Le cartilage articulaire est composé de chondrocytes et d'une matrice extracellulaire organisés de manière stratifiée dans l'épaisseur du tissu. Ce tissu ne se régénère pas de manière efficace après une lésion. L'objectif de ce travail est de construire par pulvérisation des hydrogels à base d'alginate et de film multicouches de polyélectrolytes pour créer in vitro un néotissu pouvant combler des lésions de cartilage articulaire. La méthode a été validée en observant une bonne viabilité et une synthèse matricielle par les cellules, et de meilleures propriétés mécaniques des hydrogels pulvérisés à 0,9 bar par rapport au moulage. Après la pulvérisation de cellules souches mésenchymateuses, les résultats ont montré une bonne viabilité et une différenciation des cellules. Puis, des hydrogels bistratifiés ont été construits et cultivés jusqu'à 56 jours sans dissociation des couches et sans migration des cellules. Enfin, les hydrogels ont été fonctionnalisés en modifiant la composition des couches et en y appliquant des stimulations mécaniques. Les propriétés mécaniques des hydrogels varient en fonction de leur composition et sont meilleures pour ceux stratifiés. De plus, leur stimulation mécanique a permis de potentialiser l'effet du biomatériau sur la différenciation des cellules. En conclusion, cette étude montre que des cellules souches mésenchymateuses ensemencées dans un hydrogel bistratifié pulvérisé sont fonctionnelles en termes de différenciation chondrocytaire et de synthèse matricielle. Les propriétés mécaniques des hydrogels stratifiés ne sont pas altérées. De plus, la stimulation mécanique a potentialisé la différenciation des cellules / The articular cartilage is composed of chondrocytes and of a specific extracellular matrix which are organized depth-dependently. The tissue did not have an efficient self-renewal of defects. The purpose of this study is to build up layer-by-layer a stratified hydrogel by alternating gels and multilayers polyelectrolytes film spraying, in order to obtain a neotissu in vitro to fill lesions. First, the process was validated by observing a good cells viability and matrix synthesis, and stronger mechanical behaviors of sprayed hydrogels compared to molded one. Secondly, after their spraying, mesenchymal stem cells still have a good viability and their differentiation potential. Then, bistratified scaffolds were built up and cultured up to 56 days without layers dissociation and without cells migration between layers. Finally, scaffolds were functionalized by changing biomaterial composition and by applying mechanicals stimulations. Results show us not only that the composition influences the mechanical behavior of the hydrogel, but that the stratification did not affect it. Furthermore, mechanicals stimulations improve stem cells differentiation in function of biomaterials compositions. In conclusion, this study proves not only that we are able to build up stratified scaffold seeded with mesenchymal stem cells which still have their differentiation capability and synthesize matrix, but that mechanical behaviors are improved after the biomaterial spraying and not alter by the stratification. Moreover, mechanical stimulation applied to the scaffold improves the differentiation of mesenchymal stem cells to a chondrogenic phenotype

En ingénierie tissulaire, les biomatériaux, les cellules et l'induction de la différenciation, sont des facteurs à prendre en compte. L'objectif de cette étude est de connaitre l'effet de la nature des biomatériaux et leurs propriétés mécaniques sur la différenciation des cellules souches mésenchymateuses de la moelle osseuse. Dans un premier temps, nous avons étudié l'effet d'un biomatériau de nature protéique (le collagène de type I) supplémenté en microparticules d'hydroxyaptatite (HAP). Nous avons constaté que l'ajout d'HAP améliore les propriétés mécaniques de ce biomatériau et engage la différenciation des cellules vers des phénotypes ostéoarticulaires. Dans un deuxième temps, nous avons étudié l'effet d'un biomatériau à base d'alginate supplémenté par de l'acide hyaluronique ou des microparticules d'HAP, en utilisant un plan d'expériences pour choisir les matrices convenables pour l'étude biologique en fonction de leurs propriétés mécaniques. Nous avons constaté que les composants de ce biomatériau ont un effet sur l'élasticité de ce dernier et sur la différenciation des cellules souches mésenchymateuses. En conclusion, cette étude montre que les cellules souches mésenchymateuses sont sensibles à la composition du biomatériau et ses propriétés mécaniques / In tissue engineering, biomaterials, cells and the induction of cell differentiation are factors to be studied. The aim of this study is to know the effect of biomaterials composition and mechanical properties on the differentiation of mesenchymal stem cells from bone marrow. At first, we studied the effect of a protein biomaterial (collagen type I) supplemented with hydroxyaptatite (HAP) particles. We found that the addition of HAP improves the mechanical properties of the biomaterial and conditione cell differentiation towards osteoarticular lineages. In a second step, we studied the effect of biomaterial composed of alginate supplemented with hyaluronic acid or HAP particles, using an experimental design to select suitable matrices for biological study based on their mechanical properties. We found that the components of this biomaterial have an effect on elasticity of the latter and the differentiation of mesenchymal stem cells. In conclusion, this study shows that mesenchymal stem cells are sensitive to the composition of the biomaterial and its mechanical properties

Recentes estudos em animais tem demonstrado pronunciada reabsorção da tábua óssea vestibular, após a colocação de implantes imediatos. A realização de cirurgias sem retalho para a colocação de implantes imediatos, e a utilização de enxertos ósseos nos gaps, representam alternativas viáveis para minimizar a reabsorção da tábua óssea vestibular e, dessa forma, otimizar a osseointegração. O presente trabalho se propôs a investigar a remodelação da tábua óssea vestibular, associada ou não ao enxerto ósseo sintético no gap entre a superfície do implante e a tábua vestibular, em cirurgias sem retalho. Neste contexto, implantes posicionados ao nível da tábua óssea vestibular, e 2.0 mm subcrestal foram comparados. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos sem retalho, 4 implantes foram instalados nos alvéolos de cada lado, e posicionados 2.0 mm da tábua óssea vestibular originando o gap. Os seguintes grupos foram testados: implantes equicrestal associado com o enxerto ósseo sintético (grupo teste equicrestal-GTEC), implantes equicrestal com coágulo sanguíneo (grupo controle-GCEC), implantes 2.0 mm subcrestal associado com o enxerto ósseo sintético (grupo teste subcrestal-GTSC) e implantes 2.0 mm subcrestal com coágulo sanguíneo (grupo controle-GCSC). Uma semana após as cirurgias, próteses metálicas foram instaladas. Marcadores ósseos foram administrados 1, 2, 4 e 12 semanas após as cirurgias para a análise da fluorescência. Doze semanas após a colocação dos implantes os animais foram sacrificados. Para a análise radiográfica, cortes vestíbulo-linguais foram realizados nos blocos com os implantes individualizados, e imagens digitais radiográficas foram obtidas. Para a análise histomorfométrica, lâminas foram preparadas através dos blocos seccionados. A análise radiográfica revelou que o grupo teste subcrestal apresentou maior perda da tábua óssea vestibular com diferença estatisticamente significante quando comparado com os grupos equicrestais (p<0,05). Não foi observada diferença significativa entre os grupos em relação à distância linear, e a presença ou ausência de neoformação tecidual no gap residual. A avaliação histomorfométrica demonstrou resultados significativos para o grupo teste equicrestal, sem perda da tábua óssea vestibular. Em relação aos outros parâmetros, o grupo controle subcrestal apresentou melhores resultados. A nova formação de tecido ósseo foi determinada pela quantificação da fluorescência dos marcadores em áreas adjacentes e distantes às superfícies dos implantes. As áreas adjacentes mostraram diferentes resultados entre os grupos, e pequeno um decréscimo na 12a semana, exceto para o grupo controle subcrestal, que apresentou um aumento. As áreas distantes mostraram um aumento contínuo na formação óssea. Os implantes equicrestais apresentaram insignificativa perda da tábua óssea vestibular. No entanto, os implantes subcrestais mostraram maior perda da tábua óssea vestibular, mesmo com a presença do enxerto ósseo. No entanto, a tábua vestibular manteve-se sempre coronal ao ombro do implante. Ambos os grupos equicrestais e subcrestais testes e controles foram beneficiados nas fases iniciais do processo de formação óssea como evidenciado pela análise de fluorescência. / Recent studies in animals have shown pronounced resorption of the buccal bone plate after immediate implantation. The use of flapless surgical procedures prior to the installation of immediate implants, as well as the use of synthetic bone graft in the gaps represent viable alternatives to minimize buccal bone resorption and to favor osseointegration. The aim of this study was to evaluate the healing of the buccal bone plate following immediate implantation using the flapless approach, and compare this process with sites in which a synthetic bone graft was or was not inserted into the gap between the implant and the buccal bone plate. Lower bicuspids from 8 dogs were bilaterally extracted without the use of flaps and 4 implants were installed in the alveoli in each side of the mandible and were positioned 2.0 mm from the buccal bone plate (gap). Four groups were devised: 2.0 mm subcrestal implants (3.3 x 8 mm) using bone grafts (SCTG), 2.0 mm subcrestal implants without bone grafts (SCCG), equicrestal implants (3.3 x 10 mm) with bone grafts (ECTG) and equicrestal implants without bone grafts (ECCG). One week following the surgical procedures, metallic prostheses were installed and within 12 weeks the dogs were sacrificed. The blocks containing the individual implants were turned sideways and radiographic imaging was obtained to analyze the remodeling of the buccal bone plate. In the analysis of the resulting distance between the implant shoulder and the bone crest (IS-BC), statistically significant differences were found in the SBTG when compared to the ECTG (p=0.02) and ECCG (p=0.03). For meanvalue comparison of the resulting linear distance between the implant surface and the buccal plate (GAP-L) no statistically significant difference was found between all the groups (p>0.05). The same result was observed in the parameter for presence or absence of tissue formation between the implant surface and buccal plate (GAPA). Equicrestally placed implants, in this methodology, presented little or no loss of the buccal bone. The subcrestally positioned implants presented loss of buccal bone, even though synthetic bone graft was used. The buccal bone, however, was always coronal to the implant shoulder.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-21T17:41:19Z No. of bitstreams: 1 LuanaAlves.pdf: 1633592 bytes, checksum: 785a3f9aa7f3fc08d80023603f457b39 (MD5) / Made available in DSpace on 2017-09-21T17:41:19Z (GMT). No. of bitstreams: 1 LuanaAlves.pdf: 1633592 bytes, checksum: 785a3f9aa7f3fc08d80023603f457b39 (MD5) Previous issue date: 2017-07-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / Bioactive borate glass has presented superior results then bioactive silicate glasses, when compared its conversion rates in apatite and its potential of bioactivity. In this work, new Borate glasses with the basic 60B2O3 - 4P2O5 - 18Na2O – xCaF2 – (18-xCaO), com x = 0, 5 e 10 % wt were synthesized, and investigated the influence of CaF2 addition on bioactivity of samples in vitro, in a period of 28 days in a simulator of the body fluid (SBF). This bioactivity was investigated by means of X-ray diffraction (DRX), Raman Spectroscopy, FTIR and by the measure of pH. DRX measures, prior to immersion, presented wide bands, evidencing the amorphous structure of glasses. The results of density, thermal analysis, XRD, Raman and FTIR show that the addition of CaF2, until 10 % wt, did not cause significant changes in samples network structure. The thermal stability for all samples was calculated from DSC data and all presented values up to 120°C. The results of pH of SBF solution show increase from for 24 hours, which contributes to the dissolution of the outermost layer of the glass and the precipitation of apatite. In XRD data for 60B sample soaked in SBF for 7 days, there was formation of crystalline peaks, at 26° and 32° (2ϴ), in all measures. These peaks correspond to patterns of hydroxyapatite (HA). The XRD spectra in 60B5CaF and 60B10CaF, soaked in SBF for 7 days, presented peaks in 28°(2ϴ) corresponding to fluorapatite (FA), due to the presence of CaF2 in these glasses. Raman and FTIR measurements confirmed what was measured by XRD, showing characteristic peaks of HA and FA for all the samples. Results show that the prepared samples present potential for being used as biomaterials in biomedical applications, such as orthopedics, dentistry and tissue engineering. / Vidros boratos bioativos têm apresentado resultados superiores aos vidros silicatos bioativos, quando comparamos suas taxas de conversão em apatita e seu potencial de bioatividade. Neste trabalho sintetizamos novos vidros boratos com a composição básica 60B2O3 - 4P2O5 - 18Na2O – xCaF2 – (18-xCaO), com x = 0, 5 e 10 % em massa, e investigamos a influência da adição de CaF2 na bioatividade das amostras in vitro, em um período de 28 dias, em um simulador do fluído corporal (SBF). As medidas de DRX, antes da imersão, apresentaram bandas largas, comprovando a estrutura amorfa dos vidros. E verificou-se por meio das análises de Densidade, análise térmica, DRX, Raman e FTIR, que a adição de CaF2 até 10%, em massa, não provocou grandes mudanças estruturais na amostras. Por meio da calorimetria exploratória diferencial, determinamos a estabilidade térmica dos vidros que apresentaram valores acima de 120°C. A bioatividade foi investigada por meio das técnicas de difração de raios-X (DRX), espectroscopia Raman e FTIR, e pela medida do pH da solução SBF. Os resultados obtidos mostraram que, em 24h, houve um aumento no pH da solução SBF, o que contribui para a dissolução da camada mais externa do vidro e sua conversão em apatita. Nos difratogramas (DRX) para as amostras imersas por 7 dias, houve a formação de picos cristalinos, em 26° e 32° (2ϴ), em todas as amostras medidas. Esses picos correspondem aos padrões de hidroxiapatita. A presença de CaF2 na composição das amostras imersas por 7 dias apresentou picos em 28° (2ϴ) correspondendo a fluorapatita. A intensidade destes picos apresentou um aumento em função do tempo de imersão, durante todo período estudado. As medidas de Raman confirmaram os resultados do DRX, apresentando para todas as amostras espectros característicos da hidroxiapatita em 960 cm-1 . As amostras preparadas com CaF2 apresentaram picos em 965 cm-1 , que corresponde a fluorapatita. O FTIR confirmou os resultados apresentados no DRX e Raman, em que todas as amostras apresentaram um pico centrado em 1041 cm-1 para a hidroxiapatita e 1042 cm-1 característico para a fluorapatita. Os resultados demonstram que as amostras preparadas apresentam potencial para serem usados como biomateriais em aplicações biomédicas, como ortopedia, odontologia e engenharia de tecidos.

Recentes estudos em animais tem demonstrado pronunciada reabsorção da tábua óssea vestibular, após a colocação de implantes imediatos. A realização de cirurgias sem retalho para a colocação de implantes imediatos, e a utilização de enxertos ósseos nos gaps, representam alternativas viáveis para minimizar a reabsorção da tábua óssea vestibular e, dessa forma, otimizar a osseointegração. O presente trabalho se propôs a investigar a remodelação da tábua óssea vestibular, associada ou não ao enxerto ósseo sintético no gap entre a superfície do implante e a tábua vestibular, em cirurgias sem retalho. Neste contexto, implantes posicionados ao nível da tábua óssea vestibular, e 2.0 mm subcrestal foram comparados. Metodologicamente, os pré-molares mandibulares bilaterais de 8 cães foram extraídos sem retalho, 4 implantes foram instalados nos alvéolos de cada lado, e posicionados 2.0 mm da tábua óssea vestibular originando o gap. Os seguintes grupos foram testados: implantes equicrestal associado com o enxerto ósseo sintético (grupo teste equicrestal-GTEC), implantes equicrestal com coágulo sanguíneo (grupo controle-GCEC), implantes 2.0 mm subcrestal associado com o enxerto ósseo sintético (grupo teste subcrestal-GTSC) e implantes 2.0 mm subcrestal com coágulo sanguíneo (grupo controle-GCSC). Uma semana após as cirurgias, próteses metálicas foram instaladas. Marcadores ósseos foram administrados 1, 2, 4 e 12 semanas após as cirurgias para a análise da fluorescência. Doze semanas após a colocação dos implantes os animais foram sacrificados. Para a análise radiográfica, cortes vestíbulo-linguais foram realizados nos blocos com os implantes individualizados, e imagens digitais radiográficas foram obtidas. Para a análise histomorfométrica, lâminas foram preparadas através dos blocos seccionados. A análise radiográfica revelou que o grupo teste subcrestal apresentou maior perda da tábua óssea vestibular com diferença estatisticamente significante quando comparado com os grupos equicrestais (p<0,05). Não foi observada diferença significativa entre os grupos em relação à distância linear, e a presença ou ausência de neoformação tecidual no gap residual. A avaliação histomorfométrica demonstrou resultados significativos para o grupo teste equicrestal, sem perda da tábua óssea vestibular. Em relação aos outros parâmetros, o grupo controle subcrestal apresentou melhores resultados. A nova formação de tecido ósseo foi determinada pela quantificação da fluorescência dos marcadores em áreas adjacentes e distantes às superfícies dos implantes. As áreas adjacentes mostraram diferentes resultados entre os grupos, e pequeno um decréscimo na 12a semana, exceto para o grupo controle subcrestal, que apresentou um aumento. As áreas distantes mostraram um aumento contínuo na formação óssea. Os implantes equicrestais apresentaram insignificativa perda da tábua óssea vestibular. No entanto, os implantes subcrestais mostraram maior perda da tábua óssea vestibular, mesmo com a presença do enxerto ósseo. No entanto, a tábua vestibular manteve-se sempre coronal ao ombro do implante. Ambos os grupos equicrestais e subcrestais testes e controles foram beneficiados nas fases iniciais do processo de formação óssea como evidenciado pela análise de fluorescência. / Recent studies in animals have shown pronounced resorption of the buccal bone plate after immediate implantation. The use of flapless surgical procedures prior to the installation of immediate implants, as well as the use of synthetic bone graft in the gaps represent viable alternatives to minimize buccal bone resorption and to favor osseointegration. The aim of this study was to evaluate the healing of the buccal bone plate following immediate implantation using the flapless approach, and compare this process with sites in which a synthetic bone graft was or was not inserted into the gap between the implant and the buccal bone plate. Lower bicuspids from 8 dogs were bilaterally extracted without the use of flaps and 4 implants were installed in the alveoli in each side of the mandible and were positioned 2.0 mm from the buccal bone plate (gap). Four groups were devised: 2.0 mm subcrestal implants (3.3 x 8 mm) using bone grafts (SCTG), 2.0 mm subcrestal implants without bone grafts (SCCG), equicrestal implants (3.3 x 10 mm) with bone grafts (ECTG) and equicrestal implants without bone grafts (ECCG). One week following the surgical procedures, metallic prostheses were installed and within 12 weeks the dogs were sacrificed. The blocks containing the individual implants were turned sideways and radiographic imaging was obtained to analyze the remodeling of the buccal bone plate. In the analysis of the resulting distance between the implant shoulder and the bone crest (IS-BC), statistically significant differences were found in the SBTG when compared to the ECTG (p=0.02) and ECCG (p=0.03). For meanvalue comparison of the resulting linear distance between the implant surface and the buccal plate (GAP-L) no statistically significant difference was found between all the groups (p>0.05). The same result was observed in the parameter for presence or absence of tissue formation between the implant surface and buccal plate (GAPA). Equicrestally placed implants, in this methodology, presented little or no loss of the buccal bone. The subcrestally positioned implants presented loss of buccal bone, even though synthetic bone graft was used. The buccal bone, however, was always coronal to the implant shoulder.

Material science is an interdisciplinary area of research, which in part, designs and characterizes new materials. Research is concerned with synthesis, structure, properties, and performance of materials. Discoveries in materials science have significant impact on future technologies, especially in nano-scale applications where the physical properties of nanomaterials are significantly different than their bulk counterparts. The work presented here discusses the use of ferritin, a hollow sphere-like biomolecule, which forms metal oxo-hydride nanoparticles inside its protein shell for uses as a bio-inorganic material.Ferritin is capable of forming and sequestering 8 nm metal-oxide nanoparticles within its 2 nm thick protein shell. A variety of metal-oxide nanoparticles have been synthesized inside ferritin. The work herein focuses on three distinct areas:1) Ferritin's light harvesting properties: namely band gaps. Discrepancies in the band gap energies for ferritin's native ferrihydrite mineral and non-native minerals have been previously reported. Through the use of optical absorption spectroscopy, I resolved the types of band gaps as well as the energy of these band gaps. I show that metal oxides in ferritin are indirect band gap semiconductors which also contain a direct transition. Modifications to the ferrihydrite mineral's band gaps are measured as a result of co-depositing anions into ferritin during iron loading. I demonstrate that these band gaps can be used to photocatalytically reduce gold ions in solution with titanium oxide nanoparticles in ferritin. 2) A new method for manganese mineral synthesis inside ferritin: Comproportionation between permanganate and Mn(II) forms new manganese oxide minerals inside ferritin that are different than traditional manganese oxide mineral synthesis. This reaction creates a MnO2, Mn2O3, or Mn3O4 mineral inside ferritin, depending on the synthesis conditions. 3) Ferritin as an energetic material: Ferritin is capable of sequestering various metals and anions into its interior. Perchlorate, an energetic anion, is sequestered through a co-deposition process during iron loading and is tested with energetic binding materials. Peroxide, which can be used as an oxidant, is also shown to be sequestered within apoferritin and combined with an aluminum based fuel for solid rocket propellants.

<p>This thesis describes a method of growing a highly adherent nano-porous alumina coating on titanium implant materials, a design which might be useful in hard tissue replacement. Alumina layers were formed by anodisation of aluminium, which had been deposited on titanium and titanium alloys by electron beam evaporation. Mechanical testing showed the coatings’ shear and tensile strength to be ~20MPa and ~10MPa respectively. </p><p>Human osteoblasts were cultured on purchased membranes, produced in the same way with similar characteristics as the coating mentioned above. Cell viability, proliferation and phenotype were assessed by measuring redox reactions, DNA, tritiated thymidine incorporation and alkaline phosphatase production. Results showed normal osteoblastic growth patterns with increasing cell numbers the first two weeks after which cell growth decreased and alkaline phosphatase production increased, indicating that osteoblastic phenotype was retained on the alumina. Flattened cell morphology with filipodia attached to the pores of the material was seen. </p><p>Implants frequently trigger inflammatory responses due to accumulation and activation of cells such as polymorphonuclear granulocytes (PMN), also called neutrophils. Activation and morphology of human PMN in response to nano-porous alumina with two pore sizes (20 and 200 nm) was investigated by luminol-amplified chemiluminescence, granule enzyme deposition measurement, optical and scanning electron microscopy. Activation was observed on both membrane types, however less pronounced on the 200 nm alumina. For both membranes a decrease in activation was seen after coating with fibrinogen, collagen I and serum (more pronounced for the two latter). On fibrinogen-coated alumina many flattened cells were observed, indicating frustrated phagocytosis. Finally when culturing osteoblasts on non-coated and collagen-coated membranes (after exposure to PMN) many more cells had established on the protein-coated surface after 24 h. </p><p>The overall results indicate that it might be possible to produce a novel bone implant coating by anodisation of aluminium deposited on titanium and that this material will support osteoblast adhesion and proliferation. Furthermore neutrophil activation can be suppressed when coating the alumina with collagen I, which is beneficial considering the fact that this protein also is essential for bone formation.</p>

Peripheral vascular diseases such as peripheral artery disease (PAD) and critical limb ischemia (CLI) are growing at an ever-increasing rate in the Western world due to an aging population and the incidence of type II diabetes. A growing economic burden continues because these diseases are common indicators of future heart attack or stroke. Common therapies are generally limited to pharmacologic agents or endovascular therapies which have had mixed results still ending in necrosis or limb loss. Therapeutic angiogenic strategies have become welcome options for patients suffering from PAD due to the restoration of blood flow in the extremities. Capillary sprouting and a return to normoxic tissue states are also demonstrated by the use of angiogenic cytokines in conjunction with bone marrow cell populations. To this point, it has been determined that spatial and temporal controlled release of growth factors from vehicles provides a greater therapeutic and angiogenic effect than growth factors delivered intramuscularly, intravenously, or intraarterialy due to rapid metabolization of the cytokine, and non-targeted release. Furthermore, bone marrow cells have been implicated to enhance angiogenesis in numerous ischemic diseases due to their ability to secrete angiogenic cytokines and their numerous cell fractions present which are implicated to promote mature vessel formation. Use of angiogenic peptides, in conjunction with bone marrow cells, has been hypothesized in EPC mobilization from the periphery and marrow tissues to facilitate neovessel formation. For this purpose, controlled release of angiogenic peptides basic fibroblast growth factor (FGF-2) and granulocyte-colony stimulating factor (G-CSF) was performed using tunable ionic gelatin hydrogels or fibrin scaffolds with ionic albumin microspheres. The proliferation of endothelial cell culture was determined to have an enhanced effect based on altering concentrations of growth factors and method of release: co-delivery versus sequential. Scaffolds with these angiogenic peptides were implanted in young balb/c mice that underwent unilateral hindlimb ischemia by ligation and excision of the femoral artery. Endpoints for hindlimb reperfusion and angiogenesis were determined by Laser Doppler Perfusion Imaging and immunohistochemical staining for capillaries (CD-31) and smooth muscle cells (alpha-SMA). In addition to controlled release of angiogenic peptides, further studies combined the use of a fibrin co-delivery scaffold with FGF-2 and G-CSF with bone marrow stem cell transplantation to enhance vessel formation following CLI. Endpoints also included lipophilic vascular painting to evaluate the extent of angiogenesis and arteriogenesis in an ischemic hindlimb. Tissue engineering strategies utilizing bone marrow cells and angiogenic peptides demonstrate improved hindlimb blood flow compared to BM cells or cytokines alone, as well as enhanced angiogenesis based on immunohistochemical staining and vessel densities.

This thesis describes a method of growing a highly adherent nano-porous alumina coating on titanium implant materials, a design which might be useful in hard tissue replacement. Alumina layers were formed by anodisation of aluminium, which had been deposited on titanium and titanium alloys by electron beam evaporation. Mechanical testing showed the coatings’ shear and tensile strength to be ~20MPa and ~10MPa respectively. Human osteoblasts were cultured on purchased membranes, produced in the same way with similar characteristics as the coating mentioned above. Cell viability, proliferation and phenotype were assessed by measuring redox reactions, DNA, tritiated thymidine incorporation and alkaline phosphatase production. Results showed normal osteoblastic growth patterns with increasing cell numbers the first two weeks after which cell growth decreased and alkaline phosphatase production increased, indicating that osteoblastic phenotype was retained on the alumina. Flattened cell morphology with filipodia attached to the pores of the material was seen. Implants frequently trigger inflammatory responses due to accumulation and activation of cells such as polymorphonuclear granulocytes (PMN), also called neutrophils. Activation and morphology of human PMN in response to nano-porous alumina with two pore sizes (20 and 200 nm) was investigated by luminol-amplified chemiluminescence, granule enzyme deposition measurement, optical and scanning electron microscopy. Activation was observed on both membrane types, however less pronounced on the 200 nm alumina. For both membranes a decrease in activation was seen after coating with fibrinogen, collagen I and serum (more pronounced for the two latter). On fibrinogen-coated alumina many flattened cells were observed, indicating frustrated phagocytosis. Finally when culturing osteoblasts on non-coated and collagen-coated membranes (after exposure to PMN) many more cells had established on the protein-coated surface after 24 h. The overall results indicate that it might be possible to produce a novel bone implant coating by anodisation of aluminium deposited on titanium and that this material will support osteoblast adhesion and proliferation. Furthermore neutrophil activation can be suppressed when coating the alumina with collagen I, which is beneficial considering the fact that this protein also is essential for bone formation.

Improving the functionality and reducing revision rates are important driving forces in the development of orthopaedic implants. FibMat is a fibrinogen based matrix developed towards commercialisation by the company Optovent AB. This matrix can be coated on implants and act as a local drug delivery system for bisphosphonates (BPs). BPs are drugs inhibiting bone resorption, and applied with FibMat to improve stability of implants in bone, e.g. when fixing bone fractures. In this thesis, FibMat loaded with BP (FibMat/BP) was coated on stainless-steel screws and titanium screws in order to investigate some technology properties relevant to its clinical applicability. Bone-mimicking materials were used to study scrape-off effect upon insertion. The coagulation properties of fibrinogen as well as the structural properties of BPs were studied after exposure to gamma radiation. The screws were coated with FibMat and BP (alendronate and 14C-alendronate) using standard coupling techniques. The total amount and distribution of BP after insertion was measured by liquid scintillation and autoradiography. Coagulation assays were performed in order to determine the coagulation properties of fibrinogen, exposed to doses up to 35 kGy, mixed with thrombin. The structural properties of four different BPs (alendronate, pamidronate, zoledronate and ibandronate), exposed to doses up to 35 kGy were analysed by transmission infrared spectroscopy. The results show that FibMat/BP coating on porous stainless-steel screws is virtually unaffected by insertion into bone materials. The anodised, planar titanium screws are more affected by the insertion process, but an even BP distribution in the cancellous material is indicated. The coagulation assays show that gamma-irradiated fibrinogen has a slower coagulation process compared to non-irradiated fibrinogen and form interrupted network unable to clot. The chemical structures of the BPs seem unaffected by exposure to gamma irradiation. In conclusion, the FibMat/BP is a promising technology for local distribution of BP in conjunction with bone implants.