Wnt/β-catenin And Parathyroid Hormone Signaling Pathway In Mice Role In Early Bone Repair

Fracture is a heavy social burden.Especially with the aging of the population,the related fracture morbidity and mortality are still high.It is especially important to promote the early healing of fractures and alleviate related complications and social burden.The clinical direction to promote bone repair is to identify possible causes of bone repair damage and to find ways to accelerate the healing process.Local implants and autologous bone grafting for local implantation in the surgical environment have proven effective,but presenting some complications limits the application.Stimulation of bone cells,osteoblasts and osteoconductive scaffolds/extracellular matrix and microcirculation can help new bone formation,calcium preparations and vitamin D can enhance bone density,usually for the above process,by acting on mature cells and directly increasing osteogenesis Cellular activity and inhibition of osteoclast activity are important clinical research directions.Parathyroid hormone（PTH）and drugs associated with Wnt/β-catenin signaling are two treatments for osteoporosis that have been shown to promote bone repair,but the mechanisms involved and in different parts and different age group is still not very clear.Cortical bone and trabecular bone are generally considered to be two different structures that should be treated as separate objects during bone repair.Age is an unfavorable factor in bone repair.Older people generally have a higher risk of fracture and often experience complications during bone repair,such as delayed healing and non-healing.This study produced a model of femoral bone injury in related gene mice and littermate wild mice,and then activated parathyroid hormone（PTH）and Wnt/β-catenin pathway by injection of PTH-34 and tamoxifen.To observe the morphological changes and related pathway signals of different signaling pathways in different parts of young mice,bones of different ages and metaphysis,and the changes of osteoblasts,osteoclasts and microcirculation,to explore the activation of two Different signaling pathways in different ages and different parts promote the role of bone repair and its related mechanisms,in order to further the treatment of patients of different ages and different parts of bone injury in the clinical,choose relevant drugs to provide guidance.Method:Part I:Different effects of Wnt/β-catenin activation and parathyroid hormone activation on early bone repair of femoral and metaphyseal bone in mice1.A bone injury model was established in the unilateral 0.6 mm cortical hole of Catnb^lox（ex3）mice and wild-type mice.2.Intraperitoneal injection of tamoxifen and PTH-34 activates PTH andβ-catenin signaling pathways.3.On the 3rd,6th and 14th postoperative day,the effects ofβ-catenin and PTH signaling pathways on bone repair were observed by X-ray and CT.4.RT-PCR was used to detect the mRNA expression levels ofβ-catenin,LEF-1 and PTH in the bone injury area during bone repair.5.Observing osteoblast molecular OCN,RUNX2 changes by tissue sectioning and immunohistochemistry6.Observing changes in osteoclasts during bone repair by TRAP stainingPart II:Different effects of activated Wnt/β-catenin on early bone repair in femoral diaphysis in aged and adult mice1.A bone injury model was established using Catnb^TM2Kem mice and Catnb^lox（ex3）adult and aged mice,as well as wild-type adult and aged mice,in the femoral diaphysis as a unilateral 0.6 mm cortical hole.2.Activate and inhibitβ-catenin by injection of tamoxifen.3.On the 7th,14th and 21st postoperative day,the effects of activation and inhibition ofβ-catenin signaling pathway on bone repair were observed by X-ray and CT.4.The mRNA expression level ofβ-catenin and LEF-1 was detected by RT-PCR.5.Observe the proliferation and differentiation of MMP9 and VEGF and cells by immunohistochemical staining.6.Observe the changes of osteoclasts during bone repair by TRAP staining.Part III:Different effects of Wnt/β-catenin and parathyroid hormone activation on early bone repair of femoral metaphysis in adult and elderly male mice1.Transfer the 0.6 mm single in the femoral metaphysis of Catnb^lox（ex3）adult（6 month old）and old（12 month old）mice and wild type adult（6 month old）and old（12 month old）mice.2.Activativeβ-catenin and PTH signaling pathways by intraperitoneal injection of tamoxifen and PTH1-34.3.On days 7,14 and 21 after surgery,adult CA mice,adult PTH mice and adult WT mice were observed by X-ray and CT;bone repair of aged CA mice,aged PTH mice and aged WT mice.4.RT-PCR was used to detect the levels ofβ-catenin,PTH1R and LEF-1.5.Observe angiogenesis and osteoblasts by immunohistochemistry by immunohistochemical staining.6.Observe changes in osteoclasts by TRAP.Results:Part I:Activation of Wnt/β-catenin has a greater effect on bone repair than activation of PTH signaling pathway1.In wild-type mice,the rate of repair of metaphyseal bone is faster than that of diaphysis bone.Micro-CT examination showed that the percentage of BV/TV in the area of metaphyseal injury was higher on the 6th and 14th day after surgery.2.Activation of Wnt/β-catenin and PTH signaling pathways promoted the repair of metaphyseal bone,but there was no statistical difference between the two.X-ray examination and three-dimensional reconstruction images showed that the metaphyseal bone repair in the CA group and the PTH group was faster than that in the wild type mice,especially 6 days after surgery.3.Activation of Wnt/β-catenin has a greater effect on bone healing than activation of PTH signaling pathway in diaphysis bone.X-ray examination and 3-D reconstruction images showed that the bone repair of the diaphysis bone of the CA group and the PTH group was faster than that of the WT mice.The bone repair speed of CA mice was faster than that of PTH mice.The number of RUNX2positive cells in the bone injury area of the CA group was greater than that of WT mice and PTH mice.The number of OCN positive cells in the bone injury area of PTH mice was higher than that of WT mice and CA mice.The number of TRAP positive cells in the bone injury area of the CA group was less than that of WT mice and PTH mice.PartⅡ:activation of Wnt/β-catenin signaling pathway is more effective in promoting repair of femoral diaphysis bone in aged mice than in adult mice.1.Adult WT mice have faster bone repair than older WT mice.X-ray images,3D reconstruction images and H&E staining of the bone lesion areas of adult and aged WT mice on days 7,14 and 21 postoperatively showed that the bone repair speed of adult mice was faster.RT-PCR analysis showed that the expression levels of mRNA ofβ-catenin and LEF-1in adult mice were higher than those in aged mice.2.Activation of the Wnt/β-catenin signaling pathway promotes bone repair in adult and aged mice,with a more pronounced effect in older mice.X-ray examination and three-dimensional reconstruction showed that the repair speed of femur in adult CA mice was faster than that in adult WT mice.The repair speed of adult KO mice was slower than that of adult WT mice.The repair of femur bone in aged CA mice was faster than that of aged WT mice and aged KO mice bone repair is slower than older WT mice,especially 14 days after surgery.The ratio of BV/TV of CA mice to WT mice in the aged group was higher than that in the adult group,indicating that the effect of promoting bone repair was more obvious in the aged group.3.Activation of Wnt/β-catenin promotes angiogenesis and cell differentiation in aged miceOn the 14th day after surgery,The ratio of the mRNA expression levels ofβ-catenin,LEF-1 and Osx of CA mice to WT mice in the aged group were higher than those in the adult group.Masson staining showed that collagen fibers in the defect sites of adult CA mice and aged CA mice were more than adult WT mice and aged WT mice.Fast Green/Safranin O staining showed no cartilage formation in the defect sites of the four groups.The ratio of BrdU,MMP9,VEGF,RUNX2 and OCN positive cells of CA mice to WT mice in the aged group was higher than that in the adult group.Part III:Activation of Wnt/β-catenin is better than activation of PTH signaling pathway in promoting repair of elderly metaphyseal bone1.The rate of metaphyseal repair of CA adult mice and PTH adult mice was faster than that of adult wild-type mice,but there was no statistical difference between the two group.X-ray examination,micro-CT and HE staining showed no statistically significant difference in the percentage of BV/TV in the areas of bone injury between CA adult and PTH adult mice at14 and 21 days postoperatively.2.The recovery rate of metaphyseal bone in CA old mice and PTH old mice was higher than that in WT old mice,and the bone repair speed of CA old mice was faster than that of PTH old mice.Micro-CT examination showed that the percentage of BV/TV in the femoral bone injury area of CA aged mice and PTH aged mice was higher than that of WT aged mice at 14 days after surgery,and at 14 days and 21 days after surgery,the percentage of BV/TV of CA aged mice was higher than that of PTH aged mice.HE staining showed the same trend.3.The angiogenesis-related factors VEGF,MMP9 and osteoblast-associated factors RUNX2,OCN were more expressed in CA adult mouse and PTH adult mouse bone injury areas than in WT adult mice,but there was no statistical difference between them.4.The angiogenesis-related factors VEGF,MMP9 and osteoblast-associated factors RUNX2 and OCN in CA-aged and PTH-aged mice were more than WT adult mice,and their expression was higher in CA-old mice than PTH aged mice.conclusion:1.In two-month-old mice,although activation ofβ-catenin and PTH signaling pathways promotes bone repair in the metaphyseal and diaphyseal,activation of theβ-catenin signaling pathway promotes bone repair is more effective than activation of the PTH signaling pathway.More remarkable.2.Activation of the Wnt/β-catenin signaling pathway promotes angiogenesis and cell differentiation in adult（6-month-old）and elderly（12-month-old）mouse femur bone injury areas to promote bone repair and the promote effect in aged mice is more pronounced than that in adult mice.3.Activation of Wnt/β-catenin signaling and PTH signaling pathway can promote the repair of femoral metaphysis in adult（6-month-old）and old（12-month-old）mice,and activateβ-catenin in aged mice can get better bone repair than activation of PTH.