Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture

Abstract

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure

“…21 In vitro , the addition of ethanol in chondrocyte cultures of neonatal rats drove alteration of the phenotype and synthesis activity due to the reduction of AP activity, reduction of PAS + matrix, and decrease of aggrecan expression; these effects were dose-dependent. 33 One possible explanation for the difference between the results is that methodological differences existed, since the ethanol added directly to the cell culture excludes likely alterations resulting from the metabolic processing of the substance within the organism. No other study has evaluated the effects of maternal ethanol consumption on the phenotype and in vitro synthesis activity of joint chondrocytes from offspring at the time of the present study.…”

Maternal Hyperthyroidism in Rats Alters the Composition and Gene Expression of the Matrix Produced In Vitro by Chondrocytes from Offspring with Intrauterine Growth Restriction

“…The sample values were calculated in relation to the GAPDH Ct values. The primers were as follow: forward 5 -CAACTCCCTCAAGATTGTCAGCAA-3 and reverse 5 -GGCATGGACTGTGGTCATGA-3 for Gapdh (reference sequence: NM_002046); forward 5 -CACACGCTACACACTGGACT-3 and reverse 5 -TCACACTGGTGGAAGCCATC-3 for aggrecan (reference sequence: NM 022190.1); foward 5 -GTTCACGTACACTGCCCTGA-3 and reverse 5 -AAGGCGTGAGGTCTTCTGTG-3 for Col2 (reference sequence: NM_012929.1); foward 5 -GAAACAGGTGTCTGACTTAC-3 and reverse 5 -TACTTCCAGTGGAATAGAAG-3 for Col10 (reference sequence: XM_001053056.3); foward 5 -CCCGATCTGAAGAAGGAGAGC-3 and reverse 5 -GTTCTTCACCGACTTCCTCCG-3 for Sox9 (reference sequence: NM_080403.1); foward 5 -GCGTCAACACCATCATTCTG-3 and reverse 5 -CAGACCAGCAGCACTCCATC-3 for Runx2 (reference sequence: NM_004348) [50].…”