Study Of Rarefaction-dense Wave-induced Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Chondrocytes

Objective:By investigating the best combination of wave band of the rarefaction wave and dense wave during the differentiation from bone marrow mesenchymal stem cells(BMSCs) into chondrocytes and identifying the underlying mechanism,so as to provide the experimental basis for rarefaction-dense waves therapeutic equipment used in clinic.Method:â‘ Experiments were performed at the Laboratory of Fujian Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine from October 2006 to March 2008.Totally 24 clean healthy Sprague Dawley rats aged four weeks were obtained from the Shanghai Silaike,China.Animal disposal in the experiment was accorded with the animal ethical standard.â‘¡The BMSCs were obtained from SD rats'bone marrow by the bone marrow adherence cultivation through separation,purification,cultivation in vitro and the F₃ BMSCs were used as the source for the post-researchs.â‘¢Rarefaction-dense waves emission instrument was supplied by Institute of Biomedicine Instrument,Fuzhou University. According to various proportion of the same cycle dilatational wave,there were three working modes:modeâ… :rarefaction wave:dense wave=1:1,modeâ…¡:rarefaction wave:dense wave=2:1, and modeâ…¢:rarefaction wave:dense wave=1:2.â‘£At the third passage,BMSCs were incubated in DMEM containing 15%volume fraction fetal bovine serum after inoculated for 24h by the density of 5×10⁵/ml in Tissue Culture Flask,1×10⁵/ml in 6 Cell Culture Plates pre-set Tissue Culture coverslips.According to the different working mode of the instrument or whether being induced,BMSCs were divided into four groups:group one whose proportion of rarefaction wave to dense wave is 1:1,group two whose proportion of rarefaction wave to dense wave is 2:1,group three whose proportion of rarefaction wave to dense wave is 1:2 and blank control group.Each induction group was divided into 2 subgroups:30-minutes induction group and 60-minutes induction group,once a day.After three days,investigate the effect of the rarefaction-dense waves on proliferation of the BMSCs by using methyl thiazolyl tetrazolium(MTT).And the effect of rarefaction-dense waves stimulu on the process of chonseoblast differentiation from bone marrow mesenchymal stem cells(BMSCs) was studied by using reverse transcription polymerase chain reaction(RT-PCR),toluidine blue staining and immunohistochemistry etc,after having been induced for 14 days.⑤The statistics and analysis of datas:The datas were expressed in the form of Mean and Standard Deviation,then statistically analyzed by statistics software named SPSS 13.0. Result:â‘ All of the induction groups could promote the BMSCs to proliferate. Statistical analysis showed that there were very significant differences in each groups(P＜0.01) compared with the blank group,and there was no significant difference within each treated group(P＞0.05).The experimental results showed that the rarefaction-dense waves stimulating the BMSCs to proliferate was concerned with the wave combination.The effect of proliferation got best in the group whose proportion of rarefaction wave to dense wave is 1:2 than any other group.â‘¡After having been induced for 14 days,both Cbfa1 mRNA and Sox9 mRNA were expressed in each rarefaction-dense waves group,but there was no expression in the blank control group.Statistical analysis showed that there were very significant differences in the expression of Cbfa1 and Sox9 mRNA between the group whose proportion of rarefaction wave to dense wave is 1:1 and the blank control group,the group whose proportion of rarefaction wave to dense wave is 2:1 and the blank control group as well as the group whose proportion of rarefaction wave to dense wave is 1:2 and the blank control group (P＜0.01),and the group whose proportion of rarefaction wave to dense wave is 1:2 is better then the group whose proportion of rarefaction wave to dense wave is 2:1,the group whose proportion of rarefaction wave to dense wave is 2:1 is better then the group whose proportion of rarefaction wave to dense wave is 1:1,and there was no significant difference within each treated group(P＞0.05).â‘¢Toluidine blue staining revealed different dyeing of cytoplasm and extracellular matrix,and no significant different dyeing was found in the blank control group.â‘£Immunohistochemistry exhibited that brown positive reaction was clear in cytoplasm in each induction group.Expression of typeâ…¡collagen was better in the rarefaction wave and dense wave 1:2 group than in the rarefaction wave and dense wave 2:1 group,as well as better in the rarefaction wave and dense wave 2:1 group than in the rarefaction wave and dense wave 1:1 group.Cells were negative for typeâ…¡collagen expression in the blank control group.Conclusion:â‘ All of the induction groups can promote the BMSCs to proliferate and group whose proportion of rarefaction wave to dense wave is 2:1 is the best one.â‘¡The rarefaction-dense waves can significantly promote the chondrogenic phenotype differentiation and optimize the seeding cells in cartilage tissue engineering system and improve the quality of cartilaginous tissue and postpone the changes of retrogression in cartilage.