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Doctoral Thesis
DOI
Document
Author
Full name
Gileade Pereira Freitas
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Rosa, Adalberto Luiz (President)
Faverani, Leonardo Perez
Silva, Tarcilia Aparecida da
Vieira, Eduardo Hochuli
Title in Portuguese
Efeito de injeções locais de células-tronco mesenquimais ou osteoblastos na regeneração do tecido ósseo
Keywords in Portuguese
Célula-tronco mesenquimal
Medula óssea
Osteoblasto
Regeneração óssea
Tecido adiposo
Terapia celular
Abstract in Portuguese
O osso e um tecido com grande capacidade de regeneração, mas em algumas situações a extensão da injúria impede o reparo do tecido. Nesse cenário, a terapia celular tem atraído a atenção de diversos grupos de pesquisa por ser uma alternativa promissora em relação aos tratamentos existentes. No entanto, muitos aspectos experimentais necessitam ser melhor investigados para tornar essa terapia um tratamento efetivo para a regeneração de defeitos do tecido ósseo. Sendo assim, o objetivo desse estudo foi avaliar o potencial de células-tronco mesenquimais (CTMs) derivadas de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA), bem como de osteoblastos (OBs) diferenciados a partir dessas CTMs (OBs-MO e OBs-TA), para regenerar o tecido ósseo quando injetadas diretamente em defeitos de 5mm de diâmetro criados em calvárias de ratos. Para isso, as CTMs foram obtidas de medula óssea de fêmur e de tecido adiposo inguinal de ratos, expandidas in vitro e mantidas com características de CTMs ou diferenciadas em osteoblastos. As CTMs foram caracterizadas por imunofenotipagem por citometria de fluxo para os marcadores CD29, CD31, CD34, CD45 e CD90. A diferenciacão osteoblastica foi confirmada pela avaliacão da expressão genica de runt-related transcription factor 2 (Runx2), fosfatase alcalina (Alp) e osteopontina (Opn) por PCR em tempo real. Duas semanas após a criação dos defeitos ósseos, foram realizadas as injeções das células. Foi avaliado a viabilidade das células após sua passagem pela agulha 21G utilizada nas injeções. O enxertamento e o tempo de permanência das células nos defeitos ósseos foram avaliados por bioluminescência utilizando injeção de CTMs-MO, CTMs-TA, OBs-MO e OBs-TA expressando luciferase. Quatro semanas após as injeções, os animais foram mortos e o tecido ósseo formado foi avaliado por microtomografia computadorizada, análise histológica, análise por nanoindentação e PCR em tempo real. Os dados foram submetidos ao teste de aderência a curva normal para determinar o teste estatístico adequado. O nível de significância adotado foi de 5% (p 0,05). Foi observado que a terapia celular com CTMs-MO, CTMs-TA, OBs-MO ou OBs-TA foi efetiva em injetar células viáveis, que permaneceram no local do defeito ósseo e induziram maior formação óssea em comparação com defeitos que não receberam células. Não houve diferença no tecido ósseo induzido por CTMs-MO ou CTMs-TA ou naquele induzido por OBs-MO ou OBs-TA. Apesar de algumas diferenças marcantes nas assinaturas moleculares, CTMs-MO, CTMs-TA, OBs-MO e OBs-TA foram capazes de formar tecido ósseo com as mesmas propriedades mecânicas do tecido ósseo existente na calvária
Title in English
Effect of local injections of mesenchymal stem cells or osteoblasts on bone regeneration
Keywords in English
Adipose tissue
Bone marrow
Bone regeneration
Cell therapy
Mesenchymal stem cell
Osteoblast
Abstract in English
Bone is a tissue with great capacity for regeneration, but in some situations, the extent of injury prevents tissue repair. In this scenario, cell therapy has attracted the attention of several research groups. However, many experimental aspects need to be better investigated to make this therapy an effective treatment for the regeneration of defects in bone tissue. Therefore, the aim of this study was to evaluate the potential of mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs), as well as osteoblasts (OBs), differentiated from these MSCs (BM-OBs and AT-OBs) to regenerate bone tissue when injected directly into defects of 5mm diameter created in calvaria of rats. For this, MSCs were obtained from bone marrow of femur and inguinal adipose tissue of rats, expanded in vitro and maintained with characteristics of MSCs or differentiated in OBs. The MSCs were characterized by immunophenotyping for markers CD29, CD31, CD34, CD45 and CD90 by flow cytometry. Osteoblastic differentiation was confirmed by the evaluation of the gene expression of runtrelated transcription factor 2 (Runx2), alkaline phosphatase (Alp) and osteopontin (Opn) by real-time PCR. Two weeks after the creation of the bone defects, the injections of the cells were performed. The viability of the cells after their passage through the 21 gauge (21G) needle used in the injections was evaluated. The cell tracking in bone defects was evaluated by bioluminescence using an injection of BM-MSCs, AT-MSCs, BM-OBs and AT-OBs expressing luciferase. Four weeks after the injections, the animals were killed, and the bone tissue formed was evaluated by computerized microtomography, histological analysis, nanoindentation analysis and real-time PCR. Data were submitted to the normal curve test to determine the appropriate statistical test. The level of significance was 5% (p 0.05). It was observed that cell therapy with BM-MSCs, AT-MSCs, BM-OBs or AT-OBs was effective in injecting viable cells, which remained at the site of the bone defect and induced greater bone formation compared to defects that did not receive cells. There was no difference in bone tissue induced by either BM-MSCs or AT-MSCs or in that induced by BM-OBs or AT-OBs. Although some marked differences in molecular signatures, BM-MSCs, AT-MSCs, BM-OBs and AT-OBs were able to form bone tissue with the same mechanical properties of existing bone tissue in calvaria
 
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Publishing Date
2019-10-18
 
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