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.

Keywords: alcohol; cartilage; newborn; rat.

Figure 1.

Conversion of MTT to formazan (mean ± standard deviation) in 2-dimensional cultures of articular chondrocytes from newborn rats without ethanol (control) and with different concentrations of ethanol (0.05% v/v [8.6 mM]; 0.25% v/v [42.9 mM], and 0.5% v/v [85.7 mM]) after 7, 14, and 21 days of culture. *P < 0.05 (Student t test).

Figure 2.

(A) Percentage of periodic acid Schiff+ (PAS+) areas in articular chondrocyte pellets of newborn rats without ethanol (control) and with different concentrations of ethanol (0.05% v/v [8.6 mM]; 0.25% v/v [42.9 mM], and 0.5% v/v [85.7 mM]) at 21 days of 3-dimensional culture. *P < 0.05 (Student t test). (B) Histological sections of articular chondrocyte pellets from newborn rats, under 3-dimensional culture, at 21 days. There was a significant reduction in the percentage of PAS+ areas in the group treated with 0.5% of ethanol in comparison to the control.

Figure 3.

Morphometric analysis of articular chondrocyte pellets of newborn rats cultured without ethanol (control) and with different concentrations of ethanol (0.05% v/v [8.6 mM]; 0.25% v/v [42.9 mM], and 0.5% [85.7 mM]) after 21 days of 3-dimensional culture. (A) Group treated with 0.5% ethanol exhibiting a higher percentage of chondrocyte cytoplasm in comparison with the control. (B) Group treated with 0.5% ethanol with a higher percentage of chondrocyte nuclei in comparison with the control. (C) Group treated with 0.5% ethanol with lower percentage of differentiated matrix in comparison with the control. (D) The mean diameter of chondrocytes was similar between groups. *P < 0.05 (Student t test).

Figure 4.

Expression of gene transcripts (mean ± standard deviation) of aggrecan, Sox9, and type II collagen by the real-time quantitative polymerase chain reaction (RT-qPCR) technique in articular chondrocyte of newborn rats cultured without ethanol (control) and with different concentrations of ethanol (0.05% v/v [8.6 mM]; 0.25% v/v [42.9 mM], and 0.5% v/v [85.7 mM]) after 21 days of 3-dimensional culture. (A) Group treated with 0.5% ethanol with a significant reduction of aggrecan expression as compared with control. (B) Sox9 expression showed no difference between groups. (C) Expression of type II collagen showed no difference between groups. *P < 0.05 (Student t test).