Effects Of AAPH Induced Oxidative Stress On Endochondral Ossification Of Chick Embryo

Oxidative stress has been closely associated with adverse embryo development. The building of strong bone is a key point of embryo development. However, the reports about the relationship between oxidative stress and bone development are not abundant. Based on the previous studies of our laboratory, high doses of ROS inducer could cause serious malformation of the development of limb bud. Herein, the effects of oxidative stress on bone development and the mechanisms will be further discussed.Chick embryos were administrated with different concentrations of AAPH(0.5, 1.0, 2.0, 4.0, 8.0, 16.0 μmol/egg) to establish oxidative stress model. The chick embryos were harvested on the 12.5th day of incubation after AAPH injection. Several indicators were measured to determine the appropriate AAPH dose for establishing chronic oxidative stress model. The results showed that all of the doses could induce oxidative stress in chick embryo, such as the elevated ROS production rate of chondrocytes in epiphysis and the decreased mitochondrial membrane potential. Moreover, the death rate and malformation rate of chick embryo has increased along with the increase of AAPH concentration. The LD50 was 4.8 μmol/egg. According to the principles of establishing chronic oxidative stress model that the dose should be far less than LD50, AAPH 0.5 μmol/egg was chosen for establishing chronic oxidative stress model. Meanwhile, the chick embryos injected with AAPH 0.5 μmol/egg exhibited shortened femur and tibia length.The effects of oxidative stress on endochondral ossification of embryo were further discussed based on the chronic oxidative stress model of chick embryo induced by AAPH 0.5 μmol/egg. The following experiments were performed:(1) Alcian blue&alizarin red staining were performed on the long bones of 16.5, 17.5 and 18.5 days old chick embryo to measure the length of them in the aim of observing growth rate of long bones. And the results indicated that oxidative stress could significantly slow the growth rate of long bones and decrease their length.(2) H&E staining was performed on the long bones of 16.5, 17.5 and 18.5 days old chick embryo to measure the length of chondrocyte proliferating and hypertrophy zone on the purpose of detecting the effects of oxidative stress on the key processes of endochondral ossification(chondrocyte proliferating and chondrocyte hypertrophy). The results showed that oxidative stress obviously decreased length of the two zones, and slow the proliferating rate of chondrocyte and ossification rate of hypertrophic chondrocyte. Furthermore, the total number of chondrocyte in proliferating zone decreased seriously and the same as the number of chondrocyte in division stage.(3) The m RNA expressions of Sox9, Runx2, Type II collagen and Aggrecan were detected using RT-PCR, and the results showed that oxidative stress decreased their expression levels significantly.(4) The Micro-CT was used to detect the bone related indicators of long bones to demonstrate the adverse bone ossification of chick embryo administrated with AAPH. The results showed that oxidative stress significantly decrease the bone mass and bone mineral density of chick embryo causing undesirable osteogenesis.In conclusion, AAPH-induced chronic oxidative stress led to decreases in Sox9 m RNA expression, which in turn down-regulated the gene expressions of Type II collagen and Aggrecan. These alterations affected the survival and metabolism of chondrocytes, inhibited chondrocytes proliferation and led to slow-downed growth rate of long bones. The reduced protein level of Sox9 would directly decrease the level of P-Sox9, which caused the immature progression of prehypertrophic chondrocytes. The prehypertrophic chondrocytes that were short of P-Sox9 protein would skip the hypertrophy process and enter terminal differentiation. The termination chondrocytes would go to apoptosis, thus significantly decrease the number of mature hypertrophic chondrocytes, which express Runx2. In summary, these changes could induce adverse bone ossification and decrease the bone mass and bone mineral density. This research illuminated the effects of oxidative stress on embryo bone ossification and provided insight into the relationship between oxidative stress and bone development.