Culture And Chondrogenesis Of Human Bone Marrow Derived Mesenchymal Stem Cells In Vitro

Objective To research the phenomenon and rule of multiplication and chondrogenesis of mesenchymal stem cells(MSCs)derived from Human bone marrow in vitro.Investigate the influence of TGF-β₁ (transforming growth factorβ₁)on chondrogenesis of human MSCs. Compare the influencing differences of various concentration TGF-β₁ in chondrogenesis of human MSCs by monolayer culture in vitro.Find out the best concentration of TGF-β₁ for the chondrogenesis of Human MSCs. Provide the technical bases and reference to solve the clinical problem of repairing the cartilage defect by MSCs autograft.Methods We obtained 5 ml bone marrow from 3 healthy male adult volunteers from posterior superior iliac spine by bone marrow aspiration in bacteria-free environment and then put the marrow in anticoagulation environment.Then we used the 1.077g/ml Ficoll to get the nucleated cell layer by density gradient centrifugation.The cells were Inoculated in culture bottle with 10%FBS L-DMEM culture medium at the seeding density of 5×10⁶/cm² and the bottles were placed in the 37℃incubator with 5%CO₂.After 48 hours' culture,we changed the half mount medium and abandoned the inadherent cells.The medium was changed every 2 or 3 days and the cells would be subcultured at 1:3 rate when they reached 90%fusion growth.MSCs would be basically purified after 3 passages. We inoculated the passages 3 MSCs to 96-orifice plate and selected 10 orifices to do the MTT test in 8 days just after passage.The growth curve was described according to the test result.The passage 3 MSCs were induced to chondrocyte respectively with inducing medium containing TGF-β₁ at different concentrations.We observed the cells' shape by inverted phase contrast microscope everyday.After 3 weeks' induction, every group was tested by toluidine staining,alcian blue staining, collegenâ…¡measurement with immunofluorescence method and GAG (glycosaminoglycan)measurement with alcian blue colorimetry.At last, we analyzed the differences among the groups statistically.Results(1)The primary passage was spindle shaped,small,thin and short,arranged loosely.Along with the quantity increased,there were small cell communities.The arrangement was much closer gradually and the typical long-spindle shaped cells would appear.The cells would reach 90%fusion growth after cultured 10 days.The growth speed increased after passage and MSCs would overgrow the bottom of the bottle in about 7 days.The cells were eddy-like and closely arranged.The cell shape would tend to uniform,just spindle shaped and there were long filamentary pseudopods at cells' two ends.(2)The passage 3 MSCs had about 48 hours' delitescence.MSCs started to regenerate slowly after 3 days,faster after 5days.The quantity increased significantly in 5～7 days and the arrangement tend to be regular gradually.Cells' regeneration turned to plateau phase after 7 days.(3)After induction,the cells' shape did not change significantly in the first 5 days.In the second week,MSCs turned to broad and flat gradually,polygon and multi-branch shape.We found some cells of the blank control group changed to vacuole,just like the lipocyte.The growth speed was much slower than before after induction.The number of MSCs doubled about 9 days.(4)After 3 weeks' induction,we did the toluidine staining.There were significant differences among every group.The blank control group was negative, 10μg/L and 20μg/L group were positive,5μg/L group was weak positive. (5)The alcian blue staining results were the same with toluidine staining results.(6)Collegenâ…¡immunofluorescence results:there were significant differences among every group.The blank control group had no fluorescence,5μg/L group had weak fluorescence,and 10μg/L and 20μg/L group had red fluorescence in cytoplasm and fine grained shape fluorescence in extracellular area.(7)GAG(glycosaminoglycan)contents measurement:significant differences existed among every group.The GAG contents of 5μg/L,10μg/L and 20μg/L group were different respectively and were all higher than blank control group,the differences had statistical significance.Conclusion(1)Human MSCs derived with Ficoll density gradient centrifugation could be cultured and amplified in vitro.The cells regenerated actively and could be subcultured for many passages.(2) Human MSCs regenerated slower in chondrogenesis,and the shape changed gradually,turned to be broad,flat,polygon and multi-branch.(3) Human MSCs could be induced to chondrocytes by plate culture.TGF-β₁ was the necessary condition for retaining chondrogenesis.(4)Our research showed:different concentration of TGF-β₁ had different effects in chondrogenesis,10μg/L was the best concentration for chondrogenesis.