Single Cell RNA Sequence Reveals The Cell Types During The Repair Of Distraction Osteogenesis As Well As Bone Defect And Dentification Of Mechanism Through Which PRRX1 Can Promote Distraction Osteogenesis

Objective It is still unclear whether there are substantial differences in the specific cell types during the process of osteogenesis between canine mandibular distraction osteogenesis(DO)and bone defect(BD).Canine mandibular DO and BD animal models were established and repair tissue-derived mesenchymal stem cells(MSCs)of DO and BD were isolated as well as cultured in vitro.The osteogenesis capabilities of the two tissue-derived MSCs were compared.Thereafter,the cambiums during the healing process were sequenced to construct the single cell RNA-sequence of both DO and BD.Finally,by means of difference analysis,the specific transcription factors and their regulatory genes involved the process of DO were selected and verified by in vivo experiments.The findings of this study provide a theoretical basis for further exploration of the molecular mechanisms of DO osteogenesis.Method Part 1: Analysis of differences in osteogenesis capabilities between DO and BD12 Beagle dogs were randomly divided into two different groups to establish canine mandibular DO and BD animal model.DO group received 7-day latency phase and 7-day distraction phase.The calluses were obtained on 14 days(DO14)and 56 days(consolidation 42 days,DO56)after operation.The callus of BD group was obtained at the same time point.Micro-CT,HE staining,Masson staining and immunohistochemistry staining were used to explore the process of osteogenesis in DO56 and BD56 group.The cambiums formed by healing in the animal model of DO14 and BD14 were used to isolate and culture DO-and BDoriginated MSCs.The cell population was identified by flow cytometer.CCK8,Transwell,clone formation,ALP staining,alizarin red,chondrogenic differentiation,adipogenic differentiation,PCR and WB were used to study the potential abilities of DO-/BD-MSCs.Part 2: Single cell RNA sequence in the process of canine mandibular DO and BD osteogenesis Six beagle dogs were randomly divided into two distinct groups to set up canine mandibular DO and BD animal models.DO group received 7-day latency phase and 7-day distraction phase.The calluses were obtained on 14 days(DO14).The calluses of BD group were taken at the same time point(BD14).The cambium formed by healing was prepared as a single cell suspension for 10×Genomics single cell RNA sequencing.The sequencing results were analyzed by PCA dimensionality reduction,t SNE cell clustering and visualization,cell type annotation and the proportion of cell population was determined by R language.Part 3: Characteristic Analysis of MSCs from DO The sequencing results were analyzed by R language,and the specific cell subsets of DO were analyzed.In order to verify the mechanisms through which continuous distraction stimulation can promote osteogenesis,15 beagle dogs were randomly divided into 5 different groups to establish 2 BD model groups,2conventional distraction animal model groups and a 14-day continuous distraction animal model group(continue DO,c DO).In BD group,the samples were obtained on 14 days(BD14)and 21 days(BD21).In DO group,samples were obtained on14 days after operation(immediately after distraction,DO14)and 21 days after operation(fixed for 1 week after the distraction,DO21).The samples of c DO group were collected after 7-day intermission and 14-day continuous distraction(c DO21).HE staining and immunofluorescence were used to observe the changes of EMT and neural crest stem cells marker.DO-MSCs and BD-MSCs were isolated and cultured,and the in vivo results were verified by immunofluorescence assay.Part 4: Preliminary study on the regulation of canine mandibular DO osteogenesis by PRRX1PRRX1 lentivirus was designed by using CRISPR/Cas9 technology.In vitro experiments: MSCs were cultured in vitro and transfected with PRRX1 knocked down or negative lentivirus.The potential changes in osteogenesis were observed by CCK8,Transwell,ALP standing,alizarin red standing,PCR and WB.In vivo experiments: 12 beagle dogs were randomly divided into two groups to set up an animal model of DO.PRRX1 knocked down or negative lentivirus was injected into the mandibular distraction area of the different dogs on the 6th day of latency phase,the 1st day of distraction and the 4th day of distraction respectively.The samples were obtained at 14 days(DO14)and 56 days(DO56)after operation.The silencing of PRRX1 was confirmed by PCR.The potential changes in osteogenesis were observed by Micro CT,HE staining,Masson staining and immunohistochemical staining.Result Part 1: We first successfully established the animal model of canine mandibular DO and BD.Micro-CT results showed that bone volume / total volume(BV / TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)of BD56 decreased significantly compared with DO56,but trabecular separation(Tb.Sp)increased.HE staining indicated that the new bone trabeculae in DO56 group was substantially thick and close to the new bone,whereas the gap in BD group was still fibrous connective tissue.MSCs derived from DO and BD were successfully isolated and cultured in vitro.Flow cytometry analysis showed that MSCs were positive for CD44,CD90 and CD146,negative for CD34,CD45 and CD31,and possessed the ability of three-line differentiation(osteogenesis,adipogenesis and chondrogenesis).It was found that DO-MSCs had relatively stronger ability of proliferation,migration,clone formation and osteogenesis than BD-MSCs,while the ability of cartilage formation was significantly weaker than BD-MSCs.There was no difference found in adipogenic differentiation ability between the two types of MSCs.Part 2: Single cell sequencing showed that 15120 cells were obtained from DO regenerative tissue and 8477 cells were procured from BD repaired regenerative tissue.It was found that there were 26 different cell groups.After merging,they were integrated into 6 distinct cell groups: MSC group,T cell group,B cell group,macrophage group,neutrophil group and endothelial cell group.MSC group was composed of 14 distinct subpopulations,including cell groups 0,1,2,3,6,8,9,13,14,15,16,21,23 and 24.The surface markers used were ALCAM(CD166),ITGB1(CD29),NT5E(CD73),THY1(CD90),CD44 and PRRX1.Endothelial cell group was composed of 2 subpopulations,including the cell groups 10 and20.The surface markers used were Ve-cadherin(CDH5),KDR(VEGFR2),PECAM1(CD31)and VWF.Macrophage group was composed of 4subpopulations,including cell groups 5,7,12 and 17.The surface markers used were CD86,MRC1(CD206),CD68,MSR1,MARCO and CD163.T cell group was composed of 2 subpopulations,including cell groups 11 and 25.The surface markers employed were CD3 E and CD3 D.B cell group was composed of 3subpopulations,including cell groups 18,19 and 22.The surface markers used were ENSCAFG00000030258(BRC)and FCRLA.Neutrophil group was composed of cell groups 4.The surface marker used was SELL(CD62L).Part 3: DO-MSCs had one more cell subgroup than BD-MSCs.It was found that epithelial related proteins KRT13,KRT14 and KRT15,epithelial mesenchymal transition EMT related genes TJP1,CLDN1 and CTNNB1,as well as neural crest stem cells related markers TFAP2 A,SNAI2,SOX9,DLX5,MYC,all appeared in this cell population.Immunofluorescence of the animal tissues demonstrated that the markers of EMT and the neural crest cells appeared in the conventional DO group(DO14)and continuous DO group(c DO21).After removal of the distraction stimulation,its expression gradually decreased at DO21.However,BD14 and BD21 did not exhibit similar effects.Immunofluorescence analysis of cells showed that there marker of neural crest cells was present in DO-MSCs,but not in BD-MSCs.According to gene expression data,DO-MSCs were divided into embryo-like MSCs(EMSCs),intermediate MSCs(IMSCs)and late MSCs(LMSCs).Part 4: After silencing PRRX1 in MSCs,it was observed that the abilities of proliferation,migration and osteogenesis decreased significantly.In animal experiment,the results of Micro-CT showed that after silencing PRRX1 in the distraction tissues,BV/TV,Tb.Th as well as Tb.N decreased,Tb.Sp increased and nonunion occurred at DO56.HE staining results indicated that after silencing PRRX1,there were still a large number of collagen fibers in the tissues of DO56.Immunohistochemical results demonstrated that silencing PRRX1 markedly inhibited the expression of osteogenesis protein OCN.Conclusion1.In BD repair of the same size,the osteogenic ability of DO was found to be significantly stronger than that of autologous bone repair.DO-MSCs possessed stronger ability of proliferation,migration,clone formation and osteogenesis than BD-MSCs.However,the chondrogenic differentiation ability of BD-MSCs was substantially stronger than that of DO-MSCs.There was no difference in adipogenic differentiation between the two groups.2.There were six different cell groups identified in the process of regeneration and repair of canine mandibular DO and BD,which were MSc group,T cell group,B cell group,macrophage group,neutrophil group and endothelial cell group.3.The stimulation of the continuous distraction can facilitate MSCs to acquire the characteristics of embryonic neural crest stem cells,and MSCs can display the features of EMT.4.Transcription factor PRRX1 plays an important role in the process of DO.Silencing PRRX1 could significantly inhibit the rapid osteogenesis of DO.