La régénération osseuse est un processus impliquant de nombreux types cellulaires comme les ostéoblastes, les chondrocytes ou les cellules stromales mésenchymateuses (CSM). Les CSM possèdent des capacités de différenciation suggérant leur implication dans ce processus de réparation. La régénération osseuse est le fruit de la coordination complexe de l'activité de nombreux facteurs de croissance. Parmi eux, l'« Heparin affin regulatory peptide » (HARP) est fortement exprimé dans le callus durant la régénération mais son rôle n'est pas clairement établi. Le but de ce travail de thèse a été (1) d'évaluer les effets de HARP sur les propriétés de migration, de prolifération et de différenciation des CSM in vitro ; (2) évaluer la capacité de HARP à induire une formation osseuse ou une régénération osseuse in vivo.Nos résultats démontrent que HARP est chémoattractant pour les CSM et potentialise leur prolifération. De plus, nous montrons pour la première fois que le traitement de CSM par HARP durant leur chondroinduction conduit à une différenciation chondrocytaire de type hypertrophique. Ce type cellulaire est primordial dans les derniers stades de la formation osseuse endochondrale qui se met en place durant la croissance osseuse, mais également durant la réparation. L'implantation de biomatériaux associés à HARP dans un défaut osseux de condyle fémoral a conduit à la formation de cartilage et d'os dans l'implant, reproduisant le mécanisme physiologique de formation osseuse endochondrale. Le biomatériau seul n'a été envahi que par du tissu fibreux.Durant les processus de réparation tissulaire, les glycosaminoglycannes (GAG), des chaînes polysaccharidiques sulfatées, composants majeurs de la matrice extracellulaire, participent à la modulation des effets des facteurs de croissance durant la réparation. Récemment, des mimétiques structuraux et fonctionnels des GAG ont été développés. Durant ma thèse, j'ai été associé au travail d'un doctorant de l'équipe de P.Albanese, qui a montré que des mimétiques de GAG induisent une différenciation ostéoblastique des CSM en l'absence de traitement ostéoinducteur. L'implantation sous-cutanée de biomatériaux covalemment associés aux mimétiques ont également été menées, et ont permis d'observer des potentialisations des processus de vascularisation de l'implant et de l'activité ostéoclastique. Ces resultats ont permis de valider l'interêt des GAG mimétiques dans le cadre des thérapies de régénération osseuse.Cette étude démontre pour la première fois les effets chondroinducteurs directs de HARP sur la production de molécules de la matrice cartilagineuse par les CSM in vitro, mais également sur la synthèse de tissu cartilagineux in vivo. Les effets de HARP observés sur la régénération osseuse confirment qu'il pourrait être un bon candidat en chirurgie orthopédique en permettant une régénération de type endochondrale typique de la réparation physiologique. De plus les nouvelles stratégies developpées dans le laboratoire sur la fonctionnalisation covalente de biomateriaux par des GAG mimétiques, meriteraient d'etre testées en association avec HARP, afin d'augmenter sa demi-vie et de controler son relarguage et ses activités biologiques in vivo. / Bone regeneration is a complicated process which involved many cellular types such as osteoblasts, chondrocytes and mesenchymal stromal cells (MSC). MSC can differentiate toward chondrocytes and osteoblasts, suggesting their implication in bone regeneration processes. Bone reparation involved a complex coordination of growth factors. Among them, heparin affin regulatory peptide (HARP) is found in callus during regeneration. However, its role is poorly understood. The aim of this thesis was (1) to evaluate HARP effects on proliferation, migration and differentiation of MSC in vitro, (2) to evaluate HARP ability to promote bone regeneration or bone formation.Our results demonstrate that HARP has chemoattractive and proliferative properties on human MSC. Moreover, we show for the first time that HARP commits human MSC toward hypertrophy during chondrogenesis. This is of great interest since hypertrophic chondrocytes are of primary importance in the late stage of endochondral bone formation. We further tested the association of HARP to scaffolds in a model of bone regeneration in femoral defect in rat. HARP associated scaffolds showed an invasion of cartilage and bony tissues, mimicking endochondral bone formation, whereas scaffold alone was just filled with fibrous tissue.During regenerative processes glycosaminoglycans, polysaccharides sulfated chains, are known as major components of the extracellular matrix and modulate the effects of growth factors during regenerative processes. Recently, structurally and functionally mimetics of GAG had been developed. During my PhD thesis, I was associated to the work of a doctoral student of P. Albanese who showed that GAG mimetics induce osteoblastic differentiation of MSC without any other osteoinductive treatment. The ectopic implantation of mimetic associated scaffolds didn't show effects on osteoformation but induced an enhancement of vascularization and of osteoclastic activity, both related to tissue remodeling. These results validate that GAG mimetics are of great interest in bone regenerative field.This study demonstrates for the first time the chondroinductive potential of HARP through its ability to induce cartilage specific matrix production by MSC in vitro but also by inducing cartilage tissue synthesis in vivo. The effects of HARP observed on bone regeneration, by inducing an endochondral bone formation similar to that observed in normal bone regeneration, confirm that HARP could be a good candidate in orthopedic surgery. Moreover, scaffold covalently linked with GAG mimetics should be tested in association with HARP. This strategy could increase the half life, control the release and potentiate HARP properties in vivo.

O trabalho teve como objetivo avaliar e quantificar o tecido ósseo neoformado, a distribuição e a importância das proteínas não colágenas (osteocalcina, osteopontina e osteonectina) no processo de reparação tecidual do alvéolo dental de ratos Wistar idosos após exodontia. Para sua realização, foram utilizados 80 Rattus Norvegicus albinus, linhagem Wistar, machos. Os animais foram distribuídos em dois grupos: Grupo Controle, correspondente a animais com 60 dias de vida; e Grupo Experimental correspondente aos animais com 2 anos de vida (700 dias em média). Cada grupo foi dividido em 4 subgrupos de 10 animais em cada grupo. Os animais foram submetidos à exodontia do incisivo superior direito e foram sacrificados com 05, 15, 21 e 28 dias de pós-operatório. Após a dissecção, 5 amostras foram submetidas à análise por microscopia convencional com coloração por Hematoxilina e Eosina e análise da imunohistoquímica e 5 amostras para análise por RT-PCR. Os resultados mostraram que o processo de envelhecimento não alterou a cronologia do reparo ósseo alveolar e não promoveu maior remodelação do alvéolo dental. A osteocalcina não apresentou atuação importante nos períodos pós-operatórios estudados. A osteonectina apresentou-se importante no processo de reparo, não sofrendo alterações no início da reparação óssea, apresentando marcação mais intensa durante a maturação óssea entre 21 e 28 dias de pós-operatório no grupo controle e diminuição da marcação no grupo experimental aos 28 dias de pósoperatório. O envelhecimento proporcionou uma diminuição da imunomarcação da osteonectina e demonstrou marcações positivas principalmente em osteoblastos e matriz mineralizada. A osteopontina apresentou-se importante no processo de reparo ósseo durante todos os períodos pós-operatório, apresentando marcações em osteoblastos, matriz osteóide, osteócitos e matriz mineralizada, apresentando maior marcação dos tipos celulares do no grupo experimental aos 28 de pós-operatório. Apesar desses achados, novos estudos são necessários para o melhor entendimento do processo de reparo ósseo alveolar em ratos adultos e idosos. / The aim of the present study was to evaluate and quantify the newly formed bone tissue, as well as the distribution and the importance of non-collagen proteins (osteocalcin, osteopontin and osteonectin) in the process of dental alveolar repair in Wistar aged rats submitted to tooth extraction. To perform that, about 80 male Rattus Norvegicus albinus, Wistar strain, were randomly distributed into two groups: Control Group corresponding to 60 days old rats; and Experimental Group corresponding to 2 years old animals (about 700 days old). Both groups were later subdivided into 4 subgroups consisting of 10 animals each. All animals were submitted to the upright incisive tooth extraction and were euthanized 05, 15, 21 and 28 days after the tooth extraction surgery. After the dissection, five samples from each subgroup underwent conventional microscopy analysis by hematoxylin-eosin stain as well as immunohistochemistry. Bone tissue from other five samples of each groups were subjected to Real Time RT-PCR analysis of non-collagen proteins expression The results obtained suggest that aging process was not able to change either the chronology of alveolar bone repair or the remodeling of dental alveolus. Osteocalcin did not present any important action in the post-operation periods evaluated. On the other hand, osteonectin showed an important role during the repair process, since its expression was increased in the control group and decreased in comparison to the experimental group at 28 days. Osteopontin was important in the bone repair in all times evaluated, since it was present in osteoblasts, osteiod matrix, osteocytes and mineralized matrix, being even more stained at 21 days after the surgery. Finally, besides the results obtained in the present work, other studies are necessary to better understand the alveolar bone repair in adult and aged rats.

Este trabalho avaliou a reparação e indicadores de desnutrição de ratos submetidos à fratura unilateral de côndilo mandibular e com desnutrição protéica (8% de proteína com suplemento de vitaminas e minerais). Foram utilizados 45 Rattus norvegicus Wistar, adultos machos, distribuídos em 3 grupos de 15 animais: grupo fraturado, submetido à fratura de côndilo, sem alteração de dieta (23% de proteínas); grupo fraturado desnutrido, submetido à dieta hipoprotéica por 30 dias e posterior fratura condilar; grupo desnutrido, com dieta hipoprotéica prévia por 30 dias e mantida até o final do experimento, sem fratura de côndilo. Foi documentada a quantidade de ingestão de ração e água, feita a avaliação do peso e obtido o coeficiente de eficácia alimentar (CEA). Os animais foram sacrificados nos períodos de 24 horas e 7, 15, 30 e 90 dias pós-operatório. Foram realizados os seguintes testes bioquímicos do sangue: proteínas totais, albumina sérica, cálcio sérico, fosfatase alcalina, ferro sérico e creatinina sérica; e o leucograma. A seguir, foram feitas mensurações cefalométicas por radiografias da maxila e da mandíbula. O estudo histológico compreendeu a avaliação do local da fratura e da articulação temporomandibular. Os valores numéricos foram submetidos a análises estatísticas. O consumo de ração e água foi maior no grupo fraturado desnutrido, na maioria dos períodos. Os valores do CEA foram baixos principalmente nos períodos iniciais, sendo mais significativos para o grupo fraturado desnutrido. Houve pouco ganho de peso nos períodos iniciais, exceto no grupo fraturado desnutrido que apresentou perdas significativas, havendo recuperação de peso nos demais períodos, significativamente menor neste grupo. Os testes de bioquímica do sangue mostraram queda, principalmente nos períodos iniciais, para os valores de proteínas totais e albumina, bem como para cálcio sérico em todos os períodos, significante para o grupo fraturado desnutrido. O leucograma mostrou aumento, principalmente nos períodos iniciais, de leucócitos, linfócitos e neutrófilos, mais significativo no grupo fraturado desnutrido. Houve desvio da linha média mandibular em relação à linha média maxilar, significante para o grupo fraturado desnutrido, bem como assimetria de maxila e mandíbula, em especial no período final do experimento. A análise histológica mostrou que a desnutrição protéica levou à atrofia da fibrocartilagem do côndilo. A fratura sob desnutrição comprometeu a formação do calo ósseo, bem como houve anquilose fibrosa. Foi concluído que a fratura de côndilo mandibular em ratos com desnutrição protéica promoveu alterações negativas nos valores de proteínas totais, albumina e cálcio sérico, leucocitose, bem como comprometeu a formação do calo ósseo e induziu atrofia da fibrocartilagem e anquilose fibrosa. / This study evaluated the healing and undernutrition indicatives of rats submitted to mandibular condyle fracture and with protein undernutrition (8% of protein with vitamin and minerals supplement). Forty-five adult male Wistar Rattus norvegicus were used and distributed in 3 groups of 15 animals: fracture group, submitted to condylar fracture with no changes in diet (23% of proteins); undernutrition fracture group, submitted to hypoproteic diet by 30 days and later to condylar fracture; undernutrition group, with previous hypoproteic diet by 30 days, kept until the end of experiment, without condylar fracture. The amounts of feed and water intake were registered, as well as body weight and food efficiency ratio (FER) were obtained. Animals were sacrificed at 24 hours and 7, 15, 30 and 90 days postoperatively. The following blood biochemical tests were made: total serum proteins, serum albumin, serum calcium, alkaline phosphatase, serum iron, and serum creatinine; and also white blood count. Subsequently, cephalometric mensurations by radiograms of the maxilla and mandible were made. Histological study comprised fracture site and temporomandibular joint evaluations. Numerical values were submitted to statistical analyses. Feed and water intake were higher in undernutrition fracture group, in most of periods. Values of FER were low, in special in the initial periods, being more significative to undernutrition fracture group. There was low gain of weight in the initial periods, but undernutrition fracture group which presented significative loss of weight, with recovering of weight in the remaining periods, significatively lower in this group. Blood biochemical tests showed decrease, mainly in the initial periods, of the values of total serum proteins and serum albumin, as well as serum calcium in all the periods, significative to undernutrition fracture group. White blood count showed increase, in special in the initial periods, of leukocytes, lymphocytes and neutrophils, more significative in undernutrition fracture group. There was deviation of the median line of the mandible relative to the median line of the maxilla, significative to undernutrition fracture group, as well as asymmetry of the maxilla and mandible, in special in the final period of experiment. Histological analysis showed that proteic undernutrition lead to atrophy of condylar fibrocartilage. Fracture in undernutrition impaired callus formation, as well as there was fibrous ankylosis. It was concluded that mandibular condyle fracture in rats with proteic undernutrition promoted negative alterations in values of total proteins, albumin and serum calcium, leukocytosis, as well as impaired callus formation, and induced atrophy of condylar fibrocartilage and fibrous ankylosis.

As osteotomias mandibulares são procedimentos consagrados para o tratamento de prognatismo e para outros reposicionamentos cirúrgicos mandibulares. As técnicas mais utilizadas são: a osteotomia sagital de mandíbula (OSM) e a osteotomia vertical de ramo mandibular (OVRM), cada técnica tem suas indicações e limitações. A OVRM é considerada efetiva devido ao sucesso clínico e análise imaginológica, entretanto não existem estudos que descrevam seu processo de reparação óssea, pois nesta técnica o osso mandibular fica sobreposto num contato cortical sobre cortical. O objetivo deste trabalho foi avaliar a reparação óssea cortical-cortical da técnica de osteomia vertical de ramo mandibular em mandíbulas de coelho. Após aprovação do Comitê de Ética e Pesquisa em Animais da Faculdade de Odontologia da Universidade de São Paulo (Protocolo: 014/2014) foram utilizados 12 animais, divididos em quatro grupos de três. Os animais foram submetidos a osteotomia em -L? invertido no ângulo mandibular bilateralmente, este fragmento ósseo obtido foi deslizado anteriormente e fixado com parafuso sobre a basal mandibular anterior, num dos lados de um animal de cada grupo foi realizada somente a osteotomia, sendo este utilizado como controle cirúrgico; um dos grupos foi submetido a processo de infiltração de marcadores fluorcromáticos. Os animais foram divididos em G1 (eutanasiados em 15 dias), G2 (eutanasiados em 30 dias), G3 (eutanasiados em 45 dias) e G4 (protocolo de marcadores fluorocromáticos e eutanasiados com 56 dias). Após a realização dos procedimentos cirúrgicos, os animais foram mantidos em biotério padrão, nos tempos determinados foram eutanasiados, as mandíbulas foram dissecadas, tomografadas em tomógrafo de feixe cônico e analisadas sob microscopia de fluorescência. Na análise tomográfica observou-se que existe correlação entre os valores de densidades nas áreas operadas e na região do controle, e que há correlação significativa entre as áreas de sobreposição óssea e onde foi realizado somente a osteotomia (controle cirúrgico). Com relação a análise de microscopia de fluorescência, observa-se que as proporções do marcador alizarina são superiores a calceína no osso neoformado, tanto na área operada, quanto na região onde foi feita somente a osteotomia (controle cirúrgico). Concluímos que houve consolidação óssea quando da sobreposição cortical-cortical, e que os valores de densidades tomográficas quando da sobreposição óssea ou não, são similares, e que há maior formação óssea no primeiro mês no processo de reparação. / Mandibular osteotomy is a well-established intervention for the treatment of prognathism and other conditions requiring mandibular repositioning. The most commonly used osteotomy techniques are intraoral vertical ramus osteotomy (IVRO) and sagittal split ramus osteotomy (SSRO), each of which has particular indications and limitations. Although IVRO is considered effective owing to observations of clinical success and imaging analyses, there are no studies describing the bone repair processes that follow IVRO because, in this technique, the mandibular bone is overlaid at a cortical-cortical contact site. The purpose of this study was to evaluate cortical-cortical bone repair in rabbit mandibles following IVRO. After approval of the Ethics and Research Committee on Animals of the Faculty of Dentistry of the University of São Paulo (Protocol: 014/2014), 12 rabbits, divided into four groups of three, were used. The four groups, G1, G2, G3, and G4, were euthanized 15 days, 30 days, 45 days, and 56 days post-IVRO; the animals in G4 were also infused with fluorochemical markers. All 12 animals were submitted to a bilateral inverted \"L\" osteotomy at the mandibular angle. On one side, the released bone fragment was advanced anteriorly and fixed with a screw onto the anterior mandibular basal bone; on the other side, only the osteotomy was performed to serve as a within-animal control site. After the operations, the animals were kept in a standard room until they were euthanized at the group-designated time points. Upon euthanasia, the mandibles were dissected, imaged digitally by conical beam tomography, and analyzed under fluorescence microscopy. The tomographic analysis results revealed a correlation between density values in the operated IVRO sites of bone overlap and the contralateral osteotomy-only surgical control sites. Fluorescence microscopy analysis revealed the presence of elevated alizarin marker (above calcein levels) in the neoformed bone, both in the operated side and in the osteotomy-only control side. Bone growth was greater at the 15-day and 30-day time points than at the later time points. In conclusion, bone consolidation occurred at cortical-cortical overlap sites, tomographic densities were similar between bone overlapping and nonoverlapping (control) sides, bone formation was maximal during the first month of repair.

Este trabalho avaliou a reparação e indicadores de desnutrição de ratos submetidos à fratura unilateral de côndilo mandibular e com desnutrição protéica (8% de proteína com suplemento de vitaminas e minerais). Foram utilizados 45 Rattus norvegicus Wistar, adultos machos, distribuídos em 3 grupos de 15 animais: grupo fraturado, submetido à fratura de côndilo, sem alteração de dieta (23% de proteínas); grupo fraturado desnutrido, submetido à dieta hipoprotéica por 30 dias e posterior fratura condilar; grupo desnutrido, com dieta hipoprotéica prévia por 30 dias e mantida até o final do experimento, sem fratura de côndilo. Foi documentada a quantidade de ingestão de ração e água, feita a avaliação do peso e obtido o coeficiente de eficácia alimentar (CEA). Os animais foram sacrificados nos períodos de 24 horas e 7, 15, 30 e 90 dias pós-operatório. Foram realizados os seguintes testes bioquímicos do sangue: proteínas totais, albumina sérica, cálcio sérico, fosfatase alcalina, ferro sérico e creatinina sérica; e o leucograma. A seguir, foram feitas mensurações cefalométicas por radiografias da maxila e da mandíbula. O estudo histológico compreendeu a avaliação do local da fratura e da articulação temporomandibular. Os valores numéricos foram submetidos a análises estatísticas. O consumo de ração e água foi maior no grupo fraturado desnutrido, na maioria dos períodos. Os valores do CEA foram baixos principalmente nos períodos iniciais, sendo mais significativos para o grupo fraturado desnutrido. Houve pouco ganho de peso nos períodos iniciais, exceto no grupo fraturado desnutrido que apresentou perdas significativas, havendo recuperação de peso nos demais períodos, significativamente menor neste grupo. Os testes de bioquímica do sangue mostraram queda, principalmente nos períodos iniciais, para os valores de proteínas totais e albumina, bem como para cálcio sérico em todos os períodos, significante para o grupo fraturado desnutrido. O leucograma mostrou aumento, principalmente nos períodos iniciais, de leucócitos, linfócitos e neutrófilos, mais significativo no grupo fraturado desnutrido. Houve desvio da linha média mandibular em relação à linha média maxilar, significante para o grupo fraturado desnutrido, bem como assimetria de maxila e mandíbula, em especial no período final do experimento. A análise histológica mostrou que a desnutrição protéica levou à atrofia da fibrocartilagem do côndilo. A fratura sob desnutrição comprometeu a formação do calo ósseo, bem como houve anquilose fibrosa. Foi concluído que a fratura de côndilo mandibular em ratos com desnutrição protéica promoveu alterações negativas nos valores de proteínas totais, albumina e cálcio sérico, leucocitose, bem como comprometeu a formação do calo ósseo e induziu atrofia da fibrocartilagem e anquilose fibrosa. / This study evaluated the healing and undernutrition indicatives of rats submitted to mandibular condyle fracture and with protein undernutrition (8% of protein with vitamin and minerals supplement). Forty-five adult male Wistar Rattus norvegicus were used and distributed in 3 groups of 15 animals: fracture group, submitted to condylar fracture with no changes in diet (23% of proteins); undernutrition fracture group, submitted to hypoproteic diet by 30 days and later to condylar fracture; undernutrition group, with previous hypoproteic diet by 30 days, kept until the end of experiment, without condylar fracture. The amounts of feed and water intake were registered, as well as body weight and food efficiency ratio (FER) were obtained. Animals were sacrificed at 24 hours and 7, 15, 30 and 90 days postoperatively. The following blood biochemical tests were made: total serum proteins, serum albumin, serum calcium, alkaline phosphatase, serum iron, and serum creatinine; and also white blood count. Subsequently, cephalometric mensurations by radiograms of the maxilla and mandible were made. Histological study comprised fracture site and temporomandibular joint evaluations. Numerical values were submitted to statistical analyses. Feed and water intake were higher in undernutrition fracture group, in most of periods. Values of FER were low, in special in the initial periods, being more significative to undernutrition fracture group. There was low gain of weight in the initial periods, but undernutrition fracture group which presented significative loss of weight, with recovering of weight in the remaining periods, significatively lower in this group. Blood biochemical tests showed decrease, mainly in the initial periods, of the values of total serum proteins and serum albumin, as well as serum calcium in all the periods, significative to undernutrition fracture group. White blood count showed increase, in special in the initial periods, of leukocytes, lymphocytes and neutrophils, more significative in undernutrition fracture group. There was deviation of the median line of the mandible relative to the median line of the maxilla, significative to undernutrition fracture group, as well as asymmetry of the maxilla and mandible, in special in the final period of experiment. Histological analysis showed that proteic undernutrition lead to atrophy of condylar fibrocartilage. Fracture in undernutrition impaired callus formation, as well as there was fibrous ankylosis. It was concluded that mandibular condyle fracture in rats with proteic undernutrition promoted negative alterations in values of total proteins, albumin and serum calcium, leukocytosis, as well as impaired callus formation, and induced atrophy of condylar fibrocartilage and fibrous ankylosis.

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.

Submitted by Jaqueline Bastos (keca78@yahoo.com.br) on 2016-04-26T17:00:34Z No. of bitstreams: 1 TESE JAQUELINE S BASTOS - finalizando 180316.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-04-28T17:50:25Z (GMT) No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) / Made available in DSpace on 2016-04-28T17:50:25Z (GMT). No. of bitstreams: 1 bastos_js_dr_guara.pdf: 13526973 bytes, checksum: d40891f9f50c56b18b4ee564f6453a39 (MD5) Previous issue date: 2016-01-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desse estudo in vivo foi verificar a resposta óssea de scaffolds porosos revestidos processados pela técnica de Sinterização Direta de Metais a laser (DMLS) associado à terapia de ultrassom de baixa intensidade. Os scaffolds foram processados empregando a técnica DMLS e tratados termicamente a 1000°C por 24 horas. Três tipos de tratamento de superfície foram avaliados: Alcalino, biomimético e imobilização de alendronato de sódio. Para o tratamento alcalino, as amostras foram imersas na solução de NaOH (5M) a 60ºC por 24 horas. O tratamento biomimético consistiu na imersão dos scaffolds em solução SBF (SimulatedBodyFluid) enquanto a imobilização do alendronato foi realizada a partir da imersão dos scaffolds em uma solução formada por SBF e medicamento durante 5 dias. As superfícies dos scaffolds foram avaliadas para cada etapa empregando microscopia eletrônica de varredura (MEV) e análise por difração de raios-X. Os scaffolds foram implantados na tíbia direita de 85 ratos machos da raça wistar com idade média de 12 semanas. A microtomografia computadorizada (µCT) e análise histológica foram realizadas para avaliar o reparo ósseo no defeito. As micrografias das superfícies obtidas mostraram mudanças no aspecto da superfície e composição química de acordo com o tratamento. O tratamento biomimético promoveu o crescimento da apatita sobre a superfície enquanto a imobilização com alendronato suprimiu sua formação. As imagens obtidas na microtomografia mostraram elevado valor de densidade óssea para o último grupo. No entanto, análises histológicas mostraram a formação de cápsula fibrosa em torno dos scaffolds a qual foi minimizada usando ultrassom pulsado de baixa intensidade. No entanto, mais estudos precisam ser realizados para avaliar a influência da geometria dos scaffolds na incorporação de medicamentos. / The objective of this in vivo study was to verify the bone response of coated Ti6Al4V porous scaffolds processed by Direct Metal Laser Sintering (DMLS) technique associated to low intensity pulsed ultrasound therapy. Scaffolds were processed by using Direct Metal Laser Sintering technique (DMLS) and heat treated at 1000 °C for 24 hours. Three types of surface treatments were evaluated: alkaline, biomimetic and sodium alendronate immobilization. For alkaline treatment, samples were immersed in a NaOH (5M) solution at 60ºC for 24 hours. Biomimetic treatment consisted in the immersion of the scaffolds into Simulated Body Fluid solution while for sodium alendronato immobilization the scaffolds were immersed in the solution formed by SBF plus drug during 5 days. The scaffolds surfaces were evaluated after each step employing SEM (Scanning Electron Microscopy)and X-rays diffraction analysis(XRD). Scaffolds were implanted into right tibia of 85 male Wistar rats with average age of 12 weeks. X-rays micro-computed tomography (µCT) and histological analysis were carried out to evaluate the bone repair on the defect. Micrographs analysis showed that the aspect of the surfaces and chemical composition changed according treatment. Biomimetic treatment promoted the growth of the apatite on the surface; in contrast the immobilization of alendronate suppressed apatite formation. Micro CT images showed higher value of bone density for the last group. However, histological analysis showed the formation of encapsulation fibrous around the scaffolds. This formation was minimized by using low intensity pulsed ultrasound technique, however, more studies can be carried out to evaluate the influence of scaffolds geometry in the drug incorporation.