Endogenous Parathyroid Hormone Affect Osteoclast Via Receptor Activator Of Nuclear Factor(NF)-κB Ligand(RANKL) Expressed By Osteoblast In The Process Of Fracture Healing

[Objective]:To study the effect of endogenous parathyroid hormone(PTH)deficiency on osteoclast cultured in vitro and explore the effect and mechanism of endogenous parathyroid hormone(PTH)on osteoclast in the process of fracture healing.[Mathods]:Mid-diaphyseal femur fractures were created and stabilizedwith an intramedullary pin in sixteen 8-week-old wild-type(PTH+/+)and PTH null(PTH-/-)mice.The femurs were removed at 7,14 days post-fracture.Decalcification,dehydration,paraffinembeddingand paraffin sectioning method were carrid out acoording to protocol.Then the expression of casthepsin K and RANKL were analyzed by immunohistochemistry.TRAP staining were performed to evaluate the osteoclast activity.RNA and proteins were isolated from callus tissues and osteoclast related gene and protein expression levels were evaluated by WestemblotandRT-PCR.Sixteen Eight-week-old PTH+/+ and PTH-/-mice wereused in this study.Bone marrow stem cells obtained from mouse femurs were isolated and cultured in the plate with slide or cortical bonewith macrophagecolony-stimulating factor(M-CSF)(30ng/ml)and different concentration of receptor activator of NF-κB ligand(RANKL)(50ng/ml or 100ng/ml).The tartrate-resistant acid phosphatase(TRAP)staining was performed to observe cellmorphology and TRAP-positive osteoclasts(OCs)numbers was counting and the bone resoption pits aeras was calculated after 6-day culturing and 9-day culturing.Another sixteen Eight-week-old PTH+/+ andPTH-/-mice wereused in cell culture.The BMSCs were isolated from bone marrow and induced to osteoblast with proper factors.RNA and proteins were isolated from the cell andexpression levels of RANKL were tested by Westernblot and RT-PCR.[Results]:Either at 7 days or 14 days post-fracture,the expression of RANKL and CK is higher in PTH+/+ mice than that in PTH-/-mice.And TRAP staining also show that PTH+/+ mice has stronger osteoclast activity.In the same concentration of RANKL,no changes are observed in the numbers of TRAP-positive OCs and the aeras of bone resoption pits between PTH+/+ groups and PTH-/-groups.However,high concentration of RANKL(100ng/ml)induce more TRAP-positive OCs and larger resoption areas.In the process of inducing BMSCs to differentiate into osteoblast in vitro show the osteoblast express higher RANKL level in PTH+/+ mice than that in PTH-/-mice.[Conclusion]:Endogenous Parathyroid Hormone Deficiency downregulates the RANKL expression of inducing cultured osteoblast in vitro.Endogenous Parathyroid has no effect on the numbers and Bone resorption functionof osteoclasts clutured in vitro in the presence of M-CSF and RANKL.High concentration of RANKL(100ng/ml)increased the TRAP-positive osteoclast numbers and bone resoption pits aeras compared with low concentration(50ng/ml).