This body of research investigated the bonding efficiency of self-etching primer adhesives to enamel and dentine with various surface characteristics. A series of preliminary experiments was carried out to determine the effect of operator experience, dentine tubule orientation, bond strength test method and resin composite material used on bond strengths. The results of the preliminary tests concluded that it is essential to develop skills in material handling and the test methods used; 2-step self-etching primer adhesives exhibit higher but more variable microshear bond strengths (µSBS) than ‘all-in-one’ adhesives on dentine at different depths and tubule orientations; fracture toughness and bond strength test results suggest that the fracture toughness of a resin composite may not be of significant influence on microtensile and µSBS tests outcomes for nano-filled hybrid materials. / An investigation of the bonding ability of self-etching primer adhesives under various tooth preparation conditions was carried out. Enamel and dentine specimens were prepared from human teeth and finished with various rotary cutting instruments and the erbium, chromium:yttrium, scandium, gallium, garnet laser. Specimens were bonded with two 2-step self-etching primer adhesives and two ‘all-in-one’ adhesives with a resin composite. The results showed that one of the 2-step adhesives exhibited higher but more variable µSBS than the ‘all-in-one’ adhesives and a silorane-based self-etching primer adhesive system to enamel and dentine. / The relationship between enamel microhardness and µSBS was evaluated. Enamel specimens were prepared and finished with one half of the surface tested for hardness using the Vickers test. The other half of the enamel surface was bonded using either a 2-step self-etching primer adhesive or an ‘all-in-one’ adhesive and a hybrid resin composite. Mean Vickers hardness numbers and µSBS for each enamel surface were calculated. Analysis using Pearson’s parametric test for regression analysis evaluated the correlation between Vickers hardness and µSBS. The results revealed a weak negative insignificant correlation between VHN and µSBS for the 2-step adhesive and no correlation for the ‘all-in-one’ adhesive. / The effect of conditioning and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on bonding to bleached and unbleached enamel was investigated. Four groups of enamel specimens: untreated control; bleaching with 16% carbamide peroxide gel for 90 min daily x 14 days; treated with CPP-ACP paste (Tooth Mousse, GC Corp., Japan) for 60 min daily x 7 days and bleached and CPP-ACP-treated were used. The specimens were divided into a further two groups and bonded with a total-etch adhesive or a 2-step self-etching primer adhesive. Specimens bonded with the self-etching primer adhesive were sub-divided into four conditioning subgroups before bonding: no conditioning; 30 – 40% phosphoric; 15% EDTA; 20% polyacrylic acid. Specimens were tested in shear mode until failure and analysed by 2-way ANOVA, one-way ANOVA and Tukey’s post-hoc test. The µSBS of the total-etch adhesive was not affected by enamel treatment. Bleaching reduced the µSBS of the self-etching primer adhesive but preconditioning with phosphoric acid and polyacrylic acid improved bond strengths after CPP-ACP application. Bond failure analysis revealed a predominance of adhesive failures after bleaching, but prior conditioning reduced the proportion of adhesive failures. Scanning electron microscopy (SEM) revealed that the interfacial morphology produced by the 2-step self-etching primer adhesive was independent of enamel treatment except after bleaching. Phosphoric acid etching was not inhibited by CPP-ACP treatment. Resin tag formation was observed with prior phosphoric acid and polyacrylic acid conditioning. / The effect of conditioning and CPP-ACP application on dentine bonding was also investigated. Dentine specimens with and without the smear layer were prepared and divided into a further two groups, CPP-ACP paste applied to one group for 60 min daily x 7 days and the other group was untreated. The two groups were divided into three subgroups for conditioning: no conditioning; 30 - 40% phosphoric acid; 20% polyacrylic acid. The dentine was bonded using a 2-step self-etching primer adhesive and an ‘all-in-one’ adhesive, and tested as previously described. Statistical analysis was carried out using one-way ANOVA and Tukey’s post-hoc test at α = 0.05. The results showed that the µSBS of both adhesives were not significantly affected on smear-covered dentine but was affected on smearless dentine. Conditioning did not improve bond strengths. Bond failure analysis showed more adhesive failures for the ‘all-in-one’ adhesive, particularly on smearless dentine and with prior polyacrylic acid conditioning. SEM revealed a similar morphology of the bonded interface for the 2-step self-etching primer adhesive regardless of conditioning; and areas of bond failures for the ‘all-in-one’ adhesive. / The 2-step self-etching primer adhesives exhibited higher bond strength and more regular bond integrity than the ‘all-in-one’ adhesives, as shown on the SEM observations. However, the ‘all-in-one’ adhesives exhibited less variability in bond strengths to tooth surface characteristics.

Bakgrund:Oral mukosit (OM) är en toxisk biverkan efter högdos cytostatikabehandling (HDC) och hematopoietisk stemcellstransplantation (HSCT). OM orsakar kliniska komplikationer samt negativa följder för patienten, som längre sjukhusvistelse, oral smärta, viktförlust och parenteral nutrition (PN).Syfte:Att undersöka om det föreligger skillnad i viktförändring och nutritionsstatus hos patienter som använder munsköljmedlet, Caphosol ® i tillägg till standardbehandling i jämförelse med standardbehandling vid behandling med HDC och HSCT.Metod:En randomiserad kontrollerad öppen studie där patienter > 16 år (n=40), behandlades med HDC, inför HSCT på Akademiska universitetssjukhuset, Uppsala. Patienterna randomiserades, 1:1, till oral standardbehandling och munsköljmedlet Caphosol® (EXP n=20) eller oral standardbehandling (KTR n=20). OM, oral smärta, viktförlust och dagar av PN registrerades och analyserades från baseline till 21 dagar efter avslutad HDC.Resultat:Caphosol ® hade ingen signifikant betydelse för viktförändringar mellan EXP- och KTR-grupperna. OM-smärta debuterade senare i EXP än i KTR-gruppen. KTR gruppen använde mer PN jämfört med EXP-gruppen.Konklusion:Caphosol ® hade obetydlig inverkan på förekomst, duration och svårighetsgrad av OM under HCT vid HSCT och därmed liten effekt på nutrition och vikt. Det förelåg ingen fördel att addera Caphosol ® till oral standardbehandling. / Background:Oral mucositis (OM) is a result of cytotoxic effects of high dose chemotherapy (HDCT) administered before hematopoietic stem cell transplantation (HSCT). It is a source of negative consequences for the patient, such as longer hospitalization, oral pain, weight loss, and use of parenteral nutrition (PN).Objective:To investigate whether there is differences in weight changes and nutritional status in patients receiving mouth rinse, Caphosol®, in addition to standard oral care (OC) compared to standard OC for HDCT and HSCT.Method:A randomized, controlled open study with patients > 16 years, treated with HDCT before HSCT at Akademiska University Hospital, Uppsala, Sweden. Patients randomized 1:1 to standard OC and Caphosol® (EXP, n=20) or standard OC (CTR n = 20). Oral pain, weight loss and days of PN was recorded and analysed from baseline to day 21 post HDCT.Result:Caphosol ® had no significant impact on weight changes between EXP and CTR groups. OM-pain peaked later in the EXP group than in CTR. No significance in weight change between settings. CTR group had higher use of PN compared to EXP.Conclusion:Caphosol® had little effect on frequency, duration and severity of OM and thereby little effect on nutrition and weight. There was no advantage to add Caphosol ® to standard OC.

Für die Behandlung von Knochendefekten überkritischer Größe stehen seit einigen Jahren zahlreiche resorbierbare Materialien zur Verfügung, die eine Defektheilung bis hin zur vollständigen knöchernen Regeneration erlauben. Im Fall systemischer Knochenerkrankungen, insbesondere im osteoporotischen Knochen, ist jedoch die Selbstheilungskapazität des Gewebes stark eingeschränkt, was neben der Defektbehandlung eine knochenanabole sowie resorptionshemmende Therapie erfordert. Diese kann beispielsweise durch die Gabe von Strontium-haltigen Präparaten erreicht werden, da der duale Wirkmechanismus der Strontium-Ionen zu vermehrter Knochenneubildung bei gleichzeitig verminderter Knochenresorption führt. Ziel der vorliegenden Arbeit war die Entwicklung eines Strontium-haltigen Knochenzements, welcher eine Freisetzung von Strontium-Ionen spezifisch im jeweiligen Knochendefekt und somit eine lokale Stimulation der Knochenneubildung ermöglicht. Basierend auf einem etablierten Calciumphosphat-Knochenzement wurden Strontium-haltige Zementvarianten hergestellt und ausgiebig charakterisiert. Im Gegensatz zu den meisten bislang verfolgten Methoden konnten Zemente mit deutlich verbesserten mechanischen Eigenschaften hergestellt werden, welche weiterhin Strontium-Ionen in physiologisch relevanten Konzentrationen freisetzen. Durch Zellkulturuntersuchungen an humanen Zellen sowohl der osteoblastären- als auch osteoklastären Linie konnte eine Stimulation der für den Knochenaufbau verantwortlichen Zellen sowie eine Hemmung der den Knochen resorbierenden Zellen durch die entwickelten Zemente nachgewiesen werden.

The use of injectable biomaterials is growing as the demands for minimally invasive procedures, and more easily applicable implants become higher, but their availability is still limited due to the difficulties associated to their design. Each year, more than 700,000 vertebral compression fractures (VCF’s) are reported in the US and 500,000 VCF’s in Europe due to primary osteoporosis only. VCF’s can compromise the delicacy of the spinal canal and also cause back pain, which affects the patient’s quality of life. Vertebroplasty was developed in the 80’s, and has proven to be a safe minimally invasive procedure that can, quickly and sustainably, relieve the pain in patients experiencing VCF’s. However, biomaterials for vertebroplasty still have limitations. For instance, ceramic bone cements are difficult to distinguish from the bone using X-ray techniques. On the other hand, acrylic bone cements may cause adjacent vertebral fractures (AVF’s). Large clinical studies have indicated that 12 to 20% vertebroplasty recipients developed subsequent vertebral fractures, and that 41 to 67% of these, were AVF’s. This may be attributed to the load shifting and increased pressure on the adjacent endplates reached after vertebroplasty with stiff cements. The primary aim of this thesis was to develop better injectable biomaterials for spinal applications, particularly, bone cements for vertebroplasty. Water-soluble radiopacifiers were first investigated to enhance the radiopacity of resorbable ceramic cements. Additionally, different strategies to produce materials that mechanically comply with the surrounding tissues (low-modulus bone cements) were investigated. When a suitable low-modulus cement was produced, its performance was evaluated in both bovine bone, and human vertebra ex vivo models. In summary, strontium halides showed potential as water-soluble radiocontrast agents and could be used in resorbable calcium phosphates and other types of resorbable biomaterials. Conversely, linoleic acid-modified (low-modulus) cements appeared to be a promising alternative to currently available high-modulus cements. It was also shown that the influence of the cement properties on the strength and stiffness of a single vertebra depend upon the initial bone volume fraction, and that at low bone volume fractions, the initial mechanical properties of the vertebroplasty cement become more relevant. Finally, it was shown that vertebroplasty with low-modulus cements is biomechanically safe, and could become a recommended minimally invasive therapy in selected cases, especially for patients suffering from vertebral compression fractures due to osteoporosis.

L'implantation de matériaux pour reconstruction osseuse par des techniques chirurgicales peu invasives nécessite des substituts osseux synthétiques, résorbables, injectables et radioopaques. C'est pourquoi le contrôle des propriétés de ces matériaux est primordial. Dans ce contexte, ce travail s'intéresse à la formulation de deux ciments, l'un uniquement à base de carbonate de calcium, le second composé d'un mélange de carbonate de calcium et de phosphate de calcium en quantités égales. Le traitement des phases solides pulvérulentes de ces deux ciments par les procédés de broyage et de co-broyage a été étudié. Ces procédés permettent entre autres de diminuer la taille moyenne des particules. Un mélange intime et homogène entre les constituants de la phase solide est obtenu grâce au co-broyage et les propriétés des ciments sont très significativement améliorées. Le temps de prise est diminué et l'injectabilité de la pâte est fortement augmentée (facteur 100). Cette dernière propriété a pu être évaluée grâce à la mise au point d'un dispositif et d'un protocole de mesure adaptés à un analyseur de texture. Dans le but de visualiser par radiographie aux rayons X l'introduction du ciment injectable dans le site osseux à réparer, du strontium a été introduit en tant qu'agent de contraste radio-opacifiant. Deux voies d'ajout à la formulation du ciment ont été étudiées : la première sous forme de SrCO3 dans la phase solide, la seconde sous forme de SrCl2 dans la phase liquide. Les études réalisées montrent que le co-broyage de la phase solide contenant du SrCO3 est intéressant pour homogénéiser la dispersion de l'agent de contraste et ainsi optimiser la quantité de strontium à incorporer pour atteindre le niveau de radio-opacité requis par la norme en vigueur. De plus, il a été observé que l'ajout de SrCl2 dans la phase liquide rend la pâte plus visqueuse et diminue son injectabilité. Par ailleurs, l'étude de la dissolution de ces ciments à pH physiologique a révélé une libération lente et prolongée du strontium. Enfin, des tests cellulaires in-vitro ont été réalisés sur ces ciments ; ils mettent en évidence l'excellent comportement de cellules ostéoprogénitrices vis-à-vis de ces formulations de ciment ainsi que l'intérêt d'utiliser le sel de SrCO3 plutôt que de SrCl2. La dernière partie de ce travail concerne l'étude de la cristallisation de l'aragonite, variété polymorphe du carbonate de calcium, en présence d'ions phosphate, connus pour inhiber la cristallisation du CaCO3. Grâce à une modélisation à l'aide de la technique de croissance cristalline à composition constante permettant de se placer dans des conditions proches de celles de la prise du ciment uniquement à base de carbonate de calcium in-vivo, il a été montré que la présence d'ions phosphate, même en très faible quantité (concentration < 0,5 µM) diminue à la fois la vitesse de germination et la vitesse de croissance cristalline de l'aragonite. L'ensemble de ces travaux contribue à l'optimisation des propriétés de ces ciments biomédicaux et à mieux appréhender leur comportement que ce soit au moment de leur implantation in-vivo ou de leur évolution et suivi post-opératoires. D'un point de vue fondamental, ces travaux pluridisciplinaires menés dans des conditions modèles in-vitro mais également dans le cadre d'une expérimentation in-vivo ont mis en évidence l'intérêt de confronter ces deux approches pour identifier et comprendre les phénomènes et les réactions impliqués lors de la prise des ciments à base de carbonate de calcium in-vitro et in-vivo. / Implantation of bone substitute materials using minimally invasive surgical techniques requires specific properties for the material including resorbability, injectability and adequate radio-opacity. The control of such properties of the material is of prime importance to meet a surgeon's requirements. In this context, this study deals with two different mineral cements: the first one is only composed of calcium carbonate phases and the second one is a mixture of equal amount of calcium phosphate and calcium carbonate phases. An original methodology involving complementary analytical techniques was implemented to thoroughly investigate the grinding mechanism of separated or mixed reactive powders constituting the solid phase and its effects on cement reactivity and properties. We show that co-grinding the solid phase decreases the mean size of the particles and favours both a homogeneous mixing and good contact between the components, leading to a decrease in the setting time. We also set two original protocols designed to evaluate paste injectability and phase separation during paste extrusion. Co-grinding leads to synergistic positive effects on cement injectability and radio-opacity. It allows maintaining a low and constant load during the extrusion of paste, which composition remains constant. Moreover, the cement's mechanical properties can be enhanced by lowering the L/S ratio because of the lower plastic limit. To be able to follow in situ the injection of the bone cement using X-ray radiography, strontium has been introduced as a contrast agent in the cement composition. Two different routes have been investigated: SrCO3 has been added to the solid phase or SrCl2 has been dissolved in the liquid phase. We show that co-grinding process permits to homogenise strontium distribution in the cement allowing us to optimise the minimum amount of strontium to add into the cement paste to reach the radio-opacity required by ISO 9917-1 standard. Moreover, adding SrCl2 in the liquid phase makes the cement paste more viscous and diminishes its injectability. Release tests performed on Sr-loaded cements show a sustained release of strontium at 37°C and pH 7.4. Finally, in-vitro cell tests have shown the excellent behaviour of osteoprogenitor cells, especially on cements including SrCO3. The last part of this work deals with the study of the crystallization of aragonite CaCO3 in the presence of phosphate ions, naturally present in biological fluids, to better understand the setting ability of calcium carbonate cements in-vivo. Using the constant composition crystal growth technique, we show that the presence of phosphate ions, even in very low amount (concentration < 0.5 µM) diminishes both the nucleation and the crystal growth rates of aragonite. This work contributes to the optimization of the properties of calcium carbonate-based cements and a better understanding and control of their behaviours during implantation and their evolution in-vivo. From a fundamental point of view, this multidisciplinary work performed in model conditions in-vitro and completed by preliminary in-vivo experiments have underlined the interest in combining these two approaches to identify and understand the phenomena and the chemical reactions involved during the setting of biomedical cements.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-10-18T18:02:47Z No. of bitstreams: 1 ANA CAROLINA BRASIL MARCELINO FOOK - DISSERTAÇÃO PPGCMat 2008..pdf: 7663913 bytes, checksum: 64b5bf9a06563efc6a355578e171b98e (MD5) / Made available in DSpace on 2018-10-18T18:02:47Z (GMT). No. of bitstreams: 1 ANA CAROLINA BRASIL MARCELINO FOOK - DISSERTAÇÃO PPGCMat 2008..pdf: 7663913 bytes, checksum: 64b5bf9a06563efc6a355578e171b98e (MD5) Previous issue date: 2008-02-26 / Atualmente na área de biomateriais destinados à regeneração óssea, existe um interesse no desenvolvimento de materiais capazes de estimular a resposta biológica necessária para restabelecer as funções do tecido ósseo em caso de perda, falta ou mau funcionamento. A hidroxiapatita (HA, Caio(P04)6(OH)2) que apresenta semelhança com a fase mineral dos ossos e dos dentes, é biocompatível e osteocondutiva, e tem excelente afinidade química e biológica com os tecidos ósseos. Este trabalho teve como objetivo desenvolver biocerâmicas porosas para regeneração óssea utilizando a técnica réplica da esponja polimérica. Foram utilizadas esponjas de poliuretano com densidades variadas para a obtenção dos suportes porosos. Os resultados de espectroscopia no infravermelho e difração de raios X revelaram a formação da HA monofásica e confirmaram a eficiência da reação de neutralização utilizada na obtenção da pasta cerâmica. As análises por microscopia eletrônica de varredura revelaram suportes com poros interconectados com tamanhos variados na ordem de macro (>100um) e microporosidade (1-20um) em menor ou maior grau dependendo da densidade da esponja utilizada na produção das amostras. Estes resultados foram comprovados pela análise de porosimetria de mercúrio. Os suportes porosos HAPU-28 destacaram-se por apresentar maior porosidade total em comparação com os suportes produzidos com esponja de menor densidade (HAPU-20 e HAPU-25). Os resultados apontam os suportes porosos de HA desenvolvidos nesse estudo como materiais em potencial para aplicação como substitutos ósseos por apresentarem alta porosidade (>70%), composição química, interconectividade e tamanhos dos poros adequados à regeneração óssea. / Currently in the area of biomateriais destined for bone regeneration, exists an interest in the development of materiais capable to stimulate the biological reply necessary to reestablish the functions of the bone tissue in loss, lack or bad functioning case. The hydroxyapatite (HA, Caio(P04)6(OH)2) that shows similarity with the mineral phase of bone and teeth, it is biocompatible and osteoconductive, and has excellent chemical and biological affinity with the bone tissue. This study aimed to develop porous bioceramics for bone regeneration using the replica of the polymeric sponge technique. Polyurethane sponges were used with varying densities to obtain the scaffolds. Results of infrared spectroscopy and X-ray diffraction analysis revealed the formation of the HA monophasic and confirmed the efficiency of the neutralization reaction used in obtaining the slurry ceramics. Analyses by scanning electron microscopy revealed scaffolds with interconnected pores with different sizes on the order of macro and microporosity to a lesser or greater degree depending on the density of foam used in the production of samples. These results were proven by the analysis of the mercury porosimetry. The porous scaffold HAPU28 detached by showing higher total porosity compared to scaffold produced with sponge of lower density (HAPU-20 and HAPU-25). The results indicate the porous HA scaffolds developed in this study as potential materiais for application as bone substitutes to have high porosity (> 70%), chemical composition, interconnectivity and pore sizes appropriate to the bone regeneration.

Os cimentos de fosfato de cálcio apresentam uma série de vantagens de utilização em ortopedia e traumatologia, sendo as mais destacadas a sua biocompatibilidade e bioatividade, as quais permitem a osteocondução dos tecidos ósseos e o endurecimento “in situ”, permitindo maior facilidade de manipulação. Entretanto; através dos métodos convencionais de reação no estado sólido há uma grande dificuldade em se obter a fase α- fosfato tricálcico pura. Com o desenvolvimento deste trabalho foi possível sintetizar a fase α-fosfato tricálcico nanoestruturado com elevado grau de pureza, utilizando-se o método de síntese de combustão em solução. Após os estudos da influência do pH, da natureza do combustível (uréia ou glicina) e dos teores estequiométricos de combustível (0,75; 1,0; 1,5 e 2,0) foi possível definir os melhores parâmetros de síntese (pH 1,5 e combustível uréia em teor duas vezes maior do que o estequiométrico). Com a definição dos parâmetros ótimos de reação, estudou-se a viabilidade de sua utilização como cimento de fosfato de cálcio. Nesta etapa verificou-se a influência do tamanho de partícula através do tempo de moagem. Para tempo de moagem de 180 minutos foram obtidos valores de resistência mecânica à compressão de até 30,4 MPa. Porém após a imersão em solução de plasma sanguineo simulado (SBF) em tempos crescentes de até 28 dias, ocorreu a diminuição desta propriedade o que é indicativo da alta solubilidade dos pós de α-fosfato tricálcico obtidos via síntese de combustão em solução. O ensaio de citotoxicidade In Vitro demonstrou que o CFC sintetizado neste trabalho não apresentou efeito tóxico para as células. Na sequência do trabalho, investigou-se a viabilidade da aplicação do CFC como substituto ósseo e como carga cerâmica em criogéis poliméricos para utilização em engenharia de tecidos. Inicialmente utilizou-se o sistema dimetilaminoetil metacrilato (DMAEMA) com a adição de 5% de um ácido acrílico (ácido acrílico ou ácido metacrílico), porém tal sistema se mostrou muito instável e apresentou baixa reprodutibilidade. Dessa forma, substituiu-se o ácido acrílico pelo monômero hidroxietil metacrilato (HEMA), pelo fato de o último possuir maior estabilidade química e melhores propriedades mecânicas. Com o uso deste sistema foram obtidos arcabouços porosos através do método de criopolimerização com porosidade de até 75% e poros de até 1 milímetro de diâmetro. A adição de α-fosfato tricálcico às estruturas porosas pouco influenciou nas propriedades físicas da rede polimérica e nas propriedades mecânicas dos arcabouços porosos, porém aumentou significativamente a biocompatibilidade destes, permitindo a adesão e a proliferação de células tronco mesenquimais. A presença de colágeno do tipo I e de fosfatase alcalina são bons indicativos de que as células tronco mesenquimais estão se diferenciando em tecido ósseo e demonstram o potencial destes materiais para o seu uso como biomaterial e mais especificamente como substitutos ósseos. / The calcium phosphate cements have a large number of advantages regarding its use in orthopedics and traumatology, being the most prominent its biocompatibility and bioactivity, which allows the osteoconductive of bone tissue and in situ hardening, allowing greater ease of handling. However, the use of conventionals synthesis methods, e.g. solid state reactions, brings great difficulty to the obtainment of highly pure α- tricalcium phosphate phase. In this thesis, the use of the solution combustion synthesis method allowed to synthesize nanostructured α-tricalcium phosphate with high purity. Further studies on pHs influence, fuel natures (urea or glycine) and fuel ratio (0.75, 1.0, 1.5 and 2.0) allowed to define the best synthesis parameters (pH 1.5 and urea fuel content in two times higher than the stoichiometric). After choosing the best paramaters to the obtainment of higly pure α-tricalcium phosphate, we studied the feasibility of their use as calcium phosphate cement (CFC) by studing the influence of particle size by increasing the milling time from 30 to 180 minutes. The better results were found for the milling time of 180 minutes. Compressives strength of up to 30.4 Mpa were obtained for this formulation. However, after soaking the calcium phosphate cements in simulated blood plasma (SBF) in growing times up to 28 days, a decrease in the compressive strenght was noticed, which is an indicative of the high solubility of the α-tricalcium phosphate powders obtained by solution combustion synthesis. After the obtainment of the calcium phosphate cements, its application as bone substitute and as ceramic load in polymeric cryogels for use in tissue engineering were studied. Initially, the system dimethylaminoethyl methacrylate (DMAEMA) with 5% of an acrylic acid (acrylic acid or methacrylic acid) was used, but due its great instability and lack of reproductbility this system was abandoned. Since it has greater chemical stability and good mechanical properties, the monomer hydroxyethyl methacrylate (HEMA) was chosen as pair to the DMAEMA monomer. With the use of this polymeric system, porous scaffolds with porosity of up to 75% and pores up to 1 mm in diameter were obtained by the method of cryopolymerization. The addition of α-tricalcium phosphate to the porous scaffolds did not showed a significant influence on physical properties of the polymer network nether on mechanical properties of porous structures. However, it increased significantly the biocompatibility of the scaffolds, allowing the adhesion and proliferation of mesenchymal stem cells. The presence of type I collagen and alkaline phosphatase are good indicators that mesenchymal stem cells are differentiating into bone tissue and demonstrate the potential application of these materials as biomaterials, more specifically as bone substitutes.

Made available in DSpace on 2016-08-17T18:39:44Z (GMT). No. of bitstreams: 1 4615.pdf: 2688296 bytes, checksum: 18c8fbe6fb83004856a32c02827d70f5 (MD5) Previous issue date: 2011-11-22 / Universidade Federal de Sao Carlos / Rabies is a zoonotic viral disease caused by a virus of the genus Lyssavirus that affects several species of mammals. Rabies remains a global public health threat that kills more than 55,000 people per year mainly in developing countries, this disease once established do not have a specific treatment. The RV envelope is composed of a glycoprotein, known as a unique antigen capable of conferring immune response against the rabies, and therefore, is the focus of research for development an efficient and safe recombinant vaccine based on this viral antigen. Cell line stably transfected S2 Drosophila melanogaster have been used in the production of many heterologous proteins and has been studied for the production of the rabies virus glycoprotein (RVGP) in our laboratory. This approach involves the selection of high producing cell populations; procedure that requires considerable periods of time (months), increasing management and costs of production. In this sense, in recent decades, many systems focused on the expression of heterologous proteins by transient expression of genes, were analyzed because they allow obtaining significant quantities of recombinant protein in a short period of time (weeks). For the use of transient transfection technology can be found a variety of methods and available agents, such as electroporation, cationic lipids, cationic polymers and calcium phosphate precipitated. The choice and optimization of each of them depends mainly on the cell type and protein being expressed. Thus, the objective of this study was to evaluate the transient expression of the glycoprotein gene of rabies virus (RVGP) in insect cells of Drosophila melanogaster S2 lineage, evaluating the vehicles transfection: calcium phosphate, cationic lipid (Cellfectin) and cationic polymer (ExGen500 and JetPEI). In order to determine the most efficient transfection agent, experiments were performed in 6 well plate and bottle of 100 mL of culture, which analyzed the influence of cell density, the concentration of DNA and transfection reagent volume on the expression of RVGP assessed by ELISA and fluorescence microscopy. Yields ranging from 50-90 ng/107cel were obtained in different experiments on multiwell plate, suggesting strong effect of ratio DNA: transfection agent used. Comparison of transfection agents showed no significant differences. In transfections made in suspension culture was analyzed the effect of the plasmid (whether or not the signal of BiP cell secretion) on the expression RVGP. When we used the plasmid containing the signal BiP (pMTiRVGP) were obtained 160 ng/107cel of RVGP production, and 200 ng/mL of volumetric production without significant differences between the different transfection agents. However, significant differences were found when we used the plasmid not containing the signal BiP (pMTRVGP), with the RVGP production was 60 ng/107cells in cells transfected with Cellfectin, ExGen500 and calcium phosphate, except in cells transfected with JetPEI was reached a production of 120 ng/107cells. In preliminary experiment bottle type "spinner" with a working volume of 60 mL were achieved expressions of 140 ng/107cel of RVGP in cells transfected with JetPEI and calcium phosphate. This suggests that optimization of culture conditions and transfection are possible to increase recombinant protein expression in cultured on a large scale. / A raiva é uma enfermidade causada por um vírus do gênero Lyssavirus que afeta várias espécies de mamíferos. Esta doença apresenta um alto custo social e econômico principalmente em países em desenvolvimento. Na superfície do vírus da raiva está localizada a glicoproteína do vírus, reconhecida como antígeno capaz de conferir resposta imunológica contra a raiva, sendo, o foco de pesquisas no desenvolvimento de uma vacina recombinante. Células da linhagem Drosophila melanogaster S2 estavelmente transfectadas têm sido usadas na produção de muitas proteínas heterólogas e tem sido estudada para a produção da glicoproteína do vírus da raiva (RVGP) em nosso laboratório. A abordagem para a obtenção de linhagens recombinantes estáveis envolve a seleção de populações celulares altamente produtoras; sendo um processo que requer consideráveis períodos de tempo (meses), uma elevada manipulação e altos custos de produção. Neste sentido, nas últimas décadas, muitos sistemas focados na expressão de proteínas heterólogas através da expressão transiente de genes foram analisados, porque eles permitem a obtenção de quantidades consideráveis de proteína recombinante em um curto período de tempo (semanas). Para o uso da tecnologia de transfecção transiente pode ser encontrada uma variedade de métodos e agentes disponíveis, tais como eletroporação, lipídeos catiônicos, polímeros catiônicos e fosfato de cálcio. Assim, o objetivo deste trabalho foi avaliar a expressão transiente do gene da glicoproteína do vírus da raiva (RVGP) em células de inseto da linhagem Drosophila melanogaster S2, avaliando os veículos de transfecção fosfato de cálcio, lipídeo catiônico (Cellfectin) e polímero catiônico (ExGen500 e JetPEI). A fim de determinar o agente de transfecção mais eficiente, foram feitos experimentos em placa de 6 poços e frasco de cultivo de 100mL, onde foram analisados a influência da densidade celular; a concentração de DNA, o volume do reagente de transfecção sobre a expressão da RVGP analisada através do método de ELISA. Quantidades de RVGP que variaram entre 50-90 ng/107cel foram obtidas nos diferentes experimentos feitos em placa, sugerindo um efeito da relação DNA: agente de transfecção. A comparação entre os agentes de transfecção não mostrou diferenças significativas. Nas transfecções feitas em cultura em suspensão foi analisado o efeito de transfectar o plasmídeo para expressão de RVGP contendo ou não o sinal de secreção celular BiP. Quando foi usado o plasmídeo contendo o sinal BiP (pMTiRVGP) foram atingidas valores de RVGP de 160 ng/107cel e produções volumétricas de 200 ng/mL, porém sem diferenças significativas entre os diferentes agentes de transfecção. Entretanto, foram encontradas diferenças quando foi usado o plasmídeo não contendo o sinal BiP (pMTRVGP), onde a produção de RVGP foi de 60 ng/107cells nas células transfectadas com Cellfectin, ExGen500 e fosfato de cálcio, porém as células transfectadas com JetPEI obtiveram uma produção de 120 ng/107cels de RVGP. Em experimento em frasco de cultivo tipo spinner com volume de trabalho de 60 mL, foram atingidas expressões de RVGP de 140 ng/107cel para células transfectadas com JetPEI e fosfato de cálcio, sugerindo que otimizações nas condições de cultivo e transfecção ainda podem ser testadas visando aumentar a expressão da proteína recombinante em cultivos em larga escala.

Made available in DSpace on 2016-12-08T15:56:19Z (GMT). No. of bitstreams: 1 Gisele Dalmonico.pdf: 8818399 bytes, checksum: 54cd754bf0a330bb5a3710ceaea2c8ed (MD5) Previous issue date: 2015-11-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Treatments for bone loss are research topics and involve different areas of scientific knowledge, engineering, physics, chemistry, biology and biomedicine. The biomaterials that stand out as replacement in bone structure treatments are hydroxyapatite, β and α calcium phosphate, biphasic hydroxyapatite/calcium phosphate β and α and hydroxyapatite matrix nanocomposite biomaterials. These biomaterials stand out as bone substitutes because they present a crystallography similar to that of human skeleton bone apatite, being bioactive and biocompatible. Nanostructured biphasic bioceramics are researched and show potential to be bone substitutes in surgical repairing procedures and reconstruction of bone tissue. This project was developed based on research of biomaterials of calcium phosphates, involving the synthesis of two matrices of calcium phosphates: β-calcium phosphate (β-TCP) and hydroxyapatite (HA) and the preparation of microporous granular biomaterials of β-TCP, HA and biphasic compositions HA/β-TCP. All biomaterials were characterized by different techniques: X-ray diffraction, infrared spectroscopy in Fourier transform (FTIR), Raman spectroscopy, specific surface area for BET, particle size by laser diffraction, density of helium pictometria, porosimetry mercury and hydrostatic porosity by Arthur, differential scanning calorimetry, dilatometry, scanning electron microscopy, atomic force microscopy, confocal microscopy, optical microscopy, polarized light microscopy. The interest of this research was to evaluate the performance of biomaterials in in vivo tests for the time periods of 90 and 180 days, in relation to osseointegration and the formation of neoformed bone tissue and determine which biomaterials presented potential as bone replacement for biomedical applications. The results found are encouraging and demonstrate that the granulated microporous biomaterials of calcium phosphate proves to be ability to repair and bone reconstruction for the two test times in vivo evaluated, revealing the osseointegration and bone formation similar between the compositions. / Os tratamentos de perdas ósseas são temas de pesquisa que envolve diferentes áreas do conhecimento científico, engenharia, física, química, biologia e biomédica. Os biomateriais que se destacam como substitutos em tratamentos da estrutura óssea são a hidroxiapatita, os fosfatos de cálcio-β e α, os bifásicos hidroxiapatita/fosfato de cálcio-β, α e os biomateriais nanocompósitos de matriz hidroxiapatita. O destaque destes biomateriais como substitutos ósseos, se deve, por apresentarem cristalografia similar à da apatita óssea do esqueleto humano, por serem bioativos e biocompatíveis. As biocerâmicas bifásicas nanoestruturadas são pesquisadas e demonstram ser promissoras como substitutos ósseos em procedimentos cirúrgicos de reparação e reconstrução do tecido ósseo. Este projeto se desenvolveu com base na investigação de biomateriais de fosfatos de cálcio, envolvendo a síntese de duas matrizes de fosfatos de cálcio: Fosfato de cálcio-β (TCP-β) e hidroxiapatita (HA) e elaboração de biomateriais granulados microporosos de TCP-β, HA e bifásicos HA/TCP-β. Todos os biomateriais granulados foram caracterizados por diferentes técnicas: difratometria de raios X, espectroscopia de infravermelho por transformada de Fourier (FTIR), espectroscopia Raman, área superficial específica por BET, tamanho de partícula por difração a laser, densidade por pictometria de hélio, porosimetria de mercúrio e hidrostática com método de Arthur. Com relação ao comportamento térmico utilizou-se a calorimetria exploratória diferencial, dilatometria. A caracterização microestrutural foi realizada com a microscopia eletrônica de varredura, microscopia de força atômica, microscopia confocal, microscopia óptica, microscopia de luz polarizada. Esse projeto de pesquisa envolveu a elaboração de biomateriais granulados microporosos de fosfatos de cálcio, o interesse foi avaliar o desempenho destes em teste in vivo, para os tempos de 90 e 180 dias em tíbia de ovinos, em relação ao desempenho dos mesmos sobre a osseointegração e a neoformação do novo tecido ósseo. Os resultados encontrados são animadores e demonstram que todas as composições de biomateriais granulados microporos de fosfatos de cálcio, demonstraram capacidade de reparação e reconstrução óssea para os dois tempos de teste in vivo avaliados, revelando a osseointegração e a neoformação óssea semelhante entre as composições.

Made available in DSpace on 2016-12-08T16:24:22Z (GMT). No. of bitstreams: 1 PGCA15MA177.pdf: 755005 bytes, checksum: d47e1b3be9b01d28494b49fd777da7f7 (MD5) Previous issue date: 2015-07-20 / The nanostructured calcium phosphate bioceramics have shown importance in bone repair biomaterials for its chemical and crystallographic similarity with bone tissue crystalline structure, increasing their relevance in orthopedics, dentistry and bone healing. These biomaterials have interconnected microporous nanostructure, made of thin grains and micropores, which favor their use as synthetic matrix in recuperation of injure bone. In vitro cell culture is a value tool for studying the mechanisms whereby biomaterials can cause adverse reactions in cell level, indicating their potential use at medical practice. The aim of this study was to evaluate the biocompatibility of porous calcium phosphate bioceramics with different morphology of grains and microporous by MTT assay, cell growth curve and cell-material interaction study by scanning electron microscope (SEM). The cell-material interaction study by SEM showed fibroblasts adhesion and proliferation in the biomaterials surface. In the results of MTT assay and cell growth curve it was found that the tested biomaterials were biocompatible, making them suitable candidates as bone substitutes. The biomaterial composed by 80% TCP-β/20% HA (1,67M/1100 °C/2h) showed cell viability less than 70% and descendant growth curve, indicating certain level of cytotoxicity / As biocerâmicas nanoestruturadas de fosfato de cálcio tem se destacado na área de biomateriais de reconstituição óssea pela sua semelhança química e cristalográfica com a apatita óssea do esqueleto humano, reforçando sua importância na ortopedia, odontologia, fixação de implantes e reparação do tecido ósseo. Estes biomateriais apresentam micro e nanoestruturas microporosas interconectadas, formada por finos grãos e microporos, características estas que favorecem seu uso como matriz sintética na recuperação de tecido ósseo lesado. O cultivo de células in vitro é uma valiosa ferramenta para se estudar os mecanismos pelos quais os biomateriais podem produzir reações adversas em nível celular, indicando seu potencial para uso na prática médica. Este trabalho teve como objetivo avaliar a biocompatibilidade das cerâmicas porosas de fosfato de cálcio em condições diferenciadas de morfologia de grãos e de microporos por meio do teste de citotoxicidade MTT, análise da curva de crescimento celular e estudo da interação célula-material por microscopia eletrônica de varredura (MEV). A análise da interação célula-material por MEV mostrou a adesão e proliferação dos fibroblastos na superfície das diferentes biocerâmicas de fosfato de cálcio testadas. Nos resultados obtidos por meio do ensaio colorimétrico MTT e curva de crescimento constatou-se que os biomateriais testados se mostraram biocompatíveis, possuindo grande potencial como substitutos ósseos. O biomaterial composto por 80% TCP-β/20% HA (1,67M/1100 °C/2h) apresentou viabilidade celular abaixo de 70% e curva de crescimento descendente, demonstrando grau de citotoxicidade