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.
ObjectiveTo investigate the feasibil ity of the domestic porous tantalum as scaffold material of bone tissue engineering by observing the expressions of osteogenesis related factors of MG63 cells co-cultured with domestic porous tantalum. MethodsMG63 cells were cultured with porous tantalum scaffolds (group A), with porous tantalum leaching solution (group B), and with MEM as control group (group C). The cell adhesion of group A was observed on the scaffolds at 3, 5, and 7 days after culture by scanning electron microscopy (SEM); immunohistochemistry and Western blot methods were used to detect the expressions of Runt-related transcri ption factor 2 (Runx-2), osteocalcin (OC), and fibronectin (FN). ResultsAt 3 days after culture, the cells of group A adhered the surface and pore of the porous tantalum scaffolds, with sparse cell arrangement and less protuberances; at 5 days after culture, adjacent cells connected to be a flat each other, which covered the surface and pore of the scaffold; at 7 days after culture, cells secreted plenty of extracellular matrix, covering most of the material surface. The expressions of Runx-2, OC, and FN were positive in 3 groups; darker staining of the cytoplasm was observed in group A, the expressions were significantly higher in group A than in other 2 groups. The results of immunohistochemistry and Western blot showed that the expressions of Runx-2 and OC were significantly increased in group A when compared with those in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The expression of FN had no significant difference among 3 groups (P > 0.05). ConclusionDomestic porous tantalum could promote MG63 cells adhesion and growth, and may promote the expressions of Runx-2 and OC, so it can be used as a scaffold material of bone tissue engineering.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.
Objective To explore the vitamin K level in Chinese maintenance hemodialysis (MHD) patients. Methods MHD patients and healthy subjects from our outpatient clinic were enrolled from 1 to 30 in March 2016. Demographic data was collected. Fasting serum samples from all subjects were collected for biochemistry tests and the measurement of known vitamin K-dependent proteins, i.e. matrix Gla protein (MGP), osteocalcin (OC) and uncarboxylated osteocalcin (ucOC). We also quantified the fraction of ucOC of total OC (%ucOC). Differences of these parameters between the two groups were analyzed. Results We enrolled 70 MHD patients as a test group and 70 healthy subjects as a control group. There was no significant difference in MGP between MHD group and the control group [(4.1±2.2) vs. (4.4±1.0) pg/mL, P=0.441]. The value of %ucOC was significantly higher in the MHD group than that in the control group [(79.3±19.3)% vs. (51.9±13.0)%, P<0.001]. Conclusions Deficiency of vitamin K appears common in Chinese MHD patients. Besides pathological reasons, dietary habit may also contribute to this phenomenon.
ObjectiveTo study the changes of body weight, body length, tail length, femur length, bone mineral density, serum osteocalcin content and apoptosis of bone cells in rats under intermittent hypoxia condition, so as to explore the effects of intermittent hypoxia on bone growth.MethodsForty healthy male SD rats aged 3 to 4 weeks were selected and divided into 2 groups, 20 rats in each group. Group A: normoxic control group (normal diet and normoxic environment); group B: intermittent hypoxia group (normal feeding and was put into the hypoxic chamber to establish intermittent hypoxia environment), 8 hours a day (09:00 to 17:00), 4 weeks of modeling. The body weight, body length and tail length of the two groups were measured in every morning. At the end of 4 weeks after anesthesia, the body weight, body length, tail length and right femur length were measured. The body weight growth rate, body length growth rate and tail length growth rate were calculated. Blood samples were collected from the abdominal aortic, and the content of serum osteocalcin was measured by enzyme linked immunosorbent assay; the right femur bone mineral density was measured by automatic dual-energy X-ray bone densitometer; the apoptosis of bone cells was detected by immunofluorescence staining+TUNEL.ResultsThe body weight growth rate, body length growth rate, tail length growth rate and right femur length in group A were all higher than those in group B (P<0.05); serum osteocalcin content in group A was higher than that in group B (P<0.05); bone mineral density in group A was higher than that in group B (P<0.05); the apoptotic index of bone cells in group B was higher than that in group A (P<0.05). Pearson correlation analysis showed that the serum osteocalcin content was significantly positively correlated with the growth rate of body length, femoral length and bone mineral density (P< 0.01).ConclusionIntermittent hypoxia could reduce osteocalcin secretion, inhibit bone growth and sclerosis, and induce osteocyte apoptosis, thus delay the bone growth.