In the wood industry, determination of the moisture content (mc) with a highaccuracy is of great importance. The green mc is important for optimisation of thewood drying process at sawmills, whereas the mc of dried boards is of interest for thefurther processing and final use of the boards. In the first publication in this thesis,which is a technical report written in Swedish, different measuring techniques withtheir own pros and cons are discussed. A total of 13 different measurement principlesare discussed, focusing on the ability to measure mc in the range of 7-18%. The resultsuggests that mc-meters that use electromagnetic waves in the radio- and microwaveranges have the greatest potential to become the next generation mc-meters for driedboards.The second publication, which is presented in this thesis, investigates the possibilityof measuring the green sapwood mc in pine logs by combining industrial X-rayscanners and 3D-scanners. The method is shown to be suitable for identifying batchesof logs in which the sapwood has begun to dry prior to sawing, rather than predictingthe sapwood mc with high accuracy at the level of individual logs.In a single board, one would ideally like to measure the mc profile over the entirecross section to determine the mc gradient. However, the measured result is affectedin the vicinity of the board edges. In the third publication, the existence of thisproblem is identified, and it is shown that it can be decreased by using correlationfunctions generated by finite element simulations of the measurement system.The fourth publication considers the potential to increase the measurement accuracyof the mc of single boards by combining different measurement techniques.Microwaves, X-rays and visual sorting into different wood quality classes are used. Itis shown that the measurement accuracy is increased by the addition of both the Xraymeasurements and the visual sorting. This result is interesting because visualsorting is usually already present in the final sorting of large sawmills. This is alsowhere the mc measurement preferably is to be performed.

The research is presented as a composite thesis containing a theoretical explanatory review based on four scientific articles. The thesis covers the investigation of wood modification methods, which in the future could possibly be developed further to enhance the performance of wood products in an efficient and economical way. The target applications for the studied methods were non-structural wooden materials for outdoor exposure, such as cladding, facades and decking. Therefore, weather resistance, appearance and durability were the qualities to be studied.The methods were based on the combination of heat and impregnation with liquid preservatives. The following methods were included in the study: heat treatment in steam, heat treatment in oil with subsequent oil impregnation of heat treated wood and impregnation with artificial chemicals (copper, furfuryl alcohol and phenol formaldehyde) enhanced by preheating the wood. All the treatments were performed mainly on Scots pine, but also on Norwegian spruce and European varieties of ash, aspen, birch and beech. The first article covers the investigation of methods for oil heat treatment and oil impregnation of wood. The oil treatments were designed and performed at laboratory scale. The second and third articles cover studies concerning the behaviour of steam heat treated, oil heat treated and oil impregnated wood boards in terms of their resistance to cyclical climate conditions and brown rot fungi. The climate conditions were recreated in laboratory by letting the samples to soak in water, freeze in a freezer and absorb heat from an infrared lamp. The fourth article covers the study of impregnation of preheated wood with copper-based, furfuryl alcohol and phenol formaldehyde solutions; phenol formaldehyde treated wood was tested in the accelerated weathering test in order to find out if the treatment blocks wood resin coming out from the knots.The results showed that degradation processes within wood during oil heat treatment were similar to the degradation processes during heat treatment in steam, but with the difference of a small oil uptake when heat treating in oil. When heated wood is directly placed to cool down in oil at room temperature, then it absorbs significantly more oil than during the process of heat treatment in oil; similarly, impregnation with copper-based preservative after preheating was deeper compared to impregnation without preheating. However, oil seems to be rather unstable inside wood. After the samples were impregnated with oil in the experiments, the oil migrated throughout the wood and leached out when exposed to water and heat. By visual observation it was found that colour degraded to a certain extent in all untreated and treated species exposed to the cyclical weather conditions used in the study. It could also be noticed that the method used to create the cyclical weather conditions (mentioned above), in which the wood was exposed to periods of soaking, freezing and warming, showed to be an effective method to test the wood for outdoor applications. It was observed that both heat treatment and oil impregnation improved durability of wood against brown rot fungi. However, the oil impregnation method needs, probably, to be further developed to protect wood in the long term due to oil leakage from the wood. It was found that the phenol formaldehyde treatment using hot-and-cold bath process significantly reduced the migration of resin and extractives through knot in painted wood panels.

With population expansion and industrialization, heavy metal has become one of the biggest and most toxic water pollutants, which is a serious problem for human society today. The aim of this work is to explore the potential of nanopolysaccharides including nanocellulose and nanochitin to remove metal ions from contaminated water. The above nano-polysaccharides are of interest in water purification technologies due to their high surface area, high mechanical properties, and versatile surface chemistry. Silver, copper and iron are the main metal ions targeted in the study, due to their abundance in industrial effluents. The first study shows that the effect of pH conditions, surface charge and nature of surface functional groups on native nanocellulose and nanochitin on silver ion adsorption. The highest Ag+ ion removal efficiency observed for cellulose nanocrystals (CNC), was 64 % followed by chitin nanocrystals (ChNC, 37%) and cellulose nanofibers (CNF, 27%). Wavelength dispersive X-ray analysis (WDX) and X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of silver ions on the surface of the nanocellulose and nanochitin after adsorption. This study showed that the adsorption performance of cellulose nanofibers was inferior to cellulose nanocrystals and to enhance the adsorption capacities of nanofibers, surface functionalization of nanofibers was explored in the following study.In the second study, cellulose and chitin nanofibers functionalized with carboxylate entities have been prepared by chemically modifying the industrial residues, by TEMPO mediated oxidation followed by mechanical disintegration. The increase in copper adsorption on the nanofibers correlated both with the pH and carboxylate content and reached maximum values of 135 mg g-1 and 55 mg g-1 for highly oxidized cellulose and chitin nanofibers, respectively. Furthermore, the metal ions could be easily recovered from the contaminated nanofibers through a washing procedure in acidic water. The adsorption capacity of oxidized cellulose nanofibers for other metal ions, such as Nickel (II), Chromium (III) and Zinc (II), was also demonstrated. In the third study, the adsorption selectivity and removal efficiency of metal ions (Ag+, Cu2+ and Fe3+) from model water and industrial effluents by adsorption onto native and enzymatically phosphorylated nanocelluloses were studied. Phosphorylation significantly improved the functionality and sorption behaviour of nanocellulose. The removal efficiency is considered as being driven by the nature and density of functional group on the nanocellulose surface. Generally, when the mixture of metal ions are present in water the metal ion selectivity was in the order Ag+ > Fe3+ > Cu2+, irrespective of the surface functionality of nanocellulose. Nanocelluloses showed efficient adsorption in case of industrial effluent from mirror making industry and above 99% removal of Cu2+ and Fe3+ by phosphorylated nanocellulose was observed. The work has demonstrated that nano-polysaccharides, especially after surface modification are highly promising biosorbents for capturing heavy metal ions from water and of great industrial relevance and may enable next-generation water purification technologies.

The production of fully degradable nanocomposites with biopolymers as matrix and cellulose nanofibrils with high aspect ratios as reinforcement is still a challenging task. Also, due to the large amount of hydroxyl groups on the surface of these nanofibrils, they tend to irreversibly agglomerate during drying. This process, known as hornification, decreases the aspect ratio of the nanofibrils. Consequently, their reinforcing potential in nanocomposites is lowered. Thus, the objective of this PhD project is to produce novel biopolymer composites that are reinforced by functionalised cellulose nanofibrils in powder form. A successful preparation of such bio-based composites could open up ways to new applications in e.g. medicine, bio-packaging or horticulture. In order to induce an optimal compounding of the fibrils with different biopolymers, good fibril/matrix embedding is required. Therefore, the cellulose nanofibrils have to be modified appropriately to match the hydrophilic or hydrophobic nature of the polymer matrix.The production of fully degradable nanocomposites with biopolymers as matrix and cellulose nanofibrils with high aspect ratios as reinforcement is still a challenging task. Also, due to the large amount of hydroxyl groups on the surface of these nanofibrils, they tend to irreversibly agglomerate during drying. This process, known as hornification, decreases the aspect ratio of the nanofibrils. Consequently, their reinforcing potential in nanocomposites is lowered. Thus, the objective of this PhD project is to produce novel biopolymer composites that are reinforced by functionalised cellulose nanofibrils in powder form. A successful preparation of such bio-based composites could open up ways to new applications in e.g. medicine, bio-packaging or horticulture. In order to induce an optimal compounding of the fibrils with different biopolymers, good fibril/matrix embedding is required. Therefore, the cellulose nanofibrils have to be modified appropriately to match the hydrophilic or hydrophobic nature of the polymer matrix. In the first study, water-redispersible, nanofibrillated cellulose (NFC) in powder form was prepared from refined, bleached beech pulp (RBP) by carboxymethylation and mechanical disintegration. The sequence of the treatments influenced the stability of the final products in water. When carboxymethylation was applied first, enhanced disintegration of RBP into its sub-structural elements was observed. The prepared powder of this route formed a stable gel in water without sedimentation after 20 h. SEM images affirmed a significant reduction of cellulose nanofibrils agglomeration compared to unmodified NFC. The results suggest that NFC in dry form could be used as an alternative to conventional NFC in aqueous suspensions used as starting material for derivatization and compounding with biopolymers.

O objetivo deste trabalho foi produzir hidroxiapatita (HA) contendo diferentes tipos de própolis de origem brasileira e avaliar a possível atividade antimicrobiana dos materiais. Os extratos etanólicos de própolis (EEP) vermelha, verde e marrom foram obtidos em solução alcoólica 80%. EEP verde e vermelha (8 mg/mL e 20 mg/mL) foram incorporados ao material a 10% (m/v) via atomização (spray drying), obtendo-se HA-GP8, HA-GP20, HA-RP8 e HA-RP20. Os EEP e os materiais foram caracterizados quanto ao conteúdo de compostos fenólicos e flavonoides totais. A atividade antimicrobiana dos EEP foi avaliada por difusão em ágar, concentração inibitória mínima (CIM) e bactericida mínima (CBM) frente à Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) e Escherichia coli (E. coli). Os pós de HA incorporados com própolis foram avaliados frente à S. aureus por contagem de colônias bacterianas em placa, CIM e CBM. A caracterização dos materiais foi realizada por difração de raios X (DRX), espectroscopia na região do infravermelho com transformada de Fourier (FTIR) e microscopia eletrônica de varredura (MEV). A citotoxicidade foi determinada pelo cálculo da viabilidade celular, realizada pelo método de incorporação do vermelho neutro. Para os extratos, o conteúdo de fenólicos variou entre 278,3 e 325,6 mg EAG/g ES. EEP vermelha mostrou maior conteúdo de flavonoides (112,4 mg EQ/g ES) em relação aos outros EEP (48,4 e 52,3 mg EQ/g ES), apresentando maior atividade inibitória (CIM) frente à S. aureus e S. Epidermidis (12,5 µg/mL), comparado ao EEP verde (100 e 200 µg/mL) e marrom (200 µg/mL). Para CBM, os EEP vermelha e verde foram mais efetivos (800 µg/mL) comparado ao EEP marrom (1600 µg/mL), frente às mesmas bactérias. Entretanto, não foi observada atividade frente à E coli. A caracterização dos pós incorporados ou não com própolis apresentou estrutura cristalina e morfologia aparentemente esférica, indicando diminuição no grau de aglomeração com a adição de própolis. FTIR indicou a presença de grupos funcionais característicos para HA e própolis. Os materiais apresentaram alta liberação de fenólicos (228,3 a 327,6 mg EAG/g ES) e menores quantidades de flavonoides (14,0 a 35,8 mg EQ/g ES), sendo as maiores quantidades de flavonoides atribuída à HA contendo própolis vermelha. Foi verificado efeito bactericida a partir de 0,5 h (HA-RP20 e HA-GP20) e 1 h (HA-RP8 e HA-GP8) e menor atividade inibitória (CIM) para HA-GP20 e HA-GP8 (175,4 e 182,0 µg/mL) e para HA-RP20 e HA-RP8 (51,7 e 66,8 µg/mL), comparado aos EEP. Entretanto, observou-se maior atividade bactericida (CBM) para HA-GP20 e HA-GP8 (701,5 e 728,0 µg/mL) e para HA-RP20 e HA-RP8 (206,5 e 267,0 µg/mL), em relação aos EEP. O ensaio de citotoxicidade mostrou valores para IC50 (concentração que reflete 50% da viabilidade celular) de 387,1 e 84,8 µg/mL para as amostras HA-GP8 e HA-RP8, respectivamente. Considerando os resultados obtidos neste trabalho, sugere-se que a HA incorporada com própolis (HA-GP8 e HA-RP8) pode ser utilizada como possível agente antimicrobiano, inibindo o crescimento de S. aureus, respeitando-se os valores máximos para IC50. Entretanto, não poderia ser utilizada como agente bactericida, uma vez que nestas condições os materiais apresentaram efeito citotóxico. / The aim of this study was to produce hydroxyapatite (HA) containing different types of propolis of Brazilian origin and to evaluate the possible antimicrobial activity materials. The ethanolic extracts of red, green and brown propolis (EEP) were obtained in alcoholic solution 80%. Green and red EEP (8 mg / mL and 20 mg / mL) were incorporated into the 10% (m/v) via atomization (spray drying), obtaining HA-GP8, HA-GP20, HA-RP8 and HA -RP20. EEP and materials were characterized regarding the content of phenolic compounds and total flavonoids. The antimicrobial activity of the EPS was evaluated by diffusion in agar, minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) against Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli). The HA powders incorporated with propolis were evaluated against S. aureus by plate colony counting , CIM and CBM. Materials characterization was made by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The cytotoxicity of the materials was determined by cell viability calculation, which was made by the neutral red incorporation method. For extracts, phenolic content ranged from 278.3 to 325.6 mg EAG/g ES. The red EEP showed a higher flavonoid content (112.4 mg EQ/g ES) than the other EEP (48.4 and 52.3 mg EQ/g ES), showing a higher inhibitory activity (MIC) against S. aureus and S. epidermidis (12.5 µg/mL) compared to green EEP (100 and 200 µg/mL) and brown (200 µg/mL). For CBM, red and green EEP were more effective (800 µg/mL) compared to brown EEP (1600 µg/mL) against the same bacteria. However, no activity was observed against E coli. The characterization of the powders incorporated or not with propolis presented crystalline structure and apparently spherical morphology, indicating a decrease in the degree of agglomeration with the addition of propolis. FTIR indicated the presence of functional groups characteristic for HA and propolis. The materials presented high phenolic release (228.3 to 327.6 mg EAG/g ES) and lower amounts of flavonoids (14.0 to 35.8 mg EQ/g ES), with the highest amounts of flavonoids attributed to HA containing red propolis. The bactericidal effect for all materials was observed within the interval of 0.5 (HA-RP20 and HA-GP20) to 1 hour (HA-RP8 and HA-GP8). The materials showed lower inhibitory activity (MIC) for HA-GP20 and HA-GP8 (175, 4 and 182.0 µg/mL) and for HA-RP20 and HA-RP8 (51.7 and 66.8 µg/mL) compared to EEP. However, higher bactericidal activity (MBC) was observed for HA-GP20 and HA-GP8 (701.5 and 728.0 µg/mL) and for HA-RP20 and HA-RP8 (206.5 and 267.0 µg/mL) when compared to the EEP. The cytotoxicity assay showed values for IC50 (concentration that reflects 50% of cellular viability) of 387.1 and 84.8 µg/mL for HA-GP8 and HA-RP8 samples, respectively. Considering the results obtained in this work, it is suggested that HA incorporated with propolis (HA-GP8 and HA-RP8) can be used as a possible antimicrobial agent, inhibiting the growth of S. aureus, respecting the maximum values for IC50. However, it could not be used as a bactericidal agent, since under these conditions the materials had a cytotoxic effect.

Falhas ósseas são comumente encontradas na medicina veterinária em diferentes enfermidades, principalmente por traumas em grandes animais. Frequentemente faz-se necessário auxiliar este reparo tecidual para melhor correção desta falha. Os biomateriais foram desenvolvidos para cumprir esta função. A fim de avaliar o comportamento celular perante estes substitutos ósseos, a realização de testes in vivo é fundamental. A partir da observação da unidade morfofuncional do tecido é possível avaliar a quantidade e a qualidade do tecido neoformado. Atualmente, em estudos ortopédicos, os ovinos são considerados animais de escolha como modelo experimental. Falhas ósseas foram realizadas experimentalmente em ovinos, onde foram implantados biomateriais à base de quitosana, hidroxiapatita e colágeno. Após 60 dias foi realizada biopsia no local do reparo tecidual e por meio da histomorfometria, da análise descritiva da microscopia de luz e da microscopia eletrônica de transmissão, o tecido neoformado foi avaliado e comparado. Na histomorfometria, não houve diferença significativa quanto à quantidade de tecido neorformado do grupo controle e do grupo com biomaterial, porém quando comparado ao osso pré-existente, retirado no momento da confecção da falha, a área de matriz óssea foi menor. Na descrição da microscopia de luz e em maiores detalhes na análise ultra estrutural, observou-se o biomaterial em contato íntimo com o tecido neoformado, sugerindo boa biocompatibilidade. Foi possível observar o padrão da reparação tecidual, onde o membro controle e o membro com biomaterial não diferiram quanto à arquitetura tecidual, porém nas amostras com o implante do biomaterial foi identificada a presença do mesmo. Todas estas informações sugerem que o biomaterial de estudo apresenta potencial para auxiliar na rotina ortopédica de grandes animais. / Bone defects are commonly found in veterinary medicine through different disease such as trauma, especially in large animals. Frequently it is necessary to assist this tissue repair for better regeneration. Biomaterials in general are designed to this function. In order to understand the bone cells in the presence of bone substitutes, tests in vivo are essential for these studies. The quantity and quality of new tissue formation can be assessed by observation of tissue morphofunctional unit. Currently, in orthopedic studies sheep are considerated animals of choice for experimental development. Bone defects were performed experimentally in sheep and were implanted chitosan, hydroxyapatite and collagen biomaterials. After 60 days, biopsy was performed at the site of tissue repair and the new tissue formation was described, evaluated and compared by histomorphometry and electronic transmission microscopy. After hystomorfometry analysis there was no difference between control group and biomaterial group, but when compare with pre-existent tissue, the values are smaller. In descriptive analysis was observed intimate contact with biomaterial and new tissue formation suggesting biocompatibility. It observed the pattern of tissue repair in control group and biomaterial group did not differ in the architecture tissue. However, in the biomaterial group was identified presence of the biomaterial in the specimen. All these data suggest that the biomaterial study has the potential to assistant in orthopedic routine of large animals.

Um dos grandes desafios para o tratamento de defeitos ósseos extensos na região bucomaxilofacial têm sido o desenvolvimento de um biomaterial substituto ósseo ao enxerto autógeno. No presente trabalho avaliou-se a formação óssea e a biodegrabilidade do osso desproteinizado bovino Bio-Oss® e do seu similar GenOx Inorg® e da cerâmica bifásica GenPhos® XP no processo de reparo de defeitos ósseos cranianos em coelhos, comparativamente ao osso autógeno (controle positivo) e coágulo sanguíneo (controle negativo). Foram realizados cirurgicamente defeitos bilaterais de 8-mm de diâmetro nos ossos parietais de 39 coelhos. A seguir os defeitos foram preenchidos aleatoriamente com 0,1cm3 de material ou coágulo conforme cada grupo de tratamento. Após os períodos de 4, 8 e 24 semanas os crânios foram coletados, analisados no microtomógrafo e processados histologicamente. O percentual de volume do defeito ocupado pelo material e osso neoformado foi avaliado pela microtomografia e histomorfometria, enquanto que, para a medula óssea, tegumento e tecido conjuntivo, apenas pela análise histomorfométrica. Os resultados quantitativos obtidos foram comparados estatisticamente pela ANOVA a dois critérios (período e tratamento) e teste de Tukey com p<0,05. A intensidade da associação linear dos dados microtomográficos e histomorfométricos avaliada pelo coeficiente de correlação de Pearson, mostraram correlação moderada a forte. Nos períodos iniciais de reparo (30 e 60 dias), os defeitos tratados com Bio-Oss®, GenOx® Inorg e GenPhos® XP apresentaram manutenção do volume do material enxertado (Vvi médio de 34% ) e formação óssea menor e mais imatura em relação grupo autógeno (Vvi = 22% vs. 32% no grupo autógeno). No período mais tardio (180 dias) a quantidade de formação óssea foi estatisticamente similar nos grupos Bio-Oss® (Vvi = 27%), GenOx® Inorg (Vvi = 26%) e GenPhos® XP (Vvi = 20%) porém, o GenOx® Inorg promoveu a formação de um tecido ósseo mais organizado e com maior acúmulo de biomaterial+osso+medula óssea (Vvi = 67,9%) comparado ao GenPhos® XP (Vvi =58,9%) e Bio Oss (Vvi = 55,6%) mas, inferior ao do enxerto autógeno (Vvi = 78%). Os resultados aqui obtidos permitem concluir que o osso autógeno promove rápida formação e maturação óssea, porém não consegue promover o reestabelecimento completo da díploe removida cirurgicamente. Os materiais BioOss, GenOx® Inorg e GenPhos® XP são excelentes materiais osteocondutores levando a formação óssea em toda extensão do defeito, sendo o GenOx® Inorg o que apresenta menor grau de reabsorção e maior e melhor preenchimento do defeito. / One major challenge for treatment of critical size defects in maxillofacial region has been the development of a substitute biomaterial to the autogenous bone grafts. In present study we evaluated the bone formation and biodegradability of deproteinized bovine bone Bio-Oss® and GenOx® Inorg, and biphasic calcium phosphate GenPhos XP® during bone repair process in rabbits cranial defects compared to autogenous bone (positive control) and blood clot (negative control). In parietal bone of 39 rabbits were made bilateral 8-mm diameter defects, which were filled randomly with 0,1cm3 material or clot as each treatment group. After periods of 4, 8 and 24 weeks skulls of animals were collected, analyzed the MicroCT scanner and histologically processed. The percentage of defect volume occupied by biomaterial and new-formed bone were assessed by histomorphometry and microtomography, while the bone marrow, connective tissue and tegument only by first analysis. The quantitative data were compared by two-way ANOVA analysis (time and treatment) and Tukey\'s test at p <0.05. The intensity of the linear association of MicroCT and morphometric data evaluated by the Pearson correlation coefficient, showed moderate to strong correlation. In the early repair periods (30 and 60 days), the defects treated with Bio- Oss, GenOx® Inorg and GenPhos® XP showed maintenance of the graft material volume (average Vvi of 34%) and lower and more immature bone compared autograft group (Vvi = 22% vs. 32% in the autograft group). In the later period (180 days) the amount of bone formation was statistically similar to the groups Bio-Oss® (Vvi = 27 %), GenOx® Inorg (Vvi = 26%) and GenPhos® XP (Vvi = 20%) however, the bone formation in GenOx® Inorg was more organized and with greater accumulation of particles + bone tissue + bone marrow (Vvi = 67.9%), when compared to GenPhos® XP (Vvi = 58.9%) and Bio-Oss® (Vvi = 55.6%) but lower than the autograft (Vvi = 78%). It was concluded that the autogenous bone promotes rapid bone formation and maturation, but cannot promote the complete reestablishment of diploe surgically removed. The Bio-Oss®, GenOx® Inorg and GenPhos® XP are excellent osteoconductive materials leading to bone formation in the full extent of the defects, and the GenOx® Inorg showing less absorption promotes more and better defect filling.

Os princípios biológicos da regeneração óssea guiada (ROG) têm contribuído para o desenvolvimento de membranas que, em odontologia, são utilizadas em diversas situações como tratamentos com implantes dentários, aumento de rebordo alveolar e reparo de defeitos ósseos de origem traumática e patológica. Resultados de experimentos in vitro comparando a membrana obtida pela associação do polímero poli(fluoreto de vinilideno-trifluoretileno) e da cerâmica titanato de bário (P(VDFTrFE)/ BT) à membrana de politetrafluoretileno (PTFE) mostraram uma resposta favorável de osteoblastos, fibroblastos e queratinócitos à membrana de P(VDFTrFE)/ BT. O objetivo do presente estudo foi avaliar o efeito da membrana de P(VDFTrFE)/ BT sobre a formação óssea in vivo. Foram criados defeitos ósseos com 5 mm de diâmetro em calvárias de ratos machos Wistar (peso 200-250 g), distribuídos em três grupos com relação à utilização ou não de membranas nos defeitos ósseos: (1) membrana de P(VDF-TrFE)/BT; (2) membrana de PTFE; (3) nenhum tipo de membrana. Ao final de 4 e 8 semanas os animais foram eutanasiados e as amostras foram submetidas à: (1) análise por microtomografia computadorizada (micro-CT) para avaliar volume ósseo, superfície óssea, superfície óssea específica, número de trabéculas, separação trabecular e espessura trabecular, (2) análise histológica com base em cortes histológicos não descalcificados, (3) análise por reação em cadeia da polimerase em tempo real (PCR em tempo real) para avaliar a expressão gênica dos marcadores ósseos runt-related transcription factor 2 (RUNX2), fosfatase alcalina (ALP), sialoproteína óssea (BSP), osteocalcina (OC), osteoprotegerina (OPG) e receptor activator of nuclear factor-kappa B ligand (RANKL) e (4) análise da expressão de microRNAs (miRs) pela técnica de sequenciamento na plataforma Illumina. Os dados das análises morfométricas e da expressão gênica foram submetidos ao teste de Kruskal-Wallis, seguido pelo teste de Fischer quando apropriado, e para a análise da expressão de miRs foi utilizado o teste de Mann-Whitney para comparar a membrana de P(VDF-TrFE)/BT à de PTFE. Para todas as comparações o nível de significância adotado foi de 0,05. Os defeitos que não receberam a membrana tiveram uma formação óssea insignificante. Ambas as membranas favoreceram a formação óssea, sendo a superfície óssea maior sobre a membrana de P(VDF-TrFE)/BT comparada à de PTFE em 4 (p=0,01) e 8 (p=0,001) semanas e a separação trabecular maior sobre a membrana de PTFE comparada à de P(VDF-TrFE)/BT em 4 (p=0,05) e 8 (p=0,001) semanas. A expressão gênica de BSP (p=0,01), OC (p=0,001) e OPG (p=0,001) foi maior e de RANKL (p=0,001) foi menor sobre a membrana de P(VDFTrFE)/ BT comparada à de PTFE em 4 semanas. A expressão gênica de RUNX2 (p=0,001) e OC (p=0,05) foi maior e de ALP (p=0,05), RANKL (p=0,01) e OPG (p=0,001) foi menor sobre a membrana de P(VDF-TrFE)/BT comparada à de PTFE em 8 semanas. Quarenta e cinco e 13 miRs foram regulados positivamente (>2 vezes) em 4 semanas e 8 semanas, respectivamente, e 11 e 39 miRs foram regulados negativamente (>2 vezes) em 4 semanas e 8 semanas, respectivamente, pela membrana de P(VDF-TrFE)/BT comparada à de PTFE. Os resultados indicam que a membrana de P(VDF-TrFE)/BT favorece a formação óssea quando comparada à membrana de PTFE e, portanto, pode ser considerada um biomaterial promissor para ser utilizado em procedimentos de ROG. / Biological principles of guided bone regeneration (GBR) have contributed to development of membranes that, in dentistry, are used in several situations such as treatment with dental implants, alveolar bone augmentation and repair of traumatic and pathological bone defects. Results of in vitro experiments comparing membrane obtained by the combination of poly(vinylidene fluoride-trifluoroethylene) and barium titanate ceramics (P(VDF-TrFE)/BT) with polytetrafluoroethylene (PTFE) membrane showed a favorable response of osteoblasts, fibroblasts and keratinocytes to the P(VDF-TrFE)/BT membrane. The aim of this study was to evaluate the effect of P(VDFTrFE)/ BT membrane on in vivo bone formation. Bone defects with 5 mm in diameter were created in calvaria of male Wistar rats (weight 200-250 g), distributed into three groups regarding the use or not of membranes on defects: (1) P(VDF-TrFE)/BT membrane; (2) PTFE membrane; (3) no membrane. At the end of 4 and 8 weeks, the animals were euthanized and samples were subjected to: (1) computed microtomography analysis (micro-CT) to assess bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness and trabecular separation; (2) histological analysis based on non-decalcified histological sections; (3) real time polymerase chain reaction (real time PCR) to evaluate gene expression of the bone markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC), osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL); (4) microRNAs (miRs) sequencing analysis at Illumina platform. Data from morphometric and gene expression analyses were submitted to the Kruskal-Wallis test followed by Fischer\'s test, when appropriate, and from miRs expression to the Mann-Whitney test to compare P(VDF-TrFE)/BT with PTFE membrane. For all comparisons, the significance level was 0.05. Defects without membrane exhibit a non significant bone formation. Both membranes favored osteogenesis with an increased bone formation on the P(VDF-TrFE)/BT membrane compared with PTFE at 4 (p=0.05) and 8 weeks (p=0.001). Trabecular separation was greater on PTFE membrane compared with the P(VDF-TrFE)/BT at 4 (p=0.05) and 8 weeks (p=0.001). At 4 weeks, the gene expression of BSP (p=0.01), OC (p=0.001) and OPG (p=0.001) were higher on P(VDF-TrFE)/BT membrane compared with PTFE, while RANKL (p=0.001) was lower. The gene expression of RUNX2 (p=0.001) and OC (p=0.05) were higher and ALP (p=0.05), RANKL (p=0.01) and OPG (p=0.001) were lower on the membrane of P(VDF- TrFE)/BT compared with PTFE at 8 weeks. Fortyfive and 13 miRs were up-regulated (> 2 fold) at 4 weeks and 8 weeks, respectively, and 11 and 39 miRs were negatively regulated (> 2 fold) at 4 weeks and 8 weeks, respectively, on the P(VDF-TrFE)/BT membrane compared with PTFE membrane. The results indicate that the P(VDF-TrFE)/BT membrane favors bone formation compared with PTFE membrane and, therefore, may be considered a promising biomaterial for using in GBR procedures.

As lesões em membros de grandes animais, com perda significativa de tecido ósseo é um desafio para os médicos veterinários. Isso por que muitas vezes somente técnicas de osteossíntese não são capazes de garantir resultados plenamente satisfatórios. Devido a isso, muitos pesquisadores vêm se dedicando ao desenvolvimento e estudo da biocompatibilidade de substitutos ósseos, entre eles os biomateriais, com propósito de auxiliar na reparação óssea. Os compósitos com constituintes naturais como quitosana, colágeno e hidroxipatita são uma opção, por apresentarem estruturas em sua composição semelhantes ao tecido ósseo.O objetivo deste estudo foi avaliar o comportamento biológico do ovino frente a implantação de compósito de quitosana, colágeno e hidroxiapatita após implante em osso III/IV metacarpianos. Seis ovinos fêmeas da raça Santa Inês foram submetidos à ostectomias unicorticais de sete milímetros de diâmetro na região próximal da superfície dorso-medial do III/IV metacarpianos. Em seguida, de forma randômica, foi implantado o compósito de quitosana, colágeno e hidroxiapatita em um membro torácico. No membro contralateral, foi reproduzida a mesma técnica, porém foi mantido como controle, não sendo preenchido com o compósito. Todos os animais foram submetidos à avaliação física, radiográfica, ultrassonográfica, termográfica e perfil hematológico e bioquímico previamente a cirurgia para avaliação dos padrões fisiológicos. No período pós operatório os animais foram avaliados por meio de exame físico diário, durante os 60 dias de observação, exame radiográfico, exame ultrassonográfico , exame termográfico nos momentos sete, 14, 21, 28, 35, 42, 49 e 56 dias de pós-operatório. Com 60 dias de pós-operatório, realizou-se a biópsia óssea com trefina de 3,55 mm de diâmetro na área de interface entre biomaterial-osso (membro com compósito) e osso-osso (membro controle), para realização de avaliações histológicas de material calcificado, através de microscopia de luz e eletrônica de varredura. Não foram observadas alterações no exame físico dos animais (frequência cardíaca, frequência respiratória ou temperatura retal), ou claudicação ao longo dos 60 dias, que pudessem estar relacionados ao procedimento cirúrgico ou a presença do implante. A avaliação termográfica demonstrou que as alterações de temperatura no membro com compósito e controle seguiram os mesmo padrões, retornando aos padrões fisiológicos ao fim do período de avaliação. As avaliações radiográficas e ultrassonográficas sugerem que o membro controle e com compósito se encontravam em fases semelhantes de preenchimento de falha. Na histomorfometria, através da microscopia de luz, observou-se maior porcentagem de tecido neoformado em membro controle, quando comparado ao membro com compósito.Além disso, foi possível observar tanto por microscopia de luz quanto por microscopia eletrônica de varredura, que o biomaterial apresentou comportamento osteocondutor e não foram observadas reações adversas ao implante, como formação de tecido cicatricial ou reações de corpo estranho. Com os resultados dessa pesquisa, é possível concluir que o compósito de quitosana, colágeno e hidroxipatita quando implantado em tecido ósseo ovino apresenta biocompatibilidade e perfil osteocondutor. / The lesions in limbs of large animals, with significant bone loss is a challenger for veterinarians. That\'s why frequentely only osteosynthesis are not able to fully guarantee satisfactory results. Because of this, many researchers have dedicated the projects to develop and study the biocompatibility of bone substitutes, including biomaterials, in order to assist bone repair. The composites with natural constituents such as chitosan, collagen and hydroyapatite may be an option, because they have similar structures with bone tissue. This study evaluated the biological behaviour of composite chitosan, collagen and hydroxyapatite following implantation in the ovine III / IV metacarpal bone. Six healthy female sheep Santa Inês were submitted to bilateral unicortical ostectomy (7 mm/ diameter) on the proximal aspect of the medial-dorsal surface of the III / IV metacarpal bone. One front limb was randomly selected for composite chitosan, collagen and hydroxyapatite implantation, while the contralateral limb was left untrated, not being filled. The animals were submited to daily physical examination during 60-day follow-up period. Radiographic, ultrasonographic and thermographic reassessment were performed on postoperative days seven, 14, 21, 28, 35, 42, 49 and 56. On postoperative day 60, the animals were reoperated and 3,55 mm biopsy fragment collected from the biomaterial bone or bone-new bone interface to histological evaluation by light and scanning electron microscopy. No changes in the physical examination (heart rate, respiratory rate or rectal temperature), or lameness over the 60 days, which could be related to the surgery or the presence of the implant were observed. Thermographic evaluation showed that temperature changed on the members with composite and control limb and they followed the same patterns, returning to physiological rate at the end of study. The radiographic and ultrasonographic evaluations have suggested that the control limb and limb with composite were in similar stages of bone healing. On the morphology by light microscopy, we observed a higher percentage of the new bone formation at the control limb when compared with the composite limb. However it was observed at the light and electron scanning microscopy, which showed osteoconductive biomaterial behavior and no adverse reactions to the implant, as scar tissue or foreign body reactions. With the results of this research, it can be concluded that the composite of chitosan, collagen and hidroxipatita when implanted in sheep bone tissue presents biocompatibility and osteoconductive characteristics.

Falhas ósseas são comumente encontradas na medicina veterinária em diferentes enfermidades, principalmente por traumas em grandes animais. Frequentemente faz-se necessário auxiliar este reparo tecidual para melhor correção desta falha. Os biomateriais foram desenvolvidos para cumprir esta função. A fim de avaliar o comportamento celular perante estes substitutos ósseos, a realização de testes in vivo é fundamental. A partir da observação da unidade morfofuncional do tecido é possível avaliar a quantidade e a qualidade do tecido neoformado. Atualmente, em estudos ortopédicos, os ovinos são considerados animais de escolha como modelo experimental. Falhas ósseas foram realizadas experimentalmente em ovinos, onde foram implantados biomateriais à base de quitosana, hidroxiapatita e colágeno. Após 60 dias foi realizada biopsia no local do reparo tecidual e por meio da histomorfometria, da análise descritiva da microscopia de luz e da microscopia eletrônica de transmissão, o tecido neoformado foi avaliado e comparado. Na histomorfometria, não houve diferença significativa quanto à quantidade de tecido neorformado do grupo controle e do grupo com biomaterial, porém quando comparado ao osso pré-existente, retirado no momento da confecção da falha, a área de matriz óssea foi menor. Na descrição da microscopia de luz e em maiores detalhes na análise ultra estrutural, observou-se o biomaterial em contato íntimo com o tecido neoformado, sugerindo boa biocompatibilidade. Foi possível observar o padrão da reparação tecidual, onde o membro controle e o membro com biomaterial não diferiram quanto à arquitetura tecidual, porém nas amostras com o implante do biomaterial foi identificada a presença do mesmo. Todas estas informações sugerem que o biomaterial de estudo apresenta potencial para auxiliar na rotina ortopédica de grandes animais. / Bone defects are commonly found in veterinary medicine through different disease such as trauma, especially in large animals. Frequently it is necessary to assist this tissue repair for better regeneration. Biomaterials in general are designed to this function. In order to understand the bone cells in the presence of bone substitutes, tests in vivo are essential for these studies. The quantity and quality of new tissue formation can be assessed by observation of tissue morphofunctional unit. Currently, in orthopedic studies sheep are considerated animals of choice for experimental development. Bone defects were performed experimentally in sheep and were implanted chitosan, hydroxyapatite and collagen biomaterials. After 60 days, biopsy was performed at the site of tissue repair and the new tissue formation was described, evaluated and compared by histomorphometry and electronic transmission microscopy. After hystomorfometry analysis there was no difference between control group and biomaterial group, but when compare with pre-existent tissue, the values are smaller. In descriptive analysis was observed intimate contact with biomaterial and new tissue formation suggesting biocompatibility. It observed the pattern of tissue repair in control group and biomaterial group did not differ in the architecture tissue. However, in the biomaterial group was identified presence of the biomaterial in the specimen. All these data suggest that the biomaterial study has the potential to assistant in orthopedic routine of large animals.