Experimental Study On The Impact And Mechanisms Of Endogenous Parathyroid Hormone On Fracture Healing In Mice

[Objective]:To demonstrate whether endogenous parathyroid hormone (PTH) is by BMP2 signaling pathways and/or by TGF-β signaling pathway regulates bone healing, and to further explore the mechanism of its regulation.[Mathods]:In vivo selection of 8 weeks old PTH wild-type (WT, PTH+/+) and PTH gene knockout (KO, PTH-/-) male mice as experimental subjects for model of right femur open fractures. At 7,14 and 21 days after the operation, the mice were sacrificed for measurement of bone mineral density of the callus using Micro-CT.To determine the role of PTH in fracture healing, the fracture models were assessed by histology, immunohistochemistry, X ray and Micro-CT, and the callus tissue extracted protein related bone morphogenetic protein expression by Western blot analysis methods. In vitro bone marrow mesenchymal stem cells were taken from 8-week-old male WT and KO mice,and induced into osteoblasts and chondrocytes. Using histology, morphological, immunohistochemistry and molecular biology method analyzed the functional differences between KO and WT.[Results]:Endogenous PTH deficiency impaired fracture healing, Mi-CT showed decreased in bone mineral density(BMD) at the callus area,and the proportion of non-bony callus tissue area. Endochondral mineralization diminished in PTH KO mice.(Reduction of type I collagen deposition, Runx2 protein downregulation). Runx2, BMPR2, pSMAD1/5/8 immunohistochemical staining intensity was significantly reduced in PTH gene knockout mice callus area, but Sox9, TGF-βR2,and pSMAD2/3staining intensity shown no significant difference. At the same time we found PTH gene knockout lead to significantly reduced of CREB expression in callus area.Western blot analysis in Callus protein showed the similarity results with immunohistochemistry. Bone marrow mesenchymal stem cells extracted from PTH KO mice showed no significant difference in the function of differentiation into chondrocyte. Bone marrow mesenchymal stem cells stimulate extracted from PTH KO mice showed a decline of pSmad1/5/8 levels and of pSmad 1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFβ1 stimulus. Mesenchymal stem cells induced osteoblasts, four days after the induction, Western blot detection test results showed that BMPR2, Runx2 expression decreased, dbcAMP could correctable BMPR2, Runx2 expression, and PKA inhibitor (H89) could eliminate the effect of dbcAMP.7 days after the induction showed ALP activity decreased, giving dbcAMP correctable, plus PKA inhibitor (H89) to eliminate the effect of dbcAMP. Measuring the concentration of intracellular cAMP, the results show PTH gene knockout cause the reduce of cAMP concentration in bone marrow mesenchymal stem cells and osteoblasts.[Conclusion]:These results indicate that endogenous PTH regulates BMP2 signaling pathway in the acceleration of fracture healing by increasing the intracellular cAMP concentration.