Objective To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system. Methods UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1 ∶ 1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of α-smooth muscle actin (α-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days. Results The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of α-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining. Conclusion UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
ObjectiveTo explore the relationship between the pressure level within the scope of promoting proliferation and cell injury of human bladder smooth muscle cells (HBSMCs). MethodHBSMCs in vitro were divided into the experimental group and control group. The cells in the experimental group were exposed to 40 cm H2O (1 cm H2O=0.098 kPa) pressure and those in the control group were cultured in normal condition for 24 hours. We investigated the cell morphology and cytoskeleton with indirect immunofluorescence staining for α-actin. Propidium iodide (PI) staining was applied to evaluate the level of cell apoptosis. ResultsThere was no significant difference in the cell morphology between the two groups. However, the expression of α-actin in the experimental group[(50.93±1.99)%] was significantly reduced comparing with that in the control group[(24.70±1.61)%] (t=32.404, P<0.001). The results of PI staining showed that compared with the control group[(3.50±2.12)%], the number of PI staining positive cells in the experimental group [(9.00±1.41)%] was significantly higher (t=6.110, P<0.001). ConclusionsPressure condition can promotes cell proliferation, but at the same time, it can also lead to cell injury of HBSMCs.
Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.