The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

Metallic bone implant devices are commonly used to tackle a wide array of bone failures in human patients. The success of such implants relies on the biomechanical and functional bonding between the living bone tissue and implant, a process defined as osseointegration. However, the mechanism of osseointegration is still under debate in the scientific community. One efficient method to help understand this complex process is to characterize the interface between human bones and implant devices after the osseointegration has been established, while another approach is to visualize mineralization in real-time under simulated body conditions. Both of these approaches to understand mineralization have been explored in this thesis. Firstly, due to the inhomogeneous nature of bone and complex topography of implant surfaces, a suitable sample geometry for three-dimensional (3D) characterization was required to fully understand osseointegration. Electron tomography has been proven as an efficient technique to visualize the nanoscale topography of bone-implant interface in 3D. However, resulting from the thickness and shadowing effects of conventional transmission electron microscope (TEM) lamellae at high tilt angles and the limited tilt-range of TEM holders, “missing wedge” artifacts limit the resolution of final reconstructions. In Chapter 3, the exploration of a novel sample geometry to explore osseointegration is reported. Here, on-axis electron tomography based on a needle-shaped sample was applied to solve the problem of the “missing wedge”. This resulted in a near artifact-free 3D visualization of the structure of human bone and laser-modified titanium implant, showing bone growth into the nanotopographies of the implant surface and contributing to the evolution of the definition of osseointegration towards nano-osseointegration. One of the key issues regarding the mechanism of osseointegration that remains is that of the chemical structure at the implant interface, namely distribution of calcium-based and carbon-based components at the interface and their origins. Thus, the second objective of this thesis aimed to push characterization techniques further to four dimensions (4D), by incorporating chemical information as the fourth dimension after the spatial X,Y,Z coordinates. In Chapter 4, correlative 4D characterization techniques including electron energy-loss spectroscopy (EELS) tomography and atom probe tomography (APT) and other spectroscopy techniques were used to probe the nanoscale chemical structure of the bone-implant interface. This work uncovered a transitional biointerphase at the bone-implant interface, consisting of morphological and chemical differences compared to bone away from the interface. Also, a TiN layer between the surface oxide and bulk metal was identified in the laser-modified commercial dental implant. Both findings have implications for the immediate and long-term osseointegration. Since bone formation at the implant interface is a dynamic process, which includes calcium phosphates (CaP) biomineralization as a basis of these reactions, the third objective of this work focused on exploring real-time mineralization processes. Liquid-phase transmission electron microscopy (LP-TEM) is a promising technique to enable real-time imaging with nanoscale spatial resolution and sufficient temporal resolution. In Chapter 5, by using this technique, we present the first real-time imaging of CaP nucleation and growth, which is a direct evidence to demonstrate that CaP mineralization occurs by particle attachment. Overall, this thesis has applied state-of-the-art advanced microscopy techniques to enhance the knowledge and understanding of osseointegration mechanisms by investigating established biointerfaces and real-time mineralization. The developed correlative 4D tomography workflow is transferable to study other interfacial applications in materials science and biological systems, while the LP-TEM work forms a basis for further mineralization research. / Thesis / Doctor of Philosophy (PhD)

Áreas edêntulas com severa reabsorção óssea têm sido reabilitadas com próteses fixas instaladas sobre implantes curtos, evitando que os pacientes sejam submetidos a cirurgias de reconstrução óssea. Este estudo retrospectivo descreve o comportamento de implantes curtos avaliados em um período de 7 anos. A amostra foi composta por 70 pacientes, de ambos os gêneros, que receberam 136 implantes, de 6 ou 8mm de comprimento (Straumann® bone level e tissue level standard plus) sobre os quais foram instaladas próteses unitárias e múltiplas, cimentadas e parafusadas. Foram realizadas avaliações clínicas e radiográficas mensurando a estabilidade do implante por frequência de ressonância, perda óssea marginal por meio de radiografias panorâmicas, índices de placa e de sangramento marginal, taxas de sobrevivência do implante e da prótese. Dois implantes instalados na mandíbula foram perdidos, resultando em uma taxa de sobrevivência de 98,3%. A taxa de sobrevivência das próteses foi de 100%; a média de perda óssea foi de - 0,28mm; a estabilidade média dos implantes foi de 76,515 ISQ; as médias dos índices de placa e de sangramento foram de 32,83% e 33,21%, respectivamente. A estabilidade do implante foi maior na mandíbula que na maxila (p = 0,006). Os resultados obtidos nesta pesquisa, possibilitam afirmar que os implantes curtos podem ser utilizados com segurança dentro de suas indicações específicas. / Edentulous areas with severe bone resorption have been rehabilitated with fixed prostheses installed on short implants, avoiding surgeries of bone reconstruction. This retrospective study describes the behavior of short implants evaluated during 7 years. The sample consisted of 70 patients, of both genders, who received 136 implants, 6 or 8mm in length (Straumann® bone level and tissue level standard plus) on which single and multiple, cemented and screwed prostheses were installed. Clinical and radiographic evaluations were performed by measuring implant stability by resonance frequency, marginal bone loss by panoramic radiographs, plaque and marginal bleeding rates, the implant and prostheses survival rates. Two implants installed in mandible were lost, resulting in a survival rate of 98,3%. The prostheses survival rate was 100%; mean bone loss was -0,28mm; mean implants stability was 76,52 ISQ; mean plaque and bleeding indexes were 32,83% and 33,21%, respectively. The implant stability was higher in the mandible than in the maxilla (p = 0.006). The results obtained in this research make it possible to state that short implants can be used safely within their specific indications.

The trend for designing of a titanium implant explored using different chemical compositions and crystallinity materials until people realized that the implant surface character was another important factor affecting the rate and extent of osseointegartion. Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5μm. An additional mesoporous titania top layer was created that followed the contour of the macropores and having 100–200 nm thickness and a pore diameter of 10 nm. Thus, a coherent laminar titania surface layer was obtained producing a hierarchical macro- and mesoporous surface. The interfacial bonding between the surface layers and the titanium matrix was characterized by a scratch test that confirmed a stable and strong bonding of the laminar titania surface layers upon titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy (SEM). A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, when compared with the titania surface with solo porosity scale topography. For the in vivo results of the evaluation of osseointegration, an argon ion beam polishing technique was applied to prepare the cross sections of implants feasible for the high resolution SEM investigation. The interfacial microstructure between newly formed bone and implants with four modified surfaces including the new hierarchical macro- and mesoporous implant surface retrieved after in vivo tests were characterized. By this approach it has become possible to directly observe early bone formation, the increase of bone density, and the evolution of bone structure. The two bone growth mechanisms, distant osteogenesis and contact osteogenesis, can also be distinguished. These direct observations give, at microscopic level, a better view of osseointegration and explain the functional mechanisms of various implant surfaces for osseointegration. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>

Áreas edêntulas com severa reabsorção óssea têm sido reabilitadas com próteses fixas instaladas sobre implantes curtos, evitando que os pacientes sejam submetidos a cirurgias de reconstrução óssea. Este estudo retrospectivo descreve o comportamento de implantes curtos avaliados em um período de 7 anos. A amostra foi composta por 70 pacientes, de ambos os gêneros, que receberam 136 implantes, de 6 ou 8mm de comprimento (Straumann® bone level e tissue level standard plus) sobre os quais foram instaladas próteses unitárias e múltiplas, cimentadas e parafusadas. Foram realizadas avaliações clínicas e radiográficas mensurando a estabilidade do implante por frequência de ressonância, perda óssea marginal por meio de radiografias panorâmicas, índices de placa e de sangramento marginal, taxas de sobrevivência do implante e da prótese. Dois implantes instalados na mandíbula foram perdidos, resultando em uma taxa de sobrevivência de 98,3%. A taxa de sobrevivência das próteses foi de 100%; a média de perda óssea foi de - 0,28mm; a estabilidade média dos implantes foi de 76,515 ISQ; as médias dos índices de placa e de sangramento foram de 32,83% e 33,21%, respectivamente. A estabilidade do implante foi maior na mandíbula que na maxila (p = 0,006). Os resultados obtidos nesta pesquisa, possibilitam afirmar que os implantes curtos podem ser utilizados com segurança dentro de suas indicações específicas. / Edentulous areas with severe bone resorption have been rehabilitated with fixed prostheses installed on short implants, avoiding surgeries of bone reconstruction. This retrospective study describes the behavior of short implants evaluated during 7 years. The sample consisted of 70 patients, of both genders, who received 136 implants, 6 or 8mm in length (Straumann® bone level and tissue level standard plus) on which single and multiple, cemented and screwed prostheses were installed. Clinical and radiographic evaluations were performed by measuring implant stability by resonance frequency, marginal bone loss by panoramic radiographs, plaque and marginal bleeding rates, the implant and prostheses survival rates. Two implants installed in mandible were lost, resulting in a survival rate of 98,3%. The prostheses survival rate was 100%; mean bone loss was -0,28mm; mean implants stability was 76,52 ISQ; mean plaque and bleeding indexes were 32,83% and 33,21%, respectively. The implant stability was higher in the mandible than in the maxilla (p = 0.006). The results obtained in this research make it possible to state that short implants can be used safely within their specific indications.

O grande desafio da Implantodontia é alcançar resultados a longo prazo com maior preservação dos tecidos ósseos, resposta adequada dos tecidos conjuntivos e manutenção da estabilidade secundária adequada para a retenção de próteses. Estes fatores evitam a perda indesejável tanto do tratamento reabilitador quanto dos tecidos ósseos de suporte. A região extraoral apresenta menores espessuras ósseas em comparação às regiões de maxila e mandíbula, especialmente no osso temporal. Portanto, os seguintes aspectos foram investigados: (i) Análise pelo Método dos Elementos Finitos (FEA) de 16 modelos virtuais, com aplicação de forças estáticas de tensão e compressão nos pontos médios entre os implantes. Três implantes maxilofaciais extraorais foram posicionados no osso temporal, onde um deles foi posicionado sobre o processo mastóide. Este estudo foi totalmente realizado em ambiente computacional. Foram empregados dois tipos de conexões protéticas e intermediários retos de conicidades 20º e 45º, posicionados virtualmente no modelo de osso temporal, com paralelismo entre eles. Foi desenhada uma barra virtual para retenção protética do tipo barra-clipe da prótese auricular. Foram consideradas as melhores configurações aquelas apresentando distribuição uniforme e branda das tensões de von Mises sobre o tecido ósseo adjacente aos implantes. (ii) Confecção de corpos de prova com graus variáveis de pigmentação para (a) melhorar a reprodutibilidade da coloração (b) entender e reduzir o processo de descoloração para melhorar a longevidade das próteses. Um elastômero maxilofacial tipo A foi pigmentado intrinsecamente de acordo com os seis tons da Escala de Fitzpatrick. Uma técnica de pigmentação foi desenvolvida a partir dos resultados obtidos a partir de uma Escala de Fitzpatrick impressa em papel e dos pigmentos intrínsecos de cores primárias. Este dimetil-metilvinil-siloxano reforçado por platina A 223-30 (Factor II, Inc., Lakeside, AZ, USA) apresenta dureza \"Shore-A\" 30. As amostras foram expostas a fotoenvelhecimento acelerado, e sua degradação mapeada com Espectroscopia UV/Vis/NIR. Concluiu-se que: (i) Os implantes com conexão cone Morse plataforma 3.7 tem melhor comportamento biomecânico com intermediários de 45o e altura de 2mm. Sugere-se planejamento cirúrgico visando a instalação a 3 horas para o temporal esquerdo e 9 horas para o temporal direito, mais 6 horas (mastóide). Implantes com conexão cone Morse plataforma 4.0 apresentaram comportamento biomecânico desejável com intermediários de 20o e alturas de 2mm or 4mm. Para implantes com conexão cone Morse plataforma 4.0, sugere-se a instalação a 6-9-12 horas para o temporal direito, e 12-6-3 para o esquerdo, visando comportamento biomecânico adequado. Sugere-se que os implantes extraorais Brånemark hexágono externo com plataforma 4.1 regular ou expandida sejam preferencialmente empregados com intermediários de 20o. (ii) O SiO2 5% pode ser adicionado à preparação do silicone para retardar o desbotamento da prótese maxilofacial. O TiO2 traz mudanças visuais significativas e pode ser considerado como opacificador. / Major challenges in Implantology include: achieving longevity of implants, bone tissue preservation, connective tissue suitable answer and maintenance of secondary stability in order to retain prosthesis. These factors avoid the undesirable loss of both rehabilitation treatment and supporting bone tissues. The extraoral region has less bone depth compared to the maxilla and mandible regions, especially for the temporal bone. Therefore, the following aspects were investigated: (i) Finite Elements Analysis (FEA) of 16 virtual models, with static application of tension and compression forces in medium points between implants. Three extraoral maxillofacial implants were placed on the temporal bone, which one of them was placed on the mastoid process. This study was made entirely in a computational environment. Two prosthetic connection types and straight Intermediários were employed, with conicities of 20º and 45º, placed virtually on the temporal bone model, mutually parallel. A virtual bar for bar-clip prosthetic retention of auricular prosthesis was designed. The best configurations were considered to be the ones presenting uniform and mild von Mises tension distribution over the bone tissue around the implants. (ii) Generation of samples with varying degree of pigmentation to (a) improve the reproducibility of the colouration (b) understand and minimise the discolouration process to improve prosthetics longevity. A maxillofacial elastomer type A was intrinsically pigmented according to the six tones of the Fitzpatrick Scale. A pigmentation technique was developed from spectroscopy outcomes of paper printed Fitzpatrick Scale and the primary colors pigments. This platinum reinforced dimethyl methylvinyl siloxane A 223-30 (Factor II, Inc., Lakeside, AZ, USA) presents \"Shore-A\" hardness 30. The samples were exposed to accelerated photo aging, and their degradation mapped with UV/Vis/NIR Spectroscopy. The effects of exclusion of oxygen and inclusion of nanoparticle fillers during prosthetic formulation were investigated. It was concluded that: (i) Morse taper prosthetic connection 3.7 platform implants, should be employed with 45o abutments with 2mm height. The surgical planning shall be for placement 3 hours for the left temporal bone and 9 hours for the right, and in 6 hours (mastoid). Morse taper prosthetic connection 4.0 platform implants behaved biomechanically desirably with 20o abutments in 2mm or 4mm heights. For the Morse taper connection 4.0 platform implants, their placement shall be in 6-9-12 hours, for the right temporal, and 12-3-6, for the left temporal, in order to achieve proper biomechanical behaviour. The Brånemark extraoral external hexagon implant, with 4.1 platform (normal or expanded) should preferrably be used with 20o abutments. (ii) The SiO2 5% may be added to the silicone preparation in order to slow maxillofacial prosthesis colour fading. The TiO2 brings visually significant changes and may be considered as an opacifier.

O grande desafio da Implantodontia é alcançar resultados a longo prazo com maior preservação dos tecidos ósseos, resposta adequada dos tecidos conjuntivos e manutenção da estabilidade secundária adequada para a retenção de próteses. Estes fatores evitam a perda indesejável tanto do tratamento reabilitador quanto dos tecidos ósseos de suporte. A região extraoral apresenta menores espessuras ósseas em comparação às regiões de maxila e mandíbula, especialmente no osso temporal. Portanto, os seguintes aspectos foram investigados: (i) Análise pelo Método dos Elementos Finitos (FEA) de 16 modelos virtuais, com aplicação de forças estáticas de tensão e compressão nos pontos médios entre os implantes. Três implantes maxilofaciais extraorais foram posicionados no osso temporal, onde um deles foi posicionado sobre o processo mastóide. Este estudo foi totalmente realizado em ambiente computacional. Foram empregados dois tipos de conexões protéticas e intermediários retos de conicidades 20º e 45º, posicionados virtualmente no modelo de osso temporal, com paralelismo entre eles. Foi desenhada uma barra virtual para retenção protética do tipo barra-clipe da prótese auricular. Foram consideradas as melhores configurações aquelas apresentando distribuição uniforme e branda das tensões de von Mises sobre o tecido ósseo adjacente aos implantes. (ii) Confecção de corpos de prova com graus variáveis de pigmentação para (a) melhorar a reprodutibilidade da coloração (b) entender e reduzir o processo de descoloração para melhorar a longevidade das próteses. Um elastômero maxilofacial tipo A foi pigmentado intrinsecamente de acordo com os seis tons da Escala de Fitzpatrick. Uma técnica de pigmentação foi desenvolvida a partir dos resultados obtidos a partir de uma Escala de Fitzpatrick impressa em papel e dos pigmentos intrínsecos de cores primárias. Este dimetil-metilvinil-siloxano reforçado por platina A 223-30 (Factor II, Inc., Lakeside, AZ, USA) apresenta dureza \"Shore-A\" 30. As amostras foram expostas a fotoenvelhecimento acelerado, e sua degradação mapeada com Espectroscopia UV/Vis/NIR. Concluiu-se que: (i) Os implantes com conexão cone Morse plataforma 3.7 tem melhor comportamento biomecânico com intermediários de 45o e altura de 2mm. Sugere-se planejamento cirúrgico visando a instalação a 3 horas para o temporal esquerdo e 9 horas para o temporal direito, mais 6 horas (mastóide). Implantes com conexão cone Morse plataforma 4.0 apresentaram comportamento biomecânico desejável com intermediários de 20o e alturas de 2mm or 4mm. Para implantes com conexão cone Morse plataforma 4.0, sugere-se a instalação a 6-9-12 horas para o temporal direito, e 12-6-3 para o esquerdo, visando comportamento biomecânico adequado. Sugere-se que os implantes extraorais Brånemark hexágono externo com plataforma 4.1 regular ou expandida sejam preferencialmente empregados com intermediários de 20o. (ii) O SiO2 5% pode ser adicionado à preparação do silicone para retardar o desbotamento da prótese maxilofacial. O TiO2 traz mudanças visuais significativas e pode ser considerado como opacificador. / Major challenges in Implantology include: achieving longevity of implants, bone tissue preservation, connective tissue suitable answer and maintenance of secondary stability in order to retain prosthesis. These factors avoid the undesirable loss of both rehabilitation treatment and supporting bone tissues. The extraoral region has less bone depth compared to the maxilla and mandible regions, especially for the temporal bone. Therefore, the following aspects were investigated: (i) Finite Elements Analysis (FEA) of 16 virtual models, with static application of tension and compression forces in medium points between implants. Three extraoral maxillofacial implants were placed on the temporal bone, which one of them was placed on the mastoid process. This study was made entirely in a computational environment. Two prosthetic connection types and straight Intermediários were employed, with conicities of 20º and 45º, placed virtually on the temporal bone model, mutually parallel. A virtual bar for bar-clip prosthetic retention of auricular prosthesis was designed. The best configurations were considered to be the ones presenting uniform and mild von Mises tension distribution over the bone tissue around the implants. (ii) Generation of samples with varying degree of pigmentation to (a) improve the reproducibility of the colouration (b) understand and minimise the discolouration process to improve prosthetics longevity. A maxillofacial elastomer type A was intrinsically pigmented according to the six tones of the Fitzpatrick Scale. A pigmentation technique was developed from spectroscopy outcomes of paper printed Fitzpatrick Scale and the primary colors pigments. This platinum reinforced dimethyl methylvinyl siloxane A 223-30 (Factor II, Inc., Lakeside, AZ, USA) presents \"Shore-A\" hardness 30. The samples were exposed to accelerated photo aging, and their degradation mapped with UV/Vis/NIR Spectroscopy. The effects of exclusion of oxygen and inclusion of nanoparticle fillers during prosthetic formulation were investigated. It was concluded that: (i) Morse taper prosthetic connection 3.7 platform implants, should be employed with 45o abutments with 2mm height. The surgical planning shall be for placement 3 hours for the left temporal bone and 9 hours for the right, and in 6 hours (mastoid). Morse taper prosthetic connection 4.0 platform implants behaved biomechanically desirably with 20o abutments in 2mm or 4mm heights. For the Morse taper connection 4.0 platform implants, their placement shall be in 6-9-12 hours, for the right temporal, and 12-3-6, for the left temporal, in order to achieve proper biomechanical behaviour. The Brånemark extraoral external hexagon implant, with 4.1 platform (normal or expanded) should preferrably be used with 20o abutments. (ii) The SiO2 5% may be added to the silicone preparation in order to slow maxillofacial prosthesis colour fading. The TiO2 brings visually significant changes and may be considered as an opacifier.