Objective Metal wear products cause the aseptic loosening of joint prosthesis. To investigate the effect of metal ions Co2+ and Cr3+ on the osteoblast apoptosis, cell cycle distribution, and secretion of alkal ine phosphatase (ALP), and to search a method to prevent and treat aseptic loosening. Methods The mouse calvarial osteoblasts (MC3T3-E1) were cultured in vitro to 3-5 generations (5 × 105 cells/ mL) and divided into 2 groups: the experimental group and the controlgroup. The osteoblasts were cultured in α-MEM medium containing 10%FBS (the control group), and the mixed solution ofCoCl2 and CrCl3 was added after the osteoblasts cultured in α-MEM medium containing 10%FBS attached completely (the experimental group). At 12, 24, and 48 hours after culture, the osteoblast apoptosis and the cell cycle distribution were assessed by flow cytometry; and ELISA method was appl ied to detect ALP content in serum supernatant. Results At 12, 24, and 48 hours after culture, the apoptosis rates in the experimental group (13.90% ± 0.52%, 14.80% ± 0.41%, and 13.40% ± 0.26%) were significantly higher than those in the control group (8.56% ± 0.31%, 8.19% ± 0.24%, and 2.15% ± 0.11%), (P lt; 0.05); G2M (dividing phase) distribution ratio significantly decreased and G0G1 (dormancy stage) distribution ratio significantly increased when compared with those in the control group (P lt; 0.05); and the absorbency (A) values of ALP were 0.955 ± 0.052, 0.624 ± 0.041, and 0.498 ± 0.026 in the exprimental group, and were 1.664 ± 0.041, 1.986 ± 0.024, and 2.192 ± 0.041 in the control group, showing significant differences between 2 groups (P lt; 0.05). Conclusion Metal ions Co2+ and Cr3+ have a marked effect on osteoblasts cell cycle distribution, which can make most of the cells to be in dormancy stage (G0G1), up-regulate the apoptosis rate and inhibit the releasion of ALP from osteoblasts.
Objective To further study the influence of the co-cultivation of vascular endothel ial cells (VECs) and adi pose-derived stromal cells (ADSCs) on cell osteogenic differentiation in vitro and provide experimental evidences of the probabil ity of the co-cultivation of VECs and ADSCs as the seed cells of tissue engineering. Methods The VECs derived fromcord blood and ADSCs were prepared by full-term pregnancy SD rats and 18-week-old SD rats, to carry on the morphological observation and immunohistochemical staining identification. The third generation of ADSCs and the VECs induced by conditioned medium for 6 weeks were cultured and were divided into groups A, B, and C as the experimental group according to cell ratios of 3 ∶ 1, 1 ∶ 1, and 1 ∶ 3, respectively. ADSCs or VECs was cultured alone in groups D and E as control groups. ALP and al izarin red staining were done respectively on the 7th day and 14th day; ALP and osteocalcin (OC) were detected respectively on the 4th day, 7th day, and 14th day. Results The VECs derived from cord blood showed mixed growth of short spindle and polygonal cells after 6 weeks of induction, the immunofluorescent staining result of von Willebrand factor was positive. ADSCs showed adherent mononuclear cells and spindle-shaped growth without dupl ication; the immunofluorescent staining result of CD90 was positive and no positive cells were seen in the control group. On the 7th day of cell culture, ALP staining showed that the results were negative in groups A, D, and E, and some positive cells were seen in groups B and C; on the 14th day, the results were still negative in groups D and E, and positive cells fused to sheet form in groups A, B, and C. von Kossa staining showed that the results were negative in all groups on the 7th day; few positve cells were seen in groups A, B, and C, and no positive cells were seen in groups D and E on the 14th day. The ALP contents increased gradually in all groups,which was highest in group B at every time point, showing significant difference (P lt; 0.01) between group B and other groups, between groups A, C and groups D, E. The OC value increased gradually in every group, which was highest in group B on the 7th and 14th days, showing significant difference between group B and other groups (P lt; 0.01), between group C and group D (P lt; 0.01) on the 4th and the 14th days, between groups A, C and group E (P lt; 0.05) on the 14th day. Conclusion ADSCs have potential of osteogenic differentiation by VECs in the system of co-culturing VECs and ADSCs in vitro, the influence on osteogenic differentiation is the best in a ratio of 1 ∶ 1.