Culture of mesenchymal stem cells After primary cultured for 12 hours, most of the cells were adherent; after 2-3 days, the morphology of the cells was in polygons and fusiform; after 3-5 days, distinct colony formation was observed with rapid proliferation, and the clonies were close to each other; after 10-12 days, cell integration for about 90%, at this time, the cells were arranged in relatively uniform helical or spiral shape. The passaged mesenchymal stem cells adhered rapidly, and after 3 hours, most of the cells were adherent and evenly distributed in fusiform; that of the morphology of the passaged cells was more even than that of the primary cultured cells, the passaged cell growth well with rapid proliferation, obvious nucleus, clear nucleolus and a large proportion of nucleoplasm. Generally, after cultured for 7 days, the cells could reach 90% integration with even morphology and arranged in parallel or swirling spiral. There was no significant difference after continuously passaged to the 5th generation. However, with the increased frequency of passage, the cells appeared diversity; after passaged to the 10th generation, the cells were gradually aging, presented as increased cell debris, flattened cell morphology and decreased proliferation rate till stop.       Expressions of surface antigens of mesenchymal stem cells Positive immunocytochemical staining of CD44 and CD90 of passage 3 cells was observed, the cytoplasm was brown or chocolate brown, and the CD34 staining was negative.   Growth curve of the cells in each group The number of the incubated cells in each group was counted (mean n=6), then the data were recorded in the EXCEL table to generate the curve (growth curve) automatically. The growth curves of the cells in each group were in “S” shape (Figure 1). At day 1 after incubation, the cells growth in each group was relatively flat, and presented as the latent growth curve. At day 2 after incubation, the cells in the platelet-rich plasma groups had high proliferative activity and grew rapidly, and the growth curve was significantly increased, especially in the high-dose platelet-rich plasma group; then the rapid growth period of the growth curve was constructed. The proliferation of the cells was most significant in the high-dose platelet-rich plasma group with the shortest doubling time about 30 hours, and then reached to peak at 6 days, and formed plateau phase. The cells in the control group were grew rapidly at day 3, and showed the longest doubling time about 46 hours, and after 3 days, the rapid growth period of growth curve was constructed. The cell level in the platelet-rich plasma groups was maintained in a high level after reach to plateau phase, and the number of the cells in the platelet-rich plasma groups was larger than that in the control group."

Survival and proliferation ability of mesenchymal stem cells in each group The survival and proliferation ability of mesenchymal stem cells in each group were showed in Tables 1 and 2. Methylthiazolyldiphenyl-tetrazolium bromide assay showed the absorbance values in the high-dose, middle-dose and low-dose platelet-rich plasma groups and the control group at 2 days after intervention were (0.171±0.026), (0.169±0.030), (0.165±0.034) and (0.161±0.029) respectively, and there was no significant difference (P > 0.05), that may be because of the cells growth in the stationary state, there was no significant difference in proliferation of cells. At day 3, there was significant difference in the absorbance value between platelet-rich plasma groups and the control group (P < 0.01), while there was no significant difference between platelet-rich plasma groups (P > 0.05), indicating that the platelet-rich plasma has certain effect in the proliferation of cells at 3 days after intervention without dose-dependent manner. There was significant difference in the absorbance value between high-dose platelet-rich plasma group and middle- and low-dose platelet-rich plasma groups at day 4 (P < 0.05 or P < 0.01), but there was no significant difference between middle-dose platelet-rich plasma group and the low-dose platelet-rich plasma group (P > 0.05), the results indicated that high-dose platelet-rich plasma had significant effect on cell proliferation, the low-dose platelet-rich plasma could also promote cell proliferation but not significant. At day 6, the platelet-rich plasma had significant promotion effect on cell proliferation and showed dose-dependent manner. At day 8, the platelet-rich plasma had significant promotion effect on cell proliferation and showed dose-dependent manner, but also found that the promotion effect was decreased when compared with that in the previous few day, and that may be saturated cell density. Statistical results showed that platelet-rich plasma had significant promotion effect on the proliferation of mesenchymal stem cells (since day 3), and the effect was in time-dependent manner. After cultured for several days, the effect had dose-dependent manner (since day 6), and the high-dose platelet-rich plasma had the most significant effect on cell proliferation."

Effect of platelet-rich plasma on collagen production of mesenchymal stem cells Immunochemical staining results and enzyme-linked immunosorbent assay for quantitative determination of collagen showed that mesenchymal stem cells could produce the collagen type Ⅰ and Ⅲ platelet-rich plasma which could significantly promote the collagen production of mesenchymal stem cells, and there was significant difference when compared with the control group (P < 0.05). The effect of platelet-rich plasma on the collagen production showed a dose-dependent manner (Table 2)."

Gene expression of collagen typeⅠ The relative ratio of collagen type Ⅰ density is showed in Table 3. The Tband location of collagen type Ⅰ on the electropherogram was corresponded to the designed amplification length (Figure 2). The expression of collagen type Ⅰ of mesenchymal stem cells in each platelet-rich plasma group was increased when compared with that in the control group, and there was no significant difference between groups (P < 0.01)."