Exposure To Phenobarbital Negatively Influences The Osteogenesis Of Chick Embryos

ObjectiveAntiepileptic drugs(AEDs)are commonly used in pregnant women with epilepsy.Prenatal exposure to AEDs increases the risk of congenital malformation during embryonic development.However the exact effects of these AEDs on fetal development and their precise mode of cellular mechanisms remains unclear.In this research,we employed chicken embryos,aiming to determine the teratogenic risk of phenobarbital exposure on skeletogenesis and explore the relative mechanism how phenobarbital negtively affected osteogenesis.MethodsFirstly,we exposed the chick embryos to different concentration of phenobarbital.To observe the effect of phenobarbital onthe bone skeleton in E17 days,we performed alcian blue/alizarin red staining to examine the skeletal development in detail.Afterthat,we used micromass cell culture,RT-PCR to investigate whether phenobarbital could affect mesenchyme differentiation.Then we used histological analysis,immunofluorescence staining and TUNEL staining to explore the mechanism of delayed endochondral ossification.Moreover,we used MC3T3-E1,HUVECs,chorioallantoic membrane and yolk sac membrane to further explore the inhibitory effect of phenobarbital on ossification and angiogenesis.To observe the clear mechanism of developmental defects and we also investigate the changes of key genes,such as chondrogenesis-related genes and angiogenesis-related genes.ResultsThe phenobarbital expososure caused the chick embryos to exhibit a marked defect in skeletal development,which was shown as shortened long bones and a delayed mineralization.Firstly,we found that phenobarbital retarded MSCs to differentiate into chondrocytes by limb bud mesenchymal cells in vitro culture,and inhibited chondrocyte proliferation subsequently.Next,we identified that phenobarbital influenced the growth and extension of blood vessels using the angiogenesis assessment in chorioallantoic membrane and yolk sac membrane respectively.Meanwhile,it was demonstrated that phenobarbital affected the migration and tube formation of HUVECs.Semi-quantitative RT-PCR analysis shows that the expression of HIF-1α,VEGFA,VEGF-R1 in hypertrophic cartilage were decreased after phenobarbital treatment.The results indicated phenobarbital exposure affected angiogenesis and vascularization in long bones,and ultimately led to the delayed mineralization during osteogenesis.In addition,combined in vivo histological analysis of the long bone and in vitro MC3T3-E1 cell cultures indicated that phenobarbital affected proliferation,micorstructure and mineralization of osteoblastic cells.That might be another reason of inhibited mineralization of long bones.ConclusionsIn summary,exposed to excessive phenobarbital during embryogenesis inhibited mineralization by impairing the viability of osteoblasts and vascular invasion and restricted chondrogenesis by affecting mesenchymal cells differentiation,which contributed together a shortened length of long bones during embryonic skeletogenesis...