The Experimental Research Of The Induction Of The Granula Of Penetrating Bone And Removing Pain On Chondrogenic Phenotype Differentiation Of BMSCs In Vitro

Objective:To observe the foundational experiments of the Granula of Penetrating Bone and Removing Pain on extraction,separation,purification,cultivation,proliferation and chondrogenic phenotype differentiation of bone marrow-derived mesenchymal stem cells (BMSCs)in vitro.We optimized the seeding cells in cartilage tissue engineering,improved the function of self-recovery and postponed the changes of retrogression in cartilage,to achieve the objective of preventing and treating osteoarthritis,and then to provide the substantial clinical foundations for treating osteoarthritis in TCM.Method:(1) The BMSCs were obtained from SD rats' bone marrow by the bone marrow adherence cultivation through separation,purification,cultivation in vitro.The growth conditions of the BMSCs were observed by inverted phase contrast microscope for us to investigate them in vitro.(2) The 72 SD rats aged 4 weeks were randomly divided into 3 groups, i.e.the aqueous extract group(48 rats),the alcoh extract group(16 rats) and the blank group(8 rats),and then the aqueous extract group(48 rats) were randomly divided into 3 small groups, i.e.high-dose group,midst-dose group(isoeffect-dose group) and low-dose group,16 rats for every small group.All rats were enforced intragastric administration of equivalent dose for 3 days.1 hour and 2 hours later after the last intragastric administration,the serums of the Granula of Penetrating Bone and Removing Pain were separated.Then we interfered in the obtained BMSCs for 72 hours with the serums from different density,and investigated the effect of the Granula on proliferation of the BMSCs.(3) The second generation BMSCs were randomly divided into 3 groups,i.e.the blank group,the pure chondrogenic inductor group and the group of the Granula of Penetrating Bone and Removing Pain mixed with chondrogenic inductor.We adopted pro-culture solution,pure chondrogenic induced culture solution(TGF-β₃ 10μg/L, Dex10^-7mol/L,VitC50mg/L) and the chondrogenic induced culture solution which included the serum of the Granula.All groups were cultivated in 50ml cell culture bottles.The effects of the Granula of Penetrating Bone and Removing Pain on chondrogenic phenotype differentiation of BMSCs were investigated after being cultivated for 1,2,3 weeks, then cells observed by inverted phase contrast microscope and immunocytochemical stain.(4) The statistics and analysis of datas:The datas were expressed in the form of Mean and Standard Deviation,then statistically analyzed by statistics software named SPSS14.0.Result:(1) The cultivated BMSCs grow well in vitro.We can obtain better homogeneous BMSCs.The experiment proves that the bone marrow adherence cultivation is a more successful method of separating BMSCs.It proves the success of cultivating BMSCs in vitro.(2) All of the experimental groups which are highly significant difference(P＜0.01)compared with the blank group can promote the BMSCs to proliferate.The effect of proliferation in the midst-dose group can get to the maximal(P＜0.01),but it will tend to descend if increasing the concentration.There is no significant difference between 1 hour group and 2 hours group from the midst-dose group(P＞0.05).The experiment explains that the Granula of Penetrating Bone and Removing Pain stimulating the BMSCs to proliferate is concerned with the concentration. The effect of proliferation can get to the maximal in the midst-dose group from the aqueous extract group.(3) By the induction of TGF-β₃,the BMSCs of chondrogenic phenotype differentiation mix together,grow intensively,change to the form of triangle and polygon.Both of the Granula group and the chondrogenic inductor group can promote the expression of collagenâ…¡,which is the highest in the Granula group.The is no expression of collagenâ…¡in the blank group.The experimental result proves that the Granula of Penetrating Bone and Removing Pain can effectively promote the expression of collagenâ…¡.Conclusion:(1) A perfect culture system of BMSCs is established when the BMSCs of SD rats were extracted,separated,purified,cultivated,proliferated in vitro.(2) The Granula of Penetrating Bone and Removing Pain can promote the BMSCs to proliferate.At the same time, it can significantly promote the chondrogenic phenotype differentiation,thus increasing the quantity of chondrocyte.(3) The Granula of Penetrating Bone and Removing Pain 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.MeSH Terms:Osteoarthritis,Knee/TCD therapyMesenchymal Stem Cells/drug effectsChondrocytes/drug effectsCell Culture Techniques/methodsCell Differentiation/drug effectsColorimetryTransforming Growth Factor betaCollagen Typeâ…¡@The granula of penetrating bone and removing pain...