Rozprawy doktorskie na temat „BIOACTIVE GLASS MATERIALS”

Ever since Hench et al. first discovered bioactive glass in 1969, extensive interest was created because of the materials ability to chemically bond with living tissue. In this project the bioactivity of three different compositions of the bioactive glass Na2O-CaO-SiO2 have been studied. The compositions of the different glasses were A (25% Na2O, 25% CaO and 50% SiO2), B (22.5% Na2O, 22.5% CaO and 55% SiO2) and C (20% Na2O, 20% CaO and 60% SiO2). Their bioactivity was tested through biomimetic evaluation, in this case by soaking samples of each glass in simulated body fluid (SBF) and phosphate buffered saline (PBS). After soaking, the samples were analyzed with Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray Spectroscopy (EDS), Grazing Incidence X-ray Diffraction (GIXRD) and Fourier-Transform Infrared Spectroscopy (FTIR) to analyze if hydroxyapatite formed on the glass surfaces. Both the A and B glass showed bioactivity in SBF and PBS, while the C glass did not. Further work is necessary to determine which of the A and B glass has the highest apatite formability and the reason why the C glass were not bioactive.

Thesis (Ph. D.)--Missouri University of Science and Technology, 2009.
Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 7, 2010) Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-18Bitstream added on 2014-06-13T20:25:44Z : No. of bitstreams: 1 gwinner_fpc_dr_sjc.pdf: 429388 bytes, checksum: 8f66379f177aa22a57995cbad67ae92f (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Esse estudo in vitro avaliou o efeito da adição de diferentes concentrações e composições de bioactive glass (BAG) ao cimento de ionômero de vidro modificado por resina (CIVMR) (Fuji II LC) na liberação de íons Ca2+ e PO4 3- após a imersão em simulador de fluido corpóreo (SFC), comparando com o grupo controle, sem BAG. Foram utilizados 3 tipos de BAG: BAG65 (65mol% SiO2, 31mol% CaO, 4mol% P2O5), BAG75 (75mol% SiO2, 21mol% CaO, 4mol% P2O5), BAG85 (85mol% SiO2, 11mol% CaO, 4mol% P2O5), adicionados ao CIVMR nas proporções de 25%, 35% e 50%. O BAG foi misturado ao pó do CIVMR e misturado com o líquido resinoso do CIVMR. O material foi colocado numa matriz de PVC (5mm x 1mm) e polimerizado, entre laminas de vidro, durante 40s. As amostras foram armazenadas a 37°C, com 100% de umidade, durante 24h. Após esse período as amostras foram trituradas por meio de gral e pistilo e peneiradas para a obtenção de partículas menores que 90μm. A liberação dos íons Ca2+ e PO4 3- foi mensurada após 15min, 30min, 1h, 3h, 6h e 24h de imersão de 20mg de cada material em SFC, com 5 réplicas para cada condição experimental. Para a análise estatística, foi utilizada a área sob a curva (PPM x tempo). Os dados foram submetidos à ANOVA e testes de Dunnet e Tukey (!= 0,05). Os grupos contendo BAG65 liberaram significantemente mais Ca2+ não havendo diferença entre as diferentes concentrações de BAG adicionado. Os grupos contendo BAG75 e BAG85 apresentaram resultados semelhantes, sendo que aqueles com 25% de BAG foram os que apresentaram menores valores de liberação de Ca2+ após 24h de imersão. Com relação a liberação de íons PO4 3-, os grupos contendo BAG65 apresentaram maiores valores de área...
Bioactive restorative materials may stimulate the repair of tooth structure though the release of remineralization-aiding components including calcium and phosphate. The objective of this study was to measure the ion release from resin-modified bioactive glass ionomer cement (RMBGIC) containing various formulations of bioactive glass (BAG). Three types of BAG: BAG65 (65 mol% SiO2, 31 mol% CaO, 4 mol% P2O5), BAG75 (75 mol% SiO2, 21 mol% CaO, 4 mol% P2O5), BAG85 (85 mol% SiO2, 11 mol% CaO, 4 mol% P2O5) were prepared using sol-gel method, grounded, micronized, and mixed with GIC powder (Fuji II LC, GC) in the following proportions: 1:1, 1:2, 1:3. The mixed-ionomer powders were combined with standard RMGIC liquid (Fuji II LC, GC) and light cured (40 s; Ultra Lume LED, 1100 mW/cm2) in cylindrical disk molds (5 mm x 2 mm). The cured RMBGIC specimens were grounded and sieved to 100 !m and immersed (20 mg; n=5) in 3 ml of simulated body fluid (SBF), at 37°C for 1/4, 1/2, 1, 3, 6 and 24 hours with continuous agitation. Following centrifugation and decanting, the [Ca+2] and [HxPO4 3-x] in the SBF were measured using ion specific electrode and visible spectroscopy, respectively. The amount of ion release were statistically analyzed using ANOVA/Tukey ($= 0.05). The [PO4 3-] in SBF immediately increased for all RMBGICs. The ion release slowly decreased after 1 h, yet remained higher than the original SBF over the 24 h period. The [PO4 3-] in SBF for the control GIC continuously decreased. Overall, BAG65 > BAG75= BAG85 > GIC for [PO4 3-]. The [Ca2+] release increase initially for all RMBGICs, remaining with higher values than thecontrol GIC. Resin modified glass ionomer cement... (Complete abstract click electronic access below)

Orientador: Alvaro Della Bona
Banca: Marco Antonio Bottino
Banca: Tarcisio José de Arruda Paes Júnior
Banca: Simonides Consani
Banca: Lourenço Correr Sobrinho
Resumo: Esse estudo in vitro avaliou o efeito da adição de diferentes concentrações e composições de bioactive glass (BAG) ao cimento de ionômero de vidro modificado por resina (CIVMR) (Fuji II LC) na liberação de íons Ca2+ e PO4 3- após a imersão em simulador de fluido corpóreo (SFC), comparando com o grupo controle, sem BAG. Foram utilizados 3 tipos de BAG: BAG65 (65mol% SiO2, 31mol% CaO, 4mol% P2O5), BAG75 (75mol% SiO2, 21mol% CaO, 4mol% P2O5), BAG85 (85mol% SiO2, 11mol% CaO, 4mol% P2O5), adicionados ao CIVMR nas proporções de 25%, 35% e 50%. O BAG foi misturado ao pó do CIVMR e misturado com o líquido resinoso do CIVMR. O material foi colocado numa matriz de PVC (5mm x 1mm) e polimerizado, entre laminas de vidro, durante 40s. As amostras foram armazenadas a 37°C, com 100% de umidade, durante 24h. Após esse período as amostras foram trituradas por meio de gral e pistilo e peneiradas para a obtenção de partículas menores que 90μm. A liberação dos íons Ca2+ e PO4 3- foi mensurada após 15min, 30min, 1h, 3h, 6h e 24h de imersão de 20mg de cada material em SFC, com 5 réplicas para cada condição experimental. Para a análise estatística, foi utilizada a área sob a curva (PPM x tempo). Os dados foram submetidos à ANOVA e testes de Dunnet e Tukey (!= 0,05). Os grupos contendo BAG65 liberaram significantemente mais Ca2+ não havendo diferença entre as diferentes concentrações de BAG adicionado. Os grupos contendo BAG75 e BAG85 apresentaram resultados semelhantes, sendo que aqueles com 25% de BAG foram os que apresentaram menores valores de liberação de Ca2+ após 24h de imersão. Com relação a liberação de íons PO4 3-, os grupos contendo BAG65 apresentaram maiores valores de área... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Bioactive restorative materials may stimulate the repair of tooth structure though the release of remineralization-aiding components including calcium and phosphate. The objective of this study was to measure the ion release from resin-modified bioactive glass ionomer cement (RMBGIC) containing various formulations of bioactive glass (BAG). Three types of BAG: BAG65 (65 mol% SiO2, 31 mol% CaO, 4 mol% P2O5), BAG75 (75 mol% SiO2, 21 mol% CaO, 4 mol% P2O5), BAG85 (85 mol% SiO2, 11 mol% CaO, 4 mol% P2O5) were prepared using sol-gel method, grounded, micronized, and mixed with GIC powder (Fuji II LC, GC) in the following proportions: 1:1, 1:2, 1:3. The mixed-ionomer powders were combined with standard RMGIC liquid (Fuji II LC, GC) and light cured (40 s; Ultra Lume LED, 1100 mW/cm2) in cylindrical disk molds (5 mm x 2 mm). The cured RMBGIC specimens were grounded and sieved to 100 !m and immersed (20 mg; n=5) in 3 ml of simulated body fluid (SBF), at 37°C for 1/4, 1/2, 1, 3, 6 and 24 hours with continuous agitation. Following centrifugation and decanting, the [Ca+2] and [HxPO4 3-x] in the SBF were measured using ion specific electrode and visible spectroscopy, respectively. The amount of ion release were statistically analyzed using ANOVA/Tukey ($= 0.05). The [PO4 3-] in SBF immediately increased for all RMBGICs. The ion release slowly decreased after 1 h, yet remained higher than the original SBF over the 24 h period. The [PO4 3-] in SBF for the control GIC continuously decreased. Overall, BAG65 > BAG75= BAG85 > GIC for [PO4 3-]. The [Ca2+] release increase initially for all RMBGICs, remaining with higher values than thecontrol GIC. Resin modified glass ionomer cement... (Complete abstract click electronic access below)
Doutor

Mestrado em Materiais e Dispositivos Biomédicos
Bioresorbable composites nowadays play an increasingly important role in the modern medicine, especially in orthopaedics for the fixation of bone fractures and tendons. Contrarily to the metallic counterparts, they prevent a second surgical operation to remove them, because they will be gradually integrated in the bone tissues. Finding ways to improve their physical and mechanical properties to better fit the intended specific conditions and environments has been a goal in many researches. It has already established that size, shape, aspect ratio and volume fraction of reinforcing particles are parameters which can effect on mechanical properties of a composite. The aim of this work is to investigate the effect of different proportion of particulate FastOs®BG Di70 bioactive glass filler on the mechanical properties of polycaprolactone (PCL) matrix. The selection of the PCL was based on its set of interesting properties, including the FDA approval for biomedical applications and the relatively low cost. The main drawbacks of PCL are related to its relatively hydrophobic nature and the slow degradation rate it undergoes in vivo (up to 3-4 years). The present work has a multifold purpose and aims at overcoming and/or mitigating the main identifies limitations of PCL, namely enhancing relevant mechanical properties, fastening the biodegradation rate in vivo, and turning the material bioactive. For this, FastOs®BG Di70 bioglass powder was selected as filler. This bioglass is characterised by a high biomineralisation rate in vitro, has a more hydrophilic character and higher Young modulus. The combination of PCL-FastOs®BG Di70 bioglass in different proportions is therefore expected to confer to the composites a more balanced set of properties for the intended applications. The mechanical properties of composites were assessed under different testing modes (tensile, compressive, oscillatory and torsional).
Compósitos biorreabsorvíveis desempenham hoje em dia um papel cada vez mais importante na medicina moderna, especialmente em ortopedia para a fixação de fracturas ósseas e de tendões. Contrariamente aos dispositivos metálicos, eles evitam uma segunda intervenção cirúrgica para os remover, sendo gradualmente integrados nos tecidos ósseos. Encontrar maneiras de melhorar suas propriedades físicas e mecânicas para melhor atender as condições e ambientes específicos a que se destinam tem sido uma meta estabelecida em vários trabalhos de investigação. Com base nesses trabalhos, foi possível estabelecer que o tamanho, a forma e a razão de aspecto, bem como a fracção volúmica das partículas de reforço constituem os principais parâmetros que afectam as propriedades mecânicas de um compósito. O objectivo deste trabalho é investigar o efeito da adição de diferentes proporções de partículas do vidro bioativo FastOs®BG Di70 nas propriedades mecânicas de policaprolactona (PCL) usada como matriz. A selecção desta matriz foi baseada num conjunto de propriedades interessantes que possui, incluindo o facto de ter sido aprovada pela FDA para aplicações biomédicas e ser relativamente barata. As principais desvantagens da PCL estão relacionados com a sua natureza relativamente hidrofóbica, e com uma taxa de degradação lenta in vivo (até 3-4 anos). O presente trabalho tem uma finalidade múltipla e visa a superação e / ou mitigar as principais limitações identificadas para a PCL, ou seja, melhorar as propriedades mecânicas relevantes, acelerar a taxa de biodegradação in vivo, e tornar os materiais compósitos bioactivos. Para o efeito seleccionou-se o biovidro FastOs®BG Di70 na forma de pó como material de enchimento. Este biovidro é caracterizado por uma elevada taxa de biomineralização in vitro, tem um caracter mais hidrófilo e um módulo de elasticidade mais elevado. Assim, da combinação em proporções diferentes de PCL-FastOs®BG Di70, espera-se que resultem materiais compósitos com um conjunto mais equilibrado de propriedades para as aplicações almejadas. As propriedades mecânicas dos compósitos foram avaliadas sob diferentes modos de teste (de tração, compressão, torção e oscilatórios).

Enamel damage and demineralisation are common complications associated with fixed orthodontic appliances. In particular, the clean-up of adhesive remnants after debonding is a recognised cause of enamel damage. Furthermore, fixed attachments offer retentive areas for accumulation of cariogenic bacteria leading to enamel demineralisation and formation of white spot lesions (WSLs). Bioactive glasses may be used to remove adhesives, preserving the integrity of the enamel surface, while also having the potential to induce enamel remineralisation, although their efficacy in both respects has received little attention. A systematic review evaluating the remineralisation potential of bioactive glasses was first undertaken. No prospective clinical studies were identified; however, a range of in vitro studies with heterogeneous designs were identified, largely providing encouraging results. A series of glasses was prepared with molar compositions similar to 45S5 (SylcTM; proprietary bioactive glass) but with constant fluoride, reduced silica and increased sodium and phosphate contents. These glasses were characterised in several tests and the most promising selected. This was designed with hardness lower than that of enamel and higher than orthodontic adhesives. Its effectiveness in terms of removal of composite- and glass ionomer- based orthodontic adhesives was evaluated against SylcTM and a tungsten carbide (TC) bur. This novel glass was subsequently used for remineralisation of artificially-induced orthodontic WSLs on extracted human teeth. The novel glass propelled via the air-abrasion system selectively removed adhesives without inducing tangible physical enamel damage compared to SylcTM and the conventional TC bur. It also remineralised WSLs with surface roughness and intensity of light backscattering similar to sound enamel. In addition, mineral deposits were detected on remineralised enamel surfaces; these acted as a protective layer on the enamel surface and improved its hardness. This layer was rich in calcium, phosphate, and fluoride; 19F MAS-NMR, confirmed the formation of fluorapatite. This is particularly beneficial since fluorapatite is more chemically stable than hydroxyapatite and has more resistance to acid attack. Hence, a promising bioactive glass has been developed.

Cette étude porte sur le développement et la caractérisation d’un biomatériau d’ostéosynthèse bioactif, biorésorbable et présentant une tenue mécanique la plus élevée possible. Il a pour vocation de favoriser la repousse osseuse tout en remplaçant temporairement les fonctions mécaniques de l’os. Le matériau, élaboré à base d’un polyacide lactique et de verre bioactif, doit pouvoir être transformé par injection moulage de manière à obtenir des formes complexes de petites tailles telles que des vis, des ancres ou des plaques d’ostéosynthèse. Le bioverre permet au matériau de se lier facilement à l’os tandis que le polyacide lactique apporte des propriétés mécaniques essentielles pour des applications impliquant des contraintes et l’aptitude à la mise en oeuvre. Des biocomposites à base du bioverre 45S5 existent déjà mais leurs applications sont limitées du fait d’interactions bioverre/polymères partiellement incomprises qui provoquent une stabilité thermique très faible. Un contrôle systématique de la dégradation thermique des matériaux a permis d’établir la matrice polymère, le procédé d’élaboration composite et la granulométrie du bioverre optimaux pour l’obtention d’un composite de référence à base de 45S5. Par la suite, le suivi in vitro de composites élaborés à partir de nouveaux bioverres a permis de mieux comprendre l’influence de la composition des bio-verres ainsi que les interactions polymère/bioverre. Ces essais ont permis d’identifier une nouvelle formulation permettant d’allier bioactivité (formation d’hydroxyapatite au bout de 15 jours dans du SBF) et dégradation in vitro minimisée. Cette formulation a présenté des propriétés thermiques et rhéologiques similaires à celle du polymère permettant une mise en forme de petites pièces par injection moulage bien plus aisée qu’avec le composite 45S5. En outre, au bout de 4 mois d’immersion in vitro dans du PBS, les propriétés mécaniques en traction de ce matériau s’approchent de celles du polymère et sont largement supérieures à celles du composite à base de 45S5
The elaboration and characterization of a bioresorbable and bioactive biomaterial with mechanical properties as high as possible for osteosynthesis applications is the purpose of this study. This biomaterial must promote bone healing while replacing temporarily its mechanical functions. It is made with a polylactic acid and a bioactive glass and it must be easy to process through plasturgy methods in order to obtain small complex shapes as screws, anchors or osteosynthesis plates. The bioactive glass enhances the bioactivity of the material allowing it to link with the bone and the polylactic acid brings good mechanical properties essential to the applications that imply stress support and process aptitude. Biocomposites elaborated with 45S5 bioactive glass already exist but their applications are limited because of poorly understood bioactive glass/polymer interactions implying a weak thermal stability. A systematic control of the thermal degradation of the materials allows to define the best polymer matrix, composite elaboration process and bioactive glass granulometry to obtain an optimized 45S5 composite which stands for reference composite. Then, the in vitro follow-up of composites made with new bioactive glasses enhances the comprehension of the influence of the composition of the bioac-tive glass as well as the polymer/bioactive glass interactions. Hence, a new optimal formulation was identified. This formulation showed bioactivity (hydroxyapatite formation after 15 days in SBF) and a minimized in vitro degradation. Moreover, it showed thermal and rheological properties similar to neat polymer’s, which allows the thermomanufacturing of small pieces easierly than with the 45S5 composite. Plus, after an in vitro degradation in PBS of 4 months, its tensile properties were close to polymers’ and largely superior to 45S5 composite’s

A implantação de materiais vítreos e vitrocerâmicos bioativos representa estratégia terapêutica importante para se promover a formação de matriz extracelular mineralizada em defeitos ósseos críticos. Quando expostos a fluidos biológicos, estes biomateriais sofrem alterações químicas e topográficas de superfície que afetam as interações de células com sua superfície, reduzindo o espraiamento celular e alterando o padrão de marcação de proteínas do citoesqueleto. O objetivo deste estudo foi avaliar se as alterações no padrão de marcação para as proteínas citoesqueléticas actina e tubulina observadas in vitro em células osteogênicas sobre superfícies do vidro Bioglass® 45S5 e da vitrocerâmica Biosilicato®, são decorrentes de redução quantitativa na expressão do RNAm e das proteínas correspondentes. Células osteogênicas foram obtidas a partir da digestão enzimática de calvárias de ratos Wistar recémnascidos e plaqueadas sobre superfícies de Bioglass® 45S5, Biosilicato® e borosilicato (controle bioinerte) para a avaliação dos seguintes parâmetros: 1) detecção de actina e tubulina por microscopia de fluorescência; 2) expressão de RNAm para actina e tubulina por reação em cadeia da polimerase em tempo real (Real time PCR); 3) quantificação de actina e tubulina por ensaio imunoenzimático direto (ELISA), e 4) análise da morfologia celular por microscopia eletrônica de varredura (MEV). Aos 3 e 7 dias, células crescidas sobre borosilicato exibiam padrões de marcação para actina e tubulina típicos de células aderidas e espraiadas sobre substratos planos in vitro, enquanto que sobre Bioglass® 45S5 e Biosilicato® as células apresentavam áreas circulares destituídas de marcação para essas proteínas. Nos mesmos períodos, culturas crescidas sobre os materiais bioativos apresentavam alterações significantes da expressão de RNAm para actina e tubulina, embora fossem observadas apenas discretas variações na quantidade das proteínas correspondentes em relação ao borosilicato. Além disso, apenas para culturas crescidas sobre borosilicato observava-se correlação positiva entre RNAm e proteína e correspondência entre as observações por epifluorescência e os dados quantitativos. Aos 3 dias, imagens de MEV revelaram células aderidas e espraiadas sobre os materiais bioativos, parcial ou totalmente recobertas por acúmulos de material de aspecto semelhante ao da topografia do substrato, por vezes impedindo a visualização dos limites celulares. Com base nos resultados obtidos, conclui-se que as superfícies bioativas de Bioglass® 45S5 e Biosilicato® afetam a expressão de RNAm para actina e tubulina, mas não de proteína. Assim, as alterações nos padrões de marcação por fluorescência para essas proteínas devem ser atribuídas, pelo menos em parte, a acúmulos de material sobre as células, possivelmente decorrentes das reações de superfície a que estão submetidos Bioglass® 45S5 e Biosilicato® quando em contato com fluidos biológicos.
Bioactive glasses and glass-ceramics have been successfully applied in various therapeutic strategies to promote the formation of mineralized matrix in bone defects. The exposure of these materials to biological fluids results in chemical and topographical modifications that may affect the interactions of cells with the biomaterial surface, with potential effects on cytoskeletal protein expression and/or organization and cell spreading. The aim of the present study was to evaluate whether changes in the labelling pattern for the cytoskeletal proteins actin and tubulin in osteogenic cells cultured on bioactive Bioglass® 45S5 and Biosilicate® are due to altered mRNA and protein expression levels. Osteogenic cells were obtained by enzymatic digestion of newborn Wistar rat calvarial bone and plated on Bioglass® 45S5, Biosilicate® and borosilicate (bioinert control) for periods of up to 7 days. The following parameters were assayed: i) qualitative epifluorescence analysis of actin and tubulin distribution; ii) quantitative mRNA expression for actin and tubulin by real time polymerase chain reaction (real time PCR); iii) quantitative actin and tubulin expression by enzymelinked immunoabsorbent assay (ELISA), and iv) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, cells grown on borosilicate showed typical actin and tubulin labeling patterns of adherent and spread cells on flat, rigid substrates, whereas those on Bioglass® 45S5 and Biosilicate® showed dark areas devoid of fluorescent signals for the cytoskeletal proteins. At the same time points, cultures grown on the bioactive materials showed significant changes in mRNA expression for actin and tubulin, although only slight differences in the amount of actin and tubulin were detected compared with borosilicate. Moreover, a positive correlation between mRNA and protein expression levels as well as a correspondence between epifluorescence imaging and the quantitative data were only detected for cultures grown on borosilicate. SEM analysis revealed that cells cultured on bioactive surfaces were partly or totally covered with material accumulations, whose characteristics resembled the ones for the substrate topography, and which, in some cases, prevented the visualization of the cell limits. In conclusion, Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA levels, but not the corresponding protein expression, in osteogenic cell cultures. Thus, the observed changes in the labeling pattern for these proteins should be attributed, at least in part, to the accumulation of materials on the cell surface, likely due to substrate reactions that take place on Bioglass® 45S5 and Biosilicate® when exposed to the cell culture medium.

Abstract Bone is a highly vascularized tissue which enables a close interaction between blood vessels and osteoid complexes, including osteoblasts and osteoclasts. Iatrogenic bone loss, caused by a surgical intervention or trauma, disrupts circulation in the bone and leads to hypoxia and even necrosis of the adjacent bone. The mechanisms of the bone tissue healing process by regeneration are highly specific. The most common and predictable way to manage a large-sized skull bone defect has traditionally been using the patient’s own bone tissue graft. The problem with the method is damage and pain of the donor site caused by the operation and healing problems. In paediatric patients, there is a limited availability of adequate autologous bone grafts. For this reason, efforts have been made to develop substitute materials in order to avoid the need for large bone grafts. Autologous stem cells have proven to be promising targets for the development of bone substitutes for cranio-maxillofacial bone defects. The main objective of this study was to examine, by using an experimental animal model, differences in the healing of critical-sized calvarial bone defects, similar to cranioplasty, induced with different types of autogenous bone grafts and with adipose tissue- or bone marrow-derived stem cells grown in two commonly used biomaterials, tricalcium phosphate and bioglass. We also investigated the effect of tissue adhesive, fibrin glue, on the healing process of bone defects. The effect of the stimulating growth factor proteins BMP-2, BMP-7 and VEGF on tissue transplants and ossification was also studied. The results of this study support the previously reported findings of accelerated bone graft resorption associated with autologous bone graft use. In particular, the use of fibrin tissue glue in combination with autologous particulated bone grafts reduced the formation of new bone in calvarial lesions. During the initial healing of calvarial bone defects, tricalcium phosphate granules proved to be more effective than solid bioactive glass scaffolds. Furthermore, in combination with adipose-derived stem cells, tricalcium phosphate showed better bone regeneration than the same cells in combination with a bioactive glass scaffold. Combining bone marrow-derived stem cells with biomaterial did not increase bone formation in calvarial critical-sized defects. In this study, there was no evidence of the positive effect of growth factors on cranial bone healing
Tiivistelmä Luu on runsaasti verisuonitettua kudosta, ja se mahdollistaa tiiviin vuorovaikutuksen verisuonten ja luun rakenteen perusyksikön, osteonin, sekä toiminnallisten solujen osteoblastien ja osteoklastien välillä. Kirurgisen toimenpiteen tai trauman aiheuttama luun vaurio häiritsee luun verenkiertoa ja johtaa hapenpuutteeseen ja jopa nekroosiin vaurioituneessa luun osassa. Luukudoksen paranemisen ja uudistumisen mekanismit ovat pitkälle erilaistuneita ja ne tunnetaan erittäin hyvin. Yleisin ja ennusteeltaan luotettavin tapa korjata laajoja kallon luukudoksen puutoksia on perinteisesti ollut potilaan oman luukudossiirteen käyttö. Menetelmän ongelmana on siirteen luovutuskohdan vaurio ja kipu sekä paranemiseen liittyvät ongelmat. Erityisesti lapsipotilailla on haasteena riittävän siirreluumäärän rajoitettu saatavuus. Tästä syystä on pyritty kehittämään korvaavia materiaaleja, jotta vältettäisiin suuriin luusiirteisiin liittyviä ongelmia. Autologiset kantasolut ovat osoittautuneet lupaaviksi kehitettäessä luuta korvaavia hoitomuotoja kallon luupuutosten hoitoon. Tämän tutkimuksen päätavoitteena oli tutkia kokeellisen eläinmallin avulla laajojen, kallokirurgiassa aiheutuvia luupuutoksia vastaavien vaurioalueiden paranemista. Tutkimuksessa testattiin erityyppisiä autogeenisiä luusiirteitä ja rasvakudos- tai luuydinperäisiä kantasoluja, joita kasvatettiin kahden biomateriaalin, trikalsiumfosfaatin ja bioaktiivisen lasin pinnoilla. Tutkimuksessa selvitettiin myös fibriinikudosliiman vaikutusta omakudosluusiirteen paranemiseen. Kudoskasvutekijäproteiinien BMP-2, BMP-7 ja VEGF stimuloivaa vaikutusta kudossiirteiden toimintaan ja luutumiseen selvitettiin myös. Tämän tutkimuksen tulokset tukevat aiemmin raportoituja havaintoja autologisen luusiirrännäisen käyttöön liittyvästä nopeasta resorptiosta. Erityisesti fibriinikudosliiman käytön yhdessä autologisten pienipartikkelisten luusiirteiden kanssa havaittiin heikentävän uuden luun muodostumista kallon luupuutoksissa. Kallon luupuutosten alkuvaiheen paranemisessa rakeinen trikalsiumfosfaatti osoittautui tehokkaammaksi kuin kiinteä säikeinen bioaktiivinen lasi. Samoin rasvakudosperäisten kantasolujen yhdistäminen trikalsiumfosfaattiin lisäsi merkittävästi luun muodostumista verrattuna samaan yhdistelmään bioaktiivisen lasin kanssa. Luuydinperäisiä kantasoluja yhdistettäessä vastaavaa ei voitu tässä tutkimuksessa todeta. Tutkimuksessa kasvutekijöiden käytöllä ei havaittu olevan positiivista vaikutusta kallonluiden paranemiseen

Doutoramento em Ciência e Engenharia dos Materiais
Bioactive glasses and glass–ceramics are a class of biomaterials which elicit special response on their surface when in contact with biological fluids, leading to strong bonding to living tissue. This particular trait along with good sintering ability and high mechanical strength make them ideal materials for scaffold fabrication. The work presented in this thesis is directed towards understanding the composition-structure-property relationships in potentially bioactive glasses designed in CaOMgOP2O5SiO2F system, in some cases with added Na2O. The main emphasis has been on unearthing the influence of glass composition on molecular structure, sintering ability and bioactivity of phosphosilicate glasses. The parent glass compositions have been designed in the primary crystallization field of the pseudo-ternary system of diopside (CaO•MgO•2SiO2) – fluorapatite (9CaO•3P2O5•CaF2) – wollastonite (CaO•SiO2), followed by studying the impact of compositional variations on the structure-property relationships and sintering ability of these glasses. All the glasses investigated in this work have been synthesized via melt-quenching route and have been characterized for their molecular structure, sintering ability, chemical degradation and bioactivity using wide array of experimental tools and techniques. It has been shown that in all investigated glass compositions the silicate network was mainly dominated by Q2 units while phosphate in all the glasses was found to be coordinated in orthophosphate environment. The glass compositions designed in alkali-free region of diopside – fluorapatite system demonstrated excellent sintering ability and good bioactivity in order to qualify them as potential materials for scaffold fabrication while alkali-rich bioactive glasses not only hinder the densification during sintering but also induce cytotoxicity in vitro, thus, are not ideal candidates for in vitro tissue engineering. One of our bioglass compositions with low sodium content has been tested successfully both in vivo and in preliminary clinical trials. But this work needs to be continued and deepened. The dispersing of fine glass particles in aqueous media or in other suitable solvents, and the study of the most important factors that affect the rheology of the suspensions are essential steps to enable the manufacture of porous structures with tailor-made hierarchical pores by advanced processing techniques such as Robocasting.
Os vidros e vitrocerâmicos bioactivos são uma classe de biomateriais que induzem uma resposta especial à sua superfície quando em contacto com fluidos biológicos que conduz a uma forte ligação ao tecido vivo. Esta característica particular conjugada com uma boa aptidão para a sinterização e elevada resistência mecânica torna estes materiais ideais para a fabricação de estruturas de suporte à regeneração óssea. O trabalho apresentado nesta tese pretende dar um contributo para uma melhor compreensão das relações entre composição-estrutura-propriedades em vidros potencialmente bioactivos com composições no sistema CaOMgOP2O5SiO2F, em alguns casos com a adição de Na2O. O estudo da influência exercida pela composição do vidro na estrutura molecular, capacidade de sinterização e nível de bioactividade dos vidros fosfosilicatados foi objecto de especial atenção. As composições vítreas foram concebidas no campo da cristalização primária do pseudo sistema ternário do diópsido (CaO•MgO•2SiO2) – fluorapatite (9CaO•3P2O5•CaF2) – wollastonite, e estudou-se o impacto das variações composicionais na estrutura, nas propriedades e na capacidade de sinterização destes vidros. Todos os vidros investigados neste trabalho foram preparados por fusão e fritagem e caracterizados quanto à sua estrutura molecular, capacidade de sinterização, degradação química e bioactividade, usando uma grande variedade de técnicas experimentais. Ficou demonstrado que em todas as composições de vidro investigadas a rede de silicato era dominada principalmente por unidades Q2 enquanto o fosfato se encontrava coordenado em ambiente de ortofosfato. As composições de biovidros isentas de alcalinos do sistema diópsido–fluorapatite demonstram possuir excelente capacidade de sinterização e elevados níveis de bioactividade, atributos que os qualificam como materiais promissores para a fabricação de estruturas de suporte à regeneração de tecidos ósseos, enquanto os vidros bioactivos contendo alcalinos foram mais difíceis de densificar durante a sinterização e induziram citotoxicidade in vitro, não sendo candidatos ideais para a engenharia de tecidos. Uma das nossas composições de biovidro com um baixo teor de sódio foi testada com sucesso tanto in vivo como em ensaios clínicos preliminares. Mas este trabalho precisa de ser continuado e aprofundado. A dispersão de fritas moídas em meio aquoso ou outros solventes adequados, e o estudo dos factores mais relevantes que condicionam a reologia das suspensões são etapas essenciais para viabilizar o processo de fabrico de suportes porosos com estruturas hierárquicas de poros feitas por medida através de técnicas de processamento avançadas tais como o Robocasting.

Materiais vítreos e vitrocerâmicos bioativos podem ser usados particulados ou como scaffolds em diferentes tratamentos de defeitos ósseos. Tratamentos térmicos que possibilitam o desenvolvimento de scaffolds a partir de composições de vidros bioativos introduzem fases cristalinas em sua estrutura amorfa com potencial impacto na bioatividade e biocompatibilidade do material. O objetivo do presente estudo foi avaliar, qualitativa e quantitativamente, o desenvolvimento do fenótipo osteogênico de culturas de células osteoblásticas sobre substratos vítreos e vitrocerâmicos bioativos. Células MC3T3-E1 foram cultivadas em condições osteogênicas por períodos de até 21 dias sobre superfícies de Bioglass® 45S5, de duas preparações de vitrocerâmica bioativa e altamente cristalina, Biosilicato® e Biosilicato® para scaffold, e de borosilicato (vidro bioinerte). Foram avaliados, nos períodos de 7, 12 e 21 dias, morfologia celular, formação de matriz mineralizada e expressão de genes relacionados à osteogênese. Os resultados mostraram confluência das culturas sobre as superfícies de vidros e vitrocerâmicas, com progressiva formação de multicamadas celulares. A quantificação de vermelho de Alizarina revelou aumento de mineralização para culturas sobre materiais bioativos, com os maiores valores para Biosilicato® para scaffold. Expressão diferencial de genes foi observada nos 3 períodos de culturas sobre os materiais vítreo e vitrocerâmicos bioativos em comparação ao vidro bioinerte e sobre as vitrocerâmicas em comparação ao vidro bioativo. Os resultados permitem concluir que modificações em aspectos químicos de materiais vítreos e vitrocerâmicos, com efeitos sobre sua bioatividade, resultam em alteração do potencial osteogênico e do perfil de expressão gênica de células osteoblásticas in vitro. A maior atividade osteogênica sobre o Biosilicato® para scaffold permite considerar esse material um potencial candidato para aplicações em defeitos ósseos.
Bioactive glasses and glass-ceramics have been used as bone substitutes in either particulate or scaffold forms. Various thermal treatments that allow the development of scaffolds from bioactive glasses may create varied proportions of new crystalline phases in the amorphous phase with a potential impact on the bioactivity and biocompatibility of the material. The aim of the present in vitro study was to qualitatively and quantitatively evaluate the development of the osteogenic phenotype in osteoblastic cell cultures grown on bioactive glass and glass-ceramic surfaces. MC3T3-E1 cells, subclone 14, were cultured under an osteogenic condition for periods of up to 21 days on the following disc surfaces: Bioglass® 45S5 (bioactive glass), Biosilicate® (bioactive glass-ceramic), Biosilicate® as the material for scaffold preparation (Bio-sc, bioactive glass-ceramic), and borosilicate (bioinert glass). At days 7, 12, and 21 post-plating, cell morphology, mineralized matrix formation and the expression profile of genes associated with osteogenesis were evaluated. Epifluorescence of actin cytoskeleton and DAPI DNA stain revealed confluent cell cultures at day 7 for all groups, with progressive cell multilayering formation. The quantitative analysis of Alizarin red-stained cultures at day 21 revealed significantly enhanced mineralization in cultures grown on bioactive materials compared with the ones on borosilicate and the highest absorbance intensities for the Bio-sc group. Differential gene expression profiles were detected at the three time points evaluated in cultures grown on the bioactive materials in comparison with borosilicate, and on the glass-ceramics in comparison with Bioglass® 45S5. From the results presented, it can be concluded that changes in chemical characteristics of glass and glass-ceramic that may have an impact on their bioactivity index can affect the osteogenic potential and the gene expression profile of osteoblastic cells in vitro. The highest osteogenic activity on Bio-sc renders this material a good candidate for bone defect applications.

Vertebral compression fractures (VCFs) are deformities of the vertebral body that usually do not require an open surgical approach and are often due to osteoporosis or low energy trauma. They may be healed by themselves with a conservative management: brace support and bed rest, combined with the administration of analgesics and bisphosphonates. When the non-surgical approach is not effective, VCFs usually lead to untreatable pain, spine deformity and disability with a proven increase of mortality, especially in patients with poor conditions. Vertebroplasty and kyphoplasty are known as alternative minimal invasive approaches to VCFs. They are two percutaneous spine interventions which are performed with the goal of back pain relief and, when possible, restoring of the vertebral height. Vertebroplasty involves the injection of a bone cement through the trabeculae of the fractured vertebral body. In this procedure, cement injection is performed under continuous radiological control through a needle inserted in the vertebra by a transpedicular percutaneous approach. In kyphoplasty, a balloon tamp is inserted through a vertebroplasty needle in the fractured vertebral body and then is inflated/deflated and the created cavity is filled with the bone cement injected through a needle. In randomized trials it was found that VP and KP are equally safe and significantly superior to conservative treatment. Injectable bone cements play a critical role in the effectiveness of vertebroplasty and kyphoplasty. Regarding the bone replacement, several investigations have been focused on the materials exhibiting good bioactivity, biodegradability and biocompatibility. Injectable bone cements can be divided into various categories. The first group is polymer based cements which show unique properties due to their flexibility of composition. However, their poor mechanical strength limits their applications. Metals are considered as the second group that has high wear resistance, strength and ductility. However, their high rate of corrosion and low biocompatibility make them undesirable for living tissue. In addition, allergic reactions can occur due to the high diffusion of metal ions. Another group of materials are ceramics which possess generally good biocompatibility. Although these materials have resistance to corrosion and compression, they are brittle with low fracture strength. Calcium phosphate and calcium sulphate cements could be applicable as biomaterials for vertebral stabilization and augmentation. Nevertheless, their clinical applications have been limited due to some shortcomings. Currently, the number of commercial calcium sulphate-based cements is limited. Therefore, in recent years, several approaches have been proposed to develop the synthetic calcium-based bone cements. The aim of the present research was the synthesis of a mesoporous bioactive glass (MBG) by spray-drying a mild acidic aqueous synthesis solution. These particles are able to chemically bond to the bone and can be also utilized for targeted drug delivery. In order to synthesis the MBG particles, the classical methods are time-consuming due to the additional steps to obtain the final powders. Moreover, these methods are usually performed with ethanol-based solution which is flammable and expensive. Spray-drying can be considered as a single step production technique to transform a fluid feed to a dried solid powder. In fact, the spray-drying approach can be used as an effective alternative to the standard routes with faster kinetics which allows to produce the particles with controlled size and morphology. It is reported that the spray-dried mesoporous bioactive glasses or functionalized silica have been already produced by spraying synthesis solutions based on flammable solvents (mostly ethanol) under an inert atmosphere. In general, the effective procedures involve both safety and economic constraints for a future manufacturing scale-up. In this study, MBG particles were produced by the combination of sol-gel synthesis in an aqueous medium and spray-drying technique, which can be a further improvement in terms of safety, cost and environment. The second aim of this study was to develop an innovative injectable and bioresorbable composite cements based on alpha calcium sulphate hemihydrate as a resorbable matrix, enriched with mesoporous glass particles (to impart bioactivity) and a glass-ceramic radiopaque phase. The present work was in the frame of the European Union Seventh Framework Program (FP7/2007- 2013) under grant agreement no. [280575]-Restoration. During this study, the developed injectable cements were characterized in terms of physical and mechanical properties such as setting time, injectability and compressive strength. Moreover, in vitro bioactivity and degradability of prepared composite cements were assessed in simulated body fluid (SBF). Biological tests using rat bone marrow stromal cells were also carried out in vitro. In addition, further investigations were carried out in vivo by using large animal model (sheep).

Doutoramento em Ciência e Engenharia dos Materiais
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.
Os vidros e vitrocerâmicos bioactivos fazem parte da chamada terceira geração de biomateriais, i.e., materiais que estimulam uma resposta especial quando em contacto com fluidos biológicos, capaz de conduzir ao estabelecimento de ligações fortes entre a sua superfície e os tecidos vivos. O presente estudo visou o estudo e desenvolvimento de vidros bioactivos à base de diópsido e isentos de metais alcalinos que apresentem um bom comportamento na sinterização, elevados índices de bioactividade, e taxas de dissolução / degradação compatíveis com as almejadas aplicações em regeneração óssea e em engenharia de tecidos. Procurou-se ainda entender as relações entre a estrutura e as propriedades dos vidros bioactivos estudados. De acordo com esta perspectiva, estudaram-se várias composições de vidros bioactivos pertencentes ao sistema Diópsido (CaMgSi2O6) – Fluorapatite (Ca5(PO4)3F) – Fosfato de tricálcico (3CaO•P2O5). Todas as composições vítreas foram preparados por fusão, seguida de fritagem em água fria, e caracterizados através de um conjunto de técnicas complementares de caracterização. Os vitrocerâmicos foram obtidos por sinterização das fritas de vidro moídas e compactadas, seguida de tratamento térmico adequado para promover os fenómenos de nucleação e cristalização. Além disso, algumas composições vítreas seleccionadas foram dopadas com vários iões funcionais e os seus efeitos na estrutura vítrea, na sua propensão para a sinterização, e nos comportamentos in vitro em termos de biodegradação e bio-mineralização foram avaliados. Os efeitos das mesmas variáveis no processo de devitrificação (nucleação e cristalização) dos vidros e formação de materiais vitrocerâmicos foram também investigados. Algumas composições de vítreas apresentaram taxas de bio-mineralização elevadas, expressas através da formação de camadas superficiais de hidroxiapatite após 1-12 h de imersão num fluido fisiológico simulado (SBF). Todas as composições vítreas apresentaram taxas de degradação mais baixas quando comparadas com a do 45S5 Bioglass®. Alguns vidros bioactivos revelaram comportamentos in vitro excelentes, sendo a taxa de biomineralização dos co-dopados com zinco e estrôncio dependente da dose incorporada de dopantes. Os materiais estudados demostraram boa aptidão para aplicações em regeneração óssea e para o fabrico de estruturas de suporte em engenharia de tecidos.

O sucesso no tratamento odontológico tem sido incansavelmente buscado, seja através da melhoria das técnicas ou dos materiais. Contribuindo com essa realidade, o objetivo deste trabalho foi, por meio de uma metodologia inédita, verificar e comparar a densidade óptica do hidróxido de cálcio, cimento de ionômero de vidro e de um vidro bioativo, correlacionando-as com a densidade óptica de estruturas dentais. Foi desenvolvido e confeccionado um dispositivo padronizador, que simula a situação clínica onde o material é utilizado, composta por orifícios, onde o material era depositado, e por degraus, que representavam as diferentes espessuras dos materiais, além do penetrômetro de alumínio, para que fosse obtida uma curva de padronização. Foram obtidas imagens radiográficas em tempos de exposição de 0,3; 0,4 e 0,5s. Após a leitura dos valores da densidadeópticas das imagens radiográficas em fotodensitômetro, os dados obtidos foram submetidos à análise estatística (teste 3-way ANOVA, Bonferroni, nível de significância de 95%) que permitiu verificar que houve diferença na densidade óptica dos materiais, tanto em tempo, quanto em espessuras diferentes, sejam comparados entre si ou com as estruturas dentais. Os maiores valores foram obtidos com o hidróxido de cálcio, no tempo de 0,5s com 2,0mm de espessura, enquanto que o menor valor foi apresentado pelo esmalte com 3,0mm em tempo de 0,3s. Com os resultados obtidos, concluiu-se que todos os materiais apresentaram valores menores que as estruturas dentais, sendo mais radiolúcidos.
The success of dental treatment has been continuously searched by the improvement of both the techniques and materials. In order to contribute with this reality, the purpose of this study was to verify and compare, by means of an inedited methodology, the optical densities of calcium hydroxide, glass ionomer cement and a bioactive glass, correlating them with the optical density dental tissues. A standardizing device was developed and constructed, which simulates the clinical situation in which the material is used. This device was composed of orifices in which the material was inserted and steps that represented the different thicknesses of the materials, in addition to an aluminum penetrometer to provide a standardization curve. Radiographic images were obtained using exposure times of 0.3, 0.4 and 0.5 s. After reading of the optical density values of the radiographic images in a photodensitometer, the gathered data were submitted to statistical analysis by 3-way ANOVA and Bonferroni correction at 5% significance level. There was statistically significant difference in the optical density of the material regarding both the exposure times and the thicknesses, comparing to each other as well as to the dental tissues. The greatest values were obtained for calcium hydroxide at 0.5-s exposure time and 2.0 mm thickness, whereas the lowest value was obtained for enamel with thickness of 3.0 mm and exposure time of 0.3 s. Based on the obtained results, it may be concluded that all materials presented lower optical density values than the dental structures, being more radiolucent, in addition to the suggestion of adding to the materials? instructions for use information regarding the ideal exposure time and type of x-ray equipment.

Bioactive glasses exhibit unique characteristics as a material for bone tissue engineering. Unfortunately, their extensive application for the repair of load-bearing bone defects is still limited by low mechanical strength and fracture toughness. The main aim of this work was two-fold: the reinforcement of brittle Bioglass®-based porous scaffolds and the production of bulk Bioglass® samples exhibiting enhanced mechanical properties. For the first task, scaffolds were coated by composite coating constituted by polyvinyl alcohol (PVA) and microfibrillated cellulose (MFC). The addition of PVA/MFC coating led to a 10 fold increase of compressive strength and a 20 fold increase of tensile strength in comparison with non-coated scaffolds. SEM observations of broken struts surfaces proved the reinforcing and toughening mechanism of the composite coating which was ascribed to crack bridging and fracture of cellulose fibrils. The mechanical properties of the coating material were investigated by tensile testing of PVA/MFC stand–alone specimens. The stirring time of the PVA/MFC solution came out as a crucial parameter in order to achieve a more homogeneous dispersion of the fibres and consequently enhanced strength and stiffness. Numerical simulation of a PVA coated Bioglass® strut revealed the infiltration depth of the coating until the crack tip as the most effective criterion for the struts strengthening. Contact angle and linear viscosity measurements of PVA/MFC solutions showed that MFC causes a reduction in contact angle and a drastic increase in viscosity, indicating that a balance between these opposing effects must be achieved. Concerning the production of bulk samples, conventional furnace and spark plasma sintering technique was used. Spark plasma sintering performed without the assistance of mechanical pressure and at heating rates ranging from 100 to 300°C /min led to a material having density close to theoretical one and fracture toughness nearly 4 times higher in comparison with conventional sintering. Fractographic analysis revealed the crack deflection as the main toughening mechanisms acting in the bulk Bioglass®. Time–dependent crack healing process was also observed. The further investigation on the non-equilibrium phases crystallized is required. All obtained results are discussed in detail and general recommendations for scaffolds with enhanced mechanical resistance are served.

Mestrado em Materiais e Dispositivos Biomédicos
A Engenharia de Tecidos é um domínio multidisciplinar com o objetivo de desenvolver substitutos biológicos para a regeneração, reparação ou restauração de funções de órgãos ou tecidos. Os scaffolds são materiais porosos tridimensionais que servem de matrizes para a adesão e proliferação das células, bem como para a produção de matriz extracelular, idealmente, à taxa a que decorre a degradação do scaffold. A conceção de estruturas adequadas para suportar o restauro ou melhorar a função de tecidos é um dos maiores desafios da Engenharia de Tecidos. O presente estudo teve como objetivo a produção e caracterização de scaffolds de vidro bioativo, de composição molar 38.49% SiO2 – 36.07% CaO – 19.24% MgO – 5.61% P2O5 – 0.59% CaF2, utilizando um processo de fabrico aditivo, que teve por base a construção de objetos 3-D a partir de um modelo de desenho assistido por computador, utilizando a estratégia de fabrico camada a camada. Utilizou-se para tal um equipamento de deposição robótica (3D Inks, LLC, modelo EBRD-A32_-0_). O trabalho desenvolvido incluiu diferentes etapas, tais como: (i) preparação e caracterização reológica de suspensões concentradas à base de vidro bioativo em meio aquoso, estudo do dispersante mais adequado e da respetiva quantidade para a obtenção de uma suspensão suficientemente fluida e ao mesmo tempo concentrada em sólidos para minimizar as variações dimensionais ao longo do processo; (ii) estudo dos efeitos dos aditivos de processamento e das suas quantidades necessárias para transformarem a suspensão fluida numa pasta extrudível, com rigidez suficiente para manter a forma do filamento cilíndrico após a extrusão, e a caracterização reológica da tinta; (iii) fabrico dos scaffolds através da técnica de robocasting; e por fim (iv) a caracterização das estruturas obtidas quanto às propriedades morfológicas e mecânicas. Os resultados obtidos mostraram que o vidro bioativo permite a preparação de pastas com características reológicas adequadas para a deposição camada a camada por robocasting. Os scaffolds obtidos apresentam geometrias bem definidas e distribuições de poros uniformes, confirmando a adequabilidade da técnica para o fabrico de scaffolds porosos de vidro bioativo com estruturas apropriadas para aplicações nas áreas da Engenharia de Tecidos e da Medicina Regenerativa.
Tissue Engineering is a multidisciplinary field aiming at developing biological substitutes for regeneration and repair or restore functions of organs or tissues. The three-dimensional porous scaffolds serve as supports for cell attachment and proliferation as well as the production of extracellular matrix, ideally, at the same rate as scaffold degrades. The design of structures suitable to support the restoration or improve tissue function is one of the biggest challenges of Tissue Engineering. This study aimed to the production and characterization of bioactive glass scaffolds, with molar composition of 38.49% SiO2 – 36.07% CaO – 19.24% MgO – 5.61% P2O5 – 0.59% CaF2, using an additive manufacturing process, which was based on the construction of 3-D objects from a computer-assisted design model, using the manufacturing strategy of layer by layer. For such a robocasting machine (3D Inks, LLC, modelo EBRD-A32_-0_) was used. The work included different steps such as: (i) preparation and rheological characterization concentrated aqueous suspensions of bioactive glass, and study of the most effective dispersing agents and their respective amounts for obtaining a sufficiently fluid while high solids loaded suspension to minimize dimensional variations throughout the process; (ii) studying the effects of processing additives and their required amounts for transforming the fluid suspension into an extrudable paste with enough stiffness for the cylindrical filaments to maintain the shape after extrusion, and rheological characterization of the inks; (iii) manufacturing of the scaffolds through the robocasting technique; (iv) characterization of the 3-D structures obtained for their morphological and mechanical properties. The results obtained demonstrated that the bioactive glass allows the preparation of inks with appropriate rheological characteristics to be deposited layer by layer by robocasting. The resulting scaffolds exhibit well-defined geometries and uniform pore size distributions, confirming its suitability for the fabrication of porous bioactive glass scaffolds with appropriate structures for being applied in the areas of Tissue Engineering and Regenerative Medicine

Orientador: Alessandra Nara de Souza Rastelli
Abstract: Ações preventivas têm resultado na diminuição das doenças orais infecciosas e não infecciosas, porém a prevalência de cárie permanece alta, assim como os casos de hipersensibilidade dentinária (HD) estão cada vez mais frequentes. Muitos produtos têm sido considerados na remineralização dos tecidos dentários, como aqueles que mimetizam hidroxiapatita, a base de cálcio e fosfato e flúor. O objetivo desta dissertação foi avaliar experimentalmente o efeito remineralizador de materiais bioativos (Bioglass® 45S5, Biosilicato® e F18) frente às lesões de cárie inicial (LCI) e observar o comportamento do F18 sobre hipersensibilidade dentinária em relato de caso. O estudo experimental avaliou in vitro o efeito remineralizador de materiais bioativos e produtos fluoretados sobre LCI em esmalte dental bovino. Os espécimes foram divididos em seis grupos (G1 - saliva artificial, G2 - flúor gel acidulado, G3 - verniz fluoretado, G4 - Bioglass® 45S5, G5 - Biosilicato® e G6 - F18) e tiveram sua superfície dividida em duas partes, sendo elas, esmalte hígido e desmineralizado para o grupo controle, e desmineralizado e remineralizado para os demais grupos. A indução de cárie foi feita por meio da aplicação de gel de metilcelulose e solução de ácido lático (pH=4,6) por 10 dias a 37°C. Então, foi feita a aplicação dos agentes remineralizadores e os espécimes armazenados em saliva artificial por 24 horas. Para as análises, o G1 foi considerado controle (positivo - hígido e negativo - desmineralizado... (Complete abstract click electronic access below)
Mestre

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FAPEMA
Objectives: To evaluate the effect of airborne-particle abrasion with niobium phosphate bioglass (NbG) microparticles on the bond strength (µTBS) and longevity of an adhesive system to different dentin substrates. Methods and Materials: Caries-free molars were used in this study. The dentin surfaces were evaluated in three groups: (1) Control – Healthy Dentin; (2) Partial removal of carious tissue; (3) complete removal of carious tissue. Half the teeth in each group were submitted to airborne-particle abrasion with NbG microparticles (15s/1cm/5bar). After this, the adhesive Clearfil S3 was applied and composite buildups were constructed incrementally; and specimens were longitudinally sectioned to obtain bonded sticks (1.0 mm2) to be tested in tension (0.5 mm/min) immediately or after 6 months of storage in water. The fracture patterns were evaluated by stereomicroscope (40x) and then by scanning electron microscopy (SEM). The data were analyzed by the Kruskal-Wallis (post-hoc Dunn) and Mann-Whitney tests (α=0.05). Results: Healthy dentin showed the highest bond strength (µTBS). Airborne-particle abrasion with NbG increased the µTBS values in the complete caries removal group. The bond strength values in the 24-hr period were higher than those at 6 months. In the majority of the specimens the fracture mode was adhesive/ mixed. Conclusion: The authors concluded that airborne-particle abrasion on dentin with NbG particles increased the µTBS in the group in which carious dentin was completely removed.
Objetivo: Avaliar o efeito do jateamento de micropartículas de vidro niobofosfato (NbG) na resistência de união (RU) e na longevidade de um sistema adesivo em diferentes substratos dentinários. Dezoito molares extraídos livres de cárie foram usados. As superfícies de dentina foram avaliadas em três grupos: (1) Controle – Dentina Sadia; (2) Remoção parcial do tecido cariado; (3) Remoção total do tecido cariado. Metade dos dentes de cada grupo sofreu um jateamento com partículas de NbG (15s/1cm/5bar). Em seguida o adesivo Clearfil S3 Bond Plus foi aplicado e uma coroa de resina composta foi construída. Os dentes foram seccionados para obtenção dos espécimes (1,0 mm2) e submetidos ao teste de microtração (0,5 mm/min) imediatamente e após 6 meses de estocagem em água. Os padrões de fratura foram avaliados usando estereomicroscópio (40x) e levados ao microscópio eletrônico de varredura (SEM). Os dados foram analisados pelos testes Kruskal-Wallis (pos-hoc Dunn) e Mann-Whitney (α=0.05). Resultados: A dentina sadia apresentou os maiores valores de RU. O jateamento com NbG aumentou os valores de RU no grupo remoção total. Os valores de RU no período de 24h foram superiores ao de 6 meses. A maioria das fraturas dos espécimes foi adesiva/mista. Conclusão: Os autores concluíram que o jateamento à dentina com partículas NbG aumentou a RU no grupo onde a dentina cariada foi totalmente removida.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The sol-gel process used in obtaining bioactive glasses provide suitable conditions for the precipitation of hydroxyapatite nanocrystals below the glass transition temperature (Tg), even before they were immersed in biological fluids. However, because it is an ordering short, there is a great difficulty in proving its existence via the usual methods of characterization.There is evidence that the sol-gel process used to obtain bioactive glasses provides), and even before being immersed in biological fluids. Because of its short-range order, there is a great difficulty in proving its existence via the usual methods of characterization. Thus, the aim of this study was to understand the process involved in the formation of this nanocrystalline phase on the bioactive glass using Rietveld refinement on data from X-ray diffraction and high-resolution transmission electron microscopy (HRTEM); and how this glass behave on dissolution/precipitation essays. In addition, MgO oxide was used as probe to confirm the presence of apatite phase. Nanocrystalline apatite domains were observed after thermal treatment, even at temperatures below the Tg. The formation of this phase is directly related to the initial separation of calcium nitrate and triethyl phosphate (TEP) from amorphous silicate clusters during the drying process. After heat treatment at 300oC, calcium nitrate is decomposed and calcite formed. Subsequently, calcite is decarburized, and the remaining phosphate groups react with calcium, increasing the amount of nanocrystalline apatite domains. Above the Tg, the crystallinity of these phases is increased, other phases such combeita and β-cristobalite are easily identified among the products crystallization, occurring in independent events. Therefore, it was possible to propose a mechanism for crystallization of apatite below the Tg became clear that the sol-gel process produces, in fact, suitable conditions for crystallization even during the initial formation of bioglass at low temperatures and without any contact with biological fluids. The dissolution assay showed that there is no apparent change for different levels of MgO inserted, and that along the immersion period, the glass will decompose with the release of SiO44- while an apatite layer is being formed on the surface by deposition Ca2+, Mg2+ and PO43-.
O processo sol-gel utilizado na obtenção de biovidros proporciona condições adequadas para o ordenamento de nanocristais de hidroxiapatita abaixo da temperatura de transição vítrea (Tg), antes mesmo de serem imersos em fluidos biológicos. No entanto, por se tratar de um ordenamento de curta distância, há uma grande dificuldade em se comprovar sua existência via métodos usuais de caracterização. Dessa forma, o objetivo desse trabalho foi entender o processo envolvido na formação dessa fase nanocristalina no biovidro usando refinamento Rietveld sobre dados de difração de raios X e microscopia eletrônica de transmissão de alta resolução (HRTEM); e como esse vidro se comporta em ensaios de bioatividade, através de ensaio de dissolução/precipitação. Além disso, MgO foi usado como óxido sonda para a confirmação da presença da fase apatita. Domínios de apatita nanocristalina foram observados após tratamento térmico, mesmo em temperaturas abaixo da Tg. A formação desta fase está diretamente associada à segregação inicial de nitrato de cálcio e trietilfosfato (TEP) a partir dos clusters de silicato amorfo durante o processo de secagem. Após o tratamento térmico à 300oC, o nitrato de cálcio é decomposto e a calcita formada. Posteriormente, a calcita é descarbonizada, e os grupos fosfato restantes reagem com o cálcio, aumentando a quantidade de domínios de apatita nanocristalina. Acima da Tg, a cristalinidade destas fases aumenta, e outras fases como combeita e cristobalita do tipo beta são facilmente identificadas entre os produtos de cristalização, ocorrendo em eventos independentes. Portanto, foi possível propor um mecanismo para a cristalização de uma apatita abaixo da Tg ficando claro que o processo sol-gel gera, de fato, condições adequadas para a cristalização, mesmo durante a formação inicial do biovidro em baixas temperaturas e sem qualquer contato com fluidos biológicos. O ensaio de dissolução mostrou que não há variação aparente para diferentes teores de MgO inserido, e que ao longo do período de imersão, o vidro vai se decompondo com liberação de SiO44- ao passo que uma camada de apatita vai se formando na superfície pela deposição de Ca2+, PO43- e Mg2+.

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Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA)
Objectives: To evaluate the effect of airborne-particle abrasion with niobium phosphate bioglass (NbG) microparticles on the bond strength (µTBS) and longevity of an adhesive system to different dentin substrates. Methods and Materials: Caries-free molars were used in this study. The dentin surfaces were evaluated in three groups: (1) Control – Healthy Dentin; (2) Partial removal of carious tissue; (3) complete removal of carious tissue. Half the teeth in each group were submitted to airborne-particle abrasion with NbG microparticles (15s/1cm/5bar). After this, the adhesive Clearfil S3 was applied and composite buildups were constructed incrementally; and specimens were longitudinally sectioned to obtain bonded sticks (1.0 mm2) to be tested in tension (0.5 mm/min) immediately or after 6 months of storage in water. The fracture patterns were evaluated by stereomicroscope (40x) and then by scanning electron microscopy (SEM). The data were analyzed by the Kruskal-Wallis (post-hoc Dunn) and Mann-Whitney tests (α=0.05). Results: Healthy dentin showed the highest bond strength (µTBS). Airborne-particle abrasion with NbG increased the µTBS values in the complete caries removal group. The bond strength values in the 24-hr period were higher than those at 6 months. In the majority of the specimens the fracture mode was adhesive/ mixed. Conclusion: The authors concluded that airborne-particle abrasion on dentin with NbG particles increased the µTBS in the group in which carious dentin was completely removed.
Objetivo: Avaliar o efeito do jateamento de micropartículas de vidro niobofosfato (NbG) na resistência de união (RU) e na longevidade de um sistema adesivo em diferentes substratos dentinários. Dezoito molares extraídos livres de cárie foram usados. As superfícies de dentina foram avaliadas em três grupos: (1) Controle – Dentina Sadia; (2) Remoção parcial do tecido cariado; (3) Remoção total do tecido cariado. Metade dos dentes de cada grupo sofreu um jateamento com partículas de NbG (15s/1cm/5bar). Em seguida o adesivo Clearfil S3 Bond Plus foi aplicado e uma coroa de resina composta foi construída. Os dentes foram seccionados para obtenção dos espécimes (1,0 mm2) e submetidos ao teste de microtração (0,5 mm/min) imediatamente e após 6 meses de estocagem em água. Os padrões de fratura foram avaliados usando estereomicroscópio (40x) e levados ao microscópio eletrônico de varredura (SEM). Os dados foram analisados pelos testes Kruskal-Wallis (pos-hoc Dunn) e Mann-Whitney (α=0.05). Resultados: A dentina sadia apresentou os maiores valores de RU. O jateamento com NbG aumentou os valores de RU no grupo remoção total. Os valores de RU no período de 24h foram superiores ao de 6 meses. A maioria das fraturas dos espécimes foi adesiva/mista. Conclusão: Os autores concluíram que o jateamento à dentina com partículas NbG aumentou a RU no grupo onde a dentina cariada foi totalmente removida.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Millions of bone fractures occur annually worldwide and the consequent bone repair process is complex, involving many biological events until it reaches the restoration of the tissue integrity. During that process some problems can occur due to delays in the bone healing, which does not allow the proper joining of the tissue. Thus, it is necessary to search for new technologies that work in restoring the integrity of the bone tissue and that promote the osteoconduction and the osteoinduction. In this sense, the use of bioactive materials in the bone repair process is a promising alternative. Following this, two studies (I and II) were developed in order to investigate a new fibrous glassy scaffold, and these studies were based in three lines of research: (i) the characterization of the new fibrous glassy scaffold; (ii) the biocompatibility evaluation of this bioactive material; (iii) the analysis of the biological performance of this new scaffold in the bone repair. More specifically, in the study I the developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. In regards to study II, the morphological structure of the scaffolds upon incubation in phosphate buffered saline (PBS) (via scanning electron microscope) was assessed after 1, 7 and 14 days and, also, the in vivo tissue response to the new biomaterial was evaluated using implantation in rat tibial defects. The histopathological, immunohistochemistry and biomechanical analyzes after 15, 30 and 60 days of implantation were performed to investigate the effects of the material on bone repair. The scaffolds presented interconnected porous structures (porosity of ~75%), and the precursor bioglass could mineralize a hydroxycarbonate apatite (HCA) layer in SBF after only 12 h. The PBS incubation indicated that the fibers of the glassy scaffold degraded over time. With regards to the biological investigations, the biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multi-nucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded, and an organized granulation tissue filled the area. Additionally, the histological analysis of the implants in the bone defects revealed a progressive degradation of the material with increasing implantation time and also its substitution by granulation tissue and woven bone. Histomorphometry showed a higher amount of newly formed bone area in the control group (CG) compared to the biomaterial group (BG) 15 days post-surgery. After 30 and 60 days, CG and BG showed a similar amount of newly formed bone. The novel biomaterial enhanced the expression of RUNX-2 and RANK-L, and also improved the mechanical properties of the tibial callus at day 15 after surgery. These results indicate that the new fibrous glassy scaffold is bioactive, non-cytotoxic, biocompatible and promising for using in bone tissue engineering.
Milhões de fraturas ósseas ocorrem anualmente no mundo todo e o processo de reparo é complexo, envolvendo muitos eventos biológicos até que se atinja a restauração da integridade do tecido. Problemas nessa regeneração podem ocorrer, levando a não união óssea. Assim, faz-se necessária a busca por novas tecnologias que atuem na restauração da integridade do tecido ósseo e promovam a osteocondução e a osteoindução. Para tanto, uma alternativa promissora é a utilização de materiais bioativos para o reparo ósseo. Seguindo essa linha, foram realizados dois estudos (I e II) acerca de um novo scaffold vítreo fibroso, sendo estes estudos baseados em três linhas de investigação: (i) caracterização do novo scaffold vítreo fibroso; (ii) avaliação da biocompatibilidade desse material bioativo e (iii) análise do desempenho biológico desse novo scaffold no reparo ósseo. Mais especificamente, no estudo I foi feita a caracterização dos scaffolds em termos de porosidade, mineralização e características morfológicas. Adicionalmente, fibroblastos e osteoblastos foram cultivados em contato com extratos dos scaffolds para avaliação da proliferação celular e genotoxicidade após 24, 72 e 144 h. Finalmente, nesse mesmo estudo, os scaffolds foram implantados subcutaneamente em ratos por 15, 30 e 60 dias. No que se refere ao estudo II, foram feitas avaliações da estrutura morfológica dos scaffolds (via microscopia eletrônica de varredura) imersos em tampão fosfato salino (PBS) após 1, 7 e 14 dias, além de investigações do efeito no reparo ósseo do novo scaffold utilizando implantação do mesmo em defeitos ósseos tibiais em ratos. Análises histopatológicas, imunohistoquímicas e biomecânicas foram realizadas 15, 30 e 60 dias após a implantação. Os scaffolds apresentaram estruturas altamente porosas (porosidade de ~75%) e interconectadas, e o biovidro precursor mineralizou uma camada de hidroxicarbonatoapatita (HCA) em SBF (simulated body fluid) após o curto período de 12 h. A incubação em PBS indicou que as fibras do scaffold apresentaram sinais de degradação com o passar do tempo. Sobre os testes biológicos, o novo biomaterial levou a um aumento da proliferação de fibroblastos e osteoblastos, e nenhum dano ao DNA foi observado. Os experimentos de implantação do material no subcutâneo indicaram degradação do biomaterial acompanhada do crescimento interno de tecido mole e presença de células gigantes multinucleadas ao redor do implante. Após 60 dias, os scaffolds estavam quase completamente absorvidos e um tecido de granulação organizado preenchia a área de implantação. Adicionalmente, as análises histológicas dos scaffolds em defeitos ósseos revelaram uma degradação progressiva do biomaterial e substituição do mesmo por tecido de granulação e tecido ósseo neoformado. A histomorfometria mostrou uma maior quantidade de osso neoformado no grupo controle (CG) comparado ao grupo biomaterial (BG) 15 dias após a cirurgia. No entanto, depois de 30 e 60 dias, CG e BG apresentaram quantidades similares de osso neoformado. Além disso, o novo biomaterial aumentou a expressão de RUNX-2 e RANK-L, e também melhorou as propriedades mecânicas do calo tibial 15 dias após a cirurgia. Os resultados indicam que o novo scaffold vítreo fibroso é bioativo, não-citotóxico, biocompatível e promissor para utilização na engenharia do reparo ósseo.

The function of biomaterials is to replace infected, injured or damage tissues. The first used biomaterials are bioinert, thus minimizing the formation of scar tissue at implant tissue interface. Bioglass was discovered by Dr. Larry Hench in 1969, which has the capability to bond with bone without encapsulated by fibrous tissues. It has been discovered that bioglass has a far higher bonding capacity than any other biomaterial. Its ability to form hydroxyapatite with body fluid makes it undefendable compared to virgin hydroxyapatite crystals. In addition to providing a surface for cell development, the presence of Si ions acts as a catalyst to speed up cellular proliferation. Recently, it has been discovered that bioglass is not only a good candidate for hard tissue regeneration but can also be used beyond bone regeneration such as soft tissue engineering applications. Since the late 1960s, many techniques have been developed and used to create bioglass, including the melt-quench method, the sol-gel method, flame spray synthesis, microwave synthesis, and others. Apart from these methods, a new cost effective, green synthetic route called bioinspired route has been reported by Santhiya et al in 2013. This approach was developed on admiring naturally synthesized nano-structured materials such as silica in diatoms by the guidance of bio macromolecular templates. In last two decades, the bio-inspired synthesis of nanostructured ceramic oxides below 100 C has been well established using organic templates. Santhiya et al. explored the effect of various templates on the textural and morphological properties of the bioglass particles. In the present investigation, the bio medical application of bioglass materials as inorganic-organic hybrid composites are explored. Herein, bioinspired method was adopted for in-situ mineralization of bioglass particles. Initially, the bioglass particles were synthesized using small molecules i.e., a Abstract monomer instead of high molecular weight synthetic polymers. Further, we have developed a new sustainable green synthetic method for the synthesis of bioglass particles directly utilising a natural plant extract as both a template and a source of a few inorganic metal ions. In this thesis, considering the huge importance of bioactive glass hybrid materials for both soft and hard tissue engineering applications, various in-situ mineralized bioactive glass hybrid materials are synthesized and characterized in detail. This thesis has been summarized in 6 chapters. Chapter 1 provides a general overview on soft and hard tissue engineering, generations of biomaterials, bioactive glass as third generation biomaterial and application of bioglass beyond bone regeneration. Additionally, a thorough overview of studies on the synthesis of bioactive glass and the mechanism of bioactivity in simulated body fluid is reviewed in detail. Bioactive glass hybrid materials' significance in biological applications is briefly discussed. Chapter 2 explains the fundamentals of numerous methods used to characterise bioglass materials. These tools help us to determine their structural, morphological, thermal and mechanical properties like size, charge, surface area, surface functional groups, morphology, and molecular structure. In Chapter 3, bioinspired synthesis method for mesoporous L-lysine-bioactive glass (LBG) hybrid xerogels at ambient conditions was discussed in details. L-lysine molecules were incorporated in bioactive glass (BG) network through physiochemical interaction. The step-wise addition of various BG precursors with L-lysine took place at its three pKa (2.18, 8.94, 10.54) and pI (9.74) values respectively. These LBG hybrid xerogels were thoroughly characterized before and after interaction with simulated body Abstract fluid (SBF). Interestingly, elemental analysis on xerogels reported 45S5 composition for inorganic contents of LBG_8.94 and LBG_9.74. In contrast, LBG_2.18 and LBG_10.54 contained higher SiO2 and corroborating discrete bioactivity behaviour of xerogels. Nitrogen sorption analysis confirmed the mesoporous nature of all four LBG xerogels with different pore size, pore volume and surface area. Importantly, distinctively controlled 7-Dehydrocholesterol release pattern of each LBG xerogels highlighted the importance of their tuneable textural property. Reported viscoelastic nature of freshly prepared LBG xerogels by rheological analysis promised its non invasive injectability. These new generation materials not only promise to serve nutrients for cell growth but also contain tailored textural as well as rheological characteristics for targeted bone engineering applications. In Chapter 4, we report for the first time, BGNPs synthesized using Trigonella foenum-graecum (TFG) leaf extract (TFGL_EX). Bioactive glass nanoparticles (BGNPs) have been reported in various biomedical applications such as tissue engineering, dental and bone repair, drug and gene delivery, tumour therapy and skincare. The requirement of sustainable synthesis methods is pertinent for large-scale manufacture of such popular materials. Currently, green synthesis methods have gained popularity in various nanoparticle synthesis as it is considered environmentally benign with minimal waste generation. To our knowledge, we report synthesis of BGNPs for the first time using Trigonella foenum-graecum (TFG) leaf extract (TFGL_EX). The TFGL_EX can be used both as a template and precursor for synthesizing BGNPs. The influence of the leaf extract on the composition, particle size and porosity of BGNPs has been determined using characterization techniques like ICP-MS, HR-TEM and nitrogen sorption analysis. Further, bioactivity and biocompatibility of the synthesized BGNPs could be successfully demonstrated through in-vitro studies in Simulated Body Abstract Fluid (SBF) and on model bone cell line, respectively. Native BGNPs as well as model antibacterial drug loaded counterparts demonstrates significant antibacterial properties and sustained drug release profiles. Overall, the study reports a sustainable property dependent synthesis methodology for BGNPs by utilizing organic and inorganic constitutes of TFGL_EX. In chapter 5, we address the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed that collagen and pectin molecules interacted despite having comparable net negative charges through the mechanism of surface patch binding interaction. FTIR and TGA analysis confirmed this associative interaction resulting in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. SEM, EDX and TEM results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca10(PO4)6(OH)2) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was tested against Candida albicans when infused with the model antifungal medication amphotericin-B (AmB). The AmB release kinetics of the hydrogel followed the Fickian diffusion release mechanism demonstrating direct proportionality to gel swelling behaviour. Rheological analysis revealed the viscoelastic compatibility of CPBG for mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues. Abstract In Chapter 6, ultrafine fibers and a bioactive glass mineralized fibrous mat of gelatin pectin blends were produced by electrospinning in an aqueous phase. Herein, the gelatin-pectin blend was used as a template for the in-situ mineralization of bioactive glass particles during electrospinning of gelatin-pectin based hybrid composite fiber matrix. Additionally, in-situ mineralized bioactive glass along with the fibrous mat served as a site for 7-dehydrocholesterol, i.e., a vitamin-D precursor. This engineered fibrous mat resulted in the sustained release of the drug, which is essential for fortifying the neo regenerated bones. The fibrous mat also exhibited excellent bioactivity in simulated body fluid. Additionally, hybrid composite fibre mat displayed cell proliferation on the surface of fibrous mat and had outstanding cytocompatibility with osteoblast cells.

A hybrid scaffold based on polyvinyl alcohol and bioactive glass ceramics was synthesized and characterized for its biocompatibility and its degradation behavior. Poly vinyl alcohol and glass ceramics were mixed in the ratio 70:30 by weight percentage. The glass ceramic was based on 65 % SiO2, 5 % P2O5 and 30 % CaO. Aqueous solution of PVA was prepared with 25% concentration. A glass ceramic sol containing TEOS, CaCl2, TEP was added to PVA solution. The pH of the final solution was adjusted to 2 using HF. Hybrid gel formed was then dried and characterized for phase evaluation by XRD, chemical characterization by FTIR, AP, BD, microstructure by FESEM, biodegradability and bioactivity. XRD pattern revealed that the hybrid was amorphous material. SEM microstructure showed crystallization of glass ceramic phase in polymer matrix. Incubation of glass ceramic in SBF solution showed the deposition of Calcium and Sodium containing phases on the surface of scaffold. This indicates that the hybrid scaffold is biocompatible and may be applicable for uses in bone tissues engineering.

Bioactive glasses of chemical composition 48.4SiO2 -23.8Na2O- 23.8CaO- 4.0P2O5 wt% was prepared through Sol-Gel route. It was then crystallized through thermal treatment. Porous samples of the mentioned composition using Naphthalene (0, 30, and 50 weight percentage) as pore former were made and tested for Bulk Density, Apparent porosity, Linear Shrinkage, Cold Crushing Strength, and Bi-axial flexural strength. Maximum porosity of 50% was obtained by this process. The maximum value of CCS obtained was 7.8MPa without any pore former and a minimum of 2.3 MPa for 50% Naphthalene. The Bi-axial flexural strength varied between the extremes of 16.3 MPa and 5.6 MPa. Their pore size distribution was also studied to ensure the presence of pores with size greater than 100 µm, which happens to be the critical lower limit for Angiogenesis. Samples were also prepared with a radial porosity gradient similar to the structure of the cancellous part of the Bone structure. The samples contained a central core of higher porosity and a outer concentric ring of lower porosity. These samples were also tested for the above mentioned mechanical properties. The Bioactivity of the samples was also studied by immersing them in a SBF solution for a period of 1, 3, and 7 days. These samples were then studied using XRD, SEM, EDX, and FTIR and showed significant formation of HCA ensuring their Bioactivity.

Glass-ceramics materials share many properties with both glass and more traditional crystalline ceramics. It is formed as a glass, and then made to crystallize partly by heat treatment. . Bioactive glass-ceramics describes the beneficial or adverse effects of glass-ceramic with living tissue , when placed in body. Bioactive glasses and glass-ceramics are more and more studied because of their surface chemical reactivity when in contact with body fluids [1–3]; by a complex mechanism of ions leaching and partial dissolution of the glass surface, the precipitation of bone-like apatite from the solution provides a strong chemical bonding with tissues. Since bioactive glasses and glass-ceramic are brittle materials, they are especially used in the field of small bone defects reconstruction, or as coatings on inert substrates for load-bearing prostheses. Since the discovery of bioglass by Hench et al. [4] in the early 1970s, various types of ceramic, glass and glass–ceramic have been proposed and used as bone replacement biomaterials [5-7]. Specifically, these biomaterials have found clinical applications as coating for prostheses, bone filler, vertebral substitution and, in a porous form, as bone substitutes [8-15]. Most of them are based on the SiO2–P2O5–CaO–Na2O system. Bonding between bioactive glass or glass–ceramic and the surrounding tissues takes place through the formation of a hydroxyapatite layer, which is very similar to the mineral phase of bone. When the bioactive glass is placed in contact with physiological fluids, this layer is formed through a complex ion-exchange mechanism with the surrounding fluids, known as bioactivity. This biologically-active layer of hydroxyapatite can form on the surface of glasses having a wide compositional range, and is considered as self by the surrounding living tissue; its presence is widely recognized to be a sufficient requirement for the implant to chemically bond with the living bone. Kokubo et al. [16] proposed the Tris-buffered simulated body fluid (SBF) for the in vitro study of bioactive glass and glass–ceramic, since its ion concentration is almost equal to that of human blood plasma. Since then, in vitro tests in SBF have been widely used as preliminary tests on new candidate materials showing bioactive properties. The ion leaching phenomenon involves the exchange of monovalent cations from the glass, such as Na+ or K+, with H3O+ from the solution, and thus causes an increase in the pH of the solution. It is known that osteoblasts prefer a slightly alkaline medium [17, 18], but it is also known that severe changes in pH can inhibit osteoblast activity and cause cell necrosis or apoptosis [19-21]. Different bioactive glass and glass ceramics have been synthesized in order to get desired mechanical, chemical properties by obtaining required microstructure. Some of common components used are Na2O, CaO, P2O5, SiO2 for synthesis of 45S5 and S53P4. In addition to these above components, varying composition of K2O, MgO , B2O3 are used to get 13-93, 3-04, 18-04, 23-04. There are some other glass and glass ceramics which also include ZnO, Ag and Al2O3. In current study of bioactive glass ceramic we are using rice husk ash as raw material for synthesis of silica, which is amorphous in nature. It is cheap, easily available source with high content of silica

Bone cancer treatment usually originates bone defects with residual tumour cells that can proliferate during bone regeneration. Therefore, a scaffold for bone regeneration that simultaneously kill residual tumour cells is needed. This project aims at producing a composite system composed of a bioactive glass (BAG) and magnetic nanoparticles (MNPs). This system is highly bioactive and reabsorbable due to the bioactive glass which leads to formation of a hydroxyapatite (HA) layer that bonds to bone. The system is biodegradable at an adequate rate for bone regeneration. Magnetic nanoparticles act as thermoseeds generating clinically relevant heat under an applied alternating magnetic field to kill or sensitize tumour cells. In combination with release of an anticancer drug, this composite system will effectively kill bone tumour cells whilst providing a base for bone regeneration. BAG was produced by a simple sol-gel technique assisted by EISA (Evaporation Induced Self-Assembly). Ball milling equipment was used to decrease the BAG particle size and increase its dispersibility. The powders were characterized by SEM (scanning electron microscopy), EDS (energy dispersive x-ray spectroscopy), and FTIR (Fourier Transform Infrared Spectroscopy). IONPs were produced through chemical co-precipitation and coated with oleic acid to avoid aggregation and loss of superparamagnetic properties over time. First, PVP/BAG composite membranes were produced by electrospinning and the parameters were optimized to produce smaller fibres as it translates into higher surface area and higher bioactivity. IONPs were then incorporated in the solution. The electrospun membranes were crosslinked due to the PVP water-soluble characteristic. UV and thermal crosslinking were employed, but only thermal crosslinking proved to be successful. For this to be successful TGA/DSC was helpful to find the crosslinking temperature and provided information about the thermal stability of the membranes. Water-insoluble membranes were tested for magnetic hyperthermia application and cytotoxicity assays were also performed. The IONPs proved to have superparamagnetic properties and a small temperature variation was achieved for a 10 mg membrane sample, which proved the potential of composite membranes for this application.