Objective
To construct a new composite artificial trachea and to investigate the feasibility of trachea repair and reconstruction with the new composite artificial trachea transplantation in dogs.
Methods
The basic skeleton of the new composite artificial trachea was polytetrafluoroethylene vascular prosthesis linked with titanium rings at both ends. Dualmesh was sutured on titanium rings. Sixteen dogs, weighing (14.9 ± 2.0) kg, female or male, were selected. The 5 cm cervical trachea was resected to prepare the cervical trachea defect model. The trachea repair and reconstruction was performed with the new composite artificial trachea. Then fiberoptic bronchoscope examination, CT scan and three-dimensinal reconstruction were conducted at immediate, 1 month, and 6 months after operation. Gross observation and histological examination were conducted at 14 months to evaluate the repair and reconstruction efficacy.
Results
No dog died during operation of trachea reconstruction. One dog died of dyspnea at 37, 41, 55, 66, 140, and 274 days respectively because of anastomotic dehiscence and artificial trachea displacement; the other 10 dogs survived until 14 months. The fiberoptic bronchoscope examination, CT scan and three-dimensinal reconstruction showed that artificial tracheas were all in good location without twisting at immediate after operation; mild stenosis occurred and anastomoses had slight granulation in 6 dogs at 1 month; severe stenosis developed and anastomosis had more granulation in 1 dog and the other dogs were well alive without anastomotic stenosis at 6 months. At 14 months, gross observation revealed that outer surface of the artificial trachea were encapsulated by fibrous connective tissue in all of 10 dogs. Histological examination showed inflammatory infiltration and hyperplasia of fibrous tissue and no epithelium growth on the inner wall of the artificial trachea.
Conclusion
The new composite artificial trachea can be used to repair and reconstruct defect of the trachea for a short-term. Anastomotic infection and dehiscence are major complications and problems affecting long survival.
ObjectiveTo explore the possible factors which can influence the tiered medication situation in dog-bite clinic in comprehensive hospitals and village (community) health service centers, in order to provide references for the standardization of the diagnosis and treatment for dog-bite exposed patients.
MethodsWe conducted a retrospective observational study, searched the database of Dujiangyan Center for Disease Control and Prevention, and collected all the information of dog-bite patients administrated by village (community) health service center or hospital from January 2012 to December 2015.Then we investigated the patients' satisfaction and clinical workers' familiarity of dog-bite injury treatment protocols by questionnaires in March 2016.The data were analyzed with statistical description and chi-square test, to explore the possible factors which might influence the tiered medication situation for patients exposed to dog bite in Dujiangyan city.
ResultsThere was one comprehensive hospital and 27 township (community) health centers included in this study.During the study period, the total number and proportion of dog-bite injury patients admitted in the comprehensive hospital decreased year by year, especially for the number of grade-two dog-bite exposure patients.One the contrary, the total number and proportion of patients administrated by township (community) health service centers increased year by year, especially for the number (proportion) of grade-two and grade-three dog-bite exposure patients, which increased to 1 944(44.10%) and 1 248(28.31%), respectively.The results also showed that there were significant differences between the comprehensive hospital and township health centers in the satisfaction and familiarity investigation (P < 0.05).
ConclusionsTownship and village (community) health service centers are treating many more patients than the comprehensive hospital, but with a lower satisfaction rate.The distribution of dog-bite exposure patients visiting different degrees of hospitals are generally consistent with tiered medication policy.However, township and village (community) health service centers are recommended to strengthen the staff training about how to deal with dog-bite injuries standardly in order to improve patients' satisfaction.
Objective
To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine.
Methods
Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay.
Results
Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time.
Conclusion
Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.
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.
【Abstract】 Objective To investigate the possibil ity of BMSCs seeded into collagen Ⅰ -glycosaminoglycan (CG)matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT)cross-l inked CG matrices at 1×106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscopeindicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-l ike spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimentalgroup. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.
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.
To evaluate the implantation effect of artificial vascular grafts with recombinant fibrinolytic enzyme factor II (rF II)-immobil ized lumina in animal test. Methods Four mm internal diameter (ID) polyurethane (PU) artificial vascular grafts were prepared by di pping and leaching method. The micro-pore size and morphology of the graft walls were observed by SEM. The graft lumina were immobil ized with rF II. Twenty hybrid male dogs [weighing (20 ± 1) kg] were used for animal model of carotid artery defect and were randomly divided into 3 groups: rF II -immobil ized PU group, no rF II -immobil ized PU group and expanded polytetrafluoroethylene (ePTFE) group. The vascular grafts were implanted for repairing injured segments of carotid artery in dogs. The general health state of animals was recorded. At 30 days and 60 days,the patency rate of every group was calculated. At 60 days IDs were measured, cell prol iferation in neointima was inspected by l ight microscope, morphology on neointima was observed by SEM. Results The ID of the PU vascular grafts was (3.74 ± 0.06) mm, wall thickness was 0.4-0.6 mm, the wall density was 0.25 g/cm3, the porosity was 79.8%, racical compl iance was 8.57%/100 mmHg. In the wall, micropores were well distributed and opened-pores structure was observed. Pore size was (140 ± 41) μm in the outside layer, pore size was (100 ± 3) μm in the inside layer, thickness ratio of outside / inside layers was 2 ∶ 1, the pore size was (40 ± 16) μm on the lumina surface. After operation the wounds on neck healed, all the animals survived and had no compl ication. At 30 days and 60 days after implantation, the patency rate for rF II -immobil ized PU group were 100% and 66.7%, for no rF II -immobil ized PU group were 66.7% and 33.3%, and for ePTFE group were 67.7% and 0 respectively, but at 60 days there were thrombosis at anastamotic sites of some grafts occluded. Before operation the IDs for rF II-immobil ized PU group, no rF II -immobil ized PU group and ePTFE group were (3.74 ± 0.06), (3.74 ± 0.06) and (4.00 ± 0.03) mm, at 60 days after operation the IDs were (4.51 ± 0.05), (4.31 ± 0.24) and (4.43 ± 0.12) mm respectively, showing no statistically significant differences between 3 groups (P gt; 0.05). Histological inspection indicated that at 15 days a layer of plasma protein deposited on the lumina, at 30 days some cells adhered to the lumina, at 60 days neointima could be observed on the lumina. Thickness of the neointima became larger with implantation time. At 60 days neointima thickness at proximal end, middle site and distal end ofgraft were (560 ± 22), (78 ± 5) and (323 ± 31) μm respectively for rF II -immobil ized PU group. The results of SEM showed that neointima surface consisted of flat and long cells which long axes ranged with blood flow direction and was similar to lumina morphology of carotid artery of dog. Conclusion Immobil ization of rF II to lumina of grafts could enhance fibrinolytic activity and inhibited formation of thrombo-embol ia which led to an increase in patency rate after implantation.
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 To investigate the feasibil ity of inducing canine BMSCs to differentiate into epithel ial cells in vitro with epithel ial cell conditioned medium (ECCM). Methods Five mL BMSCs were obtained from il iac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm × 4 mm after the submucosa tissue was el iminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. Results The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell prol iferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithel ial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. Conclusion The canine ECCM can induce allogenic BMSCs to differentiate into epithel ial cells in vitro.
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.
