Objective To investigate the feasibil ity of establ ishment of physiological micturition reflex arc by simultaneously reconstructing the sensory and the motorial nerve of atonic bladder after spinal cord injury. Methods Eight 1-year-old Beegle male canine were selected, weighing 7-12 kg. The left side was the experimental side, while the right side wasthe control side. Epidural microanastomosis of vertebral canal of the left L7 ventral root to S2 ventral root and L7 dorsal root to S2 dorsal root was performed to reconstruct the sensory and the motorial function of atomic bladder. The right side was used as a control without treatment. The new motor-to-motor, and sensory-to-sensory physiological bladder reflex pathway were establ ished after 12 months of axonal regeneration. Then S1-4 segmental spinal cord was destroyed for preparation of complete paraplegia. The electrophysiological examination and the bladder pressure were detected before and after paraplegia. The canine micturition was observed for 3 months after paraplegia. Nurohistological observation was performed after canine sacrifice. Results Of 8 canine, 7 canine survived. After paraplegia, canines displayed urinary incontinence and frequent micturition at first, nocturnal continence was achieved gradually without frequent micturition after 1 month. Urinary infection at different degrees occurred in 3 canines and was controlled after Norfloxacin was administered orally. The bladder pressure increased to (1.00 ± 0.13) kPa, (0.90 ± 0.12) kPa after trains of stimulation (300 mV, 0.3 ms, 20 Hz, 5 seconds) of S2 dorsal root at the experimental side before and after paraplegia respectively, showing no significant difference (P gt; 0.05). It increased to (1.90 ± 0.10) kPa after the same train of stimulation of S2 dorsal root at control side. There was significant difference between the experimental side and the control side (P lt; 0.01). Single stimulation (300 mV, 0.3 ms) of the S2 dorsal root at the experimental side resulted in evoked potentials recorded from the left S2 ventral root before and after paraplegia. Before and after paraplegia, the ampl itudes of the evoked potentials were (0.68 ± 0.11) mV and (0.60 ± 0.08) mV respectively, showing no significant difference (P gt; 0.05). It was (1.21 ± 0.13) mV while stimulating at the control side. There was significant difference between the experimental side and the control side (P lt; 0.01). Neurofibra of L7 dorsal and ventral root grew into S2 dorsal and ventral root on tissue sl ice under l ight microscope. Conclusion Reconstruction of the bladder physiological micturition reflex arc is feasible by anastomosis of sacral dorsal and ventral root below injured spinal plane with the suprasacral survival dorsal and ventral root above the plane respectively for restoration of atonic bladder after spinal cord injury.
Objective To explore an effective method to culture and purify canine bladder transitional epithelial cells.Methods Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0.125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Results Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells.Different generations of cells showed diploid cells. Conclusion A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
【Abstract】 Objective To establ ish a animal model of osteonecrosis of femoral head in canine l ike human.Methods The thermal field of canine’s femoral head was three-dimensionally analyzed with fluent 6.2 software so that the best cryosurgery patent could be designed to maximize the osteonecrosis and minimize extra surgery trauma with the cryosurgery system invented by Shanghai Jiaotong University. Liquid nitrogen was pressurized to 0.5 MPa, poured into femoral head for 6.5 minutes, rewarming to 2 for 5 minutes and then repoured into it again for another 6.5 minutes. Ten three-foot canines were conducted as the animal models of osteonecrosis of femoral head according to the method above. At the end of followup,the results were reviewed by radiologic and pathologic check. Two dogs were conducted as control group. Results In the experimental group, one of the ten canines was testified to occur osteonecrosis of femoral head after one week pathologically, cell death and vessel breakage of cavitas medullaris in the femoral head was obvious under microscope; in other nine canines beingstill under follow-up, five with three-month follow-up at least progressed to the collapse of femoral head l ike human (Ficat III). In control group, no osteonecrosis was found. Conclusion Cryosurgery for osteonecrosis of the femoral head in three-foot canine model may become a method to establ ish the animal model of osteonecrosis of femoral head l ike human.
Objective
To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice.
Methods
Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics.
Results
In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05).
Conclusion
Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.
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.
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.
Abstract： Objective To investigate the protective effects of adenosine (ADO) on lung ischemia/reperfusion injury following heart-lung transplantation in canine. Methods Canine heart-lung transplantation was performed．Canines were divided into two groups: transplant control groupand ADO group. The changes of arterial partial pressure of oxygen(PaO2) after reperfusion in two groups at 30，60，90，120 min were observed．The tissue contents of nitric oxide (NO) were measured at 10 min before ischemia, 10 min and 120 min after ischemia; 10 min and 60 min after reperfusion．The lung tissue samples were obtained 1h after reperfusion．The tissue myeloperoxidase(MPO) activity,content of malondialdehyde(MDA), content of superoxide dismutase(SOD), wet/dry ratio of lung(W/D) were measured.Microscopic examination of lungs was also conducted. Results (1)In ADO group，PaO2 were significantly higher than that in control group at 30，60，90 and 120 min after reperfusion (Plt;0.05)．(2) The tissue contents of NO at 120 min after ischemia, 10 min and 60 min after reperfusion were significantly lower than that at 10 min before ischemia(Plt;0.05). In ADO group，the tissue contents of NO at 120 min after ischemia, 10 min and 60 min after reperfusion were higher than that in control group respectively(Plt;0.05). (3)The tissue MPO activity, content of MDA, W/D in ADO group were significantly lower than those in corresponding control group. The content of SOD in ADO group were higher than that in control group(Plt;0. 05)．(4)The microscopic examination showed that there were severe leukocyte infiltration and edema formation in the alveolar space in control group, but the changes were less severe in ADO group. Conclusion Administration of ADO in canine heart-lung transplantation can protect the donor lung against ischemia／reperfusion injury．
Objective To provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passagesof cells. Methods Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs’ urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and prol iferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry. Results The cells appeared spindle in parallel rows when they grew to the degree of subconfluence, and showed the “peak-valley” structure under the inverted phase contrast microscope. The cells could be prol iferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar prol iferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells’ growth became slow, and myofilament and the dense body in cytoplasm vanished. Conclusion The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely prol iferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.
Objective To compare canine decel luarized venous valve stent combining endothel ial progenitor cells (EPC) with native venous valve in terms of venous valve closure mechanism in normal physiological conditions. Methods Thirty-six male hybrid dogs weighing 15-18 kg were used. The left femoral vein with valve from 12 dogs was harvested to prepare decelluarized valved venous stent combined with EPC. The rest 24 dogs were randomly divided into the experimental group and the control group (n=12 per group). In the experimental group, EPC obtained from the bone marrowthrough in vitro ampl ification were cultured, the cells at passage 3 (5 × 106 cells/mL) were seeded on the stent, and the general and HE staining observations were performed before and after the seeding of the cells. In the experimental group, allogenic decelluarized valved venous stent combined with EPC was transplanted to the left femoral vein region, while in the control group, the autogenous vein venous valve was implanted in situ. Color Doppler Ultrasound exam was performed 4 weeks after transplantation to compare the direction and velocity of blood flow in the distal and proximal end of the valve, and the changes of vein diameter in the valve sinus before and after the closure of venous valve when the dogs changed from supine position to reverse trendelenburg position. Results General and HE staining observations before and after cell seeding: the decelluarized valved venous stent maintained its fiber and collagen structure, and the EPC were planted on the decelluarized stent successfully through bioreactor. During the period from the reverse trendelenburg position to the starting point for the closure of the valve, the reverse flow of blood occurred in the experimental group with the velocity of (1.4 ± 0.3) cm/s; while in the control group, there was no reverse flow of blood, but the peak flow rate was decreased from (21.3 ± 2.1) cm/s to (18.2 ± 3.3) cm/s. In the control group, the active period of valve, the starting point for the closure of the valve, and the time between the beginning of closure and the complete closure was (918 ± 46), (712 ± 48), and (154 ± 29) ms, respectively; while in the experimental group, it was (989 ± 53), (785 ± 43), and (223 ± 29) ms, respectively. There was significant difference between two groups (P lt; 0.05).After the complete closure of valve, no reverse flow of blood occurred in two groups. The vein diameter in the valve sinus of the experimental and the control group after the valve closure was increased by 116.8% ± 2.0% and 118.5% ± 2.2%, respectively, when compared with the value before valve closure (P gt; 0.05). Conclusion Canine decelluarized venous valve stent combined with EPC is remarkably different from natural venous valve in terms of the valve closure mechanism in physiological condition. The former rel ies on the reverse flow of blood and the latter is related to the decreased velocity of blood flow and the increased pressure of vein in the venous sinus segment.
Objective To observe whether additional penehycl idine hydrochloride (PHC) in mechanical ventilation produces therapeutic effect on oleic acid (OA) induced acute lung injury (ALI) in canine. Methods Seventeen male canines (weighing 12-17 kg) were divided into control group (n=5), OA group (n=6) and PHC group (n=6). ALI model was developed by central venous injection of OA in canines of OA and PHC groups. ALI model was kept steady in air, all groups received mechanical ventilation 90 minutes later. Three groups received normal sal ine 0.25 mg/kg without injection of OA(control group), normal sal ine 0.25 mg/kg after injection of OA (OA group) and PHC 0.25 mg/kg after injection of OA (PHCgroup) respectively at 0 h (90 minutes after onset time of ALI/ARDS). The heart rate (HR), mean arteial pressure (MAP), mean pulmonary arterial pressure (MPAP), central venous pressure (CVP), pulmonary artery wedge pressure (PAWP), artery blood gas analysis, cardiac output (CO), extravascular lung water index (EVLWI), FiO2 and VT were observed respectively at basel ine, onset time of ALI/ARDS and 0 h, then again at 1 hour intervals for 6 hours. Besides the above, airway peak pressure (Ppeak), airway plat pressure (Pplat), mean airway pressure (Pmean) and positve end-expriatory pressure (Peep) were also observed each hour during 1-6 hours. Oxygenation index (OI), pulmonary vascular resistance (PVR), systemic vascular resistance (SVR), alveolar-arterial differences for O2 (AaDO2) and dynamic lung compl iance (DLC) were calculated and pulmonary tissue was collected for histopathologic investigation and dry wet weight ratio (WDR) test. Results The functional parameters of PHC group were improved when compared those of OA group, but there was no siginficant difference; WDR of independent region of three groups were 80.42% ± 3.48%, 82.67% ± 4.01% and 82.26% ± 1.43% respectively; WDR of dependent region of three groups were 80.51% ± 3.60%, 83.71% ± 1.98% and 82.57% ± 1.08% respectively. WDR of PHC group were obviously improved when compared with those of OA group, but there was no significant difference. Independent and dependent regions of PHC group were significantly improved when compared those of OA group in histopathologic scores, alveolar edema, inflammatory infiltration and over-distension (P lt; 0.01). Conclusion Additional PHC in mechanical ventilation produces obvious therapeutic effect on OA induced acute lung injury in canine.
