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.
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 investigate an improved large vascular reconstruction method in the canine liver transplantation and see whether it can shorten the anheptic time and thus reduce the harmful effects during the anhepatic phase. Methods Thirty-two mongrel dogs were enrolled and divided into two groups randomly:the donor group (n=16) and the acceptorgroup(n=16). The dogs in the acceptor group were divided into two groups, according to the different reconstruction methods: Group A using the magnetic rings for a large vein reconstruction in the canine liver transplantation (n=10), and Group B using a handsewing large vein reconstruction in the canine liver transplantation (n=6). The operation time, hemodymics change, anastomosis site, and survival were observed. Results The operation time was as follows: In Group A, the total operation time, the inferior vena cava anastomosistime, and the anheptic phase time were significantly shorter than those in Group B (3.24±0.49 h vs 4.12±0.51 h，5.89±2.27 min vs 28.33±6.04 min，3.89±0.73 min vs 12.16±3.72 min),with a significant difference between the two groups (Plt;0.01). The haemodymics changes were as follows: In Group A, MAP dropped during the anhepatic phase, but it soon recovered after reperfusion，and there was only 730.56±150.56 ml of fluid including the donor blood that needed to be transfused, with no pressor agent required. In Group B, blood pressure dropped during the anhepatic phase，but it slowly recovered，and there was 2241.67±390.78 ml of fluid. In Group A, all the stomas had no errhysis, twistor thrombus. The twisted stomas could be corrected by the revolving of the magnetic rings. The endangium at the site of anastomosis was smooth. In Group B, most of the stomas had errthysis. In Group A, 3 dogs survived for more than 7 days, 6dogs survived for 3-6 days, and 1 dog survived for only 12 hours. In Group B, 2 dogs survived for 3-6 days, 3 dogs survived for 1-2 days, and 1 dog survivedfor only 12 hours. Conclusion Using the magnetic rings for a large vascular reconstruction in the canine liver transplantation is an improvedmethod, which can simplify the anastomosis procedures and significantly shortenthe anheptic phase time. However, the magnetic rings have to be placed in the abdomen, so this method remains to be further improved.
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.
目的 建立犬開放性氣胸海水浸泡的實驗模型 ,探討實驗動物早期死亡原因。 方法 2 0條健康成年雜種犬隨機分為兩組。對照組 :實驗動物受傷后直接觀察 ;實驗組 :動物受傷后置入人工配制的海水中。監測血流動力學、呼吸、血液滲透壓、血液電解質、動脈血氣變化以及肺部病理改變。 結果 實驗組死亡率明顯高于對照組 ,平均生存時間為 45分鐘。實驗組經海水浸泡后有急性呼吸和循環功能衰竭、嚴重電解質平衡紊亂、高滲血癥、重度肺損傷以及嚴重代謝性和呼吸性酸中毒。 結論 開放性氣胸后海水浸泡可引起一系列嚴重的病理生理變化 ,其結果是導致實驗動物早期死亡的重要原因。
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.
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.
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.
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.