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 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.
【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 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.
Objective To make a comparison for the change of maximum tensile intensity and stiffness of a whole implant that is placed into bone tunnel with various lengths tendon, by using beagle dog’s autogenous flexor tendons to reconstruct anterior cruciate l igament (ACL). Methods Sixty male beagle dogs were included in the experiment (weighting 13-16 kg). Three dogs were used for intact flexor tendon of both knees (normal control group), 3 dogs for the intact ACL andfemur-graft-tibia complex (auto control group) and 54 dogs (108 knees) for models of reconstructed ACL (6 experimentalgroups according to different lengths of tendon: 5, 9, 13, 17, 21 and 25 mm in the bone tunnel). The tensile intensity and stiffness were measured after 45, 90 and 180 days separately after operation. Results In the normal control group, the maximum tensile intensity of the intact flexor tendon was (564.15 ± 36.18) N, the stiffness was (59.89 ± 4.28) N/ mm. In the auto control group, the maximum tensile intensity of the intact ACL was (684.75 ± 48.10) N, the stiffness was (74.34 ± 6.99) N/ mm, all ruptured through the intra-articular portion of the graft. The maximum tensile intensity of femur-graft-tibia complex in the auto control group was (301.92 ± 15.04) N, the stiffness was (31.35 ± 1.97) N/mm. After 45 days of operation, all failure occurred at the tibial or femoral insertion site. After 90 days of operation, 24 of the breakpoints were scattered in tendon-bone junction, 12 (3 in 17 mm group, 5 in 21 mm group, 4 in 25 mm group) ruptured through the intra-articular portion. After 180 days of the operation, all breakpoints were distributed inside joint of the implant. The maximum tensile intensity and the stiffness were ber in 17, 21 and 25 mm groups than in 5, 9 and 13 mm groups after operation (P lt; 0.05). Conclusion Tendon with 17 mm length, which will be implanted into bone tunnel, is an appl icable index, in reconstruction of ACL by autogenous tendons.
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.
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.
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.
