Reasearch On Differentiation Of Human Umbilical Cord Blood Mesenchymal Stem Cells Into Osteoblasts Induced By Chinese Kidney-tonifying Herbs Based On "Kidney Controlling Bones And Producing Marrow" Theory Combined With BMP-2in Vitro

Objective:To establish culture system for the human umbilical cord blood mesenchymal stem cells (UCB-MSCs), observe the biological characteristics, proliferation, osteogenic activity of UCB-MSCs induced by Chinese kidney-tonifying herbs serum combined with BMP-2in vitro, and explore its effect on transformation of UCB-MSCs into osteoblasts.Methods:30SD rats were given with Chinese kidney-tonifying herbs by gavage for7days, taking carotid arterial blood to make Chinese kidney-tonifying herbs serum, collecting human umbilical cord blood samples under sterile conditions and separating UCB-MSCs by density gradient centrifugation. The biological characteristics of cultured UCB-MSCs in vitro were observed under inverted phase contrast microscope and identified by flow cytometry.The3rd generation of UCB-MSCs were divided into eight groups:control group,5%,10%,15%Chinese kidney-tonifying herbs serum group;5%,10%,15%Chinese kidney-tonifying herbs serum combined with BMP-2group and BMP-2group. To observe the influence of each group on proliferation of UCB-MSCs on the induction of1st,3rd and5th day by MTT assay, osteoblast activity of UCB-MSCs by alkaline phosphatase and mineralized nodulesVonKossa staining method,osteocalcin (BGP) content in the culture medium measured via radioimmuno assay, the secretion of calcium in osteoblasts detected by calcium fluorescence probe,the expression of type I collagen detected by immune histochemical staining method on3,6,9,12days after induction.Results:①he primary culture cells of UCB-MSCs were round.72hours after primary culture,only a small amount of adherent cells were seen round or oval. The medium was changed for the first time on the7th, in which can be seen scattered MSCs radial growth. After two weeks the cell morphology were fusiform, colony fusion with each other after three weeks, Most of Passage cells were adherented in24hours.Generally, they can reach80%to90%confluence in3days, packed tightly, and the form is relatively uniform,mostly spindle.②Compared with the control group,5%Chinese kidney-tonifying herbs combined with BMP-2group,10%Chinese kidney-tonifying herbs combined with BMP-2group,BMP-2group was able to promote proliferation of the UCB-MSCs on the induction of3rd,5th(P＜0.05), and especially the5%Chinese kidney-tonifying herbs combined with BMP-2group was best (P＜0.01).There was significant difference between5%Chinese kidney-tonifying herbs serum combined with BMP-2group and BMP-2group (P=0.041<0.05).However, the difference was not statistically significant between Chinese kidney-tonifying herbs serum group and the control group (P>0.05).③Both BMP-2group and5%Chinese kidney-tonifying herbs serum combined with BMP-2group on the6th,9th and12th after induction can improve the content of the osteocalcin(BGP).Compared with control group,there was significant difference (P=0.049,0.012,0.021＜O.05).9days after induction,5%Chinese kidney-tonifying herbs combined with BMP-2group had statistically significant difference (P=0.004<0.01) compared with control group.④The ALP activity of each group gradually strengthened after induction. Compared with control group, BMP-2group and Chinese kidney-tonifying herbs combined with BMP-2group both could improve ALP activity on the3rd,6th,9th, and12th day after induction (P＜0.05). There was significant difference between5%Chinese kidney-tonifying herbs combined with BMP-2group and BMP-2group6days after induction (P=0.011＜0.05).The positive expression of ALP staining was weaker in7days after induction,but relatively obvious on day14. Diffuse reddish brown or coffee colored particles were expressed in the cytoplasm,but the strength showed difference among these groups. The effect of5%Chinese kidney-tonifying herbs combined with BMP-2group was superior to that of other groups.On day21after induction,the expression of ALP staining slightly decreased.⑤Black particles were found in the positive cells of Von Kossa staining of mineralized nodules14days after induction. The positive expression of Von Kossa staining was significantly obvious in BMP-2and Chinese kidney-tonifying herbs combined with BMP-2groups. The particle size was inequable. Black mineralizer deposit weren't expressed in the Von Kossa staining cells in control group.⑥luorescent probe technology showed that the content of calciumion of all the BMP-2group and Chinese kidney-tonifying herbs combined with BMP-2group were superior to that of control group and simple Chinese kidney-tonifying herbs group. The fluorescence intensity of5%Chinese kidney-tonifying herbs combined with BMP-2group was most obvious⑦Immunohistochemistry showed that the positive expression of type Ⅰ collagen was manifested as brownish yellow granules,which was mainly localized in extracellular matrix. BMP-2and5%Chinese Kidney-tonifying Herbs combined with BMP-2group all showed positive expression,but simple Chinese kidney-tonifying herbs group and control group were not obvious.Conclusion:BMP-2and Chinese kidney-tonifying herbs combined with BMP-2both are able to promote the proliferation of human umbilical cord blood mesenchymal stem cells and can induce human umbilical cord blood mesenchymal stem cell into osteoblasts.The effect of Chinese kidney-tonifying herbs combined with BMP-2is superior to that of BMP-2,but that of Simple Chinese kidney-tonifying herbs is not obvious. The results suggest that the Chinese kidney-tonifying herbs combined with BMP-2can effectively induce human umbilical cord blood mesenchymal stem cells into osteoblast, which verify the scientific nature of the theory of "kidney controlling bones and producing bone marrow" and provide a certain guiding and reference for the combination of traditional chinese medicine and tissue engineering technology,and then enrich the treatment means of the repair and reconstruction of the bone defect.