Objective-To apply self-pulmonary tissue flap to reconstruct esophagus directly or with alloy stent in this research. Methods Twenty-four dogs were divided into two groups, middle bronchus was ligated to prepare pulmonaryflap and incised, a 4 to 6 cm long and 1/2 to 2/3 perimeter defect was made in esophageal wall. Esophagus defect was repaired only with pulmonary flap (experimental group) and with pulmonary flap having self-expanded stent inside (control group). The gross appearance, histological apearance and barium X-ray films were observed at 2,4,6,8,10 and 12 weeks after operation. Results Two dogs died of anatomotic leak in experimental group, three dogs died of anatomotic leak and two dogs died of perforation of ulcer in control group. The growth of esophagus epithelium was observed from periphery area to central area after 8 to 10 weeks of operation. In pulmonary flap mass fibrous tissue proliferated and fibroblasts were active, but no necrosis occurred. Barium X-ray ofregenerated esophagus showed that mild stenosis and weakened peristalisis were observed in the middle of resophagus replacement, and that no obstruction, leakage, and dilation above anastomotic stoma occurred. Conclusion Pulmonary tissue flap can well support the mucosa crawl in the defect of esophagus. It is necessary to find a more suitable and satisfied stent for repairing segmental defect.
Abstract: Objective To observe the changes in morphology, structure, and ventricular function of infarct heart after bone marrow mononuclear cells (BMMNC) implantation. Methods Twenty-four dogs were divided into four groups with random number table, acute myocardial infarction (AM I) control group , AM I-BMMNC group , old myocardial infarct ion (OMI) control group and OM I-BMMNC group , 6 dogs each group. Autologous BMMNC were injected into infarct and peri-infarct myocardium fo r transplantation in AM I-BMMNC group and OM I-BMMNC group. The same volume of no-cells phosphate buffered solution (PBS) was injected into the myocardium in AM Icontrol group and OM I-control group. Before and at six weeks of cell t ransplantation, ult rasonic cardiography (UCG) were performed to observe the change of heart morphology and function, then the heart was harvested for morphological and histological study. Results U CG showed that left ventricular end diastolic dimension (LV EDD) , left ventricular end diastolic volume (LVEDV ) , the thickness of left ventricular postwall (LVPW ) in AM I-BMMNC group were significantly less than those in AM I-control group (32. 5±5. 1mm vs. 36. 6±3. 4mm , 46. 7±12. 1m l vs. 57. 5±10. 1m l, 6. 2±0. 6mm vs. 6. 9±0. 9mm; P lt; 0. 05). LVEDD, LVEDV , LVPW in OM I-BMMNC group were significantly less than those in OM I-control group (32. 8±4. 2 mm vs. 36. 8±4. 4mm , 48. 2±12. 9m l vs. 60.6±16.5m l, 7. 0±0. 4mm vs. 7. 3±0. 5mm; P lt; 0. 05). The value of eject fraction (EF) in OM I-BMMNC group were significantly higher than that in OM I-control group (53. 3% ±10. 3% vs. 44. 7%±10. 1% ). Compared with their control group in morphological measurement, the increase of infarct region thickness (7. 0 ± 1. 9mm vs. 5. 0 ±2.0mm , 6.0±0. 6mm vs. 4. 0±0. 5mm; P lt; 0. 05) and the reduction of infarct region length (25. 5±5. 2mm vs. 32. 1±612mm , 33. 6±5. 5mm vs. 39. 0±3. 2mm , P lt; 0. 05) were observed after transplantation in AM I-BMMNC group and OM I-BMMNC group, no ventricular aneurysm was found in AM I-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OM I-BMMNC group (0. 581±0. 013 vs. 0. 566±0.015; P lt; 0. 05). Both in AM I-BMMNC group and OM I-BMMNC group, fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescencew as irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transp lantation both in AMI and in OM I, and significant lymphocyte infiltration in AM I-BMMNC group. Conclusion BMMNC implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and st ructure, and improve neovascularization in histology, and improve the heart function.
In order to study the influence of biliary tract obstruction on enteromicroflora,we ligate the canine biliary tract to observe the acrobic and anerobic bacteria in the duodenum and ileum at intervals of post-ligation(the 10th,20th,30th days),and to study the pathogenesis and ultramicroscopic of the ileal mucosa at the same intervals.The results showed that:the population and species of enteroflora in small intestine gradually increased after biliary obstruction.Bacteria(especialy E.coli) ascended to the upper part of small intestine,from their normal habitant of lower part of small intestine.Therefore the radio of general aerobia and E.coli risen obviously in duodenum.The longer the obstruction,the more pathologic changes were observed in ileal mucosa.such as edema,leukocytes infiltration and destruction of epithelial villi.All of those changed may be the causative factor of biliary tract infection.So that,in the programs of preventing enterogenic infection at the state of biliary tract obstruction,the protection and adjusting of normal enteroflora should be adventently considered.
Objective To discuss the way of animal model building of hepaticocholedochostomy(HC) and hepaticojejunostomy(HJ) and to compare the short-term effect. Metheds Twenty-nine dogs were divided randomly into control group(n=5) and the experimental group (stenosis of left hepatic duct, n=24). After 7 weeksof stenosis of left hepatic duct,24 dogs in the experimental group were divided randomly into HC subgroup (n=12) and HJ subgroup (n=12) .The operation time and the blood loss during operation were recorded and the hepatic function was detected.Results The diameter of left hepatic duct was significantly expended after 7 week’s stenosis. Hepaticocholedochostomy took shorter time and lost less blood than hepaticojejunostomy. The dogs in HC subgroup lost less weight than thosein HJ subgroup. In HC and HJ subgroups, the mortality rates were 1/12 and 3/12;the infectious rates of incision were 3/12and 5/12 respectively. Serum levels of total bilirubin and transaminase increased significantly in the 7th week after stenosis of left hepatic duct compared with before stenosis of left hepatic duct. However, Serum levels of total bilirubin and transaminase restored to normallevels after 1 month of HC or HJ.Conclusion It is feasible to establish animal model of bile duct reconstruction on the basis of stricture of bile duct. The dogs undergoing hepaticocholedochostomy have less trauma, better results than the dogs undergoing hepaticojejunostomy. Both hepaticocholedochostomy and hepaticojejunostomy are able to relieve the obstruction of bile duct.
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 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 It is a thorny problem to reconstruct long ureteral defect in urinary surgery. To investigate the feasibil ity of intestinal sero-muscular segment with autograft of bladder mucosa as a replacement material for reconstructionof long ureteral defect. Methods Twelve adult Beagle dogs (weighing 6.5-9.3 kg and being male or female) were randomlydivided into 3 groups, each group including 4 dogs. In group A, lower segment of ureter was reconstructed by autograft of bladder mucosa to the intestinal sero-muscular segment; furthermore, the proximal and distal reconstructed ureter were anastomosed to the bladder and the upper ureter, respectively. In group B, upper segment of ureter was reconstructed by the same method as that of group A, the proximal and distal reconstructed ureter anastomosised with pelvic and lower ureter, respectively. In group C, whole ureter was reconstructed by the same method as that of group A, the proximal and distal reconstructed ureter were anastomosised with pelvic and bladder, respectively. Blood urea nitrogen, Cr2+, K+, Na+, Cl-, Ca2+ and carbon dioxide combining power were detected before operation, the general state, drainage volume, heal ing of wound, and compl ications were observed after operation. At 6 weeks, the blood biochemical indexes and intravenous urography (IVU) were detected, and the gross and histological observations of ureter were done. Results In group B, urine leakeage and infection occurred in 1 dog 2 days after operation because ureter stent prolapsed; other dogs had no complications. There was no significant difference in the biochemical indexes between before operation and 6 weeks after operation. IVU showed: in group A, hydronepherosis and ureterectasia occurred on the operation side of 1 dog; in group B, anastomotic stricture between the reconstructed ureter and lower ureter and hydronepherosis occurred in 1 dog; and in other dogs of all groups, renal function was good and the reconstructed ureter had peristalsis function. The histopathological observation showed that the reconstructed ureter had similar structure to normal ureterat 6 weeks in 3 groups; the inflammatory cells infiltrating of the reconstructed ureter was observed in 1 dog of groups A and C, respectively. Conclusion Reconstruction of ureter by intestinal sero-muscular segment with autograft of bladder mucosa has similar structure and function to the normal ureter. The results might provide an experimental basis for cl inical use.
Six dogs underwent high selective vagotomy and mucosal antrectomy (HSV+MA). The gross and histological change of dog’s stomach were observed at 4-6 months after operation. It was found that the reconstructed antrum healed well and there was no stasis and distension in the stomach .The appearance of the nerves in muscular layer of the antrum was normal. No serious gastritis and mucosal atrophy was observed. These results indicat that HSV+MA is a reasonable procedure for the treatment of duodenal ulcer.
【Abstract】 Objective To explore an effective method to cultivate esophageal mucosa epithel ial cells (EMECs)of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged l ike slabstone. Immunohistochemical staining of CK-19 of the2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. E MECs werepolygon in shape and arranged l ike slabstone, and formed a single layer on the surface of SIS. The cells were contact ed closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, andshowed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6 %FBS is a simple and feasible method. SIS shows good biocompatibil ity and can be used as a good scaffold material in th e tissue engineered esophagus.
Objective To explore the influence of different stress environmentson the growth of tissue engineering blood vessels in vivo． Methods The engineering vascular scaffolds were prepared with the porcine small intestinal submucosa(SIS) wrapping vascular endothelial cells and smooth muscle cells，which were implanted into the subcutaneous tissue(subcutaneous group), the femoral quadriceps(intramuscular group), and sheathed the femoral artery(perivascular group) respectively. Four weeks postoperatively, these cultured tissues were harvested, and evaluated by macroscopic observation and histology detection. Results The cultivated tissues in different stress environments had obvious difference in respectof the tubular configuration, cellular proliferation and tissue shape. In subcutaneous group, the wall structure integrity, seed cell proliferation and SIS scaffold decomposition were poor, lumen surface was covered without endothelial cells; in intramuscular group, integrity tubular structure had formed, seed cell proliferation was found to a certain extent, lumen surface was covered with sparseendothelial cells, and a little SIS scaffold was found, cellular and fiber structured arranged irregularly; in perivascular group, vascular-like structure formed, the seed cell growth and proliferation were good, the lumen surface was completely covered with endothelial cells, the smooth muscle cells were in good morphologicaldistribution, the antihydrostatic pressure was 247.0±35 kPa，showingsignificant differences when compared with subcutaneous group(67.0±5.8 kPa) and intramuscular group(104.0±7.6 kPa) (Plt;0.01).The total scoring of tissue engineering blood vessel formation in subcutaneous group, intramuscular group and perivascular group were 5.529±0.272，8.875±0.248 and 14.824±0.253 respectively, and the differences among them were significant (P lt; 0.05). Conclusion Stress excitation has a great influence on the cellular proliferation and the growth of tissue engineering blood vessel in vivo.