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.
A simple model of canine auto-trasplantation of liver was set up by ourselves, and the effects of hepatic artery ischemia (HAI) on ultrastructure of liver and biliary duct transplanted were observed. The results showed that the liver and biliary cells swelled slightly, mitochondrial matrix was loose and ridges were vague just often perfusion. Afte HAI for 3 hours the edema of hepatic and biliary cells aggravated cytoplasm was loose, mitochondria enlarged and partly vacuolar degenerated, ridges broke or disappeared, flocculent focal densites was seen in the matrix, endoplasmic retculum distended obviously, and the ribosome depolymerizated. So we consider that HAI causes obvious damage to hepatic and biliary cells. These indicate that HAI is one of the important factors of complication after liver transplantation, especially some biliary complications.
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。
ObjectiveTo study the preservation effect of true bone ceramics (TBC) prepared by high-temperature calcination of bovine bone on alveolar ridge of canine extraction socket.MethodsSix healthy Beagle dogs (aged 1.5-2 years) were selected to extract the second and fourth premolars of both mandibles and the second premolars of the maxilla. The left extraction socket was implanted with TBC as the experimental group, and the right side was implanted with the calcined bovine bone (CBB) as the control group, to observe the alveolar ridge preservation effect. Three dogs were euthanized after general observation at 1 and 6 months after operation respectively. After separating the maxilla and mandible, cone beam CT (CBCT) was performed to measure the average gray value of the graft site and the adjacent reference area (the area between the roots of the adjacent third premolar) and calculate the gray scale ratio between the bone graft site and the reference area. Histological observation was made on the bone graft site to evaluate the new bone formation.ResultsGeneral observation showed that the wounds of both groups were basically healed at 2 weeks after operation, and the bone graft materials were not exposed. The wounds healed well at 1 and 6 months after operation without swelling. The results of CBCT showed that the residual material was found in both groups at 1 month after operation, and no significant residual material was found in both groups at 6 months after operation, and the alveolar ridge height of the bone graft area was not significantly reduced. There was no significant difference in the bone mineral density between the experimental group and the control group. The gray scale ratios of the experimental group at 1 month and 6 months after operation were 0.97±0.14 and 0.93±0.06, respectively, and were 0.99±0.16 and 0.94±0.05 in control group, showing no significant difference between the two groups (t=?1.030, P=0.333; t=?0.770, P=0.466). HE staining observation showed that a large number of bone graft materials did not degrade and new bone formed around the grafts in both groups at 1 month after operation; the bone graft materials were absorbed and a large number of new bones were formed in both groups at 6 months after operation.ConclusionTBC can maintain bone mineral density and have good osteoconductivity in the alveolar ridge site preservation experiment of dogs, and can be used for alveolar ridge site preservation.
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 evaluate the internal fixation effect, degradation, and biocompatibility of polylactic-co-glycolic acid/hydroxyapatite (PLGA/HA) absorbable cannulated screws in treatment of lateral femoral condyle fracture of canine so as to provide the theory basis for their further improvement and clinical application. Methods Sixteen adult male Beagles (weighing, 9-12 kg) were selected to prepare the models of bilateral lateral femoral condyle fracture; left fracture was fixed with PLGA/HA absorbable cannulated screws as experimental group and right fracture with metal screws as control group. At 2, 4, 8, and 12 weeks after operation, general observation was done and X-ray films were taken for observing fracture healing; bone mineral density was measured; the histological examination was performed; and the degradation property of absorbable cannulated screws was detected. Results All animals survived to the end of the experiment. General observations showed that no fracture displacement occurred and fracture healed at 12 weeks in 2 groups; no breakage, displacement, or loosening of screws was observed in experimental group. X-ray films results showed that the absorbable cannulated screws could not be found out by X-ray in experimental group, but metal screws could be found out in control group; fracture healed with time in 2 groups. The bone mineral density reached the peak at 8 weeks in 2 groups, and no significant difference was found between 2 groups and among different time points in the same group (P gt; 0.05). Histological examination showed that 2 groups had similar fracture healing process at different time points; no obvious inflammatory reaction was found around absorbable cannulated screws in experimental group. The degradation results of absorbable cannulated screws showed that the intrinsic viscosity and molecular weight distribution obviously decreased at 2 weeks; the number average molecular weight and the weight average molecular weight markedly decreased at 4 weeks; and the maximum shear force did not decrease obviously at 8 weeks, and then decreased significantly. Significant differences were found in all indexes among different time points in the same group (P lt; 0.05). Conclusion PLGA/HA absorbable cannulated screws and metal screws show similar fracture healing process for fixing lateral femoral condyle fracture of canine, and the absorbable canulated screws have good biocompatibility. The maximum shear force of PLGA/HA absorbable cannulated screw has no obvious decrease during 8 weeks after operation, so it can ensure full healing of fracture.
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.
Objective Native extracellular matrix (ECM) is comprised of a complex network of structural and regulatory proteins that are arrayed into a tissue-specific, biomechanically optimal, fibrous matrix. The multifunctional nature of the native ECM will need to be considered in the design and fabrication of tissue engineering scaffolds. To investigate the extraction techniques of naturally derived nerve ECM and the feasibil ity of nerve tissue engineering scaffold. Methods Ten fresh canine sciatic nerves were harvested; nerve ECM material was prepared by hypotonic freeze-thawing, mechanicalgrinding, and differential centrifugation. The ECM was observed by scanning electron microscope. Immunofluorescencestaining was performed to detect specific ECM proteins including collagen type I, laminin, and fibronectin. Total collagen and glycosaminoglycan (GAG) contents were assessed using biochemical assays. The degree of decellularization was evaluated with staining for nuclei using Hoechst33258. The dorsal root gangl ion and Schwann cells of rats were respectively seeded onto nerve tissue-specific ECM films. The biocompatibil ity was observed by specific antibodies for cell markers. Results Scanning electron microscope analysis revealed that nerve-derived ECM consisted of a nanofibrous structure, which diameter was 30-130 nm. Immunofluorescence staining confirmed that the nerve-derived ECM was made up of collagen type I, laminin, and fibronectin. The histological staining showed that the staining results of sirius red, Safranin O, and toluidine blue were positive. Hoechst33258 staining showed no DNA within the decellularized ECM. Those ECM films had good biocompatibil ity for dorsal root gangl ion and Schwann cells. The cotents of total collagen and GAG in the nerve-derived ECM were (114.88 ± 13.33) μg/ mg and (17.52 ± 2.34) μg/mg, showing significant difference in the content of total collagen (P lt; 0.01) and no significant difference in the content of GAG (P gt; 0.05) when compared with the contents of normal nerve tissue [(54.07 ± 5.06) μg/mg and (25.25 ± 1.56) μg/mg)]. The results of immunofluorescence staining were positive for neurofilament 200 after 7 days and for S100 after 2 days. Conclusion Nerve-derived ECM is rich in collagen type I, laminin, and fibronectin and has good biocompatibil ity, so it can be used as a nerve tissue engineering scaffold.
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.