The implantation of any foreign material into the body automatically starts an immune reaction that serves as the first, mandatory step to regenerate tissue. The course of this initial immune reaction decides on the fate of the implant: either the biomaterial will be integrated into the host tissue to subsequently fulfill its intended function (e.g., tissue regeneration), or it will be repelled by fibrous encapsulation that determines the implant failure. Especially neutrophils and macrophages play major roles during this inflammatory response and hence mainly decide on the biomaterial's fate. For clinically relevant tissue engineering approaches, biomaterials may be designed in shape and morphology as well as in their surface functionality to improve the healing outcome, but also to trigger stem cell responses during the subsequent tissue regeneration phase. The main focus of this thesis was to unravel the influence of scaffold characteristics, including scaffold morphology and surface functionality, on primary human innate immune cells (neutrophils and macrophages) and human mesenchymal stromal cells (hMSCs) to assess their in vitro immune response and tissue regeneration capacity, respectively. The fiber-based constructs were produced either via melt electrowriting (MEW), when the precise control over scaffold morphology was required, or via solution electrospinning (ES), when the scaffold design could be neglected. All the fiber-based scaffolds used throughout this thesis were composed of the polymer poly(ε caprolactone) (PCL). A novel strategy to model and alleviate the first direct cell contact of the immune system with a peptide-bioactived fibrous material was presented in chapter 3 by treating the material with human neutrophil elastase (HNE) to imitate the neutrophil attack. The main focus of this study was put on the effect of HNE towards an RGDS-based peptide that was immobilized on the surface of a fibrous material to improve subsequent L929 cell adhesion. The elastase efficiently degraded the peptide-functionality, as evidenced by a decreased L929 cell adhesion, since the peptide integrated a specific HNE-cleavage site (AAPV-motif). A sacrificial hydrogel coating based on primary oxidized hyaluronic acid (proxHA), which dissolved within a few days after the neutrophil attack, provided an optimal protection of the peptide-bioactivated fibrous mesh, i.e, the hydrogel alleviated the neutrophil attack and largely ensured the biomaterial's integrity. Thus, according to these results, a means to protect the biomaterial is required to overcome the neutrophil attack. Chapter 4 was based on the advancement of melt electrowriting (MEW) to improve the printing resolution of MEW scaffolds in terms of minimal inter-fiber distances and a concomitant high stacking precision. Initially, to gain a better MEW understanding, the influence of several parameters, including spinneret diameter, applied pressure, and collector velocity on mechanical properties, crystallinity, fiber diameter and fiber surface morphology was analyzed. Afterward, innovative MEW designs (e.g., box-, triangle-, round , and wall-shaped scaffolds) have been established by pushing the printing parameters to their physical limits. Further, the inter-fiber distance within a standardized box-structured scaffold was successfully reduced to 40 µm, while simultaneously a high stacking precision was maintained. In collaboration with a co-worker of my department (Tina Tylek, who performed all cell-based experiments in this study), these novel MEW scaffolds have been proven to facilitate human monocyte-derived macrophage polarization towards the regenerative M2 type in an elongation-driven manner with a more pronounced effect with decreasing pore sizes. Finally, a pro-adipogenic platform for hMSCs was developed in chapter 5 using MEW scaffolds with immobilized, complex ECM proteins (e.g., human decellularized adipose tissue (DAT), laminin (LN), and fibronectin (FN)) to test for the adipogenic differentiation potential in vitro. Within this thesis, a special short-term adipogenic induction regime enabled to more thoroughly assess the intrinsic pro-adipogenic capacity of the composite biomaterials and prevented any possible masking by the commonly used long-term application of adipogenic differentiation reagents. The scaffolds with incorporated DAT consistently showed the highest adipogenic outcome and hence provided an adipo-inductive microenvironment for hMSCs, which holds great promise for applications in soft tissue regeneration. Future studies should combine all three addressed projects in a more in vivo-related manner, comprising a co-cultivation setup of neutrophils, macrophages, and MSCs. The MEW-scaffold, particularly due to its ability to combine surface functionality and adjustable morphology, has been proven to be a successful approach for wound healing and paves the way for subsequent tissue regeneration. / Die Implantation eines Biomaterials löst stets eine Immunreaktion im Körper aus, die den ersten zwingenden Schritt zur Geweberegeneration darstellt. Der Verlauf dieser anfänglichen Immunreaktion entscheidet über das Schicksal des Implantats: Entweder wird das Biomaterial in das Wirtsgewebe integriert, um anschließend seine vorgesehene Funktion (z.B. Geweberegeneration) zu erfüllen, oder aber es findet eine Abstoßungsreaktion durch Einkapselung des Implantats statt. Insbesondere Neutrophile und Makrophagen spielen für die Immunantwort eine wichtige Rolle und entscheiden daher hauptsächlich über das Schicksal des Biomaterials. Für klinisch relevante Ansätze der Gewebezüchtung können Biomaterialien sowohl in ihrer Morphologie als auch in ihrer Oberflächenfunktionalität so gestaltet werden, dass sie zum einen die Wundheilung verbessern, zum anderen auch Stammzellreaktionen während der anschließenden Geweberegenerationsphase auslösen. Der Fokus dieser Doktorarbeit lag auf der Beurteilung des Einflusses von Morphologie und Oberflächenfunktionalität fasriger Scaffolds auf die frühe Phase der Geweberegeneration. Insbesondere wurde die in vitro-Immunantwort von primären humanen Immunzellen (Neutrophile und Makrophagen) sowie die Geweberegenerationskapazität von humanen mesenchymalen Stromazellen (hMSCs) untersucht. Die hierfür verwendeten faserbasierten Poly(ε-Caprolacton) (PCL) Scaffolds wurden entweder mittels Solution Electrospinning (ES) oder Melt Electrowriting (MEW) hergestellt. Während ES eine zufällig orientierte Faserablage zur Folge hat, erlaubt MEW eine präzise Kontrolle der Scaffold-Morphologie. Zunächst wurde eine neue Strategie zur Nachahmung und Abmilderung des ersten direkten Zellkontakts während der Immunreaktion vorgestellt. Dabei wurde die Interaktion zwischen Neutrophilen mit einem Peptid-bioaktivierten Fasermaterial untersucht (Kapitel 3), wobei der sog. Neutrophilen-Angriff mittels des Enzyms Neutrophilen Elastase (HNE) nachgeahmt wurde. Das an der Faseroberfläche immobilisierte CGGGAAPVGGRGDS-Peptid verfügte über eine spezifische HNE-Schnittstelle (AAPV-Motiv), an welcher die Elastase das Peptid effizient degradieren konnte. Das Degradationsverhalten des Enzyms wurde anschließend über L929 Zelladhärenz analysiert, welche über das RGDS-Motiv im Peptid vermittelt wurde. Im Rahmen der Arbeit konnte nachgewiesen werden, dass der Neutrophilen-Angriff und die damit einhergehende Verringerung des RGDS-Motivs zu einer reduzierten Zelladhärenz führte. Die Einbettung des Scaffolds in ein Hydrogel auf der Basis von Aldehyd-haltiger Hyaluronsäure (proxHA) bot während des Neutrophilen-Angriffs einen optimalen Schutz der Peptidfunktionalität. Um diese wiederum anschließend für Adhäsionsversuche verfügbar zu machen, konnte das Hydrogelsystem derartig eingestellt werden, dass sich dieses innerhalb weniger Tage auflöste. Auf diese Weise konnte das Hydrogel den Neutrophilen-Angriff abmildern und so die Integrität des Biomaterials weitestgehend gewährleisten. Kapitel 4 behandelt die Präzisierung der Faserablage, insbesondere die Verringerung des Faserabstands, während des MEW-Prozesses. Zunächst wurde der Einfluss verschiedener Parameter (Spinndüsendurchmesser, angelegter Luftdruck und Kollektorgeschwindigkeit) auf die mechanischen Eigenschaften, die Kristallinität, den Faserdurchmesser und die Faseroberflächenmorphologie analysiert. Durch Optimierung der Druckparameter konnten innovative MEW-Designs (u.a. mit runder Porengeometrie) gedruckt werden. Der Abstand zwischen den Fasern in einem Scaffold mit standardisierter kastenförmiger Porengeometrie wurde erfolgreich auf 40 µm reduziert, während gleichzeitig eine hohe Stapelpräzision gewährleistet wurde. In Zusammenarbeit mit einer Kollegin am Lehrstuhl (Tina Tylek, die alle zellbasierten Experimente in dieser Studie durchführte) wurde nachgewiesen, dass diese innovativen MEW-Scaffolds die Polarisierung menschlicher Makrophagen in Richtung des regenerativen M2-Typs förderten. Die Makrophagen-Polarisierung ging einher mit einer Zellelongation, wobei dieser Effekt verstärkt für kleinere Porengrößen auftrat. Abschließend stand die Untersuchung der pro-adipogenen Wirkung von faserfunktionalisierten MEW-Scaffolds im Fokus (Kapitel 5), welche mit ECM-Proteinen, wie beispielsweise dezellularisiertes Fettgewebe (DAT), beschichtet wurden. Das pro-adipogene Potential dieser Materialien wurde mit Hilfe einer adipogenen Kurzzeitinduktion näher analysiert, da eine Langzeitapplikation der Differenzierungsreagenzien diesen Effekt überdeckte. Die Scaffolds mit der DAT-Beschichtung zeigten durchweg die höchste adipogene Differenzierung und boten somit für Stammzellen eine adipo-induzierende Mikroumgebung, weshalb sie für die Anwendung in der Weichgeweberegeneration sehr vielversprechend sind. An diese Arbeit anschließende Experimente sollten alle drei Projekte in einem Co-Kulturansatz von Neutrophilen, Makrophagen und MSCs kombinieren, um so einen stärkeren in vivo-Bezug herzustellen. Hierfür erweist sich das MEW-Scaffold insbesondere durch seine Kombinationsfähigkeit der Oberflächenfunktionalität und Morphologie als Ansatz für einen erfolgreichen Wundheilungsprozess und ebnet damit den Weg für eine bestmögliche Geweberegeneration.

Current articular cartilage tissue engineering endeavors, using synthetic polymers as scaffolds, have been somewhat successful. However, the use of these materials has not yielded a satisfactory, functional replacement for articular cartilage. Therefore, this project focuses on an alternative to these materials, chitosan, which is a naturally occurring biopolymer. The first project objective was to fabricate and analyze bulk, porous chitosan scaffolds, based on total porosity, average pore diameter, mechanical integrity, and degradation susceptibility. Secondly, scaffolds were evaluated in terms of their ability to support neochondrogenesis, including assessments of cell attachment and viability, cell morphology, and the biosynthesis of proteoglycan and type-II collagen-rich extracellular matrix. Results indicated that chitosan scaffolds possessing an interconnecting, porous structure could be easily created through a simple freezing and lyophilization process, and these scaffolds did support neochondrogenesis. Results suggest chitosan may be a useful alternative to synthetic polymers for use in cartilage tissue engineering applications.

No description available.

The in vivo 3D extracellular matrix provides a temporal regulatory environment of chemical cues. Understanding this dynamic environment will be crucial for efficient drug screening, diseases mechanism elucidation, and tissue engineering. Therefore, in vitro 3D cell culture systems with reversible chemical environments are required. To this end, we developed a non-cytotoxic agarose-desthiobiotin hydrogel to sequester streptavidin biomolecule conjugates (KD 10-11 M), which can then be displaced by the addition of biotin (KD 10-15 M). Streptavidin biomolecule conjugates were simultaneously and sequentially immobilized by changing media components. The time required for biochemical environment exchange was minimized by increasing the surface area to volume ratios and pore size of the hydrogels. We temporally controlled the cell adhesive properties of hydrogels with RGD modified streptavidin to influence endothelial cell tube formation. / Thesis / Master of Science (MSc)

Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses. / Doctor of Philosophy / Keratins and keratin hydrogels may exhibit several properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for payloads for tissue engineering purposes. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles that were analyzed based on drug loading characteristics. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs). Finally, we tested these products to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Particles with a porous structure were obtained. The microstructure of these particles may lend them to applications in drug release or other payload delivery. Data showed an ability to load and unload specific drug payloads. Dense cell populations were loaded onto particles. Finally, studies show that both keratin and BM-MSCs support skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.

Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new 'template plus adder' cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly α-helical adder protein to form β-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical (~10 μm diameter, ~2 GPa Young's modulus) or tape (5-10 μm height, 10-20 μm width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 μm width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' α-helices is a good predictor of high density β-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid fibers when combined with extracellular myoglobin, an adder protein. The goal of this thesis is to produce, manipulate and characterize the self-assembly of large amyloid fibers for their potential industrial biomaterial applications. The techniques used throughout this study outline various methods to design and engineer amyloid fibers of a tailored modulus and morphology. Furthermore, the mechanisms described here may offer some insight into naturally occurring amyloid forming systems. / Ph. D.

A engenharia de tecido ósseo associando células-tronco mesenquimais (CTMs) a biomateriais tem sido proposta como tratamento potencial para o reparo de defeitos ósseos, constituindo uma abordagem nova na área da medicina regenerativa e de amplo interesse para as áreas de cirurgia buco-maxilo-facial e ortopedia. A seleção das CTMs mais adequadas e o método utilizado para carreá-las nos sítios de defeitos ósseos são fatores importantes para o sucesso do tratamento. Como a osteoporose reduz a capacidade de regeneração dos ossos, seria de grande importância que a engenharia do tecido ósseo pudesse ser aplicada com sucesso nessa patologia. Assim, foi avaliado o potencial das CTMs de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) associadas ao arcabouço de vitrocerâmica BioS-2P ou a membrana de P(VDF-TrFE)/BT no reparo de defeitos ósseos em ratas osteoporóticas. A osteoporose foi induzida por ovariectomia e comprovada pela análise microtomográfica dos fêmures. Nas ratas osteoporóticas foram criados defeitos ósseos nas calvárias que foram tratados com implantação de BioS-2P associado à CTMs-MO e CTMs-TA ou com a implantação de membrana de P(VDF-TrFE)/BT combinada com a injeção de CTMs-MO e CTMs-TA. Ao final de 4 semanas, as análises microtomográficas e histológica mostraram que não houve formação óssea nos defeitos sem qualquer tratamento, mas nos defeitos tratados com implantação de BioS-2P ou membrana de P(VDF-TrFE)/BT houve formação óssea independente da presença de CTMs. Apenas os defeitos tratados com membrana de P(VDF-TrFE)/BT e injeção de CTMs-MO apresentaram maior formação óssea, mas não ocorreu a regeneração. / Bone tissue engineering based on the combination of mesenchymal stem cells (MSCs) and biomaterials, has been proposed as a potential treatment for the repair of bone defects, constituting a new approach in the field of regenerative medicine and of interest to the areas of oral and maxillofacial surgery and orthopedics. To select the most suitable MSCs and an efficient method to carry them to the bone defects are the key for the successful treatment. Considering that osteoporosis represents a challenge situation, it would be of the utmost importance that bone tissue engineering could be used in this pathological condition. Thus, the aim of this study was to evaluate the potential of MSCs harvested from bone marrow (MSCs-BM) and from adipose tissue (MSCs-AT) associated to a vitreous scaffold (BioS-2P) or to a membrane of P(VDF-TrFE)/BT in regenerate bone defects created in osteoporotic rats. Osteoporosis was induced by ovariectomy and confirmed by microtomography of the femurs. Defects created in calvaria of osteoporotic rats were implanted with either Bios-2P seeded with MSCs-BM and MSCs-AT or a membrane of P(VDF-TrFE)/BT combined with injection of MSCs-BM and MSCs-AT. After 4 weeks, microtomography and histological analyses showed that there was no bone formation in untreated defects but in those treated with BioS-2P or membrane of P(VDF-TrFE)/BT there was bone formation irrespective of the presence of MSCs. Only defects treated with membrane of P(VDF-TrFE)/BT and MSCs-BM injection resulted in greater bone formation but there was not full bone regeneration.

A engenharia de tecido ósseo associando células-tronco mesenquimais (CTMs) a biomateriais tem sido proposta como tratamento potencial para o reparo de defeitos ósseos, constituindo uma abordagem nova na área da medicina regenerativa e de amplo interesse para as áreas de cirurgia buco-maxilo-facial e ortopedia. A seleção das CTMs mais adequadas e o método utilizado para carreá-las nos sítios de defeitos ósseos são fatores importantes para o sucesso do tratamento. Como a osteoporose reduz a capacidade de regeneração dos ossos, seria de grande importância que a engenharia do tecido ósseo pudesse ser aplicada com sucesso nessa patologia. Assim, foi avaliado o potencial das CTMs de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) associadas ao arcabouço de vitrocerâmica BioS-2P ou a membrana de P(VDF-TrFE)/BT no reparo de defeitos ósseos em ratas osteoporóticas. A osteoporose foi induzida por ovariectomia e comprovada pela análise microtomográfica dos fêmures. Nas ratas osteoporóticas foram criados defeitos ósseos nas calvárias que foram tratados com implantação de BioS-2P associado à CTMs-MO e CTMs-TA ou com a implantação de membrana de P(VDF-TrFE)/BT combinada com a injeção de CTMs-MO e CTMs-TA. Ao final de 4 semanas, as análises microtomográficas e histológica mostraram que não houve formação óssea nos defeitos sem qualquer tratamento, mas nos defeitos tratados com implantação de BioS-2P ou membrana de P(VDF-TrFE)/BT houve formação óssea independente da presença de CTMs. Apenas os defeitos tratados com membrana de P(VDF-TrFE)/BT e injeção de CTMs-MO apresentaram maior formação óssea, mas não ocorreu a regeneração. / Bone tissue engineering based on the combination of mesenchymal stem cells (MSCs) and biomaterials, has been proposed as a potential treatment for the repair of bone defects, constituting a new approach in the field of regenerative medicine and of interest to the areas of oral and maxillofacial surgery and orthopedics. To select the most suitable MSCs and an efficient method to carry them to the bone defects are the key for the successful treatment. Considering that osteoporosis represents a challenge situation, it would be of the utmost importance that bone tissue engineering could be used in this pathological condition. Thus, the aim of this study was to evaluate the potential of MSCs harvested from bone marrow (MSCs-BM) and from adipose tissue (MSCs-AT) associated to a vitreous scaffold (BioS-2P) or to a membrane of P(VDF-TrFE)/BT in regenerate bone defects created in osteoporotic rats. Osteoporosis was induced by ovariectomy and confirmed by microtomography of the femurs. Defects created in calvaria of osteoporotic rats were implanted with either Bios-2P seeded with MSCs-BM and MSCs-AT or a membrane of P(VDF-TrFE)/BT combined with injection of MSCs-BM and MSCs-AT. After 4 weeks, microtomography and histological analyses showed that there was no bone formation in untreated defects but in those treated with BioS-2P or membrane of P(VDF-TrFE)/BT there was bone formation irrespective of the presence of MSCs. Only defects treated with membrane of P(VDF-TrFE)/BT and MSCs-BM injection resulted in greater bone formation but there was not full bone regeneration.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-16T14:41:47Z No. of bitstreams: 1 PGCA17MA222.pdf: 4155710 bytes, checksum: b20faa3c60a0a388927aaabec4041811 (MD5) / Made available in DSpace on 2018-03-16T14:41:47Z (GMT). No. of bitstreams: 1 PGCA17MA222.pdf: 4155710 bytes, checksum: b20faa3c60a0a388927aaabec4041811 (MD5) Previous issue date: 2017-02-17 / Capes / Several research studies related to bone substitutes have been conducted in order to find the ideal material for this purpose, with similar qualities to the autogenous bone regarding its application of repairing bone defects, whether it results from fracture, degeneration or neoplasias. Accordingly, surgical experiments were carried out in rabbit calvaries, aiming to analyse and compare different micro-nanostructured biomaterials in relation to their capacity of osteoinduction and osteoconduction. Fourteen male New Zealand rabbits, eight months old and with a mean weight of 3.43 ± 0.53kg, were used. The animals were proven to be healthy through clinical examination and haematological analysis. The animals were submitted to inhalation anesthesia with isoflurane for the surgical procedure. From the bone exposure of the calvary region, four cylindrical polyamide guides were fixed with stainless steel screw. Each guide was filled with a pre-determined biomaterial composed of: hydroxyapatite with 5% silica (HA/SiO2 5%), hydroxyapatite with β-tricalcium phosphate (β-TCP/HA - 26.27%/73.53%), β-tricalcium phosphate (β-TCP) and Bio-Oss® (control). The animals were randomly assigned into two groups: G60 (n=7) and G90 (n=7), which were submitted to euthanasia at 60 and 90 days postoperatively, respectively, each calvary being removed and conditioned in buffered formaldehyde for 15 days. Afterwards, the calvaries were sectioned in four quadrants, each containing the biomaterial, being kept in buffered formaldehyde for another 15 days and then sent to histological analysis and scanning electron microscopy (SEM). From the results obtained by the methodology of this study, it can be concluded that all applied biomaterials show osteoinduction and osteoconduction capacity, despite different degrees. Through the analysis performed, it was observed that the Bio-Oss® presented smaller neoformation of bone tissue. The biphasic HA/SiO2 5% composition did not show incorporation on the interface between the recipient bed and the bone graft in most animals, inducing the formation of fibrous tissue for the most part. The β-TCP and the biphasic β-TCP/HA 73,53/26,27%, were the most promising biomaterials of this study, inducing a greater bone neoformation. β-TCP has been shown to be the biomaterial with the fastest rate of formation and maturation of bone tissue. The biphasic β-TCP/HA presented results very close to the β-TCP alone. However, it presented a greater amount of phagocytic cells, which in a longer period of evaluation could present a higher rate of absorption of the biomaterial / Diversas pesquisas relacionadas a substitutos ósseos têm sido realizadas na busca de um material ideal, que tenha qualidades próximas ao osso autógeno quanto à sua aplicação para reparação de um defeito ósseo, seja ele decorrente de fratura, degeneração ou neoplasias. Com esta finalidade, foram realizados experimentos cirúrgicos guiados em calvárias de coelhos onde se teve por objetivo analisar e comparar diferentes biomateriais micronanoestruturados em relação à sua capacidade de osteoindução e osteocondução. Foram utilizados 14 coelhos da raça Nova Zelândia, machos, com oito meses de idade e com peso médio de 3,43 ± 0,53kg. Os animais foram comprovadamente hígidos através de exame clínico e análise hematológica. Os animais foram submetidos à anestesia inalatória com isoflurano para realização do procedimento cirúrgico. A partir da exposição óssea da região da calvária, foram fixadas quatro guias cilíndricas de poliamida com parafuso de aço inoxidável. Cada guia foi preenchida por biomaterial pré-determinado, compostos por: Hidroxiapatita com Sílica a 5% (HA/SiO2 5%), Hidroxiapatita com Fosfato tricálcico- β (HA/TCP-β: 73,53/26,27%), Fosfato tricálcico (TCP-β) e Bio-ss® (controle). Os animais foram distribuídos aleatoriamente em dois grupos: G60 (n=7) e G90 (n=7), que foram submetidos à eutanásia com 60 e 90 dias de pós-operatório, respectivamente, sendo cada calvária removida e acondicionada em formaldeído tamponado por 15 dias. Após, as calvárias foram seccionadas em quatro quadrantes, cada qual contendo o biomaterial, sendo mantidos em formaldeído tamponado 10% por mais 15 dias e então encaminhados para análises histológica e de microscopia eletrônica por varredura (MEV). A partir dos resultados obtidos pela metodologia deste estudo, pode ser concluído que todos os biomateriais aplicados, apresentam capacidade de osteoindução e osteocondução, mesmo que em graus variados. Observou-se que o Bio-Oss® apresentou menor neoformação de tecido ósseo. A composição bifásica HA/SiO2 5% não apresentou incorporação na interface entre o leito receptor e o enxerto ósseo na maior parte dos animais, induzindo à formação de tecido fibroso em sua maior parte. O TCP-β e o bifásico HA/TCP-β 73,53/26,27%, foram os biomateriais mais promissores deste estudo, induzindo a uma maior neoformação óssea. O TCP-β demonstrou ser o biomaterial com velocidade mais rápida de formação e maturação do tecido ósseo. O bifásico HA/TCP-β apresentou resultados bem próximos ao TCP-β isolado. No entanto, apresentou maior quantidade de células fagocitárias, o que num período maior de avaliação, poderia apresentar maior velocidade de absorção do biomaterial

Made available in DSpace on 2015-03-26T13:47:13Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2356825 bytes, checksum: d31053454fc83d68df0097e746529fd9 (MD5) Previous issue date: 2013-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The loss of bone tissue is an important problem both in human and in veterinary medicines. For cranial bone defects, surgical intervention is necessary since, if not correct, these defects result in high number of cerebral lesions and neurological dysfunctions as well as esthetic sequelae. In such context, autogenous and alogenous grafts have given place to resorbable osteoconductive biomaterials. The objective of the present study was to evaluate a bone cement made from a solid phase (β-tricalcium phosphate and calcium sulfate dihydrate) and a liquid one (phosphoric acid 2M). These components were mixed in a stainless steel cube at a proportion of 1:1 and the material while still moldable, was applied in 15mm critical size defects in the cranial bone of rabbits, Scanning electron microscopy of the cement itself showed a complex surface with non-interconnected pores of 19.5±5.92 μm in diameter. Animals were separated in three experimental moments based on the time after surgical procedure (7, 28 and 90 days), comparing to defects that did not receive the material. Hematological and serum biochemistry revealed normal values during the entire experiment. Dehiscence, edema or signs of infection were not observed. In treated animals, alterations were observed on skin (hyperemia and necrosis) and brain (circular lesions of green color with vascular congestion) that evolved to healing at 90 days. These alterations may be related related to a low pH of the biomaterial during the implantation (3.38), resulted from the proportion of liquid:powder used in the present study (1:1). As suggested by computerized tomography and confirmed by histology, new bone formation occurred in control group from the margin of the defect and inside it. In treated group, bone growth was apparently less intense at 90 days when compared to the control one, limited to the margin of the defect and not observed inside it. Osteointegration was confirmed and neither signs of undesirable chronic inflammation nor the formation of a fibrous capsule around the material was observed. Thereby, the biomaterial was characterized as osteoconductor, osteointegrated, degradable in vivo and histologically biocompatible. The alterations in the color of nervous tissue and skin adjacent to the implant, although with no clinical relevance, indicate the need for a modification on the proportion of the biomaterial components. Possibly, reducing the relation liquid:powder may allow its application. / A perda de tecido ósseo é um problema grave tanto na medicina humana quanto na veterinária. No caso das falhas ósseas cranianas, a intervenção cirúrgica se faz necessária porque, se não forem corrigidas, provocam altos índices de lesões cerebrais e disfunções neurológicas, bem como graves sequelas estéticas. Nesse contexto, enxertos autógenos e alógenos têm sido substituídos por biomateriais osteocondutores e reabsorvíeis. O objetivo desse trabalho foi avaliar a utilização de um cimento ósseo formulado a partir de uma fase sólida (β-fosfato tricálcico e sulfato de cálcio di-hidratado) e uma fase líquida (ácido fosfórico 2M). Esses componentes foram misturados na proporção 1:1 e o material, ainda moldável, foi aplicado em defeitos ósseos de tamanho crítico no crânio de coelhos, com 15 mm de diâmetro. A análise do cimento pela microscopia eletrônica de varredura revelou superfície complexa com poros de 19,5 (±5,92) μm não interconectados. Os animais foram divididos em três momentos experimentais com base no tempo transcorrido após o procedimento cirúrgico (sete, 28 e 90 dias), onde os defeitos tratados foram comparados aos defeitos que não receberam o biomaterial. As análises hematológicas e da bioquímica sérica evidenciaram valores normais durante todo o experimento. Não foram observados deiscência, edema ou sinais de infecção. Nos animais tratados, foram observadas alterações na pele (hiperemia e necrose) e no cérebro (lesões circulares de coloração esverdeada com congestão vascular), que evoluíram para a cicatrização aos 90 dias. Essas alterações podem ser atribuídas ao baixo pH do biomaterial no momento de sua implantação (3,38), resultante da relação líquido/pó utilizada no presente trabalho (1:1). Foi sugerido pela tomografia computadorizada e confirmado pela análise histológica crescimento de osso novo, que no grupo controle ocorreu a partir das bordas e no interior do defeito. Já nos animais do grupo tratado esse crescimento foi menor aos 90 dias, quando comparado ao controle na mesma data, e limitou-se às bordas do defeito, não ocorrendo no interior. Foi demonstrada a osteointegração do biomaterial, não sendo observados sinais de inflamação crônica indesejável ou a formação de cápsula fibrosa ao seu redor. Após análise dos dados obtidos o biomaterial foi caracterizado como osteocondutor, osteointegrável, possuindo degradação in vivo e histologicamente biocompatível. As alterações na coloração no tecido nervoso na região do implante, mesmo sem comprometimento clínico, e as lesões de pele, indicam a necessidade de modificação da proporção dos componentes. Possivelmente, a redução da relação líquido/pó viabilizará sua aplicação.