Introdução: Pacientes com câncer na região de cabeça e pescoço normalmente são tratados por combinação de cirurgia, radioterapia (RT) e quimioterapia. Em muitos casos, a reabilitação oral com próteses implanto-suportadas representa a melhor opção para uma recuperação funcional adequada. Entretanto, em pacientes irradiados procedimentos como exodontias e instalações de implantes dentários são fatores de risco ao desenvolvimento de osteorradionecrose. Diversos estudos experimentais têm demonstrado que o uso de células-tronco mesenquimais (CTMs) associadas a fatores de crescimento como plasma rico em plaquetas (PRP) proporciona melhora no reparo ósseo e na osseointegração, podendo ser considerada uma alternativa viável para defeitos ósseos ou injúria. Objetivos: Isolar e caracterizar as CTMs da medula óssea (MO) de miniporcos brasileiros (Minipigs BR-1), avaliar a interferência da RT e o efeito da associação de CTMs-MO+PRP no processo de osseointegração de implantes instalados em alvéolos frescos em mandíbulas de miniporcos, por análise histológica e histomorfométrica da interface osso-implante. Métodos: CTMs-MO de 12 miniporcos adultos machos foram isoladas da crista ilíaca. Após 21 dias de cultura, o potencial de diferenciação celular foi avaliado por meio de coloração e RT-PCR. O perfil imunofenotípico foi caracterizado por citometria de fluxo. Os animais foram divididos em três grupos: Grupo A (grupo controle, sem RT), Grupo B (implantes instalados 15 dias antes da RT) e Grupo C (implantes instalados três meses após a RT). A dose total de radiação para cada lado da mandíbula foi de 24 Gy, divididos em três doses de 8 Gy com intervalo de 7 dias entre as doses, a qual equivale biologicamente a aproximadamente 56 Gy, com 28 exposições de 2 Gy cada. Quatro implantes de titânio foram instalados nos alvéolos frescos, imediatamente após as extrações dos terceiro e quarto pré-molares, totalizando 48 implantes controles e 48 experimentais (uso de CTM-MO+PRP). Os animais foram eutanasiados 90 dias pós-implantação. Foram analisados o percentual de implantes perdidos (PIP), o contato osso-implante (COI), e a densidade óssea no interior das roscas (DOIR). Resultados: A eficiência de isolamento das CTMs-MO foi de 100% e em cultura as células apresentaram morfologia fibroblastóide. As células foram positivas para CD90 (88,6%), CD29 (89,8%), CD44 (86,9%) e negativas para CD34 (1,6%), CD45 (1,8%), CD14 (1,8%) e MHC-II (2,7%). As células foram diferenciadas em adipócitos, demonstrado pela presença de vacúolos lipídicos no interior das células; osteoblastos, pela mineralização da matriz extracelular e condrócitos pela presença de lacunas ao redor dos condrócitos jovens. A maior expressão gênica de AP2, ALP e COL II em células induzidas também confirmou o potencial de diferenciação (p < 0,001; p < 0,001; p = 0,031; respectivamente). Os PIP nos lados controle e experimental foram respectivamente 25,0% e 18,7% no grupo A (p = 0,686), 31,2% e 25,0% no grupo B (p=0,686) e 68,7% e 68,7% no grupo C (p =1,000). Na comparação entre os três grupos, o PIP apresentou diferença estatisticamente significante tanto no lado controle (p=0,041) como no experimental (p = 0,047). Os percentuais de COI nos lados controle e experimental foram respectivamente 39,0 e 27,7 no grupo A (p = 0,110); 20,9 e 16,7 no grupo B (p=0,347) e 16,0 e 7,1 no grupo C (p = 0,310), com diferença estatística entre os grupos tanto no lado controle (p = 0,033) quanto no experimental (p=0,046). As DOIR nos lados controle e experimental foram respectivamente 46,8 e 36,5 no grupo A (p = 0,247); 29,3 e 24,1 no grupo B (p = 0,379) e 21,0 e 11,6 no grupo C (p = 0,421), porém com diferença estatisticamente significante entre os três grupos somente no lado controle (p=0,025). Conclusões: As CTMs-MO de Minipigs BR-1, obtidas com o protocolo utilizado neste estudo são células-tronco, podendo ser aplicadas em ensaios pré-clínicos na medicina regenerativa. Os resultados mostraram um efeito negativo da radiação ionizante na neoformação óssea periimplantar tanto no grupo B quanto no C. Após a RT a perda de implantes foi três vezes maior que em osso não irradiado. A RT realizada 15 dias após a instalação dos implantes não interferiu na perda de implantes, sugerindo que este seria o melhor momento para reabilitação bucal com implantes dentários. Com a metodologia empregada, o uso da associação CTMs-MO+PRP previamente à instalação de implantes não apresentou efeito positivo significante na neoformação óssea periimplantar / Introduction: Patients with head and neck cancer are usually treated by a combination of surgery, radiotherapy (RT) and chemotherapy. In many cases, oral rehabilitation with implant-supported prostheses is the best option for a proper functional recover. However, in irradiated patients procedures as dental extractions and implants are risk factors for developing osteoradionecrosis. Several experimental studies have shown that the use of mesenchymal stem cells (MSCs) associated with growth factors such as platelet-rich plasma (PRP) provides improvement in bone regeneration and osseointegration, being considered an alternative to bone defects or injury. Objectives: To isolate and characterize MSCs from bone marrow (BM) of Brazilian minipigs (Minipigs BR-1), to evaluate the effect of RT and of BM-MSCs+PRP in the osseointegration of implants placed in fresh sockets, by histological and histomorphometric analysis of the bone-implant interface. Methods: BM-MSCs from 12 adult male minipigs were isolated from the iliac crest. After 21 days of culture, cell differentiation potential was assessed by staining and RT-PCR. The immunophenotypic profile was characterized by flow cytometry. The animals were divided into three groups: Group A (control group, no RT), Group B (implants placement 15 days before RT) and Group C (implants placement three months after RT). The total radiation dose for each side of the mandible was 24 Gy, divided into 3 doses of 8 Gy with a 7 dayinterval for each dose, which is biologically equivalent to approximately 56 Gy, with 28 sections of 2 Gy each. Four titanium implants were installed in the alveoli fresh, immediately after the extraction of the third and fourth premolars of each hemimandible, totalling 48 implants on the control side and 48 on the experimental side (using BM-MSCs+PRP). The animals were euthanized 90 days post-implantation. The implant loss rate (ILR), the bone-implant-contact (BIC) and bone density inside the threads (BDIT) were determined in each group. Results: The efficiency of the isolation of BM-MSCs was 100%, and in culture, the cells showed fibroblastoid morphology. Cells were positive for CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.6%), CD45 (1.8%), CD14 (1.8%) and MHC-II (2.7%). Cells were differentiated into adipocytes, as demonstrated by the presence of lipid vacuoles in the cells, osteoblasts, by mineralization of extracellular matrix and chondrocytes, by the presence of gaps around the young chondrocytes. The higher gene expression of AP2, ALP and COL II at induced cells also confirmed the differentiation potential (p < 0.001, p < 0.001, p=0.031; respectively). The ILR in control and experimental sides were respectively 25.0% and 18.7% in group A (p=0.686), 31.2% and 25.0% in group B (p = 0.686) and 68.7% and 68.7% in group C (p =1.000), with a statistically significant difference between the three groups at the control side (p = 0.041) and at the experimental side (p = 0.047). The percentage of BIC in control and experimental sides were respectively 39.0 and 27.7 in group A (p = 0.110), 20.9 and 16.7 in group B (p = 0.347) and 16.0 and 7.1 in group C (p = 0.310), with statistical significance between the groups at the control side (p = 0.033) and at the experimental side (p = 0.046). The BDIT in experimental and control sides were respectively 46.8 and 36.5 in group A (p = 0.247), 29.3 and 24.1 in group B (p = 0.379) and 21.0 and 11.6 in group C (p = 0.421), with statistical significance between the three groups only at the control side (p = 0.025). Conclusions: The BM-MSCs of Minipigs BR-1 obtained with this protocol can be used in pre-clinical regenerative medicine since they are stem cells. The results showed a negative effect of RT in peri-implant bone regeneration in group B and C. The implant loss was three times higher in irradiated bone than in non-irradiated one. The RT performed 15 days after implant placement did not interfere with implant loss, suggesting that this would be the best time for oral rehabilitation with dental implants. With this methodology, the use of BM-MSCs+PRP before the implant placement did not show any significant positive effect on peri-implant bone regeneration

O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5&micro;m sob microscopia Confocal. Constatamos o homing das CTIPDh nos órgãos derivados das linhas germinativas endodermais, ectodermais e mesodermais. No estômago e intestinos as CTIPD/GFP+ foram identificadas tanto no espaço intraglandular, como na camada muscular da mucosa; no fígado no parenquima hepático; no coração especialmente no tecido muscular do miocárdio; no cérebro nos vasos da substância branca, e cerebelo entre células de Purkinje. Na placenta estas células foram encontradas especialmente junto aos vasos. Quantificamos as CTIPD GFP+ utilizando a citometria de fluxo. Comparativamente dentre os órgãos analisados, obtivemos resultados expressivos do homing celular no miocárdio (~50%), no baço e fígado. Nossos resultados foram confirmados através das análises de imunohistoquímica e imunofluorescência utilizando os anticorpos Anti-núcleo (HuNu), Anti-CTIPD e Anti-GFP humanos. Concluímos que as CTIPDh apresentam grande potencial migratório e proliferativo após o TIUCT em fetos caninos. Estas células indiferenciadas demonstraram homing, especialmente nos tecidos: hematopoiéticos fetais (placenta, fígado e baço), tecido epitelial e glandular de órgãos, bem como de nichos perivasculares de CT. Estes dados sugerem que as CTIPD através do TIU, é uma alternativa viável, segura e promissora para o tratamento de doenças genéticas, congênitas, hematológicas e imunológicas. / Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs. All experimental procedures were approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of São Paulo University and were performed under appropriate anesthesia. 1x106 of undifferentiated GFP-positive human IDPSCs were transplanted following laparotomy and intraperitoneal injection under intra-operative ultrasound control into 5 fetuses at the 45 days of gestation. Five fetuses, which did not receive IDPSCs, were used as a control. Ultrasound analyses were performed daily before collection of the fetuses. After 7 days ovarian hysterectomy was performed, fetuses were collected; organs and tissues were isolated and fixed in 4% paraformaldehyde or cryopreserved. Biodistribution of IDPSCs within the organs and tissues were analyzed on cryosections (5&micro;m) under Confocal Microscopy. Homing of IDPSCs was observed in organs derived from three germ lines, endoderm, ectoderm and mesoderm. In stomach and in intestine GFP IDPSCs were found in intraglandular space as well as in muscularis mucosae. In liver they appeared in hepatic parenchyma; in heart in myocardium and in brain in bold vessels, in cerebellum within Purkinje cells. Using Flow cytometry assay GFP IDPSCs graft was quantified. Among the different organs an expressive homing was observed in myocardium of heart (~50%), in spleen and liver. The IDPSCs were also found in canine placenta, especially in blood vessels. These data were confirmed using anti-human nucleus (HuNu), anti-GFP and anti-IDPSCs anti-bodies. Human IDPSCs showed high migration and proliferation potential after IUSCT in dog fetuses. Undifferentiated IDPSCs demonstrated homing in fetal hematopoietic (placenta), epithelial (gastric glands) and perivascular stem cells niches. Our data suggest that IDPSCs is a new promising source for genetic, congenital, hematological, and immunological treatment for those diseases through IUSCT.

Notre équipe a développé une stratégie innovante de fonctionnalisation d’implants nanofibreux synthétiques à base de nanoréservoirs actifs pour la médecine régénérative osseuse. Notre objectif essentiel est de proposer un implant synthétique, biodégradable, et nanostructuré permettant d’accélérer la réparation du tissu osseux. Ces nouveaux implants synthétiques représentent un choix alternatif aux membranes de collagène d’origine animale. Notre stratégie consiste à construire des nanoréservoirs de chitosane, contenant des facteurs ostéoinducteurs tels que la BMP-2 afin d’enrober les nanofibres de nos implants. L’implant synthétique et biomimétique a été conçu à partir du le poly(ε-caprolactone) (PCL),polymère biocompatible et biodégradable approuvé par la FDA, et élaboré grâce à la technique de l’electrospinning afin de mimer la matrice extracellulaire. L’optimisation de ce procédé a permis la mise en oeuvre d’implants d’épaisseurs différentes (jusqu’à 10mm). La double fonctionnalisation de l’implant a permis de le rendre bioactif et vivant en utilisant la combinaison de facteur de croissance et de cellules souches mésenchymateuses. L’efficacité de la double fonctionnalisation des implants de PCL a ainsi été mise en évidence par l’accélération de la régénération osseuse in vivo.L’activité de ces implants fonctionnalisés de nanoréservoirs bioactifs est en cours d’analyse dans le cadre de tests précliniques pour une application maxillo-faciale, parodontale et orthopédique en vu d’obtenir un marquage CE. De plus, une start-up (ARTiOS NanoMed) basée sur cette nanotechnologie a été crée. En conclusion, nous pensons que la technologie développée par notre laboratoire a permis une avancée dans le domaine de la régénération osseuse et que cette technologie présente un fort potentiel d’application en clinique. / Our team has developped a novel and unique strategy to functionnalize nanofibrous and synthetic implants based on active nanoreservoirs for bone regeneration. We propose a new synthetic biodegradable and nanostructured implant to accelarate restoration of bone tissue. These new implants could replace collagen membranes from animal origin. The nanoreservoirs are based on chitosan containing osteoinductive growth factors such as BMP-2. Poly(ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer approved by FDA and has been used to produce the synthetic and biomimetic implants by electrospinning in order to mimic the bone extracellular matrix. Optimization of this process has allowed the elaboration of nanofibrous implants with different thicknesses reaching 10 mm. Using the combination of growth factors and mesenchymal stem cells in a double functionalization created a bioactive and living implant. This strategy has been validated in vitro and in vivo thanks to bone site implantation in murin model. Acceleration of bone regeneration in vivo has brought to light the efficiency of the double functionalization onto the PCL implants.The functionalized implants bioactivity is still currently in study for pre-clinical trials in order to obtain authorization for applications in maxillo-facial, parodontal, and orthopaedic fields. Moerover, astat-up (ARTiOS NanoMed) based on this nanotechnology has been founded.To conclude, we believe that our nanotechnology could lead to a new generation of engineered bone implants which has a great potential to be used in the clinic.

Une approche de la médecine régénérative du système nerveux consiste à développer des substituts biologiques avec une fonction réparatrice en utilisant des cellules souches et des biomatériaux qui peuvent être recouverts des molécules de la matrice extracellulaire. Nous avons ainsi développé des microcarriers pharmacologiquements actifs, MPA. Ce sont des microsphères (MS) polymériques à base de PLGA, biodégradables et biocompatibles, recouvertes des molécules d’adhérence qui fournissent un support en 3-dimensions aux cellules. Les microcarriers ainsi associés aux cellules souches permettent, après implantation, d’augmenter la survie et de maintenir l’état de différenciation des cellules qu’ils portent,renforçant leurs effets de réparation tissulaire. Ces MPA peuvent également libérer des facteurs de croissance encapsulés et afin d’améliorer le relargage de protéines encapsulées une nouvelle combinaison de polymère : PLGA-Poloxamer188 (P188) -PLGA a été développé dans notre laboratoire. Il a aussi été montré que les MPA de PLGA-P188-PLGA fonctionnalisées avec de la fibronectine et poly-D-lysine induisaient une meilleure prolifération de cellules souches mésenchymateuses que les MPA de PLGA.Ces cellules sont très largement utilisées en médecine régénérative car elles sont faciles à prélever, se trouvant dans la moelle osseuse, et capables de se différencier vers le lignage chondrogénique, ostéogénique et dans certaines conditions, neuronale. Nous travaillons avec une sous population de ces cellules appelées cellules MIAMI (marrow isolated adult multilineage inducible) qui s’engagent vers une différenciation en cellule neuronale après un traitement avec 2 facteurs de croissance (EGF/ bFGF) et sur un support matriciel de laminine. Dernièrement, il a été mis en évidence que les propriétés physicochimiques des supports polymériques régissent également le comportement des cellules souches(adhésion, survie et différenciation). L’objectif de cette étude est d’étudier l’effet des propriétés physicochimiques et mécaniques des surfaces i) des MS sur l’adsorption de laminine et poly-D-lysine et ii) des MPA sur l’adhérence et la différenciation neuronale des cellules MIAMI. Nous avons montré que la présence du bloc hydrophile « poloxamère 188 » dans la composition du polymère PLGA-P188-PLGAdiminue l’adsorption de molécules d’adhérence en formant une couche sur ces surfaces. Sur les MPA de PLGA, les molécules d’adhérence s’adsorbent bien quelle que soit la charge globale des molécules. Cesdeux MPA ont une charge globale positive et permettent l’attachement de cellules à leur surface. Cependant, l’adhérence à court terme de cellules est plus forte sur les MPA de PLGA comparé aux MPA de PLGA-P188-PLGA mais à la longue les cellules finissent par adhérer aux deux supports. Le PLGAP188-PLGA présente une forte énergie libre de surface et ces MPA présentent une surface moins rigide que les MPA de PLGA. Nos résultats suggèrent que ces caractéristiques de surface permettent aux cellules d’adhérer malgré la faible quantité de laminine sur ces supports. A long terme les cellules présentent le même comportement quel que soit le type du support. Elles se différencient en cellule de type neuronal exprimant des marqueurs de neurone mature comme le neurofilament et nous trouvons le même nombre de cellules adhérées à leur surface. En outre, nous avons montré que les cellules sont capables de sécréter de la même manière des molécules de la matrice extracellulaire sur les deux types de MPA expliquant probablement la similitude de comportement à long terme. / An approach to regenerative nervous system medicine is to develop biological substitutes with restorative function using stem cells and biomaterials that can be coated with extracellular matrix molecules. We have developed pharmacologically active microcarriers, PAMs. These are PLGA based, biodegradable and biocompatible polymeric microspheres (MS) coated with adhesion molecules that provide 3-dimensional support for cells. The microcarriers thus associated with the stem cells make it possible, after implantation, to increase the survival and maintain the state of differentiation of the cells they carry, reinforcing their tissue repair effects. These PAMs can also release encapsulated growth factors and to enhance the release of encapsulated proteins a new polymer combination: PLGA-Poloxamer188 (P188) -PLGA has been developed in our laboratory. It has also been shown that PLGA-P188-PLGA PAMs functionalized with fibronectin and poly-Dlysineinduce better proliferation of mesenchymal stem cells than PLGA PAMs. These cells are very widely used in regenerative medicine because they are easy to collect, found in the bone marrow, and able to differentiate towards the chondrogenic lineage, osteogenic and under certain conditions,neuronal. We are working with a subpopulation of these cells called MIAMI cells (marrow isolated adult multilineage inducible) that engage in neuronal cell differentiation after treatment with 2growth factors (EGF / bFGF) and on a laminin matrix support. Recently, it has been demonstrated that the physicochemical properties of polymeric supports also regulate the behavior of stem cells (adhesion, survival and differentiation). The objective of this study is to study the effect of physicochemical and mechanical properties of surfaces i) MS on laminin and poly-D-lysineadsorption and ii) PAMs on adhesion and neuronal differentiation of MIAMI cells. We have shown that the presence of the hydrophilic "poloxamer 188" block in the PLGA-P188-PLGA polymer composition decreases the adsorption of adhesion molecules by forming a layer on these surfaces.On PLGA PAMs, the adhesion molecules adsorb well regardless of the overall charge of the molecules. These two PAMs have a positive overall charge and allow the attachment of cells to their surface. However, in short-term cell adhesion is stronger on PLGA PAMs compared to PLGA-P188-PLGA PAMs, but in the long-term the cells eventually adhere to both supports. PLGA-P188-PLGAhas a high free surface energy and these PAMs have a less rigid surface than PLGA PAMs. Our results suggest that these surface characteristics allow cells to adhere despite the low amount of laminin on these supports. In the long-term the cells exhibit the same behavior whatever the type of PAMs. They differentiate into neuronal cells expressing mature neuron markers such as the neurofilament-M and we find the same number of cells adhered to their surface. Furthermore, we have shown that cells are able to secrete extracellular matrix molecules in the same way on both types of PAMs, probably explaining the similarity of the behavior in long-term.

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INTRODUÇÃO: Os enxertos de gordura tem se mostrado como uma poderosa técnica cirúrgica em reconstrução mamaria secundária e os enxertos enriquecidos com células-tronco, além de suas ações parácrinas, vem apresentando resultados encorajadores no que tange a persistência volumétrica. OBJETIVO: Este estudo clínico teve como objetivo analisar comparativamente quantitativa e qualitativamente enxertos de gordura enriquecidos com células da fração vásculoestromal em reconstrução mamária secundária e a incidência de complicações. MÉTODO: Nós desenvolvemos um método que produz enxertos de gordura, na sala de cirurgia, em uma taxa de enriquecimento maior que os já publicados (2:1). Este estudo clínico prospectivo e controlado analisou qualitativa e quantitativamente enxertos de gordura com (GT - grupo tronco) e sem (GC - grupo controle) adição das células da fração vásculo-estromal fresca em reconstrução mamária secundária; através de volumetria mamária por RNM de mamas, imunofenotipagem e contagem celular. Também foram estudados os resultados estéticos, a satisfação das pacientes e as complicações. RESULTADOS: A persistência volumétrica no GT foi 78,9% e 51,4% no GC, entretanto não houve diferença estatisticamente significativa entre os grupos. CD90 foi o marcador mais expresso e que alcançou diferença significante e ao mesmo tempo apresentou correlação positiva entre a sua expressão e a persistência volumétrica (r=0.651, p=0.03). Necrose gordurosa ocorreu, isoladamente em 4 pacientes do GT submetidas à radioterapia e nenhuma paciente do GC apresentou este evento. Desta forma, pacientes do GC mostraram tendência de estar mais satisfeitas com o enxerto de gordura. Nos dois grupos, os resultados estéticos foram iguais e não foram observadas recidivas loco-regionais. CONCLUSÃO: Os resultados do enriquecimento em uma taxa maior que as já publicadas são encorajadores, apesar de a persistência volumétrica não ter alcançado diferença estatisticamente significante entre os grupos. Enxertos de gordura enriquecidos na proporção 2:1 podem não ser indicados para pacientes submetidas à radioterapia apesar de terem se mostrados seguros num tempo de seguimento de 3 anos / BACKGROUND: Fat grafting is a tremendous tool in secondary breast reconstruction. Stromal vascular fraction (SVF) enriched fat grafts have been presenting promising results regarding volume maintenance. OBJECTIVE: The main purpose of this study was to analyze comparatively SVF-enriched fat grafts in secondary breast reconstruction: volumetric persistence, expression of surface markers and complications. METHODS: We developed a method that produces a superior SVF enrichment rate (2:1) in the operating theatre. This prospective and controlled trial analyzed quantitatively and qualitatively fat grafts with (stem cells group - SG) and without (control group - CG) SVF enrichment in secondary breast reconstruction, through MRI-based volumetry, immunophenotyping and cell counting. Also, patient satisfaction, aesthetic outcomes and complications were analyzed. RESULTS: Volumetric persistence in the SG was 78,9% and 51,4% in the CG, however it did not reach statistical significant difference. CD90 was the only marker highly expressed in the SG and showed a positive correlation with volumetric persistence (r=0.651, p=0.03). Fat necrosis occurred in 4 patients in the SG and in none in the CG. Patients in the CG showed a trend to be more satisfied. Considering aesthetics, both groups presented improvements. No locoregional recurrences were observed. CONCLUSIONS: Results are encouraging despite the fact that SVF enrichment in a higher supplementation rate did not improve, with statistical significance, fat graft volumetric persistence. Enriched fat grafts have proven to be safe in a 3-years follow up, however they do not seem suitable for patients that received radiotherapy

A lesão medular (LM) é uma patologia incapacitante que resulta em déficits sensoriais e motores. No Brasil, a incidência anual é de 30 novos casos de lesão medular a cada 1 milhão de indivíduos e, infelizmente, a LM permanece sem um tratamento eficaz. Células-tronco derivadas do dente decíduo humano estão entre as potenciais fontes de células-tronco para transplante após a lesão medular, cujo objetivo é de promover a proteção ou a recuperação da lesão na medula espinal. Buscou-se nesta tese avaliar os efeitos do transplante, uma hora após a lesão, das células tronco de dente decíduo humano (SHED) no período agudo, subagudo e crônico sobre a neuroproteção, proteção tecidual e recuperação funcional em ratos Wistar submetidos à lesão medular por contusão. Os principais objetivos foram: a) investigar os efeitos do transplante das SHED sobre a recuperação funcional, volume da lesão e morte neuronal; b) verificar os efeitos do transplante sobre as células progenitoras, formação da cicatriz glial e modificações astrocitárias após o modelo de contusão medular Observou-se a melhora na recuperação funcional, redução do volume da lesão e morte neuronal na medula espinal dos animais que receberam o transplante de SHED após a lesão medular. As SHED aumentam o número de células precursoras na medula espinal, no período subagudo, reduzem a expressão da proteína fibrilar glial ácida (GFAP) e aumentam a expressão do canal retificador de influxo de potássio 4.1, ambas proteínas astrocitárias. Concluímos que o transplante de células-tronco derivadas do dente decíduo humano após a lesão medular promove a recuperação funcional a partir do efeito neuroprotetor iniciado na fase aguda, confirmado pelo maior número de neurônios motores presentes seis semanas após a contusão. As SHED são capazes de aumentar o número de células precursoras e de produzir modificações astrocitárias na medula espinal de ratos lesados na fase subaguda, reduzindo a formação da cicatriz glial. / Spinal cord injury (SCI) is a disabling condition that results in sensory and motor deficits. The estimated annual incidence in Brazil is of 30 new cases of spinal cord injury per 1 million of individuals; unfortunately SCI remains without an effective treatment. Stem cells from human exfoliated deciduous teeth (SHED) are one among potential sources of stem cells for transplantation after spinal cord injury in order to promote protection or tissue and functional recovery after spinal cord injury. The aim of this Thesis was to evaluate the effects of stem cells from human exfoliated deciduous teeth (SHED) transplantation, one hour after lesion, in the acute, subacute and chronic phases on neuroprotection, tissue protection and functional recovery in Wistar rats submitted to spinal cord injury by contusion The main goals were: a) to investigate the effects of SHED transplantation on functional recovery, lesion volume, and neuronal death; b) to verify the effects of the transplantation on the progenitor cells number, glial scar formation and astrocytic modifications after spinal cord contusion. Improvement of functional recovery, reduction of lesion volume and neuronal death were observed in the spinal cord of animals submitted to spinal cord injury and SHED transplantation. SHEDs increased the number of precursor cells in the spinal cord in the subacute period, reduced the expression of glial fibrillary acidic protein (GFAP) and increased the expression of the potassium influx rectifier channel 4.1, both astrocyte proteins. We conclude that transplantation of stem cells from human exfoliated deciduous teeth after spinal cord injury promotes functional recovery from the neuroprotection effect, which starts in the acute phase and is confirmed six weeks after the contusion with a higher number of motor neurons in the ventral horn of spinal cord. SHEDs are able to increase the number of precursor cells and produce astrocyte modifications in the spinal cord of injured rats in the subacute phase, reducing glial scar formation.

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No. of bitstreams: 1 brock_rs_dr_bot.pdf: 138052465 bytes, checksum: f5b517e489dbadd6cf6b83fbef881ecd (MD5) Previous issue date: 2017-01-31 / As deformidades craniofaciais decorrentes de traumas, ressecções de tumores ou malformações congênitas são freqüentes na prática médica e o tratamento destas necessitam de cirurgia reparadora, com técnicas especializadas e profissionais qualificados para corrigir os defeitos e proporcionar melhor qualidade de vida, aprimorar a fala, respiração, mastigação e deglutição. Há diversas técnicas descritas para corrigir os defeitos ósseos, cada uma com vantagens e desvantages, escolhidas de acordo com o tipo de deformidade. Este estudo avaliou a formação óssea em um retalho tubular vascularizado, gáleo-periostal, enriquecido com o uso de pó de osso, plasma rico em plaquetas, células-tronco mesenquimais e ácido hialurônico, em coelhos, que tenha capacidade de substituir o enxerto ósseo nas reconstruções, principalmente nos defeitos faciais. No estudo, utilizou-se 98 coelhos divididos em doze grupos, submetidos à cirurgia para confecção do retalho em calota craniana. Foram realizados retalhos tubulares com o periósteo voltado para dentro e preenchidos com pó de osso, plasma rico em plaquetas (PRP), células-tronco mesenquimais (CTM) e ácido hialurônico. O Grupo 1 não foi manipulado. No Grupo 2 foi realizado o retalho tubular e mantido vazio. O Grupo 3 teve o retalho preenchido com pó de osso, no Grupo 4 o retalho foi mantido vazio. O Grupo 5 teve o retalho preenchido com PRP. No Grupo 6 o retalho foi preenchido com PRP e pó de osso. O Grupo 7 foi preenchido com CTM. O Grupo 8 teve o retalho preenchido com CTM e pó de osso. O Grupo 9 teve o retalho preenchido com CTM e PRP. No Grupo 10, o retalho tubular foi preenchido com PRP, CTM e pó de osso. O Grupo 11 foi mantido vazio e o Grupo 12 foi preenchido com ácido hialurônico. Os resultados foram avaliados através de métodos de imagem e avaliação histológica. Os resultados demonstraram que, no modelo experimental utilizado, os grupos com apenas periósteo, isto é, retalho tubular vazio, apresentaram formação óssea pequena e irregular. No grupo com PRP também houve a formação óssea irregular e imatura. Quando o PRP foi associado ao pó de osso houve uma formação mais regular e organizada. O grupo com célula-tronco mesenquimal também apresentou formação óssea, com características teciduais organizadas, próprias do tecido ósseo maduro. Quando associada ao pó de osso e ao PRP, as características histológicas apresentaram-se com tecido organizado, regular, maduro com células bem formadas e organizadas. O uso de materiais com fatores de crescimento celular ósseo melhoram a qualidade e organização do tecido neoformado. Quanto maior o número de fatores de enriquecimento usados, melhores foram os resultados quanto a qualidade tecidual neoformada. / Craniofacial deformities caused by traumas, tumor ressections or congenital malformation are frequent in medical practice, and their treatment with reconstructive surgeries are common, especially in plastic surgery, which aim to provide the patients with better quality of life and functional improvement of speach, breathing, chewing and swallowing. Many different techniques are described to correct bone defects. They have advantages and disadvantages, chosen according to the type of deformity. This study evaluated a vascularized galeal and periosteum flap filled with bone fragments, platelet rich plasma, mesenchymal stem cells and hyaluronic acid, using rabbits, which could possibily substitute the bone graft in reconstructive surgery, especially for facial defects. It was an experimental study, with 98 rabbits divided into twelve groups, submitted to a surgical procedure to construct a calvaria flap. A tubular flap with the periosteum inside was constructed and filled with bone fragments, platelet rich plasma (PRP), mesenchymal stem cells (MSC) and hyaluronic acid. Group 1 was not manipulated. In Group 2, the tubular flap was maintained empty. Group 3 had the flap filled with bone fragments, in Grupo 4 the flap was maintained empty. Group 5 had the flap filled with PRP. In Group 6 the flap was filled with PRP and bone fragments. The Group 7 was filled with MSC. Group 8 had the flap filled with MSC and bone fragments. The Group 9 had the flap filled with MSC and PRP. In Group 10, the tubular flap was filled with PRP, MSC and bone fragments.The Group 11 was maintained empty and Group 12 was filled with hyaluronic acid. The results were evaluated using image methods and histological analysis. The results demonstrated that, in the experimental model used, the groups with only periosteum, this is the empty tubular flap, presented small and irregular bone formation. In the group with PRP, it also had irregular and imature bone formation. When the PRP was associated to bone fragments it had a more regular and organized formation. The group with mesenchymal stem cell also presented bone formation, with organized tissue characteristics, proper of mature osseous tissue. When associated to bone fragments and PRP, the histological characteristics presented organized, regular, mature tissue with organized and well formed cells. The use of materials with osseous cellular growth factors improves the quality and organization of the neoformed tissue. The more enrichment factor used, the better the neoformed tissue quality result was.

Multipotent mesenchymal stem cells (MSCs), found in many adult tissues, may be useful for regenerative medicine applications. Their identification and purification have been difficult due to their low frequency and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove contaminating hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOp) population that was also enriched for other mesenchymal precursors, including MSCs (Itoh and Aubin, 2009). To further enhance enrichment, we positively selected BMSCs and HipOps for CD73, a putative MSC marker, which resulted in no significant additional enrichment for osteoprogenitors when the population was tested in vitro. However, we also found that HipOps were enriched in vascular endothelial cells, and that removing these cells by further negative selection with CD31/PECAM resulted in a CD31(-) HipOp population with higher osteogenic capacity than HipOps in vitro and in vivo.

Multipotent mesenchymal stem cells (MSCs), found in many adult tissues, may be useful for regenerative medicine applications. Their identification and purification have been difficult due to their low frequency and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove contaminating hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOp) population that was also enriched for other mesenchymal precursors, including MSCs (Itoh and Aubin, 2009). To further enhance enrichment, we positively selected BMSCs and HipOps for CD73, a putative MSC marker, which resulted in no significant additional enrichment for osteoprogenitors when the population was tested in vitro. However, we also found that HipOps were enriched in vascular endothelial cells, and that removing these cells by further negative selection with CD31/PECAM resulted in a CD31(-) HipOp population with higher osteogenic capacity than HipOps in vitro and in vivo.