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 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.
Objective To observe the histological structure and cytocompatibility of novel acellular bone matrix (ACBM) and to investigate the feasibility as a scaffold for bone tissue engineering. Methods Cancellous bone columns were harvested from the density region of 18-24 months old male canine femoral head, then were dealt with high-pressure water washing, degreasing, and decellularization with Trixon X-100 and sodium deoxycholate to prepare the ACBM scaffold. The scaffolds were observed by scanning electron microscope (SEM); HE staining, Hoechst 33258 staining, and sirius red staining were used for histological analysis. Bone marrow mesenchymal stem cells (BMSCs) from canine were isolated and cultured with density gradient centrifugation; the 3rd passage BMSCs were seeded onto the scaffold. MTT test was done to assess the cytotoxicity of the scaffolds. The proliferation and differentiation of the cells on the scaffold were observed by inverted microscope, SEM, and live/dead cell staining method. Results HE staining and Hoechst 33258 staining showed that there was no cell fragments in the scaffolds; sirius red staining showed that the ACBM scaffold was stained crimson or red and yellow alternating. SEM observation revealed a three dimensional interconnected porous structure, which was the microstructure of normal cancellous bone. Cytotoxicity testing with MTT revealed no significant difference in absorbance (A) values between different extracts (25%, 50%, and 100%) and H-DMEM culture media (P gt; 0.05), indicating no cytotoxic effect of the scaffold on BMSCs. Inverted microscope, SEM, and histological analysis showed that three dimensional interconnected porous structure of the scaffold supported the proliferation and attachment of BMSCs, which secreted abundant extracellular matrices. Live/dead cell staining results of cell-scaffold composites revealed that the cells displaying green fluorescence were observed. Conclusion Novel ACBM scaffold can be used as an alternative cell-carrier for bone tissue engineering because of thoroughly decellularization, good mircostructure, non-toxicity, and good cytocompatibility.
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.
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 Using chemically extracted acellular methods to treat extracranial section of the canine whole facial nerve, to evaluated its effects on nerve structure and the removal extent of Schwann cells and myel in. Methods Twenty whole facial nerves were exposed from 10 canines [weighing (18 ± 3) kg]. The extracranial trunk of canine facial nerve and its branches (temporal branch, zygomatic branch, buccal branch, marginal mandibular branch, and cervical branch) were dissected under l ight microscope. Twenty facial nerves were divided into the experimental group (n=12) and control group (n=8) randomly. In experimental group, the nerve was extracted with the 3%TritonX-100 and 4% sodium deoxycholate. In control group, the nerve was not extracted. HE staining and immunofluorescence histological stainings for Hoechst33258, P75, Zero, and Laminin were performed. Results After histological staining, it was found that myel in and Schwann cells were removed from the facial nerve while the basal lamina tube remained intact. The whole canine facial nerves (one nerve trunk and multiple nerve branches) had the similar result. Conclusion The canine whole facial nerve has natural structure (one nerve trunk and multiple nerve branches) by extracted with chemically extracted acellular methods, so it is an available graft for repairing the defect of the whole facial nerve.
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 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 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.
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.