The Functions Of Fibroblast Growth Factor-23 On Bone Resorptionand Bone Formation In Osteoclast-osteoblast Co-culture System

Bone is very important for vertebrates and human beings.To maintain its strength and structure,bone requires continuous metabolism,which is dependent on bone reconstruction.Bone reconstruction maintains its balance by means of bone absorption mediated by osteoclast?OC?and bone formation mediated by osteoblast?OB?throughout the life span.Various bone diseases,such as osteoporosis,bone sclerosis et al.,will seriously harm economic benefits and the quality of life when the balance is broken.Fibroblast growth factor?FGF?23,a hormone found recently,mediates a negative feedback loop composed of parathyroid,kidney,bone and vitamin D.It affects bone metabolism indirectly by regulateing bone mineral metabolism through the"bone-kidney-parathyroid"endocrine axis.FGF23 is closely related to bone diseases because when its secretion,activity and intracellular processes are abnormal,the metabolic disorders of bone minerals can lead to utosomal dominant hypophosphatemic rickets?ADHR?,X-linked hypophosphatemicrickets?XLH?,tumor-induced osteomalacia?TIO?,chronic kidney disease-mineral and bone disorder?CKD-MBD?,osteoporosis,and bone sclerosis.However,little is known about the mechanism of the direct effect of FGF23 on bone.Therefore,the study about the differentiation and function of OC and OB will help to understand its direct role on bone,and provide reference and theoretical support for the pathogenesis,prevention and treatment of bone diseases related to FGF23.In this study,the osteoclast-osteoblast co-culture system in vitro was constructed at first.The method was:mouse mononuclear/macrophage line RAW264.7 and osteoblast like cell line MC3T3-E1 were inoculated in transwell co-culture device,while added with 1?,25?OH?₂D₃ and macrophage colony-stimulating factor?M-CSF?for 6 days,then detected tartrate-resistant acid phosphatase?TRAP?activity,TRAP staining and toluidine blue staining to identify whether RAW264.7 was differentiated into mature OC,detect Alkaline phosphatas?ALP?activity to identify whether MC3T3-E1 was differentiated into mature OB.Then added FGF23 into the co-culture system,detect indexes related to OC and OB respectively.The indexes of OC include the TRAP activity on 2nd,4th,6th and 8th days,the areas of bone resorption lacunae on 6th day,and the mRNA relative expression of factors related to OC on 2nd,4th,6th and 8th days;The indexes of OB include the ALP activity on2nd,4th,6th and 8th days,the Alizarin Red adsorption on 6th and 17th days,the mRNA relative expression of factors related to OC on 2nd,4th,6th and 8th days.The results showed that at the end of the construction of co-culture system,the TRAP activity of co-culture OC was significantly enhanced?p<0.05?,TRAP staining is positive,and the bone resorption lacunaes appeared after toluidine blue staining compared with single culture OC.The ALP activity of co-culture OB was extremely significant enhanced compared with single culture OB?p<0.01?.After adding FGF23,the TRAP activity of the experimental group was significantly?p<0.05?or extremely significant?p<0.01?increased compared with the control group in every days,the areas of bone resorption lacunae were increased extremely significant?p<0.01?.The mRNA relative expression of V-H⁺-ATPase?ATP6i?,cathepsin K?Cts K?,TRAP,matrix metalloproteinase-9?MMP-9?,fibroblast growth factorreceptor?FGFR1?,FGFR3,FGFR4 and Klotho,which could promote the differentiation and maturation of OC,and platelet-derived growth factor-bb?PDGF-bb?,Sphingosine kinase1?SPHK1?,bone morphogenic protein2?BMP2?,BMP6 and BMP7,which can promote the differentiation of OB was significantly?p<0.05?or extremely significant?p<0.01?up-regulated.The ALP activity of the experimental group was significantly?p<0.05?or extremely significant?p<0.01?increased.The adsorption of alizarin red was extremely significant?p<0.01?declined on 17th day when OB is at the stage of mineralization period,and the mRNA relative expression of M-CSF,receptor activator of nuclear factor?B ligand?RANKL?and FGF23which can promote the differentiation and maturation of OC was significantly?p<0.05?or extremely significant?p<0.01?up-regulated,the mRNA relative expression of OPG and osteoclast inhibitory lectin?OCIL?which can inhibit the differentiation and maturation of OC was significantly?p<0.05?or extremely significant?p<0.01?down-regulated.The results indicated that in the transwell co-culture system,a few of RAW264.7 had been differentiated into multi-core mature OC with the function of bone absorption.The differentiation activity of MC3T3-E1 was obviously enhanced,and it had been differentiated into mature OB.The system was successfully constructed and could be used in the subsequent experiment.FGF23 may promote the differentiation,maturation of OC and bone absorption mediated by OC.FGF23 can promote differentiation of osteoblast during non mineralizing period,but in mineralization period,OB mineralization and bone formation is inhibited.The functions of FGF23 on OC and OB may be through the dialogic factor between OC and OB.