Apoptosis and Proliferation of Growth Plate Chondrocytes in Rabbits

Abstract

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL-and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased.We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate.J Bone Joint Surg [Br] 1997;79-B:483-6. Received 3 September 1996; Accepted after revision 22 November 1996 In tissues, homeostasis is achieved by the balance between cell death and proliferation. When a population of cells is changing, however, the proportion of proliferating to dying cells may be expected to vary, and should occur in chondrocytes during closure of the growth plate in endochondral ossification. It has been suggested that apoptosis 1 is closely related to closure and endochondral ossification. 2-5 Our aim was to elucidate the kinetics of apoptosis in chondrocytes in closing growth plates by comparing the ratios of dying to proliferating chondrocytes using markers for apoptosis and proliferation. MATERIALS AND METHODSWe used eight Japanese white rabbits, two of which were killed by intravenous injection of pentobarbital sodium at each of 5, 10, 15 and 20 weeks of age. The epiphyseal growth plate with metaphyseal and epiphyseal bones was obtained from the middle of the femoral head of each rabbit.The growth plates were sectioned coronally into blocks 5 mm thick. These were fixed in 10% formalin for three days, decalcified by EDTA at 4°C for ten days, embedded in paraffin and cut into slices 2.5 m thick.For in situ visualisation of apoptotic cells, we performed terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling (TUNEL). 6 To show the proliferative portion of the tissues, we used immunostaining for proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase. After removal of the paraffin and dehydration, the first slice from each specimen was stained with haematoxylin and eosin for histological differentiation of the three zones of the growth plate: resting, proliferating and hypertrophic (Fig. 1). The second slice was stained by the TUNEL method. The reaction time was 20 minutes for protease K and 100 minutes for both TdT and biotin-dUTP. We performed PCNA immunostaining on the third slice using the labelled streptavidin-biotin (LSAB) method with a reaction time of 60 minutes for anti-PCNA antibody (PC-10, Code No. M879, DAKO, Denmark) at a dilution of 1:50. Counterstaining was carried out by methyl green in both instances. Some sections used for TUNEL staining were pretreated wit...

Evidence for genomic and nongenomic actions of estrogen in growth plate regulation in female and male rats at the onset of sexual maturation

“…In the E 2 -supplementation experiment, and to a lesser extent in the OVX experiment, PCNA-positive nuclei were abundantly observed in the pre-and hypertrophic zone. In a study by Aizawa and co-workers, the same phenomenon has been described in rabbit growth plates (Aizawa et al 1997). It appears that subtle changes in the fixation procedure as well as the fact that chondrocytes have a long G1 phase in relation to the S, G2 and M phase contribute to the relatively high number of PCNA-positive hypertrophic chondrocytes (Aizawa et al 1997).…”

“…In a study by Aizawa and co-workers, the same phenomenon has been described in rabbit growth plates (Aizawa et al 1997). It appears that subtle changes in the fixation procedure as well as the fact that chondrocytes have a long G1 phase in relation to the S, G2 and M phase contribute to the relatively high number of PCNA-positive hypertrophic chondrocytes (Aizawa et al 1997). In addition, we speculate that E 2 may have a sexually dimorphic effect on the cell cycle G1 phase duration in chondrocytes (Leung & Potter 1987, Takahashi & Noumura 1987.…”

Normal Bone Growth Requires Optimal Estrogen Levels: Negative Effects of Both High and Low Dose Estrogen on the Number of Growth Plate Chondrocytes

“…The epiphyseal growth plate was taken from the center of the femoral head of each rabbit as in our previous studies (Aizawa et al 1997(Aizawa et al , 1998(Aizawa et al , 1999Irie et al 2005). The growth plates were sectioned coronally into blocks 5 mm thick.…”

Aberrant death in dark chondrocytes of the avian growth plate

“…It is still a point of controversy whether all chondrocytes undergo apoptosis (Farnum and Wilson, 1990;Gibson et al, 1995;Hatori et al, 1995;Bronkers et al, 1996;Aizawa et al, 1997) or whether some survive to become bone-forming cells (Cancedda et al, 1995;Roach et al, 1995). Using morphological criteria, any chondrocyte with a condensed nucleus is often considered unviable or apoptotic.…”