Effects Of Different Serums On Proliferation And Osteogenic Induction Of Human Bone Marrow Mesenchymal Stromal Cells In Vitro

BackgroundThe reconstruction of large bone segments remains a significant clinical problem. Present therapeutic approaches include bone graft transplants(autologous,homologous or heterologous implants of different biomaterials or bone transport methods (Ilizarov technique), but none has proven to be fully satisfactory.Bone tissue engineering is a promising therapy for clinical bone defects and to achieve the transition from basic research to clinical stage of the preliminary application. Safety of the treatment is the first to be considered because of the difference of clinical studies and animal experiments. It should be careful attention that the choice of culture of the appropriate cells, appropriate biomaterial scaffolds and the implantation of the composites in vivo.Bone mesenchymal stem cells (BMSCs), have been demonstrated to be an ideal cell source for bone tissue engineering applications due to their ability to be easily isolated and expanded, low immunogenicity, be easily transducted exogenous gene without effecting proliferation and differentiation of them. Animal and clinical studies have shown that BMSCs are capable of repairing damaged skeletal tissue or large bone segmental defects.At the moment, however, most isolation and expansion protocols for clinical-scale production of MSCs and use culture media supplemented with fetal bovine serum (FBS). Consequently, a host of potential problems such as viral and prion transmission or immunological reactions can arise. Many researchers used serum-free culture methods, but the price is too high, and the culture cycle is longer, about 40d-45d; application of inducing agents and cytokines are also used, but the costs are also considerable.To find a suitable FBS substitute and to established human bone marrow mesenchymal stromal cells (hBMSCs) culture system in vitro which has been standardized, safe and efficient has just started. In this study, the culture media supplemented of FBS, pooled human AB serum (ABS), or autologous serum (AS) was used which to investigate the effects of different serums on proliferation and osteogenic induction of mesenchymal stromal cells from bone marrow in vitro.ObjectivesTo investigate the effects of different serums on proliferation and osteogenic induction of mesenchymal stromal cells from bone marrow in vitro and initially explore the mechanism of osteogenesis.Materials and Methods1. Preparation of pooled human AB serum and autologous serumAbout 50ml of peripheral venous blood was collected without anticoagulants and allowed to clot overnight at 4℃from bone marrow donations of 6 donors and blood donations of 6 AB-blood-group-typed donors under sterile conditions. Subsequently, AB serum was prepared from 6 AB-blood-group-typed which was pooled together. Autologous serum was prepared separately from bone marrow donations of 6 donors which was not pooled together. Serum was separated by twice centrifugation, inactivated and filter sterilization, and add into DMEM basal medium or stored at-80℃until use.2. Isolation and culture hBMSCshBMSCs were obtained from lipoaspirates of 6 donors. Three groups was divided according to different serum. hBMSCs were isolated and cultured using the whole bone marrow culture method is vitro. Cells was subculture at 1:3 ratio when cells achieved 80% confluence. The passage 3 hBMSCs were inoculated into 6-well plates for osteogenic and adipogenic induction.3. Identification of hBMSCs The passage 3 hBMSCs were used for flow cytometric analyses. Surface marker expression contained CD 14、CD29、CD34、CD44、CD45 and CD 105 was detected by flow cytometric analyses. ALP and oil red O staining were used at 10d. ALP positive cells stained blue-violet and oil Red O-positive lipid droplets in cells stained red. Calcium staining were used at 21d, calcium nodules stained reddish brown.4. Proliferation assays of hBMSCsThe passage 3 hBMSCs (1000cells/well) were inoculated into 96-well plates for proliferation assays.100μ1 of complete medium was added into each wells, and 5 wells in each group for time point.10μ1 of AlamarBlue working solution was added into each wells at days 1,3,5,7,9,11. After 4h incubation, fluorescence intensity was determinated on a microplate reader. The cell apoptosis of the passage 3 hBMSCs was determinated by flow cytometry through Annexin V-FITC reagents.5. Osteogenic induction assays of hBMSCsAt 4,7,14 and 21 days after culture, fluorescence analysis of calcein incorporated into mineralized bone matrices and ALP activity were measured among all serum groups. The specific osteogenic genes [ALP, osteopontin (OPN), osteocalcin (OCN)] expression were also measured by real-time quantitative PCR analysis during these time points.6. Statistical analysisRepeated measures was used to compare the overall repeated measures analysis of variance of data.The same group at different time points compared using repeated measures analysis. One-Way ANOVA method was used for statistical analysis and LSD method for multiple comparisons of data in each group; if by the Levene test, the data does not meet the homogeneity of variance, the approximate method of analysis of variance Dunnet’s T3 method for statistical analysis, the significance level a=0.05.Results1. Preparation of pooled human AB serum and autologous serumInactivated by the second centrifugation, autologous serum and AB serum was translucent, light yellow, no floe.2. Isolation and culture hBMSCs There has been a lot of disc-shaped red blood cells suspended in the bottom of the medium delay when three groups of primary cell culture in serum medium was changed after 4d. But the bottom flask can be seen in several clusters of adherent cell clones growing colony, cells become spindle. After 7-9d culture, adherent cells significantly increased the number of cloned colonies; after 12-14d culture, the cells can reach 80 to 90% confluence. After passage, cells were completely adhered, spread, once again become a long spindle cells strong after 24h. After passage 3, hBMSCs were more homogeneous, hybrid cells gradually disappeared. 3. Identification of hBMSCsAfter osteogenic induction, three group of hBMSCs were morphologically similar, the gradual emergence of stacked cells, the growth gathered momentum, the integration of the cell layer and showed "grid" change, cells became larger, polygonal shape, and a large, clear nucleoli, Increase in intracellular granules, extracellular matrix secretion of strong, visible in the culture medium and a large number of brown-black particulate matter was appeared. After adipogenic induction, the cells in serum gradually with small lipid droplets appeared, mainly in the nucleus around with time, the gradual accumulation of small lipid droplets into large fat bubbles, the original spindle-shaped cells into round or angle. Cell surface markers showed that: three groups of hBMSCs:CD29, CD44 and CD 105 were strongly positive expression (above of 98%), the cells were CD 14, CD34 and CD45 was negative (lower than 2%), the group have no Statistically significant (P> 0.05). Staining results showed that: alkaline phosphatase, oil red O, alizarin red staining was positive in three groups of hBMSCs.4. Proliferation assays of hBMSCsWith the culture time, the fluorescence intensity of each serum group increased gradually, reached the highest at day 7. At days of 3,5,7,9, and 11, AB serum group were higher than the fluorescence intensity of fetal bovine serum group and the autologous serum group (P<0.05). AB serum promote the most efficient for hBMSCs proliferation. Apoptosis rate by flow cytometry showed that apoptosis of fetal calf serum group was (4.34±0.59)%, autologous serum group was (4.31±0.70)%, AB serum was (4.33±0.76)%. No statistical significance between the groups (P> 0.05). The cells grew well in these serums.5. Osteogenic induction assays of hBMSCsAfter days 4,7,14 and 21 of osteogenic induction, the DNA content of each well was measured in each group. The same time, each group showed no significant difference (P> 0.05). The measured DNA content and alkaline phosphatase activity normalized to alkaline phosphatase activity in each 100μg DNA. The same time, alkaline phosphatase activity in AB serum group were higher than fetal bovine serum and autologous serum, and reached the peak at 7d, alkaline phosphatase activity in autologous serum group was the lowest, the group differences were statistically significance (P<0.05). The cells were outside the mine when the mineral deposit has not yet formed at 4d, mineral deposits were scattered when the point distribution after 7d, and significantly increased when mineral deposition, were little clusters like distribution 14d through fluorescence microscope. A great quantity of mineralized bone matrices were produced in AB serum which was higher than that in fetal bovine serum or autologous serum through fluorescence microscope at 21 days after culture. ALP activity was higher in AB serum than that in fetal bovine serum or autologous serum at each time point (P<0.05). The gene expression of ALP, OPN and OCN were higher in AB serum than that in fetal bovine serum or autologous serum at 7,14 and 21 days after culture (P<0.05). ALP gene expression reached a peak at 7d, OPN gene expression reached a peak at 14d, OCN gene expression reached a peak at 21d.Conclusion1. We isolated the BMSCs from iliac of human. This experiment confirmed that with AB serum and autologous serum, after cultured in growth to 3 generations, hBMSCs was of higher purity and thriving. the hBMSCs had a great potentiality of proliferation and osteogenic and chondrogenic differentiation in vitro. hBMSCs are an ideal seed cell for tissue engineering and it has broad application prospects, which provide an experimental foundation for hBMSCs being used in bone and cartilage tissue engineering in vivo.2. AB serum was found to be had a higher osteogenic capability than fetal bovine serum and autologous serum so as to establish a new and safer culture model in clinical cytotherapy of bone repair.