Osteogenic And Osteoclastic Differentiation Ability Of MSCs Derived From Congenital Pseudarthrosis Of Tibia

Objective: Isolating the Mesenchymal stem cells(MSCs)form periosteum,and analyzing the osteogenic and osteoclastic differentiation ability of MSCs from CPT.Then identifing the differential express m RNA between CPT and normal periosteum by High-volume sequencing technology to explore the possible pathogenesis of CPT.Methods:The first part: The MSCs was isolated form 5 CPT and normal periosteum and identified by attachment dependence,cell surface antigen and differentiation ability.Then the MSCs were differentiated by osteogenesis and osteoclast,and the differences of differentiation ability was compared(3 cases in each group).The second part: The MSCs,isolated form 5 CPT and normal periosteum,was sequenced by High-throughput sequencing technology.The differential express m RNA between CPT and normal periosteum was identified.Then the differential express m RNA was analyzed by GO and KEGG.Results:The first part:(1)A large number of cells was obtained form the periostrum,including some adherent growth cells.Flow cytometry analysis show that the rate of cell which express the marker of CD31 and CD34 were under 1.5%,and which express the marker of CD44 and CD90 cells were more than 95%.All that indicating that the isolated cells were MSCs.(2)ALP staining showed that the alkaline phosphatase activity of MSCs,which isolated form CPT tissue,was lower than normal periosteum,and Alizarin red staining showed that the count of mineralized nodules,which isolated form CPT tissue,was less than normal periosteum,after two weeks for osteogenic induction.The level of Osterix,Runx2,OCN and OPN from CPT was lower than normal periosteum.The difference was statistically significant.(3)After osteoclast induction for 7 days,the number of osteoclasts that produced from CPT was more than normal periosteum,and Western blots showed the level of CTSK,CALCR and ITGβ3 from CPT was more than normal periosteum.The second part:(1)During the research,the count of m RNAs,which sequenced form the MSCs that isolated form 5 CPT and normal periosteum in the first part,was 1472.The quantity of up-regulated m RNAs was 691,while down-regulated was 781.(2)During GO analysis,we found that the m RNAs,which was up-regulated or down-regulated in MSCs,involved in the biological processes,cell composition and molecular functions were similar.(3)KEGG enrichment analysis of differentially expressed m RNAs showed that the most obvious enrichment pathway was endocytosis,and thyroid hormone signaling pathway was one of the enrichment pathways.Conclusion :1.CPT tissue contain MSCs with lower osteogenic and higher osteoclastic differentiation ability.2.A total of 1472 differentially expressed m RNAs were obtained during High-throughput sequencing.And 691 m RNAs were up-regulated and781 m RNAs were down-regulated compared with normal periosteum.3.The differentially expressed m RNAs may related to the lower osteogenic differentiation ability of MSCs derived from congenital pseudarthrosis of tibia.