Effects Of Concentrated Growth Factors On Proliferation And Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells In Vitro

Aims:Concentrated Growth Factors (CGF) fibrin which is obtained by differental centrifugation of autologous, as a new generation of palatelet concentrate. It can promote the proliferation, differentiation, and chemotaxis of cell and stimulate vascularization to accelerate the repair of hard and soft tissue. It is a new technology in the regenerative medicine. The bone mesenchymal stem cells in vitro rats as the research object, observe the effects of CGF on proliferation and differentiation of bone mesenchymal stem cells in vitro. For bone tissue engineering this provides the experimental basis as growth factors source.Methods:1、The isolation and culture of bone marrow mesenchymal stem cellsWistar rats which were healthy and 4-week old were choosed for experiments.The tibia and femurs were removed from them after operation. The mesenchymal stem cells were separated and purified by the method of whole bone marrow adherent culture. BMSCs were used at passages 2 to 4 for subsequent experiments.2、The identification of BMSCs(1)The observation of cells morphology by a phase-contrast microscope.(2)The cells surface antigen (CD34、CD44) were detected by Immunocy to chemical staining.3、The preparation of CGFWistar rats which were healthy and 4-week old were selected in the study.5ml venous blood from each rats were placed into the drum of the Medifuge centrifugal acceleration machine, to separate CGF fibrin gel, according to the preparation process.4、Groups of experimentThis cell proliferation experiment was divided into two groups (standard medium and CGF group,control group),according to the standard medium was join the CGF or not, and selected the four time points (1th,3rd,5th and7thday), respectively observed cell proliferation of two groups. This cell differentiation experiment was divided into four groups(standard medium and CGF group, osteogenic induction medium group, osteogenic induction medium and CGF group,control group),and selected the two time points (7thand 14th day), respectively observed osteogenesis induced of four groups in every time.5、MTT method was used to observe concentrated growth factors to act on bone mesenchymal stem cells proliferation.6、Alkaline phosphatase kits were adopted to detect CGF on the effects of BMSCs of alkaline phosphatase activity.7、At 14 days of culture, Alizarin red S staining and semi-quantification of mineralized matrix nodules ware performed in osteogenic induction condition.8、Real-time quantitative PCR method was adopted to assay mRNA expression of alkaline phosphatase (ALP), I type collagen (COL-1), Osteopontin (OPN) and osteocalcin (OCN) of rat bone marrow mesenchymal stem cells.9、Statistical analysisAll all used SPSS 17.0 statistical software analysis on the experimental data, the data results were expressed as a mean±tandard error(mean±E). P<0.05 was considered as the level of statistical significance.Results:1、BMSCs grew well in vitro and most of them were generally elongated and spindle shaped. Cells immunohistoehemistry showed that BMSCs were CD44 positive and CD34 negative.2、After centrifuging,the venous blood was divided into three layers. The upper layer was platelet-poor plasma.The bottom layer was red blood cells.The middle layer was CGF.3、 MTT results showed that:on the 1 st day and 3rd day,there was no significant difference between the absorbanee of the standard medium and CGF group and that of control group (P>0.05); in the 5th day and 7th day,there was significant difference between the absorbance of the standard medium and CGF group and that of control group (P<0.05).4、After 7d and 14d, ALP activity in osteogenic induction medium and CGF group was much higher than that other groups, followed by ostoegenic induction medium group. ALP activity of the control group is the lowest in the all groups. There were significant difference among four groups (P<0.05).5、Alizarin red staining and semi-quantification of mineralized matrix nodules:The mineralized nodules were observed by an inverted phase contrast microscope, and the amount of nodules was much more in osteogenic induction medium and CGF group than that of other groups. However, mineralized nodules in the control group was the lowest in the all groups. After cetylpyridiniumchloride dissolved mineralized matrix nodules, the absorption values of four groups were 0.6914±0.0136 (standard medium and CGF group),1.1607±0.0651 (ostoegenic induction medium group),1.6685±0.0214(ostoegenic induction medium and CGF group),0.2102±0.0231 (control group), respectively. There were significant difference among four groups (P<0.05).6、Gene expression of ALP, Col-I, OPN and OCN in the cells stimulated by standard medium and CGF group showed lower than those of cells stimulated by ostoegenic induction medium group,but higher than those of cells stimulated by standard medium group only. Gene expression of ALP, Col-I, OPN and OCN in the cells stimulated by ostoegenic induction medium and CGF group were much higher than other groups (P<0.05)Conclusions:1、In this study, we isolated BMSCs successfully. Surface markers of the cultured cells were detected by immunocytochemistry staining.The results indicated that the cultured cells were indeed mesenchymal stem cells.2、CGF can stimulate the proliferation of bone mesenchymal stem cells in vitro.3、CGF can prompt the differentiation of bone mesenchymal stem cells in vitro. It can also enhance osteogenic capability of osteogenic induction medium.4、CGF is expected to become a source of growth factors in reconstruction of bone tissue engineering,which can stimulate the proliferation and differentiation of seed cells.Because it contains a large number of cytokines.