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.
【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 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.
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 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.
ObjectiveTo compare the difference of rotator cuff healing between different types of injury and between different repair methods, and to explore the animal model to accurately simulate the restorative process after repair of rotator cuff injury. MethodTwelve adult male beagle dogs (weighing, 10-15 kg) were divided into 3 groups (n=4) according to different processing methods:acute rotator cuff injury+Mason-Allen suture repair (group A), huge rotator cuff injury+Mason-Allen suture repair (group B), and huge rotator cuff injury+Mason-Allen combined with autogenous semitendinosus expansion suture repair (group C). The external fixation was used for immobilization after repair. After operation, the general situation of the animals was observed, and the infraspinatus tendon was harvested for gross observation at 6 weeks after operation. The biomechanical test of limit load and histological observation of tendon fibers were carried out. ResultsAll the animals survived to the end of the experiment. All incisions healed well and no infection occurred. Gross observation showed more scar tissues at the end of infraspinatus muscle tendon than normal tendon in group A; no obvious tendon tissue was observed at the end of infraspinatus muscle tendon in group B; the infraspinatus muscle tendon was covered with some white scar tissue, but the tendon and the general direction could be observed in group C. The limit load of groups A, B, and C were (223.75±24.28) , (159.25±34.87) , and (233.25±14.24) N respectively, group B was significantly lower than groups A and C (P<0.05) , and no significant differnce was found between group A and group C (P>0.05) . Histological observation showed normal arrangement of tendon fibers in group A; tendon fibers arranged disorderly in group B and tendon cells were significantly less than those of group A; tendon fibers arranged in neat in group C and tendon cells were more than those of group B. ConclusionsCanine autologous semitendinosus expansion repair of massive rotator cuff injury immobilization model can better simulate the clinical rotator cuff injury healing process, so it can be used as an ideal animal model for related research.
Objective To investigate the result of the transplantation of frozen canine phalangeal joint allografts perforated and incorporated with autogenic bone marrow. Methods A proximal interphalangeal joint defect of 1.5 cm was prepared at bilateral sides of twenty-four adult healthy out-bred dogs. Three different types of allografts were applied to repair the defects: fresh autogenic phalangeal joints (group A,n=16), frozen phalangeal joint allografts perforated and incorporated with fresh autogenic bone marrow(group B, n=16), and frozen phalangeal joint allografts(group C, n=16). Radiographic and histological study wereused to evaluate the survival of transplanted joints. The observation was done 1, 3, 6 and 12 months after operation respectively. Results Based on the radiographic and histological changes of the transplanted joints, the osteoarthropathy of transplanted canine phalangeal joints could be divided into 3 degrees: mild degeneration, moderate degeneration and severe degeneration. Mild degeneration was observed in group A from 3 to 12 months. Mild degeneration was also found in group B from 1 to 6 months, and the endochondral ossification was obvious within the drilled bony holes.However, some joints in group B underwent moderate degeneration 12 months after operation. Group C joints in the first month had moderate degeneration, which progressed to severe egeneration 3 months after operation. Conclusion Transplantation of frozen canine phalangeal joint allografts perforated and incorporated with autogenic bone marrow can effectively delay the degeneration of transplanted osteoarticular allografts at the early and middle stage.
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 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.