[Experimental studies on canine bladder smooth muscle cells cultured on acellular small intestinal submucosa in vitro].
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 enzyme-treatment 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 muscle cells 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 vitro for 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 spindle-shaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the well-developed actin filaments in the cytoplasm and the dense patches in the cell membrane under the transmission electron microscope. The immunohistochemical staining showed the canine bladder smooth muscle cells with positive reacting alpha-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 single-layer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were (16.85 +/- 0.79) x 10(5), (39.74 +/- 2.16) x 10(5) and (37.15 +/- 2.02) x 10(5), respectively. The cell counts in the control group were (19.43 +/- 0.54) x 10(5), (34.50 +/- 1.85) x 10(5) and (33.07 +/- 1.31) x 10(5), respectively. There was a significant difference between the two groups at 5 days (P < 0.05).


CONCLUSIONS
With 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.
Han
Ping
P
Division of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, PR China. hanp2001a@sohu.com
Yang
Zhiming
Z
Zhi
Wei
W
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D004285:Dogs; D007413:Intestinal Mucosa; D007421:Intestine, Small; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000648:ultrastructure; D023822:Tissue Engineering / Q000379:methods*; D001743:Urinary Bladder / Q000166:cytology*