013-11-19T12:20:34Z (GMT) No. of bitstreams: 1 Tese_ODONTO_Luiz Guilherme Pinheiro Soares.pdf: 3345860 bytes, checksum: 283f739a64a95083e8cecd801d43636c (MD5) / Made available in DSpace on 2013-11-19T12:20:34Z (GMT). No. of bitstreams: 1 Tese_ODONTO_Luiz Guilherme Pinheiro Soares.pdf: 3345860 bytes, checksum: 283f739a64a95083e8cecd801d43636c (MD5) / CNPq / Ao lado dos biomateriais, as fototerapias laser e LED têm obtido resultados positivos como terapias auxiliares ao processo de reparação óssea, principalmente quando envolvem grandes perdas teciduais. O objetivo deste estudo foi avaliar; por meio de análise histológica através de microscopia de luz e espectroscopia Raman, a influência da fototerapia, laser ou LED, no processo de reparo de defeitos ósseos em fêmur de ratos, com ou sem implante de hidroxiapatita. Sessenta ratos Wistar albinus foram divididos em seis grupos, cada um deles subdividido em dois subgrupos de acordo com o período de sacrifício (15 e 30 dias). Sob anestesia geral, um defeito ósseo crítico com 2mm2 foi criado no fêmur esquerdo de cada animal. No grupo Coágulo, o defeito foi preenchido apenas por coágulo sanguíneo, no grupo Biomaterial o defeito foi preenchido com o implante de HA + ß-TCP, no grupo LED o defeito foi preenchido por coágulo sanguíneo e irradiado com LED (λ = 850 ± 10 nm, P = 150 mW, CW, 20,4 J/cm2 por sessão, 142,8 J/cm2 por tratamento), no grupo LED + Biomaterial, o defeito foi preenchido com implante de HA + ß-TCP e irradiado com LED, no grupo Laser, o defeito foi preenchido por coágulo sanguíneo e irradiado com laser (λ = 780 nm, P = 70 mW, CW, 20,4 J/cm2 por sessão, divididos em 4 pontos NSLO de 5,1 j/cm2, 142,8 J/cm2 por tratamento), no grupo Laser + biomaterial, o defeito foi preenchido com implante de HA + ß-TCP e irradiado com laser. Os protocolos de irradiação foram realizados a cada 48 horas durante 15 dias. A morte dos animais ocorreu após 15 e 30 dias. As amostras foram divididas em duas metades, uma foi analisada por espectroscopia Raman, para avaliar o grau de mineralização óssea através das intensidades dos picos de Raman do conteúdo inorgânico (~960, ~1070 cm-1) e orgânico (~1454 cm-1) do tecido ósseo. A outra metade foi processada e avaliada qualitativamente através de microscopia de luz. Histologicamente, a presença das linhas basofílicas, indicou que o grupo Laser + Biomaterial encontrava-se em estágio mais avançado de reparo. Espectroscopicamente, as fototerapias, laser e LED, aumentaram a deposição de HA e, consequentemente, a mineralização do osso neoformado. Concluiu-se que a fotobiomodulação Laser ou LED foram eficazes na melhora no processo de reparo ósseo de defeitos ósseos confeccionados em fêmur de ratos, submetidos ou não, a implante de fosfocerâmica bifásica de hidroxiapatita e -Fosfato tricálcico.