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Doctoral Thesis
DOI
https://doi.org/10.11606/T.23.2021.tde-27102021-181959
Document
Author
Full name
Tais Carvalho de Oliveira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2021
Supervisor
Committee
Chavez, Victor Elias Arana (President)
Gómez, Inmaculada Casado
Marques, Jose Luiz da Silva Lage
Prado, Karina Fittipaldi Bombonato
Title in Portuguese
Estudo comparativo da clastogênese entre células MDPC-23 e células da medula óssea de camundongos
Keywords in Portuguese
Calcitriol
Clastogênese
LPS
MDPC-23
Medula óssea
Abstract in Portuguese
Reabsorções internas das paredes dentinárias levantam dúvidas sobre a origem de células clásticas na polpa dentária em casos idiopáticos. Estudos recentes sugeriram que as células MDPC-23 (odontoblast-like) podem se diferenciar em células clásticas, contrariando estudos anteriores. O presente estudo teve como objetivo observar a influência do calcitriol (1,25-dihidroxivitamina D3) e do lipopolissacarídeo (LPS) na indução da diferenciação de células MDPC-23 (semelhantes a odontoblastos) em células semelhantes a clastos. Como as células MDPC-23 têm origem ectomesenquimal e células clásticas possuem origem hematopoiética, o estudo foi proposto para comparar, in vitro, o potencial clastogênico de dois modelos de origem embrionárias distintas frente a diferentes estímulos. Dois grupos, MDPC-23 e células da medula óssea de camundongos, foram cultivados e tratados com LPS ou 1,25-dihidroxivitamina D3 (calcitriol). No sexto dia, o ensaio de metiltiazolil-tetrazólio (MTT) foi realizado para observar a viabilidade celular diante dos tratamentos. Em seguida, o ensaio citoquímico foi executado para identificar células positivas para TRAP. Adicionalmente foi feito estudo da expressão gênica dos marcadores da clastogênese, OPG, RANK, RANKL, Csf1r, M-Csf1 e catepsina K, através da PCRq. No ensaio de MTT, a viabilidade celular não foi alterada com os tratamentos em comparação com os subgrupos controle. Células TRAP positivas estavam presentes apenas nos subgrupos medula óssea, induzidos com LPS ou calcitriol. O grupo medula óssea apresentou amplificação para todos os genes alvos mencionados. Já para os subgrupos do grupo MDPC-23 apresentaram expressão gênica significativa, diante dos tratamentos, apenas para os genes Csf1r e catepsina K. Concluiu-se, portanto, que embora apresente expressão relativa para os genes Csf1r e catepsina K, sob tratamento, as células MDPC-23 não foram capazes de se diferenciar em células clásticas.
Title in English
Effects of lipopolysaccharide and calcitriol on mdpc-23 differentiation into osteoclasts
Keywords in English
Bone Marrow
Calcitriol
Clastogenesis
LPS
MDPC-23
Abstract in English
Resorption of internal dental walls raises doubts about the origin of clastic cells in idiopathic cases. Recent studies have suggested that MDPC-23 cells (odontoblast-like) can differentiate into clast-type cells, contrasting to previous finding. The present study aimed to observe the influence of calcitriol and lipopolysaccharide (LPS) to induce the differentiation of MDPC-23 (odontoblast-like) cells into clast-like cells. As MDPC-23 cells have ectomesenchymal origin and clastic cells hematopoietic origin, the study was proposed to compare, in vitro, the clastogenic potential of two different models of embryonic origin against different stimuli. Two groups, MDPC-23 and mouse bone marrow cells were cultured in LPS and 1.25-dihydroxyvitamin D3 (calcitriol). On the sixth day, after treatments, methyl tetrazolium (MTT) assay was performed to observe cell viability. Then, a cytochemical assay was performed to identify TRAP positive cells. In addition, gene expression of clastogenesis markers, OPG, RANK, RANKL, Csf1r, M-Csf1 and cathepsin K, was performed using the PCRq. Cell viability wasnt disturbed at cited concentrations treatments compared to the control subgroups with the MTT assay. There were no TRAP positive cells in all MDPC-23 subgroups or in the control subgroup of bone marrow. TRAP positive cells were present only in bone marrow induced subgroups LPS and calcitriol. Bone marrow subgroups had gene expression amplification for all targets mentioned. Otherwise, MDPC-23 group had significant gene expression only for Csfr and cathepsin K. The others specific targets had no amplification in MDPC-23 subgroups. It was concluded that although the treatments were not toxic and MDPC-23 cells had significant gene expression for Csf1r and cathepsin K, these cells did not present TRAP positive cells, and therefore were not able to differentiate into clastic cells.
 
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Publishing Date
2022-01-28
 
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