Cebpb Regulates Osteogenic Differentiation Of Skeletal Stem Cells And Fracture Healing Through WNT/β-Catenin Pathway

Objective:Fracture is the most common traumatic organ injury,and fracture complications such as nonunion and bone defect are on the rise.At present,autologous iliac crest bone graft transplantation is the gold standard for the treatment of the above-mentioned fracture complications.However,autologous iliac crest bone graft transplantation has complications in the donor area and high medical costs.Skeletal stem cells(SSCs)are stem cells with long-term self-renewal ability and osteo-lineage differentiation ability in bone,which play a central role in the maintenance of bone homeostasis,bone regeneration and bone repair.Studying the signaling pathways and key molecules that affect the osteogenic differentiation of SSCs can help promote the fracture healing process involving SSCs and treat fracture complications such as nonunion and bone defect.Methods:Using single-cell RNA sequencing data of SSCs at different differentiation stages(stem and progenitor),the R Seurat software package was used to analyze the differential genes between different stages during the osteogenic differentiation of SSCs,the R Cluster Profiler software package was used for GO enrichment analysis,and the transcription factor activity analysis was performed using python py SCENIC package.Intersections of differentially expressed genes and differentially active transcription factors were used to explore key signaling pathways and transcription factors during the osteogenic differentiation of SSCs.The SSCs were sorted by multi-label(CD45~-Ter119~-Tie2~-CD51~+Thy~-6C3~-CD105~-)flow cytometry,and the in vitro proliferation experiments,colony formation experiments and osteogenic differentiation experiments were performed to confirm their osteogenic stem cell characteristics.SSCs were transfected with lentiviral vectors to knock down Cebpb protein.The expression of osteogenesis-related proteins(Runx2,Sp7 and Bglap2)was detected by western blot and q PCR.Alkaline phosphatase staining and Alizarin red staining were used to evaluate the role of Cebpb in the osteogenic differentiation of SSCs.In order to exclude off-target effects,lentiviral vectors containing Cebpb c DNA were transfected in SSCs,and the expressions of osteogenesis-related proteins(Runx2,Sp7 and Bglap2)were detected by western blot and q PCR,further confirmed the role of Cebpb in the osteogenic differentiation of SSCs.After knocking down Cebpb in SSCs,western blot was used to detect the changes of marker genes(Hes1、Gli1、p-Erk1/2、p-Smad1/5/9 andβ-catenin)in Notch,Hedgehog,MAPK,BMP2/SMAD,and WNT/β-catenin signaling pathways.On the basis of Cebpb knockdown in SSCs,different concentrations of WNT/β-catenin signaling pathway inhibitors IWR-1 were used to detect the expressions of osteogenesis-related proteins(Runx2,Sp7 and Bglap2)by western blot,and alkaline phosphatase staining,alizarin Red staining,which confirmed that Cebpb regulates the osteogenic differentiation of SSCs through the WNT/β-catenin signaling pathway.We constructed a femoral monolayer cortical defect model in six-week-old C57/BL6 mice,loaded the treated SSCs on collagen scaffolds,and implanted them into the model.Micro CT was used to detect the microstructure,three-point bending test was used to detect the biomechanics,and immunohistochemistry was used to detect the expression of osteogenic and chondrogenic proteins.Results:Bioinformatics analysis of single-cell sequencing data of SSCs and osteoprogenitors(OPs)revealed that stemness genes were highly expressed in SSCs,while osteogenic differentiation genes were highly expressed in OPs.Through GO enrichment analysis,it was found that the differential genes were enriched in the signal pathways of cartilage development,ossification,biomineralization,osteoblast differentiation,and cell aggregation.Taking the intersection of differential genes and differentially active transcription factors,we found that Cebpb was highly expressed and had enhanced transcriptional activity in SSCs.We knocked down Cebpb in SSCs and found that the expression of osteogenesis-related genes at the transcriptome and protein levels,the calcified nodules and alkaline phosphatase activity was enhanced during osteogenic differentiation.Overexpression of Cebpb in SSCs yielded opposite results to Cebpb knockdown.Through the detection of Notch,Hedgehog,MAPK,BMP2/SMAD and WNT/β-catenin signaling pathway marker genes,we found that knockdown of Cebpb in SSCs can activateβ-catenin of WNT/β-catenin signaling pathway.Knockdown of Cebpb combined with IWR-1 treatment in SSCs significantly attenuated the expression of osteogenesis-related genes,decreased alkaline phosphatase activity,and reduced calcium nodule production.In a mouse femoral defect model with SSCs implanted,knockdown of Cebpb can increase the bone volume fraction,improve the biomechanical properties(elastic modulus,maximum load,and failure energy)of fractured femurs,and increase the expression of osteogenic and chondrogenic proteins in the callus.Conclusions:Stemness genes decrease and osteogenic differentiation-related genes increase during the osteogenic differentiation of SSCs.Inhibition of the transcription factor Cebpb by regulating the activity ofβ-catenin affects the expression of WNT/β-catenin pathway target genes(such as Runx2 and Sp7),and promotes the osteogenic differentiation of SSCs.Knockdown of Cebpb can promote the fracture healing process involving SSCs.Our study confirmed the value of Cebpb in the osteogenic differentiation of SSCs and fracture healing.Our study provided a new biological therapeutic target for fracture treatment.