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.
Objective To establish and evaluate a hydrocephalus model in dogs. Methods Twelve healthy adult male mongrel dogs (weight, 10-15 kg) were randomly divided into the control group (n=6) and the experimental group (n=6). All the dogs were given CT and neurological examination to exclude congenital ventricular enlargement and neurological abnormity before they received hydrocephalus induction. Surgical procedures included the exposing of the foramen magnum area, the opening of the atlantooccipita anadesma, and the injecting of silicone oil (0.3 ml/kg) into the fourth ventricle through a silicone tube. Normal saline was injected in the control group. The Tarlov neurological fitness assessment and the Evan’s ratio were used to evaluatethe degree of hydrocephalus at 3, 14 and 56 days after operation. Results In the experimental group, the dogs were dull and unsteady in walking,and they drank and ate less. The lateral ventricle began to expand 3 days afteroperation, and then the temple horn of the lateral ventricle and the third ventricle were also affected 14 days after operation. The ventricles were enlarged progressively after operation. The Tarlov scores measured at 3, 14 and 56 days afteroperation had a significant difference at the same time point between the control group(5.83±0.75，6.50±0.55，6.00±0.63) and the experimental group (4.00±0.89，4.83±1.17，4.50±1.05，P＜0.01), but had no significant difference within the same group at different time points (P＞0.05). The Evan’s ratios measured at 3, 14 and 56 days after operation were 0.33±0.04,0.39±006，0.44±0.03，respectively,in the experimental group; and were 0.27±0.06，0.25±0.09, 0.26±0.05,respectively,in the control group. There was a significant difference atthe same time point between the two groups, and at different time points within the experimental group (P＜0.05).Conclusion The dog model of hydrocephalus induced by the injecting of silicone oil into the fourth ventricle has a highsuccess rate, and the model is appropriate for the studies on diagnosis and therapy of hydrocephalus.
Objective To establish dog model of testicular autotransplantation with a modified technique.Methods Testicular autotransplantations were performed on the right side of 30 male dogs, whose ages ranged from 1.5 to 2.0 years old and weights ranged from 14 to 17 kg. After the spermatic artery with a cuff of abdominal aorta and spermatic vein and with a cuff of inferior vena cava were detached, the testis was perfused and kept at icing temperature. An end-to-side anastomosis of the spermatic vessels to the external iliac vessels was conducted subsequently. The survival conditions of the auografts were assessed by digital subtraction arteriography (DSA). Histological examination and detection on the serum levels of follicle stimulating hormone (FSH), luteotrophic hormone (LH), and testosterone (T) were made at two weeks intervals. Results Of the 30 testicular autotransplantations performed, 27 cases were successful. The success rate was 90%. The time of heat ischemia, cold ischemia, anastomosis of spermatic vessels, and total operation was 4.5±0.9 minutes, 50.0±10.0 minutes, 35.5±5.5 minutes, and 3.5±0.5 hours respectively. DSA proved that the testis survived well. No morphological abnormality was found at different stages of the spermatogenic cells. The LH level was higherthan that before operation, being statistically different (Plt;0.05);however, the levels of FSH and T did not changed significantly (Pgt;0.05). Conclusion A stable and feasible model of testicular autotransplantation is established and it provides a reliable experimental platform for human testicular transplantation.