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Previous issue date: 2016-12-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The present research aims to characterize the mesenchymal stem cells of adipose origin (MSCAs) and to evaluate its use on the scaffold of hyaluronic acid (HA) as a cellular compound for bone tissue engineering. Firstly, cell characterization was performed through the collection of epididymal adipose tissue, isolation, culture and in vitro expansion. Through the morphological analysis of MSCAs culture it was possible to confirm elongated fusiform characteristic, centralized nucleus, extensions and adhesion to the plastic bottle. Expansion analysis also demonstrated a high cell proliferative index during the 26 days of in vitro culture. The Flow Cytometry test allowed the identification of the main surface markers that characterize the mesenchymal stem cells (CD29 and CD90) and negative for hematopoietic markers (CD31 and CD45). Moreover, MSCAs when induced to adipogenic and osteogenic media showed plasticity, since they were able to differentiate into adipocytes and osteoblasts respectively. The cell viability test was also performed in vitro, through the M.T.T (mitochondrial activity) of MSCAs on the HA scaffold. Concentrations of 100%, 75%, 50%, 25% and 15% were evaluated on the HA scaffold at times of 24, 48 and 72 hours. The results have shown cellular viability above 60% in almost all times and concentrations. Then, 50 critical bone defects were performed in the femoral region with 2 mm in diameter (one defect per femur) in 25 Lewis rats. The grafting treatments were divided as follows: I-negative control / only the defect (C); II-HA Scaffold; III- MSCAs; IV- MSCAs + HA and V- MSCAs previously osteoinduced + HA. After 23 days the rats were euthanized and had 5 femurs used for the microtomographic (?-CT) and histomorphometric analysis and 5 femurs used for the RT-PCR (Real-Time Polymerase Chain Reaction) analysis. The results for the ?-CT tests in the volume of osseous tissue parameter (VOT) and the percentage of osseous tissue (POT) did not present statistical differences among all groups. However, on the osseous contact surface (SCO) and osseous surface density (DSO) parameters, we had groups IV, III and V with higher indexes and differing statistically from the negative control groups I and group II. In histomorphometry we also had groups IV, V and III with greater area of regenerated bone tissue and differing with significance from groups I and II. The results were analyzed statistically by Analysis of Variance one way (ANOVA) and the level of significance was 5% (p <0.05). Regarding RT-PCR, a statistically significant difference was observed only when we evaluated osteonectin (ON) in which group II and V were more expressive in relation to groups III and IV. Regarding osteopontin (OP) and Type I collagen (Col1A), no differences were identified among the treated groups. The results were analyzed using Kruskal-Wallis non-parametric test and the significance level set was 5% (p <0.05). / A presente pesquisa tem por objetivo caracterizar as c?lulas-tronco mesenquimais de origem adiposa (CTMAs) e avaliar seu uso sobre a matriz de ?cido hialur?nico (AH) como composto celular para engenharia tecidual ?ssea. Primeiramente, foi realizada a caracteriza??o celular atrav?s da coleta do tecido adiposo epididimal, isolamento, cultivo e expans?o in vitro. Atrav?s da an?lise morfol?gica da cultura das CTMAs foi poss?vel confirmar caracter?stica fusiforme alongada, n?cleo centralizado, prolongamentos e ades?o ? garrafa pl?stica. A an?lise de expans?o tamb?m comprovou um alto ?ndice proliferativo celular, durante os 26 dias de cultura in vitro. J? o teste de citometria de fluxo permitiu a identifica??o dos principais marcadores de superf?cie que caracterizam as c?lulas-tronco mesenquimais (CD29 e CD90) e sendo negativo para marcadores hematopoi?ticos (CD31 e CD45). As CTMAS tamb?m quando induzidas aos meios adipog?nico e osteog?nico mostraram plasticidade, j? que foram capazes de diferenciarem-se em adip?citos e osteoblastos, respectivamente. Ainda in vitro, foi realizado o teste de viabilidade celular atrav?s do MTT (atividade mitocondrial), ap?s o contato das CTMAs sobre a matriz de AH. As concentra??es de 100%, 75%, 50%, 25% e 15% foram avaliadas sobre a matriz de AH nos tempos de 24, 48 e 72 horas. Os resultados comprovaram uma viabilidade celular acima de 60% em quase todos os tempos e concentra??es. Em seguida, foram realizados 50 defeitos ?sseos cr?ticos (DOC) na regi?o femoral com 2 mm de di?metro (um defeito por f?mur) em 25 ratos Lewis. Os tratamentos de enxertia realizados foram divididos da seguinte forma: I-controle negativo / apenas o defeito (C); II- matriz AH; III-CTMAs; IV-CTMAs + AH e V- CTMAs osteoinduzida + AH. Ap?s 23 dias os ratos sofreram eutan?sia e tiveram 5 f?mures utilizados para as an?lises microtomogr?ficas (?-CT) e histomorfom?trica e 5 f?mures utilizados para a an?lise por RT-PCR (Rea??o em cadeia da Polimerase em tempo Real). Os resultados para os testes por ?-CT no par?metro volume de tecido ?sseo (VTO) e porcentagem de tecido ?sseo (PTO) n?o apresentaram diferen?as estat?sticas entre todos os grupos. J?, nos par?metros superf?cie de contato (SCO) e densidade de superf?cie ?ssea (DSO) tivemos o grupo IV, III e V com maiores ?ndices e diferindo estatisticamente dos grupos controle negativo I e do grupo II. Na histomorfometria tamb?m tivemos os grupos IV, V e III com maiores ?reas de tecido ?sseo regenerado e diferindo com signific?ncia dos grupos I e II. Os resultados foram analisados estatisticamente pela An?lise de Vari?ncia de uma via (ANOVA) o n?vel de signific?ncia estabelecido foi de 5% (p < 0,05). Em rela??o ao RT-PCR, observou-se diferen?a estatisticamente significante apenas quando foi avaliada osteonectina (ON), sendo que o grupo II e V apresentaram maior express?o em rela??o aos grupos III e IV. Em rela??o ? osteopontina (OP) e ao col?geno Tipo I (Col1A) n?o foram identificadas diferen?as entres os grupos tratados. Os resultados foram analisados atrav?s do teste n?o param?trico de Kruskal-Wallis e o n?vel de signific?ncia estabelecido foi de 5% (p < 0,05).
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/7412 |
| Date | 19 December 2016 |
| Creators | Boeckel, Daniel Gon?alves |
| Contributors | Teixeira, Eduardo Rolim, Sesterheim, Patr?cia |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Odontologia, PUCRS, Brasil, Faculdade de Odontologia |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | -8096554818733665164, 600, 600, 600, 600, 4673435736271820140, -2070498469879244349, 2075167498588264571 |
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