Exploring The Mechanism Of Glucocorticoid-induced Femoral Head Lesion Based On Bone Development In Rats And Mice

Objective:1.To compare the changes of bone mass in the epiphysis between femoral head and tibia during the development of rats and mice of inbred and closed group.To explore the difference development of epiphysis bone between femoral head and tibia of rats and mice of different strains.2.To observe the effect of methylenolone on bone mass and bone metabolism related factors in the epiphysis of femoral head and tibia of C57BL/6J mice.To explore the possible mechanism of glucocorticoid induced femoral head lesion.Methods:1.Preparation of bone development of rats and miceThe female closed group SD and the inbred wistar rats(3-month old and 6-month old)and the female and male half of C57 and BABL/c mice(1 1.5 2 3 4 4.5 5 6 7 8 9 months old)were selected respectively and each of them were 5 rats or mice.The right and left femoral head and the proximal tibia cancellous bone were taken respectively,and bone morphometry were used to compare the changes of epiphysis of femoral head and the proximal tibia cancellous bone in different strains rats and mice during different growth and development stages.2.Effect of methylprednisolone(Met)on the epiphysis of femoral head and proximal tibia cancellous bone in C57 miceC57 mice of 4 months old and 6 months old were randomly divided into two groups,each of which were 16: control group(4M-C,6M-C)and Met group(4M-Met,6M-Met).In the first week,the Met group were intramuscularly injected with Met 40 mg / kg / d,then injected with Met 20 mg / kg / d for 3 weeks,The control group were intramuscularly injected with normal saline 0.1ml/30g/d.The day after withdrawal,the right and left femoral head and proximal tibia cancellous bone of the mice were taken respectively,and bone morphometry were used to compare the effects of met on the bone structure and bone mass of femoral head and tibia epiphysis.Paraffin sections were taken to detect the changes of protein content of Matrix metalloproteinase 13(MMP13),Osteoprotegerin(OPG)and Collagen type I(COLI)by immunohistochemistry.Results:(1)The percentage of total tissue area(%T.Ar)in the epiphyseal region of femoral head of SD rats at aged 3 months were(48.42 ±1.61)%,which were(94.56± 1.59)% at 6 months old;%T.Ar in the epiphysis of the proximal tibia at 3 months old were(99.16±0.95)%,and which were(94.58±0.36)% at 6 months old.(2)%T.Ar of femoral head epiphysis in Wistar rats of 3 months old were(42.42±2.39)%,and which were(92.89±2.40)% at 6 months old;%T.Ar in the epiphysis of the proximal tibia at 3 months old were(98.26±0.77)%,which were(99.53±0.37)% at 6 months old.(3)Epiphyseal subchondral region of femoral head: the epiphysis of femoral head of C57 and BABL/c mice were chondrocytes before 4 months old,but no ossification were found,the bone trabeculae in C57 mice of 6 months old were intact and no chondrocytes were found.The corresponding changes occurred in BABL/c mice at 8 months old.In the same strain,after 4 months old,The age of male femoral head epiphysis ossification were earlier than that of female.(4)Closure of the epiphyseal plate of the femoral head in C57 mice at 6 months old,The BABL/c epiphyseal plate were closed until to 8 months old.(5)Epiphyseal subchondral region of tibia: The bone trabeculae were intact and no chondrocytes in the tibia epiphysis of C57 and BABL/c mice of 1.5 month-old.(6)Compared with 4M-C group,the percent trabecular area(%Tb.Ar),trabecular thick(Tb.Th),trabeculae number(Tb.N)and trabecular separation(Tb.Sq)in epiphysis of femoral head were significantly decreased,the Tb.Sq increased in 6M-Met group.(7)Compared with 4M-C group,the expression of COLI,OPG protein in the epiphysis of femoral head in 4M-Met group were down-regulated(p<0.05)and the expression of MMP13 were up-regulated,compared with 6M-C group,the expression of COLI in 6M-Met group were up-regulated.The expression of COLI,OPG and MMP13 in tibial epiphysis were No significant change in 6M-Met group and 4M-Met.Conclusion:1.During the development process,the epiphysis of femoral head and tibia in rats and mice were different,in which the ossification of tibia were completed earlier than that of femoral head.2.The epiphyseal ossification of femoral head were basically completed in SD and Wistar rats and C57 mice at the 6-month-old.In addition,the epiphyseal plate disappeared in 6-month-old of C57 mice.The epiphyseal plate of BABL/c mice disappeared at the age of 8 months old.Therefore,the C57 mice of 6-month-old were more suitable than rats and BABL/c mice to make Steroid-induced avascular necrosis of the femoral head animal model.3.Before the ossification of epiphyseal cartilage of femoral head was completed,the pathological changes of femoral head caused by glucocorticoid were mainly manifested as inhibition of ossification,but there was no significant change in bone mass in the epiphyseal region of tibia.The mechanism were related to the COLI and OPG protein were down-regulated and MMP13 protein were up-regulated in epiphysis of femoral head.