Alkali-free bioactive glasses for bone regeneration

Bioactive glasses and glass-ceramics are a class of third generation biomaterials which elicit a special response on their surface when in contact with biological fluids, leading to strong bonding to living tissues. The purpose of the present study was to develop diopside based alkali-free bioactive glasses in order to achieve good sintering behaviour, high bioactivity, and a dissolution/ degradation rates compatible with the target applications in bone regeneration and tissue engineering. Another aim was to understand the structure-property relationships in the investigated bioactive glasses. In this quest, various glass compositions within the Diopside (CaMgSi2O6) – Fluorapatite (Ca5(PO4)3F) – Tricalcium phosphate (3CaO•P2O5) system have been investigated. All the glasses were prepared by melt-quenching technique and characterized by a wide array of complementary characterization techniques. The glass-ceramics were produced by sintering of glass powders compacts followed by a suitable heat treatment to promote the nucleation and crystallization phenomena. Furthermore, selected parent glass compositions were doped with several functional ions and an attempt to understand their effects on the glass structure, sintering ability and on the in vitro bio-degradation and biomineralization behaviours of the glasses was made. The effects of the same variables on the devitrification (nucleation and crystallization) behaviour of glasses to form bioactive glass-ceramics were also investigated. Some of the glasses exhibited high bio-mineralization rates, expressed by the formation of a surface hydroxyapatite layer within 1–12 h of immersion in a simulated body fluid (SBF) solution. All the glasses showed relatively lower degradation rates in comparison to that of 45S5 Bioglass®. Some of the glasses showed very good in vitro behaviour and the glasses co-doped with zinc and strontium showed an in vitro dose dependent behaviour. The as-designed bioactive glasses and glass–ceramic materials are excellent candidates for applications in bone regeneration and for the fabrication of scaffolds for tissue engineering.

Development and characterization of Poly(L-lactic acid) (PLLA) platforms for bone tissue engineering

The development of scaffolds based on biomaterials is a promising strategy for Tissue Engineering and cellular regeneration. This work focuses on Bone Tissue Engineering, the aim is to develop electrically tailored biomaterials with different crystalline and electric features, and study their impacts onto cell biological behavior, so as to predict the materials output in the enhancement of bone tissue regeneration. It is accepted that bone exhibits piezoelectricity, a property that has been proved to be involved in bone growth/repair mechanism regulation. In addition electrical stimulations have been proved to influence bone growth and repair. Piezoelectric materials are therefore widely investigated for a potential use in bone tissue engineering. The main goal is the development of novel strategies to produce and employ piezoelectric biomaterials, with detailed knowledge of mechanisms involved in cell-material interaction. In the current work, poly (L-lactic) acid (PLLA), a synthetic semi-crystalline polymer, exhibiting biodegradibility, biocompatibility and piezoelectricity is studied and proposed as a promoter of enhanced tissue regeneration. PLLA has already been approved for implantation in human body by the Food and Drug Administration (FDA), and at the moment it is being used in several clinical strategies. The present study consists of first preparing films with different degrees of crystallinity and characterizing these PLLA films, in terms of surface and structural properties, and subsequently assessing the behavior of cells in terms of viability, proliferation, morphology and mineralization for each PLLA configuration. PLLA films were prepared using the solvent cast technique and submitted to different thermal treatments in order to obtain different degrees of crystallinity. Those platforms were then electrically poled, positively and negatively, by corona discharge in order to tailor their electrical properties. The cellular assays were conducted by using two different osteoblast cell lines grown directly onto the PLLA films:Human osteoblast Hob, a primary cell culture and Human osteosarcoma MG-63 cell line. This thesis gives also a comprehensive introduction to the area of Bone Tissue Engineering and provides a review of the work done in this field in the past until today, in that same field, including the one related with bone’s piezoelectricity. Then the experimental part deals with the effects of the crystallinity degrees and of the polarization in terms of surface properties and cellular bio assays. Three different degrees of crystallinity, and three different polarization conditions were prepared; which results in 9 different configurations under investigation.

Assessment of biocompatibility of selected ferroelectric ceramics

Desde há muitas décadas que é sabido que os organismos vivos, em especial os tecidos, reagem fisicamente a estímulos eléctricos, podendo esses efeitos reproduzirem-se numa libertação de químicos endógenos, ou deformar a sua estrutura física. O tecido ósseo por si só é considerado um material/tecido piezoeléctrico, deformando-se mecanicamente quando lhe é induzido um estímulo eléctrico e vice-versa, ou seja, produz um potencial eléctrico quando sofre uma tracção ou compressão mecânica. A hipótese de que um material ferroeléctrico possa vir a produzir efeitos no desempenho deste tipo de tecidos é então proposta, como por exemplo, para uma melhor, mais rápida e eficaz regeneração óssea. Estes mesmos materiais ferroeléctricos podem porventura alterar as cargas de superfície dos tecidos vivos de modo a atrair, atrasar ou até impedir o fluxo iónico de elementos químicos específicos responsáveis pelo processo de regeneração. São escolhidos então o niobato de lítio e o tantalato de lítio como cerâmicos ferroeléctricos e foi estudada pela primeira vez a sua bioactividade in vitro, esperando-se encontrar pistas relativas à sua bioactividade in vivo. Estes cerâmicos ferroeléctricos foram seleccionados devido às suas importantes propriedades piezoeléctricas e ferroeléctricas. Estas propriedades podem abrir um novo e importante leque de aplicações biomédicas caso estes cerâmicos sejam bioactivos. Este trabalho foi dividido em 3 fases: (i) sintetização dos pós de niobato de lítio e tantalato de lítio, (ii) caracterização dos pós e (iii) preparação das amostras e (iv) estudo da bioactividade destes cerâmicos ferroeléctricos. Os pós foram produzidos através de um processo simples de mistura/moagem seguido de calcinação. Foram estudadas as fases cristalinas presentes através de Difracção de raios-X (DRX) e avaliadas as características morfológicas destes pós, nomeadamente o diâmetro de partículas e área superficial específica. De modo a simular o ambiente do plasma humano, foi produzido sinteticamente um “Simulated Body Fluid” (SBF). Seguidamente as amostras foram imersas nesse ambiente líquido por 1, 3, 7, 15 e 21 dias. Após remoção dos pós foram realizadas uma série de análises de modo a estudar a sua bioactividade. De entre estes testes destacam-se a microscopia electrónica de varrimento (SEM/EDS), DRX e espectroscopia de Infravermelho por transformada de Fourier com reflectância total atenuada (FTIR-ATR). Embora não tenham sido detectadas alterações no DRX realizado aos pós, verificou-se a formação de aglomerados de fosfato de cálcio na superfície dos pós através do SEM, resultados estes, reforçados pelo EDS e FTIR-ATR. Estes precipitados de fosfato de cálcio indiciam a capacidade destes pós cerâmicos ferroeléctricos se comportarem como bioactivos em contacto com tecidos ósseos in vivo.

Avaliação do desempenho in vivo do biovidro FastOs™

O conceito de bioatividade surgiu com a descoberta, no início década de 70, de que algumas composições vítreas (ex.: 45S5 Bioglass®), tinham a capacidade de estabelecer uma ligação direta e estável com os tecidos vivos. Desde então, este grupo de biomateriais tem vindo a receber uma atenção cada vez maior por parte dos investigadores, tendo como motivação principal a busca de novas composições com propriedades mais adequadas para a regeneração óssea do que as composições comercialmente disponíveis. Na presente tese, avaliou-se o desempenho in vivo de duas composições de biovidro do sistema diopsite (CaMgSi2O6) - fluorapatite (Ca5(PO4)3F) - fosfato tricálcico (3CaO•P2O5) aplicados em defeitos ósseos de tamanho não crítico em carneiros, tendo também sido avaliada a biocompatibilidade dos biomateriais através da aplicação subcutânea de placas dos mesmos vidros. O trabalho realizado também incluiu a avaliação dos materiais in vitro, através de estudos de biomineralização em fluido corporal simulado e estudos de degradação. Os biomateriais foram comparados com o biovidro 45S5 Bioglass®, sendo que em termos de bioatividade in vitro, as duas composições investigadas apresentaram um maior potencial bioativo, levando à formação de uma camada superficial de hidroxiapatite carbonatada, em contraste com a formação de calcite na composição comercial, sob condições idênticas. Os testes de degradação in vitro também apresentaram resultados melhores para as duas novas composições, traduzidos por variações de pH e taxas de degradação menores do que os observados no caso do 45S5 Bioglass®. A avaliação in vivo dos implantes subcutâneos permitiu apurar a biocompatibilidade dos biovidros testados, tendo sido considerados ligeiramente irritantes. Os resultados relativos à aplicação dos pós de vidro bioativo nos defeitos ósseos não foram obtidos em tempo útil de modo a poderem ser incluídos na presente tese. Considerando o desempenho in vitro e a biocompatibilidade dos materiais estudados, estes podem apontar-se como materiais promissores para aplicações em engenharia de tecidos, particularmente na regeneração do tecido ósseo.

Compósitos CNTs/biocerâmico para a estimulação elétrica óssea in situ

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of ~500 mg/day. The deposition parameters were optimised to obtain high pure CNTs ~99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.

Functional alkali tantalate 2D structures for microelectronics and related applications

Alkali tantalates and niobates, including K(Ta / Nb)O3, Li(Ta / Nb)O3 and Na(Ta / Nb)O3, are a very promising ferroic family of lead-free compounds with perovskite-like structures. Their versatile properties make them potentially interesting for current and future application in microelectronics, photocatalysis, energy and biomedics. Among them potassium tantalate, KTaO3 (KTO), has been raising interest as an alternative for the well-known strontium titanate, SrTiO3 (STO). KTO is a perovskite oxide with a quantum paraelectric behaviour when electrically stimulated and a highly polarizable lattice, giving opportunity to tailor its properties via external or internal stimuli. However problems related with the fabrication of either bulk or 2D nanostructures makes KTO not yet a viable alternative to STO. Within this context and to contribute scientifically to the leverage tantalate based compounds applications, the main goals of this thesis are: i) to produce and characterise thin films of alkali tantalates by chemical solution deposition on rigid Si based substrates, at reduced temperatures to be compatible with Si technology, ii) to fulfil scientific knowledge gaps in these relevant functional materials related to their energetics and ii) to exploit alternative applications for alkali tantalates, as photocatalysis. In what concerns the synthesis attention was given to the understanding of the phase formation in potassium tantalate synthesized via distinct routes, to control the crystallization of desired perovskite structure and to avoid low temperature pyrochlore or K-deficient phases. The phase formation process in alkali tantalates is far from being deeply analysed, as in the case of Pb-containing perovskites, therefore the work was initially focused on the process-phase relationship to identify the driving forces responsible to regulate the synthesis. Comparison of phase formation paths in conventional solid-state reaction and sol-gel method was conducted. The structural analyses revealed that intermediate pyrochlore K2Ta2O6 structure is not formed at any stage of the reaction using conventional solid-state reaction. On the other hand in the solution based processes, as alkoxide-based route, the crystallization of the perovskite occurs through the intermediate pyrochlore phase; at low temperatures pyrochlore is dominant and it is transformed to perovskite at >800 °C. The kinetic analysis carried out by using Johnson-MehlAvrami-Kolmogorow model and quantitative X-ray diffraction (XRD) demonstrated that in sol-gel derived powders the crystallization occurs in two stages: i) at early stage of the reaction dominated by primary nucleation, the mechanism is phase-boundary controlled, and ii) at the second stage the low value of Avrami exponent, n ~ 0.3, does not follow any reported category, thus not permitting an easy identification of the mechanism. Then, in collaboration with Prof. Alexandra Navrotsky group from the University of California at Davis (USA), thermodynamic studies were conducted, using high temperature oxide melt solution calorimetry. The enthalpies of formation of three structures: pyrochlore, perovskite and tetragonal tungsten bronze K6Ta10.8O30 (TTB) were calculated. The enthalpies of formation from corresponding oxides, ∆Hfox, for KTaO3, KTa2.2O6 and K6Ta10.8O30 are -203.63 ± 2.84 kJ/mol, - 358.02 ± 3.74 kJ/mol, and -1252.34 ± 10.10 kJ/mol, respectively, whereas from elements, ∆Hfel, for KTaO3, KTa2.2O6 and K6Ta10.8O30 are -1408.96 ± 3.73 kJ/mol, -2790.82 ± 6.06 kJ/mol, and -13393.04 ± 31.15 kJ/mol, respectively. The possible decomposition reactions of K-deficient KTa2.2O6 pyrochlore to KTaO3 perovskite and Ta2O5 (reaction 1) or to TTB K6Ta10.8O30 and Ta2O5 (reaction 2) were proposed, and the enthalpies were calculated to be 308.79 ± 4.41 kJ/mol and 895.79 ± 8.64 kJ/mol for reaction 1 and reaction 2, respectively. The reactions are strongly endothermic, indicating that these decompositions are energetically unfavourable, since it is unlikely that any entropy term could override such a large positive enthalpy. The energetic studies prove that pyrochlore is energetically more stable phase than perovskite at low temperature. Thus, the local order of the amorphous precipitates drives the crystallization into the most favourable structure that is the pyrochlore one with similar local organization; the distance between nearest neighbours in the amorphous or short-range ordered phase is very close to that in pyrochlore. Taking into account the stoichiometric deviation in KTO system, the selection of the most appropriate fabrication / deposition technique in thin films technology is a key issue, especially concerning complex ferroelectric oxides. Chemical solution deposition has been widely reported as a processing method to growth KTO thin films, but classical alkoxide route allows to crystallize perovskite phase at temperatures >800 °C, while the temperature endurance of platinized Si wafers is ~700 °C. Therefore, alternative diol-based routes, with distinct potassium carboxylate precursors, was developed aiming to stabilize the precursor solution, to avoid using toxic solvents and to decrease the crystallization temperature of the perovskite phase. Studies on powders revealed that in the case of KTOac (solution based on potassium acetate), a mixture of perovskite and pyrochlore phases is detected at temperature as low as 450 °C, and gradual transformation into monophasic perovskite structure occurs as temperature increases up to 750 °C, however the desired monophasic KTaO3 perovskite phase is not achieved. In the case of KTOacac (solution with potassium acetylacetonate), a broad peak is detected at temperatures <650 °C, characteristic of amorphous structures, while at higher temperatures diffraction lines from pyrochlore and perovskite phases are visible and a monophasic perovskite KTaO3 is formed at >700 °C. Infrared analysis indicated that the differences are due to a strong deformation of the carbonate-based structures upon heating. A series of thin films of alkali tantalates were spin-coated onto Si-based substrates using diol-based routes. Interestingly, monophasic perovskite KTaO3 films deposited using KTOacac solution were obtained at temperature as low as 650 °C; films were annealed in rapid thermal furnace in oxygen atmosphere for 5 min with heating rate 30 °C/sec. Other compositions of the tantalum based system as LiTaO3 (LTO) and NaTaO3 (NTO), were successfully derived as well, onto Si substrates at 650 °C as well. The ferroelectric character of LTO at room temperature was proved. Some of dielectric properties of KTO could not be measured in parallel capacitor configuration due to either substrate-film or filmelectrode interfaces. Thus, further studies have to be conducted to overcome this issue. Application-oriented studies have also been conducted; two case studies: i) photocatalytic activity of alkali tantalates and niobates for decomposition of pollutant, and ii) bioactivity of alkali tantalate ferroelectric films as functional coatings for bone regeneration. Much attention has been recently paid to develop new type of photocatalytic materials, and tantalum and niobium oxide based compositions have demonstrated to be active photocatalysts for water splitting due to high potential of the conduction bands. Thus, various powders of alkali tantalates and niobates families were tested as catalysts for methylene blue degradation. Results showed promising activities for some of the tested compounds, and KNbO3 is the most active among them, reaching over 50 % degradation of the dye after 7 h under UVA exposure. However further modifications of powders can improve the performance. In the context of bone regeneration, it is important to have platforms that with appropriate stimuli can support the attachment and direct the growth, proliferation and differentiation of the cells. In lieu of this here we exploited an alternative strategy for bone implants or repairs, based on charged mediating signals for bone regeneration. This strategy includes coating metallic 316L-type stainless steel (316L-SST) substrates with charged, functionalized via electrical charging or UV-light irradiation, ferroelectric LiTaO3 layers. It was demonstrated that the formation of surface calcium phosphates and protein adsorption is considerably enhanced for 316L-SST functionalized ferroelectric coatings. Our approach can be viewed as a set of guidelines for the development of platforms electrically functionalized that can stimulate tissue regeneration promoting direct integration of the implant in the host tissue by bone ingrowth and, hence contributing ultimately to reduce implant failure.

Cimentos injetáveis à base de fosfatos de cálcio para vertebroplastia

O principal objectivo desta investigação foi o desenvolvimento cimentos de fosfatos de cálcio com injetabilidade melhorada e propriedades mecânicas adequadas para aplicação em vertebroplastia. Os pós de fosfato de tricálcico (TCP) não dopados e dopados (Mg, Sr e Mn) usados neste estudo foram obtidos pelo processo de precipitação em meio aquoso, seguidos de tratamento térmico de forma a obter as fases pretendidas, α− e β−TCP. A substituição parcial de iões Ca por iões dopantes mostrou ter implicações em termos de estabilidade térmica da fase β−TCP. Os resultados demonstraram que as transformações de fase alotrópicas β↔α−TCP são fortemente influenciadas por variáveis experimentais como a taxa de arrefecimento, a presença de impurezas de pirofosfato de cálcio e a extensão do grau de dopagem com Mg. Os cimentos foram preparados através da mistura de pós, β−TCP (não dopados e dopados) e fosfato monocálcico monidratado (MCPM), com meios líquidos diferentes usando ácido cítrico e açucares (sucrose e frutose) como agentes retardadores de presa, e o polietilenoglicol, a hidroxipropilmetilcelulose e a polivinilpirrolidona como agentes gelificantes. Estes aditivos, principalmente o ácido cítrico, e o MCPM aumentam significativamente a força iónica do meio, influenciando a injetabilidade das pastas. Os resultados também mostraram que a distribuição de tamanho de partícula dos pós é um factor determinante na injetabilidade das pastas cimentícias. A combinação da co-dopagem de Mn e Sr com a adição de sucrose no líquido de presa e com uma distribuição de tamanho de partícula dos pós adequada resultou em cimentos de brushite com propriedades bastante melhoradas em termos de manuseamento, microestrutura, comportamento mecânico e biológico: (i) o tempo inicial de presa passou de ~3 min to ~9 min; (ii) as pastas cimentícias foram totalmente injectadas para uma razão liquido/pó de 0.28 mL g−1 com ausência do efeito de “filter-pressing” (separação de fases líquida e sólida); (iii) após imersão numa solução durante 48 h, as amostras de cimento molhadas apresentam uma porosidade total de ~32% e uma resistência a compressão de ~17 MPa, valor muito superior ao obtido para os cimentos sem açúcar não dopados (5 MPa) ou dopados só com Sr (10 MPa); e (iv) o desempenho biológico, incluindo a adesão e crescimento de células osteoblásticas na superfície do cimento, foi muito melhorado. Este conjunto de propriedades torna os cimentos excelentes para regeneração óssea e engenharia de tecidos, e muito promissores para aplicação em vertebroplastia.