In vitro proliferation of achondroplastic and normal mouse chondrocytes, before and after basic fibroblast growth factor stimulation

Abstract

Achondroplasia in mice is a recessive genetic disorder, characterized by disproportionate dwarfism with reduced bone growth. The cause of this chondrodystrophy is unknown. In this study normal and achondroplastic mouse chondrocytes were cultured in monolayer primary culture, their differentiation was verified by immunofluorescence and their growth was compared. The results showed that achondroplastic cells exhibited a higher proliferative activity than control cells of the same age, confirmed also by a thymidine incorporation assay. Furthermore, basic fibroblast growth factor treatment was found to induce a strong increase in growth of normal mouse chondrocytes, while it did not stimulate statistically significant proliferation of achondroplastic mouse cells. We suppose that this different growth rate could play a role in achondroplastic phenotype development.

Figure 1

Comparison of growth curve of normal and achondroplastic cultured chondrocytes. Cells were seeded at 1 × 10⁵per 35‐mm dish in complete medium. Cell number was determined as described under Materials and Methods. Values presented are averages ±SD for triplicate samples.

Figure 2

Incorporation of [³H] thymidine in normal and achondroplastic chondrocyte cultures. Cells were seeded at a density of 1 × 10³/ml and confluent cultures were pulsed with 185 kBq/ml of [³H] thymidine for 2 h intervals during 24 h. The figure shows more representative experimental points. Triplicate dishes were determined for every point. The standard deviation in the different determinations did not exceed 5% of the mean.

Figure 3

Immunofluorescence micrographs of achondroplastic chondrocyte cultures, stained with appropriate antibodies to (a) type II collagen and to (b) chondroitin sulphate. Micrographs were taken at confluence (magnification 250×).

Figure 4

Effect of bFGF on the proliferation of NOR (black symbols) and ACH (white symbols) chondrocytes. Cells were seeded at 1 × 10⁴ per 35‐mm dish and treated (squares) or not treated (triangles) by bFGF (1.0 ng/ml). Cell counts were determined as described under Materials and Methods. Values presented are averages ±SD for triplicate samples.