An In Vitro Study Of Cranial Base Synchondrosis Chondrocytes After Cts Loading

Objectives:This study was designed to establish a method for culturing rat CBS（cranial base synchondrosis）chondrocytes in vitro,study the change of extracellular matrix synthesis of CBS chondrocytes with CTS（Cyclic tensile strain）loading and detect the differentially expressed miRNAs after CTS loading.Methods:Two-step-digestion method was used to isolate CBS chondrocytes obtained from 1 week old SD rats.IHC staining of type II collagen of and Sox9 were made to identify chondrocytes.The expression of Sox9,Col1a1,Col2a1 and Col10a1mRNA in the process of passage were quantitatively detected by RT-PCR and the protein level of COL2A1 and SOX9 were detected by western blotting.A CTS of 1Hz,10%elongation with sin waveforms was applied to the P2 generation chondrocytes by FX-5000^TM tension system and the loading time was 24h,while the control group kept static at the same time.The expression change of Acan,Col1a1,Col2a1,Col10a1Sox9,Ihh and PTHrP mRNA were quantitatively detected by RT-PCR and the protein level of COL2A1 and SOX9 were detected by western blotting.High-throughput sequencing of miRNAs was conducted in control and experimental groups.The three main steps included DNA library preparation,cluster formation and sequencing.The differentially expressed miRNAs were obtained through data analysis.Results:IHC staining results of type II collagen and Sox9 showed strongly positive within 4 generations The expression of Col2a1 decreased in passage 1 and picked up in passage 2,and then remained stable but a little bit lower in passage 4;The change of Sox9 expression showed same tendency as Col2a1,however the expression levels were higher than primary chondrocytes from passage 2;The expression of Col10a1 dropped to near zero after passages;The expression of Col1a1declined about 50 percents in passage 1 and 2 and increased drastically from passage3.The Western blotting results showed positive expression of COL2A1 and SOX9 in P0⁴ chondrocytes with a little bit lower expression in P1.With the loading of CTS,the expression of Acan,Col2a1 and Col10a1 significantly increased in cranial base synchondrosis chondrocytes（P<0.05）.mRNA for Col2a1 elevated two folds after stretching while mRNA for Col1a1 changed little（P>0.05）.With the loading of CTS,the expression of Sox9 and Ihh significantly increased in cranial base synchondrosis chondrocytes with more than 6 folds elevation of Sox9 and less than 2 folds of Ihh（P<0.05）.The expression of PTHrP remained stable（P>0.05）.The protein level of COL2A1 and SOX9 increased significantly after CTS loading.24 differentially expressed miRNAs were found with 10 up-regulated and 14 down regulated（P<0.05）.Among these miRNAs,miR-140-5p and miR-125b-5p were most important candidates.Conclusion:Rat CBS chondrocytes were successfully isolated in this study and P2,P3 and P4 cells could be used in subsequent experiments.A CTS of 1Hz and 10%elongation could promote cartilage matrix synthesis of CBS chondrocytes.24differentially expressed miRNAs might play roles in this process with miR-140-5p and miR-125b-5p being most important.