In cases of compromised bone remodeling like osteoporosis, insufficient osseointegration occurs and results in implant failure. Implant retention relies on proper secondary fixation, which is developed during bone remodeling. This process is disrupted in metastatic bone diseases like osteoporosis. Osteoporosis is characterized low bone mass and bone strength resulting from either accelerated osteoclast-mediated bone resorption or impaired osteoblast-mediated bone formation. These two processes are not independent phenomena. In fact, osteoporosis can be viewed as a breakdown of the cellular communication connecting bone resorption to bone formation. Because bone remodeling occurs at temporally generated specific anatomical sites and at different times, local regulators that control cross-talk among the cells of the BRU are important. Previous studies show Ti implant surface characteristics like roughness, hydrophilicity, and chemistry influence the osteoblastic differentiation of human MSCs and maturation of OBs. Furthermore, microstructured Ti surfaces modulate the production of factors shown to be important in the reciprocal communication necessary for the maintenance of healthy bone remodeling. Semaphorin signaling proteins are known to couple the communication of osteoblasts to osteoclasts and are capable of stimulating bone formation or bone resorption depending on certain cues. Implant surface properties can be optimized to exploit these effects to favor rapid osseointegration in patients with osteoporosis.

Orientador: Francisley Ávila Souza / Banca: Idelmo Rabgel Garcia Junior / Banca: Osny Ferreira Júnior / Resumo: Objetivo: Avaliar o comportamento biológico do tecido ósseo ao redor de implantes manufaturados em liga Ti6Al-4V diante das superfícies modificadas por jateamento de óxido de alumínio seguido condicionamento ácido (SBAS) e a superfície usinada (MS). Material e Métodos: Dez coelhos receberam aleatoriamente 20 implantes de hexágono externo com dimensões de 4,0 mm de diâmetro e 10 mm de comprimento em suas tíbias direita e esquerda, sendo um implante de cada superfície em cada tíbia. Os implantes de ambos os grupos foram analisados através de microscopia eletrônica de varredura (MEV-EDX) e microscopia de força atômica (AFM). Discos com as mesmas superfícies foram submetidos à análise de rugosidade média (Ra) e secção transversal. Nos períodos de 3 e 6 semanas foi mensurado o coeficiente de estabilidade primária por meio da análise de frequência por ressonância (ISQ), seguido da eutanásia dos animais. As peças obtidas foram submetidas ao processamento de inclusão em resina fotopolimerizável e levadas ao micrótomo para corte calcificado. Foi mensurada em porcentagem a extensão linear de contato entre tecido ósseo mineralizado e implante (ELCOMI) e área óssea neoformada (AON). Os dados foram submetidos à análise de variância e ao teste t de Tukey. Resultados: MEV e AFM mostraram diferenças na topografia das superfícies. Os valores médios obtidos pelas análises de frequência por ressonância, Ra e secção transversal para SBAS foram estatisticamente superiores (p<0,05) quando comparad... (Resumo completo, clicar acesso eletrônico abaixo) / Objective: Evaluate the biological behavior of bone tissue around implants manufactured in Ti-6Al-4V alloy before surfaces modified by aluminum oxide blasting followed by acid etching (SBAS) and machined surface (MS).Material and Methods: Ten rabbits randomly received 20 external hexagon implants with dimensions of 4.0 mm in diameter and 10 mm in length on theirright and left tibiae, one implant of each surface on each tibia. The implants of both groups were analyzed by scanning electron microscopy (SEM-EDX) and atomic force microscopy (AFM). Disks withthe same surfaces were submitted to analysis of average roughness (Ra) and cross section. In the periods of 3 and 6 weeks, the primary stability coefficient was measured by means of resonance frequency analysis (ISQ), followed by euthanasia of the animals. The obtained pieces were submitted to the inclusion processing in photopolymerizable resin and taken to the microtome for cutting without decalcification. The bone interface contact (BIC) and neoformed bone area (NBA) was measured in percentage. Data were submitted to analysis of variance and Tukey t test. Results: SEM and AFM showed differences in surface topography. The mean values obtained by the resonance, Ra and cross sectionanalyzes for SBAS were statistically higher (p <0.05) when compared to MS. The mean values in percentage of BICfor SBAS were 69.65 and 71.56 respectively for 3 and 6 weeks, while for MS the mean values were 54.06and 55.99 respectively for the same periods. The mean values of NBAin the SBAS group were 69.86 and 79.59 for the 3 and 6 week periods, while forMS the mean values were 59.59and 74.32, respectively, in the same groups. There were statistically superior differences (p <0.05) in SBAS implants for BICin the 3 and 6 week periods and AON in the 3 week period. Conclusion: The surface modification of the SBAS implants providedchanges... (Complete abstract electronic access below) / Mestre

In this master thesis the connection between the two major parts of an osseointegrated prosthesis system for lower limb amputees has been investigated by finite element (FE) analysis. The prosthesis system is developed by Integrum and the current design consists of a fixture, which is integrated in the residual bone, an abutment that penetrates the skin and an abutment screw that holds the parts together. The connection between the fixture and the abutment has a hexagonal section and a press-fit section that together form the connection. Due to wear and fracture problems it is desired to improve the connection. A tapered connection could be an alternative and three different taper angles, the effect of the length of the taper and the smoothness of the outer edge of a tapered fixture have been investigated. The results show that the taper has potential to function well and that a longer connection will give lower stresses in the system, but further investigations are needed.

An understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is critical in the design of next generation implants to improve osseointegration and clinical success rates. Critical to the process of osseointegration is the rapid establishment of a patent neovasculature in the peri-implant space to allow for the delivery of oxygen, nutrients, and progenitor cells. The central aim of this thesis is to understand how biomaterials regulate cellular and host tissue response to elicit a pro-angiogenic microenvironment at the implant/tissue interface. To address this question, the studies performed in this thesis aim to 1) determine whether biomaterial surface properties can modulate the production and secretion of pro-angiogenic growth factors by cells, 2) determine the role of integrin and VEGF-A signaling in the angiogenic response of cells to implant surface features, and 3) to determine whether neovascularization in response to an implanted biomaterial can be modulated in vivo. The results demonstrate that biomaterial surface microtopography and surface energy can increase the production of pro-angiogenic growth factors by osteoblasts and that these growth factors stimulate the differentiation of endothelial cells in a paracrine manner and the results suggest that signaling through specific integrin receptors affects the production of angiogenic growth factors by osteoblast-like cells. Further, using a novel in vivo model, the results demonstrate that a combination of a rough surface microtopography and high surface energy can improve bone-to-implant contact and neovascularization. The results of these studies also suggest that VEGF-A produced by osteoblast-like cells has both an autocrine and paracrine effect. VEGF-A silenced cells exhibited reduced production of both pro-angiogenic and osteogenic growth factors in response to surface microtopgraphy and surface energy, and conditioned media from VEGF-A silenced osteoblast-like cell cultures failed to stimulate endothelial cell differentiation in an in vitro model. Finally, the results show that by combining angiogenic and osteogenic biomaterials, new bone formation and neovascularization can be enhanced. Taken together, this research helps to provide a better understanding of the role of material properties in cell and host tissue response and will aid in the improvement of the design of new implants.

Osseointegration, or the direct integration of an implant into bone tissue, is necessary for implant success. Titanium is commonly used clinically in dental and orthopaedic implants because of its passivating oxide layer, which facilitates osseointegration, and its mechanical properties such as a modulus of elasticity similar to bone. Diverse studies have shown that surface microtopography, chemistry, and surface energy affect osteoblast behavior. The problem with these studies is that they access the average behavior of a culture in response to a substrate and not the behavior of individual cells. The objective of this study was to develop a method for correlating the behavior of individual cells with the characteristics of the surface underneath them. More specifically, this work developed a method to correlate integrin beta-1 (β1) expression with the surface characteristics under individual cells. Integrins are cell surface receptors that bind to specific proteins in the extracellular matrix adsorbed on the implant surface. Previous work has shown that expression of certain integrins is increased when osteoblasts on titanium substrates develop a more differentiated phenotype, and that integrin β1 is necessary for osteoblast response to roughness on titanium substrates. This study used molecular beacons specific to integrin β1 to quantify integrin β1 expression of MG63 cells cultured on titanium disks. A template was designed to coordinate the location of cells using fluorescence microscopy and scanning electron microscopy (SEM) in reference to laser etchings on the disks. After live cell imaging, cells were fixed, dried, and critical point dried for focused ion beam (FIB) milling. Transmission electron microscopy (TEM) sections of cells identified with high and low integrin β1 molecular beacon intensity were milled, and cells with high and low integrin β1 molecular beacon intensity were also serial sectioned. While our TEM results were inconclusive, SEM images from serial sectioning showed contact points between the cell body and the substrate, consistent with previous results. Cells cultured on pretreatment (PT) or sandblasted acid etched (SLA) titanium surfaces were also serial sectioned, showing that cells on SLA surfaces have more regions of contact between the cells and the substrate than cells on PT surfaces. This work is significant as it is the first study to develop a method to correlate individual cell behavior with the substrate surface characteristics under the individual cells. Previous studies have reported the average cell behavior in response to their substrates, while this work allows for the study of substrate surface characteristics that positively affect integrin β1 expression in individual cells. Further optimization of the fluorescence imaging process and FIB milling process could be done, and the method developed in this study could be used in future studies to investigate surface characteristics after using other fluorescent analyses of cell behavior, such as immunocytochemistry.

Modifications of biomaterial surface properties are employed to increase osteoblast differentiation and bone formation. Microtextured metallic surfaces promote osteoblast differentiation and high surface energy- achieved by controlling surface hydrocarbon contamination- increases osteoblast differentiation and peri-implant bone formation. Recombinant human bone morphogenic protein 2 (BMP2) is approved to induce bone formation in a number of applications. It is used clinically in combination with biomaterials to improve peri-implant bone formation and osseointegration. The amount of BMP2 that is required is large and inflammatory (swelling/seroma) and bone-related (ectopic bone/bone resorption) complications have been reported after BMP2 treatment. The aim of this study was to examine potential deleterious effects of BMP2 on the inflammatory environment and apoptosis of osteoblasts. Surface roughness and energy decreased pro-inflammatory interleukins and increased anti-inflammatory interleukins. In contrast, BMP2 abolished the surface effect, increasing pro-inflammatory interleukin (IL) 6, IL8, and IL17 in a surface roughness-dependent fashion and decreasing anti-inflammatory IL10 on rough surfaces. 5Z-7-Oxozeaenol and Dorsomorphin, but not H-8, blocked the effect of BMP2 on IL1A expression. There was an increase in expression of IL6 when treated with BMP2 for the control and H-8 groups, but both 5Z-7-Oxozeaenol and Dorsomorphin blocked the effect. Both 5Z-7-Oxozeaenol and H-8 blocked the effect of BMP2 on IL10 expression. BMP2 treatment had little effect on apoptosis in human mesenchymal stem cells (MSCs). Exogenous BMP2 had no effect on TUNEL. Caspase-3 activity was increased only at 200ng/ml BMP2. BAX/BCL2 decreased in MSCs treated with 50 and 100ng/ml BMP2. In contrast, BMP2 increased caspase-3 activity and TUNEL at all doses in normal human osteoblasts (NHOst). BAX/BCL2 increased in NHOst treated with BMP2 in a dose-dependent manner. Cells treated with 200 ng/ml BMP2 had an 8-fold increase in BAX/BCL2 expression in comparison with untreated cells. Similarly, BMP2 increased DNA fragmentation in NHOst cells. The BMP2-induced increase in DNA fragmentation was eliminated by 5-Z7-Oxozeaenol and Dorsomorphin. The results suggest that while surface features modulate an initial controlled inflammatory response, the addition of BMP2 induces a pro-inflammatory response. The effect of BMP2 on apoptosis depends on cell maturation state, inducing apoptosis in committed osteoblasts. BMP2 together with microtextured orthopaedic and dental implants may increase inflammation and possibly delay bone formation. Dose, location, and delivery strategies are important considerations in BMP2 as a therapeutic and must be optimized to minimize complications.

M. Tech. Biomedical Technology. / Aims to gain more insights into the effect of a specific human derived BMP-complex, standardised according to the human BMP-2 content, by loading it at concentrations higher than the concentration of the morphogen found in human (hDBM), in order to determine changes in the osteogenic response.

Current orthopedic implant technologies used suffer from slow rates of osseointegration, short lifetime, and lack of mechanical integrity as a result of poorly controlled cell-surface interactions. Recent biologically-inspired surface strategies (biomimetic) have focused on mimicking the biofunctionality of the extracellular matrix (ECM) by using short, adhesive oligopeptides, such as arginine-glycine-aspartic acid (RGD) present in numerous ECM components. However, these strategies have yielded mixed results in vivo and marginal bone healing responses. The central goal of this dissertation project was to engineer bioactive surfaces that specifically target integrin receptors important for osteogenic functions in order to improve bone tissue repair. In order to create integrin-specific interfaces, integrin-specific ligands reconstituting the fibronectin (FN) secondary/tertiary structure were first engineered and functionalized on material surfaces using several robust presentation schemes. We demonstrated that FN-mimetic-functionalized surfaces that directed α5β1 binding enhanced osteoblast and stromal cell integrin binding and adhesion, osteogenic signaling, and osteoblastic differentiation compared to various other RGD-based ligand-functionalized surfaces. Next, we investigated the effect of integrin-specific biointerfaces to modulate bone healing in a rat tibia implant bone model. We demonstrated, using a robust polymer brush system, that bioactive coatings on titanium implants that conferred high α5β1 integrin specificity in vitro enhanced bone formation and implant integration in vivo. Moreover, we showed that integrin specificity can be engineered using different immobilization schemes, including clinically-relevant ligand dip-coating, and promote the same robust in vivo effect. Furthermore, we investigate the synergistic roles of integrin specificity and ligand clustering on cell response by engineering biointerfaces presenting trimeric and pentameric "heads" of FNIII7-10 with nanoscale spacing. Integrin-specific ligand clustering supported α5β1-specific binding and cell adhesion and enhanced implant osseointegration in vivo compared to monovalent FNIII7-10 or non-functionalized biointerfaces. In summary, the FN-mimetic integrin-specific biointerfaces engineered in this thesis provide a clinically-relevant material surface strategy to modulate tissue healing responses. In addition, these results contribute to our greater understanding of how two specific material design parameters, integrin binding specificity and clustered ligand presentation, contribute individually and synergistically toward directing cell and tissue function.

Bone response is one of the key determining factors in the overall success of biomaterials intended for bone regeneration and osseointegration. Understanding the formation of bone at an implant surface may lead to the improved design of biomaterials for the future. However, due to the inhomogeneity of bone tissue at an interface, two-dimensional images often lack detail on the interfacial complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar nano length scale. While current analysis methods, such as X-ray tomography, transmission electron microscopy, focused ion beam microscopy and scanning electron microscopy, provide a basis for analysing biomaterials and biointerfaces, they are incapable of doing so with both nanometre resolution and three-dimensional clarity. In contrast, electron tomography may be used to characterize the three-dimensional structure of biomaterials and their interfaces to bone with nanometre resolution. In this work, hydroxyapatite scaffolds, and laser-modified titanium and Ti6Al4V implants were studied in contact with human or rabbit bone. Z-contrast electron tomography revealed that the orientation of collagen in bone apposing hydroxyapatite, titanium and Ti6Al4V implants is consistently parallel to the implant surface, where the bioactive layer that precipitates on HA is oriented perpendicular to the implant surface. With this method, complete three-dimensional nanoscale osseointegration of titanium-based implants was also established. The extension of this technique from interfacial analyses to the design of biomaterials provided an understanding of the pore structure of mesoporous titania. In further investigations, the open three-dimensional pore network, as revealed by electron tomography, showed promise as a coating that improves implant osseointegration and enables site-specific drug-delivery from an implant surface. In summary, it was demonstrated that two-dimensional characterization techniques were insufficient for the investigation of nanostructured biomaterials, as well as their interfaces to bone. Visualizing biointerfaces and biomaterials with nanometre precision in three-dimensions can expose new fundamental information on materials properties and bone response, enabling better design of biomaterials for the future.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.