The Role And Mechanism Of Wnt/?-catenin Signaling Pathway In Terminal Differentiation Of Osteoblasts And Remodeling Process Of Fracture Healing

Backgrounds:Now accumulating evidence suggest that Wnt/?-catenin signaling plays an important role in the process of bone homeostasis.It was shown that the inactivation of ?-catenin in mesenchymal progenitors,osteoblasts and osteocytes results in low bone mass,whereas the stabilization of ?-catenin in mesenchymal progenitors and osteoblasts could increase bone mass.These studies revealed that the modification of Wnt/?-catenin signaling shows promise in the treatment of low bone volume conditions such as osteoporosis.Osteoblasts play an important role in controlling bone mass because they are the main cells responsible for bone formation,and osteocytes make up more than 95% of all bone cells in the adult skeleton.Previously,we reported that constitutive ?-catenin activation in either osteoblasts or osteocytes potentially had adverse effects on the bone quality,although it could increase bone mass.However,the mechanism of bone strength decreased is unknown currently.Therefore,our first aim was to observe the effect of constitutive activation of ?-catenin on terminal differentiation of osteoblasts.Now more and more evidence suggest that Wnt/?-catenin signaling also plays an important role in the process of fracture repair.Activation of Wnt signaling were found to improve bone healing in mice.In contrast,the inhibition of Wnt signaling by treatment impaired fracture healing.Secondary bone healing is the most common type of fracture healing clinically.Fracture healing can be divided into three overlapped phases(i.e.,inflammatory,reparative and remodeling phases);disadvantageous factors present during each phase can hinder the healing process,resulting in delayed bone fracture healing or non-union.Especially,improper remodeling can not only result in delayed bone healing but can also lead to re-fractures or recurrent fractures,which are regularly reported in clinical scenarios.However,most studies have focused on the inflammatory and reparative phases,and little attention has been paid to the remodeling phase.And,the role of the Wnt/?-catenin signaling pathway during the remodeling phase of bone fracture healing is currently unknown.In the present study,?-catenin was high level activated ?-catenin activated,low level activated or deleted in mice,and the effects of ?-catenin on the bone remodeling phase and eventual bone fracture healing quality were investigated.Taken togather,in the present study,we aimed to: 1.to observe the effect of constitutive activation of ?-catenin on terminal differentiation of osteoblasts;2.to observe the role of Wnt/?-catenin pathway in remodeling phase during fracture healing.Methods:Part I: the effect of ?-catenin on terminal differentiation of osteoblastsI-1: the effect of constitutive activation of ?-catenin in osteocytes(ocCA-?-catenin)on bone mass1.X-Ray and Micro-CT were used to observe bone structure and analyze bone mass;2.Three-point bending test was used to test biomechanical properties of femur;3.Immunohistochemisty were used to analysis the expression of Col1a1 and FGF23.I-2: the effect of constitutive activation of ?-catenin in osteoblasts(ob CA-?-catenin)on terminal differentiation of osteoblasts1.Three-point bending test was used to test biomechanical properties of femur;2.Masson and Sirius Red staining were employed to evaluate the content and structure of collagen;3.Undecalcified skeleton sections were stained with Von Kossa to observe bone mineralization;4.BrdU immunohistochemisty were used to analysis the proliferation of osteoblasts and TUNEL assay was used to evaluate hypertrophic cell apoptosis;5.The expression of Run X2?Osterix?Osteocacin?DMP1?Col1a1?c-myc?cyclinY and Dkk1 were analysed by immunohistochemisty;6.Cell culture system was used to evaluate the differentiation of osteoblasts and Alizarin red staining was used to observe osteocytes mineralization;7.Reverse transcription PCR(RT-PCR)and Western Blot(WB)were used to analysis the expression of Run X2?Osterix?Osteocacin?DMP1?Col1a1?c-myc?cyclin Y and Dkk1.Part II: the effect of Wnt/?-catenin signaling pathway on fracture remodeling process1.Bone fractures were generated in 8-week-old male mice and the following experiments would be taken at eight weeks after operation;2.To high level activate ?-catenin(HA-?-catenin)? low level activate ?-catenin LA-?-catenin and knockout ?-catenin(?-catenin-KO)though tamoxifen(TM)injected;And reverse transcription PCR(RT-PCR)was used to analysis the expression of ?-catenin of HA-?-catenin?LA-?-catenin??-catenin-KO and control mice;3.Histological sections of mice were stained with H&E,X-Ray and Micro-CT were used to observe bone structure and analyze bone mass,Safranin O staining was employed to evaluate cartilage component content,Masson and Sirius Red staining were employed to evaluate the content and structure of collagen;4.Three-point bending test was used to test biomechanical properties of tibia;5.Undecalcified skeleton sections were stained with Von Kossa to observe bone mineralization;6.TRAP staining was used to observe osteoclast activities;7.Immunohistochemisty were used to analysis the expression of ALP?Runx2 ?OSX?OCN;8.Reverse transcription PCR(RT-PCR)was used to analysis the expression of ALP?Runx2 ?OSX?OCN?DMP1?RANKL?OPG;9.Concentration of serum biomarkers of osteoblasts(ALP)and osteoclasts(CTX?)was measured by ELISA.Results:Part I: Terminal osteoblast differentiation was impaired in the obCA-?-catenin miceI-1: bone strength and bone growth impaired,although cancellous bone mass increased in the oc CA-?-catenin mice.1.The cancellous bone volume significantly increased in ocCA-?-catenin mice;2.Bone strength decreased in oc CA-?-catenin mice;3.Collagen arrangement disorganized in oc CA-?-catenin mice;4.The exorssion of Col1a1 and FGF23 decreased in oc CA-?-catenin mice.I-2: The terminal differentiation of osteoblasts was impaired when ?-catenin was constitutively activated and that DKK1 plays a pivotal role in this process.1.Bone strength decreased in ob CA-?-catenin mice;2.The mineralization level decreased,collagen arrangement disorganized anddown-regulated Col1 expression;3.The early differentiation markers of osteoblasts such as alkaline phosphatase and osterix,were up-regulated,while the expression level of osteocalcin and Dmp1 the markers for the terminal differentiation of osteoblasts,decreased in ob CA-?-catenin mice;4.Osteoblast was maitained in highly proliferative state in the ob CA-?-catenin mice;5.The expression of c-myc?cyclinD1?cyclinY?CDK14 increased in obCA-?-catenin mice;6.The levels of c-myc,cyclin D1,CDK14 and cyclin Y were reversed in the osteoblasts that were isolated from obCA-?-catenin mice after DKK1 was added to the culture medium,and the mineralization level of osteoblasts that were isolated from ob CA-?-catenin mice was almost restored to that from control mice after the adding of recombinant DKK1.Part II: Wnt/?-catenin signaling should be kept at proper level to ensure better bone fracture remodeling and better bone quality1.X-ray examination revealed that the calluses were over-absorbed in ?-catenin-KO mice,there have a larger callus remained in the fracture site in HA-?-catenin mice that only a small callus remained at the fracture site in LA-?-catenin mice;However,?CT analysis indicated that the BV/TV decreased significantly in ?-catenin-KO mice,slightly decreased in HA-?-catenin mice,slightly increased in LA-?-catenin mice when compared to control mice.2.Fast Green/Safranin O staining revealed the presence of a large amount of residual cartilage matrix in the un-remodeled callus in HA-?-catenin mice;3.Collagen was disorganized in the HA-?-catenin mice and ?-catenin-KO mice,but collagen organization of LA-?-catenin mice did not significantly differ from that of control mice as confirmed by Sirius red staining;4.The bone strength of LA-?-catenin mice was significantly higher than that of HA-?-catenin mice and ?-catenin-KO mice and was higher than that of control mice;5.The mineralization levels of HA-?-catenin mice and ?-catenin-KO mice were significantly decreased compared with control mice;However,the mineralization levels of LA-?-catenin mice did not significantly differ from that of control mice;6.The osteoclast activities of HA-?-catenin mice was significantly lower than that of and ?-catenin-KO mice and control mice,However,LA-?-catenin mice did not significantly differ from that of control mice as confirmed by TRAP staining and ELISA testing;7.The exprssion of ALP?Runx2?OSX in HA-?-catenin mice were significantly higher than other group,While the expression level of OCN in LA-?-catenin mice was the highest as confirmed by IHC staining and RT-PCR.Conclusions:1.Trabecular bone mass increased significantly in CA-?-catenin mice;however,bone strength decreased;osteoblasts were maintained in a highly proliferative state when ?-catenin was constitutively activated,and highly proliferative state would decreased osteoblast terminal differentiation.2.DKK1 played a pivotal role in the impaired terminal differentiation of osteoblasts in ob CA-?-catenin mice.Wnt signaling must be down-regulated by increased DKK1 expression levels to promote mineralized matrix formation.3.Wnt/?-catenin signaling pathway play an important role during the remodeling phase of bone fracture healing,but either too much or too little ?-catenin can be detrimental to bone healing at this stage,it should be precisely regulated to allow complete fracture healing.