Dissertação
Avaliação in vitro de condrócitos da cartilagem articular e da diferenciação osteogênica das células tronco da medula óssea extraídas da prole de ratas tratadas com etanol
Fecha
2020-02-28Autor
Kênia Mara Magalhães Campos Cardoso
Institución
Resumen
Two studies were performed to evaluate the effects of maternal ethanol consumption on
articular cartilage chondrocytes and on osteogenic differentiation of offspring bone
marrow mesenchymal stem cells (BMMSCs). Thirteen adult female Wistar rats were
divided into two experimental groups, the control group and the ethanol-treated group.
The rats of the ethanol and control group received by daily gavage, from the ninth day
of gestation, 40% alcohol solution and distilled water, respectively until the thirtieth day
of lactation. On the day of delivery, four newborns were euthanized per female and, at
30 days of lactation, three pups per female. The first study evaluated in vitro the effect
of maternal ethanol consumption on articular cartilage chondrocytes. Chondrocytes
were extracted from articular cartilage and cultivated in chondrogenic medium for
seven, 14 and 21 days. MTT conversion tests, alkaline phosphatase activity, percentage
of cells per field and percentage of PAS+ areas in 3D culture were performed. At 21
days, the quantification of gene transcripts for agrecan, Sox-9, collagen type II, collagen
X, Runx-2 and VEGF was performed by real-time RT-PCR. The second study
evaluated the effect of maternal ethanol consumption during pregnancy and lactation on
BMMSCs osteogenic differentiation of pups at weaning. The BMMSCs were extracted
from the pelvic limbs and cultured in osteogenic medium for seven, 14 and 21 days.
MTT conversion, alkaline phosphatase activity and cell percentage per field tests were
performed. At 21 days, the average size of the mineralized nodules was quantified and
the gene transcripts were quantified for osteopontin, osteocalcin, BMP-2, Runx-2 and
collagen I by real-time RT-PCR. Means were compared by Student's t-test and
differences were considered significant if p <0.05. Neonates of mothers receiving
ethanol were less heavy when compared to control neonates. The chondrocyte culture of
the ethanol group showed higher MTT conversion, higher alkaline phosphatase activity
and higher percentage of cells compared to the control group in all periods. There was
no difference between both groups in the percentage of PAS+ areas in 3D culture. There
was higher expression of collagen type II and lower expression of Sox-9 in the ethanol
group compared to the control group. The weight of pups at weaning of mothers
receiving ethanol was significantly lower when compared to control. BMMSCs ethanol
group showed lower MTT conversion after seven days of osteogenic differentiation but
higher alkaline phosphatase activity, increased percentage of cells larger average size of
mineralized nodules as well as increased expression of BMP-2, osteopontin and
osteocalcin. In conclusion, maternal ethanol consumption during pregnancy and
lactation alters, in vitro, the phenotype and chondrocyte synthesis activity of neonates,
as well as increases the osteogenic differentiation of BMMSCs from weaning offspring