Anat Cell Biol.  2016 Jun;49(2):107-115. 10.5115/acb.2016.49.2.107.

5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

• Haq SH

• KMID: 2308917
• DOI: http://doi.org/10.5115/acb.2016.49.2.107

Abstract

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.

Keyword

Hypomethylation; Endochondral ossification; Extracellular matrix; Scanning electron microscopy; Transmission electron microscopy; Rough endoplasmic reticulum

MeSH Terms

Alkaline Phosphatase
Apoptosis
Cartilage
Cell Size
Chondrocytes*
Chromatin
Collagen Type X
Cytoplasm
DNA
Endoplasmic Reticulum
Endoplasmic Reticulum, Rough
Extracellular Matrix
Fibroblasts
Hypertrophy*
Microscopy, Electron, Scanning
Microscopy, Electron, Transmission
Phenotype
Vacuoles
Alkaline Phosphatase
Chromatin
Collagen Type X
DNA

Figure

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  Fig. 1 Effect of 5-aza-2'-deoxycytidine on cell size with time in culture. The diameter of 5-aza-2'-deoxycytidine treated and untreated cells were measured at each indicated time point. Their cell volumes were calculated on the assumption that chondrocytes were spherical in shape. One hundred cells were measured at each time point in each culture. This was repeated in 10 cultures. Value represent mean±SEM (100 cells were measured for each time point in 10 experiments).

• 

  Fig. 2 Morphological effect of decitabine on chondrocyte morphology. The control and pretreated with 5-aza-2'-deoxycytidine caudal region chick sternal chondrocytes at day 7 in culture were stained with alcian blue, acridine orange and toluidine blue. (A) The control cultures stained with Acridine orange. (B) The pretreated chondrocytes cultures stained with Acridine orange (A and B, ×80). (C) The control cells stained with Alcian blue. (D) The pretreated cultures stained with Alcian blue (C and D, ×103). (E) The control cells stained with toluidine blue. (F) The pretreated cultures stained with toluidine blue (E and F, ×80). The arrows indicated the toluidine blue staining negative cells in the cultures.

• 

  Fig. 3 Scanning electron microscopic photomicrographs of the chondrocytes in culture. (A) Control chondrocytes at day 7 in culture. (B) Pretreated chondrocytes at day 7 in culture. (C) Extracellular matrix secreted by control chondrocytes at day 14 in culture. (D) Extracellular matrix secreted by pretreated chondrocytes at day 14 as described in "Materials and Methods." Note the presence of electron dense particles extensively present in the pretreated cultures. Scale bars=4.90 µm (A), 4.99 µm (B), 2 µm (C, D).

• 

  Fig. 4 Histogram showing the relative proportion of alkaline phosphatase (ALP) enzyme activity in the control and pretreated cultures at day 7 and day 14. ALP enzyme assay was done on triplicate samples as described in "Materials and Methods" section and expressed as per microgram of DNA.

• 

  Fig. 5 Transmission electron microscopic photomi crograph of the control and treated chondrocytes. The control and pretreated chondrocytes at day 14 were examined under the transmission electron microscope. (A) Control chondroc ytes. (B) Pretreated chondrocyte. (C) The control chondrocytes. (D) Pretreated chondrocytes. Note the presence of extensive and well defined rough endoplasmic reticulum (RER) and euchromatic nucleus and condensed chromatin in the pretreated cultures. Scale bars=2.01 µm (A, B), 2 µm (C, D). N, nucleus; V, vesicle; M, mitochodria.

• 

  Fig. 6 (A) Immunolocalization of type X collagen in chondrocyte cultures. Caudal region sternal chondrocytes pretreated with 5-aza-2'-deoxycytidine (5-aza-dC) and grown for day 14 in culture were fixed and the type X collagen localized in the culture by treating with a monoclonal antibody known to recognize the helical domain of type X collagen. Bound antibody was reacted with a second antibody IgG-linked to alkaline phosphatase and after a subsequent color reaction the site of type X collagen in the culture could be identified by red staining. (B) Western blotting with type X monoclonal antibody and fibronectin antibody. Medium proteins of the 14-day control and pretreated cultures were recovered after 30% ammonium sulphate precipitation and run on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto a nitrocellulose membrane and immune-blotted with fibronectin and type X monoclonal antibody.

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