Different Bone Metabolism Levels Of Trabecular And Cortical Bone In Response To Changes In Wnt/?-catenin Signaling In Mice

Backgrounds:The vertebrate skeleton comprises trabecular bone(also called spongy,or cancellous bone)and cortical bone(also called compact bone).Generally speaking,trabecular bone is the type of bone mainly found in the core of short and flat bones,as well as in the metaphyses of long bones,whereas compact bone constitutes the diaphyses of the long bones and the cortical shell of short and flat bones.The two types of bone differ not only in their structure but also in their mechanical properties and metabolic activity.Structurally,cortical tissues are solid and dense and consist of osteons and plexiform lamellae aligned in the longitudinal direction of the bone.By contrast,trabecular tissues are relatively loose and contain trabecular bone packets with boundary layers at various angles compared with the longitudinal axis of the trabecula.In additional,trabecular bone has a larger surface area and trabeculae are in closer contact with vascular spaces.The different structures of the two types of bone tissue lead to differences in their mechanical properties.Cortical bone tissue has a higher Young's modulus compared with trabecular bone tissue,and the higher degree of mineralization of cortical bone contributes to a stiffer,but more brittle tissue compared with single trabeculae.In addition,cortical bone and trabecular bone show different metabolic behaviors in many physiological and pathological processes,such as glycation,pharmacokinetics,and their response to diet and implant insertion.The difference in metabolic activity is best demonstrated by their different behaviors during the bone remolding process,which has significant clinical relevance for the treatment of osteoporosis.,Trabeculae,as thin plates,have a low matrix volume and a large surfa ce area;therefore,signals within the matrix can readily traverse the matrix volume to locate a surface to initiate trabecular remodeling.By contrast,cortical bone has a large matrix volume and a small surface area;therefore,signals deep within the ma trix may not locate a surface as readily to initiate remodeling,allowing for the accumulation of microdamage,particularly in interstitial bone that is less remodeled.Furthermore,different mechanoresponsiveness levels in cortical and trabecular bone lea d to different bone formation abilities in response to the mechanical load on the bone.The different bone metabolism processes between cortical and trabecular bone may be the reason for the relative lack of efficacy of anti-osteoporotic drugs on nonvertebral fractures.The underlying mechanisms responsible for the different properties between cortical and trabecular bone are not fully understood.It was generally believed that the function of an organ or tissue depends on its cellular function,which is regulated by various hormones and locally acting growth factors through different signaling pathways.Specifically for bone,all factors influencing the composition,structure,and metabolic differences between cortical and trabecular bone do so through the final common cellular and signaling pathways.Based on the central role of the Wnt/?-catenin signaling pathway in bone development,bone growth and bone metabolism,as shown by accumulating evidence,we hypothesized that the expression levels of molecules in the Wnt/?-catenin signaling pathway in the cortical and trabecular compartments are different and that the different expression levels lead to different bone metabolism levels between cortical and trabecular bone.In the present study,the transcriptional and protein expression levels of Wnt/?-catenin signaling-related molecules were examined in the metaphyseal trabecular and diaphyseal cortical bone of the tibia in mice.Then,the biological functions of osteoblasts and osteoclasts along with bone metabolism processes were compared between the metaphyseal trabecular and diaphyseal cortical bone after ?-catenin was either stabilized or deleted in mice.Methods:Part I: To investigate the difference of Wnt/?-catenin pathway and associating factors expression in cortical and trabecular bone.1.?-catenin and associating factors such as ?-catenin?TCF(T cell factor)?LEF-1(lymphoid enhancer-binding factor 1)in cortical and trabecular bone were examined by real time PCR.2.?-catenin and associating factors such as ?-catenin,Runx2(Runt-related transcription factor 2),Osterix and RANKL(Receptor activator of nuclear factor-kb ligand)in cortical and trabecular bone were examined by Immunohistochemical staining on mice.3.TRAP staining was used to observe osteoblast and osteoclast activities.4.Osteoblasts were cultured,and ?-catenin and associating factors such as ?-catenin,Runx2,Osterix and RANKL in cortical and trabecular bone were examined by Cellular immune fluorescence.5.The ratio of ?-catenin enterning nuclear in vitro wereexamined by western bloting.6.The protein of LRP6(Low-density lipoprotein receptor–related protein 6),?-catenin,ALP(Alkaline phosphatase),Runx2,cyclin D1 and OCN(Osteocalcin)in vivo were examined by western bloting.Part II: The constitutive activation of ?-catenin(CA-?-catenin)and ?-catenin knock out(?-catenin-KO)models were used to investigate the difference of bone metabolism levels in trabecular and cortical bone in response to changes in Wnt/?-catenin signaling in mice.1.Bone structure and bone mass were obserbed and analyzed by Micro-CT in 3.2cre-CA-?-catenin,3.2 Cre-?-catenin-KO and wild-type models.2.In above models,?-catenin and associating factors such as ?-catenin?TCF?LEF-1 in cortical and trabecular bone were seperated examined by real time PCR.3.In above models,?-catenin and associating factors such as ?-catenin?ALP?Runx2 and cyclinD1 in cortical and trabecular bone were seperated examined by western bloting.4.TRAP staining was used in three models to observe osteoblast and osteoclast activities.Results:Part I:Different expression levels of Wnt/?-catenin signaling molecules and bone metabolism-related molecules in the metaphyseal trabecular and diaphyseal cortical bone of tibia in mice.1.The mRNA expression levels of Wnt 3a,Wnt5 a,?-catenin,TCF,LEF-1,WISP(WNT1 inducible signaling pathway protein 1),axin-2,cyclin D1,c-Myc(cellular-myelocytomatosis viral oncogene),and RANKL were higher in metaphyseal trabecular bone than in diaphyseal cortical bone.The m RNA expression levels of OCN and ALP,two bone formation markers,were also higher in metaphyseal trabecular bone than in diaphyseal cortical bone.2.For bone resorption,the ratio of RANKL to OPG(Osteoprotegerin)was higher in metaphyseal trabecular bone than in diaphyseal cortical bone,and TRAP(Tartrate resistant acid phosphatase)staining revealed that the number of TRAP-positive cells and osteoblasts was higher in metaphyseal trabecular bone than in diaphyseal cortical bone.3.The ratio of ?-catenin protein entering nuclear were higher in trabecular bone were examined by western bloting.4.the protein levels of LRP6,?-catenin,ALP,Runx2,cyclin D1 and OCN in trabecular bone were higher than those in cortical bone,which was confirmed by western bloting.5.The results of Cellular immune fluorescence and Immunohistochemical staining were identical with the real time PCR.Part II:Trabecular and cortical bone respond differently to CA-?-catenin.1.After ?-catenin was stabilized in osteoblasts,the changing levels of the genes noted above in metaphyseal trabecular bone and diaphyseal cortical bone were determined.The m RNA expression levels of ?-catenin,TCF,LEF-1,WISP,axin-2,cyclin D1,c-Myc,ALP and OCN were significantly up-regulated in trabecular bone,and the degree of up-regulation of the same genes in cortical bones was not as significant as in trabecular bone.2.The same trends of ?-catenin,ALP,Runx2 and cyclin D1 protein expression level were confirmed by Western blotting examination.3.The ratio of RANKL to OPG decreased in both trabecular bone and cortical bone in CA-?-catenin compared with wild-type mice,and the extent of decrease was more striking in trabecular bone.Consistently,a decreased number of osteoclasts in both the trabecular bone and cortical bone were observed in CA-?-catenin mice compared with wild-type mice;however,the extent of changes was greater in the trabecular bone than in the cortical bone.4.Regarding the number of osteoblasts,it increased significantly in trabecular bone of CA-?-catenin mice,and only increased slightly in cortical bone of CA-?-catenin mice compared with wild-type mice.The extent of change is more striking in trabecular bone than in cortical bone after stabilization of ?-catenin.5.Micro-CT revealed that the metaphyseal trabecular BV/TV(Bone volume/tissue volume)increased approximately 2.4 times in CA-?-catenin mice compared with wild-type mice.By contrast,the cortical bone mass only increased 0.2 times in CA-?-catenin mice compared with wild-type micePart III: Trabecular and cortical bone respond differently after gene deletion of ?-catenin.1.After ?-catenin was knocked-out in mice,the m RNA expression levels of ?-catenin,TCF,LEF-1,wisp,axin,cyclin D1,c-Myc,ALP and OCN were significantly down-regulated in trabecular bone compared with wild-type mice,and the degree of down-regulation of the same genes in cortical bones was not as significant as in trabecular bone.2.The same trends of ?-catenin,ALP,Runx2 and cyclin D1 protein expression level were confirmed by Western-blotting examination.3.The ratio of RANKL to OPG increased in both trabecular bone and cortical bone in ?-catenin knockout mice compared with wild-type mice,and the increment was more significant in trabecular bone.Consistently,an increased number of osteoclasts was observed in both the trabecular bone and cortical bone in ?-catenin knockout mice when compared with wild-type mice.Again,the extent of these changes was greater in trabecular bone than cortical bone.4.Regarding the number of osteoblasts,it decreased significantly in trabecular bone of ?-catenin knockout mice,and only decreased slightly in cortical bone of ?-catenin knockout mice compared with wild-type mice.The extent of change is more striking in trabecular bone than in cortical bone after deletion of ?-catenin.5.MicroCT examination showed that metaphyseal trabecular BV/TV decreased approximately 0.47 times in ?-catenin knockout mice compared with wild-type mice.By contrast,the cortical bone mass only decreased 0.19 times in ?-catenin knockout mice compared with wild-type mice.Conclusions:the expression levels of Wnt/?-catenin signaling molecules and bone metabolism-related molecules were different in cortical bone and trabecular bone and that the bone metabolism level in trabecular bone was more readily affected by changes in the Wnt/?-catenin signaling pathway.