In Vitro Study Of The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Chondrocytes

Objective:To observe the effect of yam on the differentiation of rabbit bone marrow mesenchymal stem cells into chondrocytes in vitro and to verify the role of yam in the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)for tissue engineering in the treatment of intervertebral disc degeneration in rabbits.The body experiment provides the experimental basis to explore the potential and practical value of traditional Chinese medicine in tissue engineering repair technology;at the same time,it provides the clinical efficacy of yam,"Yijing fills the marrow,tonic,kidney qi",and the traditional Chinese medicine theory of kidney and bone marrow.Methods: Experiment I: Rabbit bone marrow mesenchymal stem cells were isolated and cultured by using whole bone marrow adherent culture method and density gradient centrifugation.The differences between the two culture methods were compared and immunohistochemistry and three-way differentiation cells were identified.Twelve New Zealand rabbits were divided into four groups with 3 rats in each group.After intragastric administration with different concentrations of traditional Chinese medicines,concentration-gradient yam drug-containing serum was collected.MTT assay was used to examine the effects of different concentrations of yam serum on cell growth.Different concentrations of yam medicine-containing serum were added to the culture medium to culture bone marrow mesenchymal stem cells,and the effects of different concentrations of yam medicine-containing serum on cell growth and proliferation were observed.Experiment 3: Differentiation of bone marrow mesenchymal stem cells into chondrocytes was induced under monolayer culture conditions.21 days,the induction results were detected by toluidine blue staining;bone marrow mesenchymal stem cells were induced to differentiate into chondrocytes with complete culture medium under micromass culture conditions,and cell growth status wasobserved and stained with toluidine blue and safranin O,respectively.The results of differentiation were detected.The chondrocytes cultured in micelles of different concentrations were induced to differentiate into chondrocytes for 5 days.The results of differentiation were detected by toluidine blue and safranin O staining methods,respectively.The chondroblastic microspheres stained by toluidine blue were obtained.Scoring criteria were used to score the induction;DMMB was used to detect the GAG content in different concentrations of yam serum after differentiation.Results: Experiment 1: Rabbit bone marrow mesenchymal stem cells with good growth state can be obtained by whole bone marrow adherence method and density gradient centrifugation.The whole bone marrow culture method has the advantages of strong cell proliferation and regeneration,rapid growth,and high success rate of culture.The obtained cells contained more impurity cells;the target cells obtained by density gradient centrifugation had high purity,uniform morphology,and orderliness,but cell growth was slow.Experiment 2: Different concentrations of yam medicine serum can significantly promote the growth and proliferation of bone marrow mesenchymal stem cells,especially with the concentration of yam contained drug serum is the most significant.Experiment 3: The results of toluidine blue staining under single-layer culture conditions showed that the yam medicine-containing serum induced differentiation of bone marrow mesenchymal stem cells into chondrocytes was not significant;in the case of micromass conditioning culture,yam medicine-containing serum could significantly promote bone marrow The mesenchymal stem cells differentiated into chondrocytes and formed cartilage microspheres.Scores of chondroblastic microspheres after toluidine blue staining showed that the yam group score was significantly higher than that of the control group(P<0.05).The difference was statistically significant;DMMB assay The results of intracellular GAG after induction showed that GAG content in the drug-induced group was significantly higher than that in the control group(P<0.05).Conclusion:Experiment one: The cells cultured with whole bone marrowadherence method and density gradient separation method are all rabbit bone marrow mesenchymal stem cells with good growth and proliferation state;experiment two: Different concentration of yam medicine serum can promote bone marrow mesenchymal stem cells Growth value,especially the concentration of yam drug-containing serum has the best effect.Experiment 3: The yam medicine serum alone promotes the transformation of bone marrow mesenchymal stem cells into chondrocytes under monolayer culture conditions;in micelle culture conditions,the yam medicine serum can significantly promote and accelerate the progression of bone marrow mesenchymal stem cells.Chondrocyte differentiation.In order to further use the traditional Chinese medicine in tissue engineering technology to treat rabbit intervertebral disc degeneration in vivo experiment provides the experimental basis.