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.
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.
To find the relation between the damage of gastric remnant mucosal barrier and the precancerous lesion of gastric remnant mucosa, in the process of the canine gastric remnant precarcinogenesis induced by N-methyN’-nitro-N-nitrosoguanidine (MNNG), we performed regularly the esophagogastroscopy and the mucosal biopsy.At the same time, we also measured gastric transmucosal potential difference and intracellular DNA content of remnant mucosa.We found that the more severe the damage of gastric remnant mucosal barrier was , the greater the malignant capacity of gastric remnant mucosal was.Our study suggests that the damage of gastric remnant mucosal barrier plays an important role in the gastric remnant mucosal precarcinogenesis.
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.
The purpose of the study was to observe effect of chinese medicine “Qing Yi Tang” on the repair of injury of intestinal mucosa in acute necrotizing pancreatitis (ANP). Dogs ANP model were induced by injection of 5% sodium taurocholate (0.5 ml/kg) with 3 000 u/kg trypsin into the pancreatic duct. Diamine oxidase and anylase activity in blood, protein and MDA levels of ileal mucosa were to be determined in ANP and after treatment of “Qing Yi Tang”. Intestinal permeability was also to be studied, LPS and bacteria translocation (BT) were obseved. All animals were sacrificed on day 7, the tissue of ileal mocosa was collected for histological and ultrastructural studies. The results showed that after treatment with Chinese medicine “Qing Yi Tang”, the injury of intestinal mucosa in ANP reduced. The length, height, area and protein of ileal mucosa increased significantly, intestinal permenbility decreased, the levels of LPS reduced in 1-2 times, and organ BT rate also reduce in 50%. The results indicated that chinese medicine “Qing Yi Tang” had good effect on improving repair of intestinal mucosa injury, protecting gut barrier function, reducing the incidence of LPS and bacteria translocation.
To evaluatae the role of cathepsin B in the pathogenesis of acute pancreatitis. The experimental dogs were divided into three groups based on the severity. An acute edematous pancreatitis group (n＝11), an acute hemorrhagic necrotizing pancreatitis group (n＝12), and a control group (n＝7). Distribution of cathepsin B in pancreatic aciner cell was studied. Results: there was a redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction. The results indicated that cathepsin B play a important role in the pathoagenesis of acute pancreatitis.
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 investigate the antibacterial and osteogenic capabil ities in vivo of hydroxyapatite (HA)/silver (Ag) coating. Methods HA/Ag coating (Ag qual ity percentage was 3%) and HA coating were deposited to external fixator Schanz screws. The tibial fracture model was establ ished in right hindl imb of 18 adult male Beagle dogs (weighing 15-20 kg). Thetibia was stabil ized with an external fixator and 2 Schanz screws of HA coating at proximal tibia (control group, n=18) and HA/Ag coating at distal tibia (experimental group, n=18), and every screw incision was infected with Staphylococcus aureus. Infection in screw holes and the changes of bone-screw interface were observed by wound grading and X-ray films. Results In control group, wounds infection became worse with time (χ2=13.492, P=0.001), while in experimental group, no obvious change was observed (χ2=0.208, P=0.901). The wound grading of experimental group was significantly better than that of the control group at 1, 2, and 3 weeks (P lt; 0.05). Laser scanning confocal microscope showed that there was bacterial adhesion on the surface of screws in 2 groups, viable becteria mainly in control group and non-viable becteria mainly in experimental group. The scanning electron microscope (SEM) observation results of the fractured sclerous tissue section showed that an obvious transparent boundary between screw and bone in control group, but no obvious boundary in experimental group. The osseointegration ratios were 76.23% ± 15.54% in control group and 93.42% ± 5.53% in experimental group, showing significant difference (t=8.843, P=0.000). The SEM observation showed that HA/Ag coating integrated with new bone and the surface of implant was filled with new bone in experimental group; obvious interspace was seen between the HA coating and new bone in control group. Conclusion HA/Ag coating has good antibacterial and osteogenic capabil ities, so it can take effects in preventing infection in screw holes and loosening of implants.
Objective To verify adhesion and growth ability of canine esophageal epithelial cells (EECs) on the poly (lactic-co-glycolic acid) (PLGA), a three-dimensional biodegradable polymer scaffold, and to reconstruct the canine esophagus by the tissue engineering. Methods Free canine EECs isolated from adult dogs by esophagoscopy were seeded onto the PLGA scaffolds precoated with collagen type Ⅳ after the first passage by the in vitro culture. Then, the composites of the cell-scaffold were respectively cultured invitro and in the abdominal cavity of the dog in vivo. After different periods, the cell-seeded scaffolds were assessed by histological HE staining, scanning electron microscopy, and immunohistochemical analysis. Results The cells displayed a cobblestone-shaped morphology that was characteristic of the epithelial cells and were stained to be positive for cytokeratin, which indicated that the cells were EECs. The canine EECs were well distributed and adhered to the PLGA scaffolds, and maintained their characteristics throughout the culture period. After the culture in vivo for 4 weeks, the cell-seeded scaffolds looked like tissues. Conclusion PLGA scaffolds precoated with collagen type Ⅳ can be suitable for adhesion and proliferation of EECs, and can be used as a suitable tissue engineering carrier of an artificial esophagus.