Doctor of Philosophy / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Doctor of Philosophy / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Human dental tissues are fascinating nano-structured hierarchical materials that combine organic and mineral phases in an intricate and ingenious way to obtain remarkable combinations of mechanical strength, thermal endurance, wear resistance and chemical stability. Attempts to imitate and emulate this performance have been made since time immemorial, in order to provide replacement (e.g. in dental prosthodontics) or to develop artificial materials with similar characteristics (e.g. light armour). The key objectives of the present project are to understand the structure-property relationships that underlie the integrity of natural materials, human dental tissues in particular, and the multi-scale architecture of mineralized tissues and its evolution under thermal treatment and mechanical loading. The final objective is to derive ideas for designing and manufacturing novel artificial materials serving biomimetic purposes. The objectives are achieved using the combination of a range of characterization techniques, with particular attention paid to the synchrotron X-ray scattering (Small- and Wide-Angle X-ray Scattering, SAXS and WAXS) and imaging techniques (Micro Computed Tomography), as well as microscopy techniques such as Environmental Scanning Electron Microscopy (ESEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). Mechanical properties were characterized by nanoindentation and photoelasticity; and thermal analysis was carried out via thermogravimetric analysis (TGA). Experimental observations were critically examined and matched by advanced numerical simulation of the tissue under thermal-mechanical loading. SAXS and WAXS provided the initial basis for elucidating the structure-property relationships in human dentine and enamel through in situ experimentation. Four principal types of experiment were used to examine the thermal and mechanical behaviour of the hierarchical structure of human dental tissue and contributed to the Chapters of this thesis: (i) In situ elastic strain evolution under loading within the hydroxyapatite (HAp) in both dentine and enamel. An improved multi-scale Eshelby inclusion model was proposed taking into account the two-level hierarchical structure, and was validated against the experimental strain evaluation data. The achieved agreement indicates that the multi-scale model accurately reflects the structural arrangement of human dental tissue and its response to applied forces. (ii) The morphology of the dentine-enamel junction (DEJ) was examined by a range of techniques, including X-ray imaging and diffraction. The transition of mechanical properties across the DEJ was evaluated by the high resolution mapping and in situ compression measurement, followed by a brief description of the thermal behaviour of DEJ. The results show that DEJ is a narrow band of material with graded structure and mechanical properties, rather than a discrete interface. (iii) Further investigation regarding the thermo-mechanical structure-property relationships in human dental tissues was carried out by nanoindentation mapping of the nano-mechanical properties in ex situ thermally treated dental tissues. (iv) In order to understand the details of the thermal behaviour, in situ heat treatment was carried out on both human dental tissues and synthetic HAp crystallites. For the first time the in situ ultrastructural alteration of natural and synthetic HAp crystallites was captured in these experiments. The results presented in this thesis contribute to the fundamental understanding of the structure-property integrity mechanisms of natural materials, human dental tissues in particular. These results were reported in several first author publications in peer-reviewed journals, conference proceedings, and a book chapter.

Este trabalho teve o objetivo de avaliar a resistência adesiva de sistemas adesivos baseados no condicionamento prévio com ácido fosfórico (etch-and-rinse) e auto-condicionantes (de um e dois passos) ao substrato dental, submetido a diferentes condições de irradiação com laser de Er,Cr:YSGG. Amostras de esmalte e dentina foram obtidas do terço médio da face vestibular de 216 incisivos bovinos, divididos aleatoriamente em 18 grupos (n=12), de acordo com o substrato, (esmalte e dentina), sistema adesivo (etch-and-rinse, auto-condicionante de um passo e auto-condicionante de dois passos) e condições de irradiação (sem irradiação ? controle, irradiado com refrigeração ar-água e irradiado sem refrigeração ar-água). Em seguida foi realizada inclusão e polimento, sob refrigeração, com lixas de carbeto de silício de granulação decrescente 180, 320, 400 e 600 (por um minuto para padronização da camada de esfregaço). Para os grupos irradiados com laser de Er,Cr:YSGG (?=2,79 ?m, taxa de repetição de 20 Hz), com densidade de energia de 2,8 J/cm2, 12,5 mJ por pulso, potência média de 0,25W, com a ponta S75 de diâmetro de 750 ?m a 1 mm da superfície do substrato. A condição de refrigeração da irradiação foi realizada com 11 mL/min. Os corpos-de-prova foram confeccionados com a resina composta Filtek Z250, em forma de cone invertido para realização do teste de resistência adesiva. Após 24 h de armazenagem em água destilada a 37ºC, fez-se o ensaio de tração a 0,5 mm/min. Os dados foram comparados estatisticamente pelo método de ANOVA com significância de 5%. Os resultados obtidos mostraram que para o tecido esmalte foi observada diferença estatisticamente significante para os fatores adesivo e condições de irradiação empregadas, sem significância para a interação entre os fatores. A resistência de união do sistema adesivo auto-condicionante de dois passos e do sistema adesivo etch-and-rinse apresentam desempenho semelhante, sendo superiores ao sistema adesivo auto-condicionante de passo único. Ainda para esmalte, o efeito das condições de irradiação mostrou que os grupos sem irradiação com laser de Er,Cr:YSGG apresentaram maior resistência adesiva do que os grupos irradiados com refrigeração e sem refrigeração. Para o substrato dentina, houve diferença estatisticamente significante para os fatores de variação principais: sistemas adesivos, condição de irradiação com laser de Er,Cr:YSGG e para a interação adesivo X condição de irradiação. Quanto à comparação entre os sistemas adesivos testados na condição sem irradiação, os sistemas adesivos auto-condicionantes, de dois passos e o de um passo, não foram estatisticamente diferentes entre si, porém ambos forneceram resultados de resistência adesiva estatisticamente menores do que o sistema adesivo etch-and-rinse. Para as condições de irradiação com laser de Er,Cr:YSGG, com refrigeração e sem refrigeração, obteve-se valores de resistência adesiva maiores para o sistema auto-condicionante de dois passos do que para os demais sistemas. Deste modo, conclui-se que a irradiação com laser de Er,Cr:YSGG reduz a adesão ao esmalte independentemente do sistema adesivo utilizado e da condição de irradiação. Adicionalmente, observa-se que a irradiação de dentina com laser de Er,Cr:YSGG somente não altera a resistência adesiva do sistema auto-condicionante de dois passos, sendo deletéria aos demais adesivos testados. / The aim of this study was to assess the bond strength to dental substrate of adhesives based on previous etching with phosphoric acid (etch-and-rinse) and self-etching (one- and two-step) under different conditions of Er,Cr:YSGG laser irradiation. Enamel and dentin samples were obtained from the middle third of the buccal face of 216 bovine incisors, randomly divided in 18 groups (n=12), according to the substrate (enamel and dentin), adhesive system (etch-and-rinse, one-step self-etching and two-step self-etching) and irradiation condition (without irradiation ? control, irradiated with cooling, and irradiated without cooling). Next, the samples were included and polished, under cooling, with silicone carbide abrasive papers of decreasing grit #180, 320, 400 and 600 (for one minute to standardize the smear layer). For the irradiated groups with Er,Cr:YSGG laser, an energy density of 2.8 J/cm2, 12.5 mJ per pulse, mean power of 0.25W was selected. During laser irradiation, cooling was performed with 11 mL/min. Inverted cone-shaped test specimens were built-up with resin composite Filtek Z250, for performing the bond strength test. After 24 h storage in distilled water at 37ºC, the tensile test was performed at 0.5 mm/min. The results obtained show, for enamel, statistically significant difference for the factors adhesive and irradiation conditions used, without significance for the interaction among the factors. The bond strength of the 2-step self-etching and the etch-and-rinse adhesive system presented similar performance, being superior to that of one-step self-etching system. For enamel, the effect of the irradiation conditions showed that the groups without Er,Cr:YSGG laser irradiation presented higher bond strength than the groups irradiated with and without cooling. For the dentin substrate there was statistically significant difference for the main variation factors: adhesive systems, condition of Er,Cr:YSGG laser irradiation and for the interaction adhesive X irradiation condition. As regards the comparison among the adhesive systems tested under the condition without irradiation, the one-step and two-step self-etching adhesive systems did not differ statistically between them, but both provided statistically lower bond strength results than the etch-and-rinse adhesive system. For the condition of Er,Cr:YSGG laser irradiation with and without cooling, higher bond strength values were obtained for the two-step self-etching system than for the other systems. Therefore, it was concluded that Er,Cr:YSGG laser irradiation reduces the bond strength to enamel irrespective of the adhesive system used and the irradiation condition. Furthermore, it was observed that dentin irradiated with Er,Cr:YSGG laser did not alter the bond strength of the two-step adhesive system only, and it was deleterious to the other adhesives tested.

Este trabalho teve o objetivo de avaliar a resistência adesiva de sistemas adesivos baseados no condicionamento prévio com ácido fosfórico (etch-and-rinse) e auto-condicionantes (de um e dois passos) ao substrato dental, submetido a diferentes condições de irradiação com laser de Er,Cr:YSGG. Amostras de esmalte e dentina foram obtidas do terço médio da face vestibular de 216 incisivos bovinos, divididos aleatoriamente em 18 grupos (n=12), de acordo com o substrato, (esmalte e dentina), sistema adesivo (etch-and-rinse, auto-condicionante de um passo e auto-condicionante de dois passos) e condições de irradiação (sem irradiação ? controle, irradiado com refrigeração ar-água e irradiado sem refrigeração ar-água). Em seguida foi realizada inclusão e polimento, sob refrigeração, com lixas de carbeto de silício de granulação decrescente 180, 320, 400 e 600 (por um minuto para padronização da camada de esfregaço). Para os grupos irradiados com laser de Er,Cr:YSGG (?=2,79 ?m, taxa de repetição de 20 Hz), com densidade de energia de 2,8 J/cm2, 12,5 mJ por pulso, potência média de 0,25W, com a ponta S75 de diâmetro de 750 ?m a 1 mm da superfície do substrato. A condição de refrigeração da irradiação foi realizada com 11 mL/min. Os corpos-de-prova foram confeccionados com a resina composta Filtek Z250, em forma de cone invertido para realização do teste de resistência adesiva. Após 24 h de armazenagem em água destilada a 37ºC, fez-se o ensaio de tração a 0,5 mm/min. Os dados foram comparados estatisticamente pelo método de ANOVA com significância de 5%. Os resultados obtidos mostraram que para o tecido esmalte foi observada diferença estatisticamente significante para os fatores adesivo e condições de irradiação empregadas, sem significância para a interação entre os fatores. A resistência de união do sistema adesivo auto-condicionante de dois passos e do sistema adesivo etch-and-rinse apresentam desempenho semelhante, sendo superiores ao sistema adesivo auto-condicionante de passo único. Ainda para esmalte, o efeito das condições de irradiação mostrou que os grupos sem irradiação com laser de Er,Cr:YSGG apresentaram maior resistência adesiva do que os grupos irradiados com refrigeração e sem refrigeração. Para o substrato dentina, houve diferença estatisticamente significante para os fatores de variação principais: sistemas adesivos, condição de irradiação com laser de Er,Cr:YSGG e para a interação adesivo X condição de irradiação. Quanto à comparação entre os sistemas adesivos testados na condição sem irradiação, os sistemas adesivos auto-condicionantes, de dois passos e o de um passo, não foram estatisticamente diferentes entre si, porém ambos forneceram resultados de resistência adesiva estatisticamente menores do que o sistema adesivo etch-and-rinse. Para as condições de irradiação com laser de Er,Cr:YSGG, com refrigeração e sem refrigeração, obteve-se valores de resistência adesiva maiores para o sistema auto-condicionante de dois passos do que para os demais sistemas. Deste modo, conclui-se que a irradiação com laser de Er,Cr:YSGG reduz a adesão ao esmalte independentemente do sistema adesivo utilizado e da condição de irradiação. Adicionalmente, observa-se que a irradiação de dentina com laser de Er,Cr:YSGG somente não altera a resistência adesiva do sistema auto-condicionante de dois passos, sendo deletéria aos demais adesivos testados. / The aim of this study was to assess the bond strength to dental substrate of adhesives based on previous etching with phosphoric acid (etch-and-rinse) and self-etching (one- and two-step) under different conditions of Er,Cr:YSGG laser irradiation. Enamel and dentin samples were obtained from the middle third of the buccal face of 216 bovine incisors, randomly divided in 18 groups (n=12), according to the substrate (enamel and dentin), adhesive system (etch-and-rinse, one-step self-etching and two-step self-etching) and irradiation condition (without irradiation ? control, irradiated with cooling, and irradiated without cooling). Next, the samples were included and polished, under cooling, with silicone carbide abrasive papers of decreasing grit #180, 320, 400 and 600 (for one minute to standardize the smear layer). For the irradiated groups with Er,Cr:YSGG laser, an energy density of 2.8 J/cm2, 12.5 mJ per pulse, mean power of 0.25W was selected. During laser irradiation, cooling was performed with 11 mL/min. Inverted cone-shaped test specimens were built-up with resin composite Filtek Z250, for performing the bond strength test. After 24 h storage in distilled water at 37ºC, the tensile test was performed at 0.5 mm/min. The results obtained show, for enamel, statistically significant difference for the factors adhesive and irradiation conditions used, without significance for the interaction among the factors. The bond strength of the 2-step self-etching and the etch-and-rinse adhesive system presented similar performance, being superior to that of one-step self-etching system. For enamel, the effect of the irradiation conditions showed that the groups without Er,Cr:YSGG laser irradiation presented higher bond strength than the groups irradiated with and without cooling. For the dentin substrate there was statistically significant difference for the main variation factors: adhesive systems, condition of Er,Cr:YSGG laser irradiation and for the interaction adhesive X irradiation condition. As regards the comparison among the adhesive systems tested under the condition without irradiation, the one-step and two-step self-etching adhesive systems did not differ statistically between them, but both provided statistically lower bond strength results than the etch-and-rinse adhesive system. For the condition of Er,Cr:YSGG laser irradiation with and without cooling, higher bond strength values were obtained for the two-step self-etching system than for the other systems. Therefore, it was concluded that Er,Cr:YSGG laser irradiation reduces the bond strength to enamel irrespective of the adhesive system used and the irradiation condition. Furthermore, it was observed that dentin irradiated with Er,Cr:YSGG laser did not alter the bond strength of the two-step adhesive system only, and it was deleterious to the other adhesives tested.