Resumo: Injúrias traqueais são problemas na prática cirúrgica, pois apresentam dificuldades no tratamento. Atualmente, a engenharia tecidual (ET) é a única técnica para substituição traqueal que fornece promessa real. O uso de suportes naturais apresenta vantagens biológicas, mas faz-se necessário a descelularização desses materiais.Assim, o objetivo do presente trabalho é a descelularização de traquéias, com posterior aplicação das células epiteliais respiratórias (CERs) na face interna das traquéias descelularizadas; bem como, a diferenciação das células-tronco mesenquimais (CTMs) em condrócitos. O presente estudo foi efetuado com modelo em coelhos da raça Botucatu, pesando entre 3,0 e 4,0kg, provenientes do Biotério Central da Unesp. A utilização desses animais foi aprovada pelo Comitê de Ética em Experimentação Animal. Foram realizados 54 diferentes protocolos de descelularização, que envolvem métodos físicos (congelamento, agitação, pressão mecânica-PM, irradiação eletromagnética não ionizante-LED 475nm e LED 630nm), associados com métodos químicos (Triton X-100, SDS e DS) e enzimáticos (DNase e RNase). Paralelamente, também foi realizado cultura celular das CTMs, com posterior diferenciação em condrócitos. A expansão ex vivo das CERs foi realizada por diferentes protocolos: fragmentos traqueais, lavado traqueal, raspado traqueal, punch dermatológico de 2mm e digestão por tripsina. As células obtidas foram aplicadas na face interna das traquéias descelularizadas. Com relação a PM, não se observa contribuição nos protocolos de descelularização. A irradiação com LED 475nm provoca remoção das células condrogênicas; já a irradiação com LED 630nm provoca aumento da proliferação celular. Sob o critério custo-benefício, a utilização dos processos enzimáticos não foi validada. Em relação aos detergentes, ocorre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tracheal injuries are a problem in surgical practice once they present difficulties in their treatment. Nowadays, tissue engineering (TE) is the only technique for tracheal replacement that provides real promise. There are biological benefits in using natural scaffolds, but the decellularization of such materials is necessary. Thus, the objective of this study is tracheal decellularization with subsequent application of respiratory epithelial cells (RECs) on the inner side of the decellularized cells, as well as the differentiation of mesenchymal stem cells (MSCs) into chondrocytes. This study was carried out with model in Botucatu rabbits weighing between 3.0 to 4.0kg from the Animal Colony at Unesp. The use of such animals was approved by the Ethics Committee of Animal Experiments. Fifty-four different decellularization protocols were used; they involved physical methods (freezing, agitation, mechanical pressure - MP, non-ionizing electromagnetic irradiation - LED 475nm and LED 630nm), associated with chemical methods (Triton X-100, SDS and DS) and enzymatic ones (DNase and RNase). At the same time, a cell culture of the MSCs was done as well, with subsequent differentiation into chondrocytes. The ex vivo expansion of RECs was performed with different protocols: tracheal fragments, tracheal wash, scraped trachea, 2mm dermatological punch, and trypsin digestion. The cells obtained were applied to the inner side of the decellularized tracheae. Regarding MP, no contribution to the decellularization protocols is observed. Irradiation with LED 475nm causes the removal of chondrogenic cells whereas the irradiation with LED 630nm increases cell proliferation. Under the cost-benefit criterion, the use of enzymatic processes was not validated. As for the detergents, there is more destruction of the extracellular matrix of the tracheae treated with SDS when compared to those treated with... (Complete abstract click electronic access below) / Orientador: Elenice Deffune / Coorientador: Raul Lopes Ruiz Júnior / Banca: João Tadeu Ribeiro Paes / Banca: Paulo Francisco Guerreiro Cardoso / Mestre

Human adult mesenchymal stem cells (MSCs) derived from bone marrow have the capacity to self-renew and to differentiate into a variety of cells and tissues. They can leave their niche to migrate to remote tissues where they play a critical role in angiogenesis, wound repair and tissue regeneration. A major goal in adult stem cell research is to define how MSC fate is controlled by the pericellular extracellular matrix (ECM) and soluble factors that largely constitute their tissue-specific niches. Defining crucial regulatory signals that control the fate and function of MSCs in vitro will contribute to the development of therapeutic strategies to improve tissue regeneration. The objective of this study was to investigate the molecular relationships between cell-ECM integrin receptors and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, which are crucial in modulating MSC expansion, recruitment, and differentiation towards a number of different cell lineages. This study reports that ECM-directed cross-talk between PDGFR-β and alpha5β1 integrin controls the migration of MSCs. Cell adhesion to fibronectin induced integrin alpha5β1-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with integrin alpha5 and co-localised with alpha5β1 in a transient tidemark of focal adhesions. Adhesion to fibronectin also strongly potentiated platelet-derived growth factor (PDGF)-BB-stimulated PDGFR-β phosphorylation, in an alpha5β1-dependent manner. PDGFR-β-activated phosphatidylinositol 3 ́-kinase (PI3-kinase) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin engagement of alpha5β1 integrin. This synergistic relationship between integrin alpha5β1 and PDGFR-β is a fundamental determinant of mesenchymal cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of tissue remodelling.

It has been repeatedly proven that cell therapies can address many current unmet clinical treatment needs and also improve on current treatment options for various diseases, from neurological disorders to bone repair (Rosset et al. 2014; Corey et al. 2017). Though the potential of cell therapies has been demonstrated at a relatively small scale, the realisation of bringing cell based treatments to a larger market is hindered by the complexity of the product along with safety concerned and high production cost. Safety concerns can be informed with more in-depth analytical analysis of the product, however this in turn increase the costs involved in producing a cell therapy (Davie et al. 2012). Consequently the cost of analytical techniques also needs to be reduced, to address this need the area of microfluidic based bioanalytics holds much promise (Titmarsh et al. 2014). The culturing of human mesenchymal stem cells (hMSC) was used as a proof of concept model to demonstrate where improved bioanalytical and bioassay methods could be utilised in the production of cell therapies. Cells from four donors were cultured under three different oxygen environments and the conditioned medium assessed for pro-angiogenic capabilities using a tube formation bioassay and a proportion of the cytokine secretome profile measured using Luminex technology. Thorough secretome analysis it was shown that predicting cytokine levels based solely on the donor was not possible as the handling of the cells also had an influence on the secretome profile. The donor expression profiles did not behave in the same manner across all oxygen environments, for example in some donors IL-8 levels increased per cell at lower oxygen where as other donors showed a decrease per cell. While the tube formation assay showed some differences between donors in pro-angiogenic capabilities it also highlights the challenges with interpreting large data sets. The feasibility of using a microcapillary film (MCF) based enzyme-linked immunosorbent assay (ELISA) to detected two relevant cytokines, IL-8 and hepatocyte growth factor (HGF) was investigated. Following on from this work the development of a combined MCF ELISA assay with hMSC cell culture to produce a fully closed cell screening system was initiated. It was shown that it was feasible to measure IL-8 and HGF using the MCF ELISA platform but further work would need to be done to make the system more compatible with the manufacturing environment. In order to adapt the MCF to also be an hMSC culture platform the first challenge was to functionalise the Fluorinated Ethylene Propylene (FEP) surface of the MCF. It was concluded that a poly (vinyl- alcohol) (PVA) and gelatin mixture produced a homogenous coating to which a consistent level of hMSC would attach. This work was carried out on a flat surface; therefore steps were taken to adapt this knowledge into the MCF, while there was evidence of hMSCs present inside the MCF more work will need to be done to bring this concept to an established platform.

Periosteum and bone marrow (BM) contain cells that, after isolation and culture-expansion, exhibit properties of mesenchymal stromal/stem cells (MSCs). However, these cells have not been identified and characterised in situ due to the lack of specific markers. This study aimed to identify and phenotypically characterise long-term label-retaining cells (LT-LRCs), thought to include stem cells (SCs), in mouse periosteum and BM. Two mouse models were used: nucleoside-analogue labelling, and doxycycline (Dox)-inducible expression of histone 2B–green fluorescent fusion protein (H2B-GFP). LRCs were identified and phenotypically characterised by immunostaining, and microscopy or by flow cytometry (FCM). LRCs were detected throughout the periosteum with no apparent focal concentration, and subsets of cells displayed a phenotype compatible with MSCs but not pericytes. Osteoblasts were also labelled, but osteocalcin-expressing osteoblasts were distinct from Low-affinity nerve growth factor receptor (LNGFR)/P75-expressing MSCs. Similarly, BM contained LRCs expressing MSC markers that were distinct from pericytes. For FCM analyses, two cell isolation methods were compared, which revealed that crushing and collagenase digestion of long bones yielded a higher percentage of LRCs compared with flushing. BM analysed 40 days after the end of nucleoside administration showed that LRCs both within the CD45- and CD45low population were enriched for cells expressing Platelet-derived growth factor receptor α (PDGFRα) together with Stem cell antigen-1 (Sca-1) as well as cells expressing LNGFR/P75+. Furthermore, the CD45-PDGFRα+Sca-1+ population showed an increase in the percentage of LRCs with an increasing washout period, suggesting PDGFRα together with Sca-1 is most suitable to identify stromal LRCs in mouse BM. Comparison of the nucleoside label-retaining model with the H2B-GFP-label-retaining transgenic model showed a good correlation between nucleoside and H2B-GFP-label retention, suggesting the suitability of the H2B-GFP model for identification of stromal LRCs in BM. Future studies characterising the MSC niche in-vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., lapatinib). For patients with TNBC, chemotherapies such as taxanes and anthracyclines are standard of care therapies. However, for each breast cancer subtype, a significant number of patients develop resistance to these therapies and eventually die from metastasis, a process which accounts for ~90% of breast cancer mortality. Currently, metastatic breast cancer is incurable, and the short median survival of 3 years for patients with metastatic breast cancer has not significantly changed in over 20 years. Therefore identification of new molecules that are involved in breast cancer metastasis and development of more precisely targeted therapeutic strategies are urgently needed to improve the clinical outcome for this disease. The transforming growth factor pathway beta (TGFβ) pathway has been show to play a key role in metastasis through induction of epithelial-mesenchymal transition (EMT), cell migration and invasion. Over more than a decade, this pathway has been studied across several cancers and it is now better established that it has context-dependent tumor suppressive and oncogenic qualities. In the early stages of breast cancer, TGFβ pathway is a suppressor of benign and early stage tumor growth. However, as disease progresses and corresponding levels of TGFβ ligands become elevated, a “switch” will take place and promote oncogenic phenotypes like EMT and cancer cell stemness which drive metastasis. Long noncoding RNAs (lncRNAs) are an emerging subclass of RNA molecules in cancer biology. LncRNAs are >200nt and can influence target gene expression locally in “cis”, or along a distant chromosome in “trans”, through various mechanisms and interactions with other biological molecules. The contribution of TGFβ-regulated lncRNAs to associated phenotypes like EMT and cancer cell stemness has not been very well studied. The aim of this doctoral dissertation is to address the functional and mechanistic roles of lncRNAs in these processes. Using a well-established TGFβ-induced EMT model (e.g., mouse mammary epithelial cell NMuMG treated with TGFβ, we have identified 3 conserved lncRNAs (lncRNA-HIT, WDFY3-AS2 and TIL) that are significantly upregulated upon TGFβ-induced EMT. They all mediate TGFβ-induced EMT, cell migration and invasion. Overexpression of these lncRNAs is frequently detected during the breast cancer progression and is associated with high grade and late stage of breast cancer as well as metastatic lesion. We have also demonstrated that lncRNA-HIT positively regulates HOXA13 through “cis” mechanism and that WDFY3-AS2 induces WDFY3 and STAT3 expression at mRNA level by direct interaction with hnRNP-R. Interestingly, TIL stimulates C-MYC protein but not mRNA expression by promoting Akt phosphorylation of NF90 leading to its translation from the nucleus to cytosol where NF90 binds to C-MYC mRNA and enhances C-MYC translation. Importantly, we have shown that knockdown of lncRNA-HIT and WDFY3-AS2 significantly reduces breast cancer growth and lung metastasis in orthotopic breast cancer model. These findings indicate that these TGF-induced lncRNAs play critical role in EMT, metastasis, and are relevant in human patient tumors. Therefore, it is important to consider utilizing these molecules for clinical applications like diagnosis, monitoring recurrence, predicting a response to therapy, and even as a direct target for therapeutic intervention.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Mark L. Weiss / The traditional cell culture method after isolation from the body involves growing cells in 2 dimensions on plastic culture plate. However, the natural structure and physiology is 3 dimensions. To mimic in vivo environment, there has an increasing interest to find the way to maintain physiological properties. Here, we describe culturing human umbilical cord mesenchymal stromal cells (HUC-MSCs_in 3D setting using human platelet lysate gel. This gel is a fibrin-based structure like a blood clot. The preparation step of human platelet lysate (HPL) is by freeze- thaw cycles in order to release factors important for cells to grow and expand. Using of HPL to substitute for fetal bovine serum reduces potential cross contamination between species and xenogenicity. To maintain HPL media as a liquid, we add the anticoagulant heparin. Without adding anticoagulant, the gel will form. The aim of this study is to retrieve HUC-MSCs from HPL gel using Nattokinase, to characterize HUC-MSCs following the International Society for Cell Therapy’s MSC criteria, and to test a 3D invasion model with HPL-gel based structure. The result shows that using 1.75% Nattokinase at 60 minutes can recover the cells without reducing cell number and viability. After Nattokinase treatment, cells are able to attach to plastic and to increase in number. Moreover, they are able to differentiate into fat, bone, and cartilage no different from cells grown in 2D culture. However, to test surface markers by flow cytometry, all MSC markers are positive except CD 105. They are also positive of cell surface markers that should be negative. When seeded back to 2D culture for an additional passage, the MSCs meet ISCT criteria the same as control.

Orientador: Sandra Helena Penha de Oliveira / Banca: Gustavo Pompermaier Garlet / Banca: Willian Fernando Zambuzzi / Resumo: Células-tronco mesenquimais (CTMs) obtidas a partir da medula óssea são capazes de se diferenciarem, sobretudo, em condrócitos, adipócitos e osteoblastos. Durante a osteogênese in vitro, alguns parâmetros são utilizados para caracterizar este processo, tais como atividade da fosfatase alcalina (FAL), mineralização e expressão de proteínas associadas à osteoblastos. Ratos espontaneamente hipertensos (SHR) são um modelo animal de hipertensão essencial humana e desenvolvem hipertensão após 4 semanas de idade. Esta linhagem apresenta alterações significativas no metabolismo ósseo. O objetivo do presente estudo foi investigar se, o genótipo hipertensivo poderia interferir na diferenciação osteoblástica das CTMs de ratos SHR e qual mecanismo está alterado quando comparadas com a linhagem progenitora, ratos Wistar. Para isso, nós obtivemos CTMs da medula óssea de ratos Wistar e SHR com 4 semanas de idade, sem a hipertensão estabelecida, afim de avaliar somente o possível efeito do genótipo hipertensivo na diferenciação osteogênica in vitro. Nós induzimos, ou não, a diferenciação osteogênica in vitro por meio da utilização dos indutores osteogênicos: ácido ascórbico, β-glicerofosfato e dexametasona. Os resultados demonstraram que, CTMs indiferenciadas de SHR (SHRC) demonstraram taxa de proliferação aumentada em comparação a CTMs, na mesma condição, de Wistar (WC), e após a indução da osteogênica, a taxa de proliferação apresentou uma diminuição acentuada no grupo SHR (SHRMO) do que no grupo Wistar na mesma condição (WMO). Embora não fora observada diferença significativa na atividade da FAL entre SHRMO e WOM no 7° dia, a mineralização e a diferenciação osteoblástica foram menores no grupo SHRMO no mesmo período experimental. Os fatores de transcrição Osterix e β-catenina parecem estar envolvidos na diferenciação reduzida no grupo SHRMO... / Abstract: Mesenchymal stem cells (MSCs) from bone marrow are able to differentiate mainly into chondrocytes, adipocytes and osteoblasts. During in vitro osteogenesis, some parameters are used to characterize this process, such as the activity of alkaline phosphatase (ALP), mineralization and osteoblast-associated proteins expression. Spontaneously hypertensive rats (SHR) is an animal model of human essential hypertension. This animals developing hypertension after 4 weeks of age. This strain shows significant changes in bone metabolism. The aim of this study was to investigate whether the hypertensive genotype could influence the osteoblastic differentiation of MSCs from SHR and which mechanism are altered when compared to the parental strain, Wistar rats. For that, we have obtained bone marrow MSCs from Wistar and SHR rats at 4 weeks of age, without hypertension established in order to evaluate only the possible effect of hypertensive genotype on osteogenic differentiation in vitro. We induced or non-osteogenic differentiation in vitro using osteogenic inducers: ascorbic acid, dexamethasone and β-glycerophosphate. The results demonstrate that undifferentiated MSCs SHR (SHRC) showed increased proliferation rate compared to MSCs, in the same condition Wistar (WC) and after osteogenic induction, proliferation rate showed a marked decrease in SHR (SHRMO) than in Wistar group in the same condition (WMO). Although it was not observed significant difference in ALP activity between WMO and SHRMO on day 7, mineralization and osteoblast differentiation were lower on group SHRMO in the same experimental period. The transcription factors Osterix and β-catenin appear to be involved in reduced differentiation in SHRMO group because they showed lower expression in this experimental group. Furthermore, the decreased... / Mestre

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:50:10Z : No. of bitstreams: 1 evaristo_tc_me_botfm.pdf: 1944919 bytes, checksum: f9a5a93860c79f6554bb319a05d333fe (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Injúrias traqueais são problemas na prática cirúrgica, pois apresentam dificuldades no tratamento. Atualmente, a engenharia tecidual (ET) é a única técnica para substituição traqueal que fornece promessa real. O uso de suportes naturais apresenta vantagens biológicas, mas faz-se necessário a descelularização desses materiais.Assim, o objetivo do presente trabalho é a descelularização de traquéias, com posterior aplicação das células epiteliais respiratórias (CERs) na face interna das traquéias descelularizadas; bem como, a diferenciação das células-tronco mesenquimais (CTMs) em condrócitos. O presente estudo foi efetuado com modelo em coelhos da raça Botucatu, pesando entre 3,0 e 4,0kg, provenientes do Biotério Central da Unesp. A utilização desses animais foi aprovada pelo Comitê de Ética em Experimentação Animal. Foram realizados 54 diferentes protocolos de descelularização, que envolvem métodos físicos (congelamento, agitação, pressão mecânica-PM, irradiação eletromagnética não ionizante-LED 475nm e LED 630nm), associados com métodos químicos (Triton X-100, SDS e DS) e enzimáticos (DNase e RNase). Paralelamente, também foi realizado cultura celular das CTMs, com posterior diferenciação em condrócitos. A expansão ex vivo das CERs foi realizada por diferentes protocolos: fragmentos traqueais, lavado traqueal, raspado traqueal, punch dermatológico de 2mm e digestão por tripsina. As células obtidas foram aplicadas na face interna das traquéias descelularizadas. Com relação a PM, não se observa contribuição nos protocolos de descelularização. A irradiação com LED 475nm provoca remoção das células condrogênicas; já a irradiação com LED 630nm provoca aumento da proliferação celular. Sob o critério custo-benefício, a utilização dos processos enzimáticos não foi validada. Em relação aos detergentes, ocorre... / Tracheal injuries are a problem in surgical practice once they present difficulties in their treatment. Nowadays, tissue engineering (TE) is the only technique for tracheal replacement that provides real promise. There are biological benefits in using natural scaffolds, but the decellularization of such materials is necessary. Thus, the objective of this study is tracheal decellularization with subsequent application of respiratory epithelial cells (RECs) on the inner side of the decellularized cells, as well as the differentiation of mesenchymal stem cells (MSCs) into chondrocytes. This study was carried out with model in Botucatu rabbits weighing between 3.0 to 4.0kg from the Animal Colony at Unesp. The use of such animals was approved by the Ethics Committee of Animal Experiments. Fifty-four different decellularization protocols were used; they involved physical methods (freezing, agitation, mechanical pressure - MP, non-ionizing electromagnetic irradiation - LED 475nm and LED 630nm), associated with chemical methods (Triton X-100, SDS and DS) and enzymatic ones (DNase and RNase). At the same time, a cell culture of the MSCs was done as well, with subsequent differentiation into chondrocytes. The ex vivo expansion of RECs was performed with different protocols: tracheal fragments, tracheal wash, scraped trachea, 2mm dermatological punch, and trypsin digestion. The cells obtained were applied to the inner side of the decellularized tracheae. Regarding MP, no contribution to the decellularization protocols is observed. Irradiation with LED 475nm causes the removal of chondrogenic cells whereas the irradiation with LED 630nm increases cell proliferation. Under the cost-benefit criterion, the use of enzymatic processes was not validated. As for the detergents, there is more destruction of the extracellular matrix of the tracheae treated with SDS when compared to those treated with... (Complete abstract click electronic access below)

Orientador: Ana Liz Garcia Alves / Coorientador: Fernanda da Cruz Landim e Alvarenga / Banca: Anna Paula Balesdent Barreira / Banca: Brunna Patricia Almeida da Fonseca / Banca: Rogério Martins Amorim / Banca: Carlos Alberto Hussni / Resumo: Lesões tendíneas, especialmente do tendão flexor digital superficial dos membros torácicos, são importantes enfermidades que acometem a espécie equina, e apresentam alto índice de recidivas e afastamento da atividade atlética. Os avanços médicos e pesquisas relacionadas têm mostrado crescente interesse na utilização da terapia celular em tratamentos dessas lesões que apresentam uma cicatrização lenta ou ineficaz. Existem dúvidas quanto à persistência e comportamento das células tronco mesenquimais (CTM) implantadas no local da lesão e de sua migração a outros locais inflamados. Sendo assim, este estudo teve como objetivo identificar CTM marcadas com nanocristais antes e após o implante em lesões experimentais do tendão flexor digital superficial (TFDS) de eqüinos, observando a possibilidade de migração das CTM para outro foco de lesão realizado no membro contralateral do animal. Para isso, foram utilizados cinco equinos, submetidos à indução de lesões no TFDS em ambos os membros torácicos e, após sete dias, foram implantadas as CTM autólogas marcadas com nanocristais Qtracker 655 em um dos membros do animal. Sete dias após o implante, foram realizadas biópsias dos tendões, congelados em nitrogênio, sendo posteriormente realizados cortes histológicos para visualização em microscópio de fluorescência. Paralelamente, células marcadas com nanocristal foram mantidas em cultivo por 24 horas e, após este período, avaliou-se a sua viabilidade. Nas lesões que receberam células marcadas, foram visualizados sinais fluorescentes nas amostras, enquanto estes eram ausentes nas lesões dos membros contralaterais. As CTM marcadas e injetadas no tecido tendíneo mantiveram sua fluorescência sete dias após o implante in vivo, não sendo observado migração para o membro contralateral. As células mantiveram sua viabilidade após 24 horas de incubação com o marcador Qtracker / Abstract: Tendon lesions, especially of the superficial digital flexor tendon (SDFT) on forelimb, are important injuries in equine, because have high reinjury incidence and can cause retirement of athletic activities. Medical researches have shown growing interest on using stem cell therapy in the treatment of lesion with slow or ineffective repair. There are doubts regarding the persistency of the mesenchymal stem cells (MSC) implanted on the lesion site and of its migration to other injury sites. This study aims to identify marked MSC with nanoparticles quantum dots (QD) before and after the implant in equine induced tendinitis and to verify if there is a migration to another injury site on the contralateral. Five equines have a SDFT tendinitis induced on both forelimb and, after seven days, have seeded autologous MSC, marked with quantum dots Qtracker 655 in one of the limbs. Seven days after the implant, biopsies of the tendons were conducted, in which histological cuts were made for visualization in fluorescence microscope. The viability of the cells marked with nanoparticles and kept in cultivation was evaluated after 24 hours of incubation. Fluorescent signs were visualized on samples of the lesions that received marked cells, while they were absent on the lesions of the contralaterals. The marked MSCs kept their fluorescent emission, making it possible to identify them after seven days of in vivo implant, not occurring migration to the opposite limb. The cells maintained their viability in cultivation after 24 hours of incubation with the Qtracker marker / Doutor

Orientadores: Ângela Cristina Malheiros Luzo, Joaquim Murray Bustorff Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T07:41:35Z (GMT). No. of bitstreams: 1 Volpe_BrunoBosch_M.pdf: 3151194 bytes, checksum: afd54e9f5fdcdd55a24ddfa4b90d02a7 (MD5) Previous issue date: 2013 / Resumo: As fístulas enterocutâneas (FE) são de difícil cicatrização e seu tratamento cirúrgico frequentemente falha, fazendo com que a fistula volte a abrir. Estudos recentes têm demonstrado que a terapia celular pode ser uma nova forma de tratamento nesta área. As células tronco mesenquimais (MSCs) são capazes de se auto- renovar tem alta capacidade proliferativa, podendo se diferenciar em várias linhagens celulares. Ainda apresentam capacidade imunomodulatória. A medula óssea, o sangue de cordão umbilical e o tecido adiposo são as principais fontes de MSCs. O tecido adiposo (TA) é de fácil acesso, sendo que o procedimento de lipoaspiração é um procedimento comum. O tratamento de fístulas enterocutâneas com AT-MSCs já foi testado algumas vezes, porém a fístula em sua grande maioria não se fecha totalmente. O objetivo deste estudo foi analisar o potencial terapêutico das MSCs aderidas a fios de sutura no intuito de melhorar a cicatrização e recuperação no tratamento de FE. O TA foi obtido através do procedimento de lipoaspiração. O TA foi submetido ao processo de digestão com colagenase. As células ficaram em meio de cultura DMEM com baixa glicose e com SFB durante 3 dias. Quando atingiram 80% de confluência, as células aderentes foram tratadas com tripsina e ressuspendidas com o meio citado acima. Na 4ª passagem essas células foram caracterizadas com citometria de fluxo, microscopia confocal e diferenciadas nas três linhagens mesodérmicas para confirmação que realmente são MSCs. Após, a confirmação de que as células eram realmente células tronco mesenquimais, elas foram aderidas em fios de sutura de poliglactina (4-0 Poly Vicryl / Poliglactina 910). Foram gotejadas 106 MSCs em cima de cada fio de sutura e logo em seguida foi adicionada a cola de fibrina (20uL Fibrinogênio, 30uL Trombina e 10uL Cloreto de Cálcio) para ajudar na fixação das células. Os fios de sutura com MSCs aderidas ficaram em meio de cultura durante 24 horas para proliferação celular. As amostras foram analisadas por microscopia confocal de imnunofluorescência e eletrônica de varredura. O experimento animal utilizou ratos Wistar com 10 semanas de vida que foram distribuídos em três grupos: Grupo Controle (GC) que incluiu 5 animais onde a formação da fístula foi através de cirurgia sem nenhum tipo de suporte. Grupo Injeção (GI) que incluiu 8 animais que receberam uma injeção de 106 AT-MSCs ao redor do local de formação da fístula. Grupo Sutura (GS) que incluiu 9 animais que receberam sutura de 4-0 Vicryl (poliglactina) com 106 AT-MSCs aderidas com a ajuda de cola de fibrina. Após a exposição do ceco foi realizada enterotomia de 5mm, sendo então realizada a sutura da abertura a abertura da parede abdominal (superfície interna da pele) com 4 pontos separados com 4-0 Vicryl - Poly J-304 Polyglactin 910. No grupo GS, o fio para confecção da fístula, como descrita acima, continha 106 AT-MSCs aderidas. As fístulas foram fotografadas no dia da cirurgia e no 3°, 6°, 9°, 12°, 15°, 17°, 19° e 21° dias, onde foram anestesiados e sacrificados. O tamanho da área da fístula foi mensurado através do software ImageJ. Foram utilizados dois métodos estatísticos para analisar os dados do modelo animal. O primeiro método foi o Two-way Anova, fazendo a análise comparativa da cicatrização entre os três grupos, e o segundo o One-way analysis of variance, fazendo a análise comparativa da cicatrização entre os três grupos nos dias D0, D12 e D21. As microscopias confocal de imunofluorescência e eletrônica de varredura demonstraram a presença de AT-MSCs aderidas aos fios de sutura. No experimento animal mostrou que a média de redução do tamanho da área da fístula no 21º dia foi de 46,54% no GC, 71,80% no GI e 90,34% no GS (p<0,05), demonstrando que as MSCs foram eficazes na cicatrização das fístulas enterocutâneas tanto no grupo que recebeu a injeção de células quanto no grupo que utilizou as células aderidas ao fio de sutura. As MSCs foram capazes de se fixarem nos fios de sutura. Quando as fístulas enterocutâneas receberam a sutura com MSCs aderidas, elas mostraram uma melhor recuperação e cicatrização da fístula. AT-MSCs aderidas a fios de sutura pode ser uma nova e efetiva forma no tratamento de fístulas enterocutâneas / Abstract: Enterocutaneous fistulas (EF) are difficult to resolve and surgical failure is frequent. Cell therapy could be a new approach in this area. Mesenchymal stem cells (MSCs) have high proliferative capacity, can differentiate into several lineages and have immunomodulatory capacity. Adipocyte tissue (AT) is an easy source of them. Enterocutaneous fistula (EF) treatment with MSCs was yet performed but sometimes, the fistula did not close completely. The aim of this study is to analyze if AT-MSCs could attached in the suture filament in order to be used for EF treatment. AT obtained from lipoaspirate procedure was submitted to collagenase digestion. Cells were cultured in DMEM low glucose medium, with FBS during 3 days. At the 4ª passages, cells were characterized by flow cytometry, confocal microscopy, differentiated to mesodermal lineages to confirm MSCs and telomerase enzyme activity and karyotype analysis. The experiments were performed with polyester suture filament. MSCs, 106 cells, were fixed in the suture filament by adding fibrin glue.Filaments was led in the medium described above during 3 days. Samples were analyzed by confocal and scanning electron microscopy. The animal experiments were performed on 10 weeks old male Wistar rats divided into 3 groups. Control Group (CG): 5 animals undergoing fistula formation alone. Injection Group (IG):8 animals receiving 106 AT-MSCs injected around the suture line. Suture Filament Group (SG): 9 animals in which suture were performed using 4-0 Vicryl with 106 MSCs attached in the filament with fibrin glue. The cecum was accessed through a standard 7mm stab incision on the lower left side of the abdomen. Upon exposure, a 5mm enterotomy was performed and sutured to the abdominal wall in order to produce the fistula. To ensure normal closure of the fistula the opening in the cecum wall was fixed to the internal surface of the skin, without maturation, using four separate 4-0 Vicryl stitches (Poly J-304 Polyglactin 910 Ethicon). The fistulas were photographed on the day of operation and on the 3°, 6°, 9°, 12°, 15°, 17°, 19° and 21° day, in which they were anesthetized and sacrificed. Measure of the size of the fistula was performed using ImageJ software. Statistic comparison between the groups was performed by ANOVA. Confocal and scanning electronic microscopy results demonstrated that the cells were able to attach to the suture filaments. Animal experiments showed that the average size reduction of the fistula area at 21th day was: control group, 46.54%; injected group 71.80% and sutured group 90.34% (p<0,05). MSCs were able to attach to the suture filaments. When the fistulas were sutured with filaments containing MSCs they showed better recovery and healing than the injected and control group. Adipocyte tissue MSCs adhered to suture filament might be a new and effective approach for enterocutaneus fistulas treatment / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências