【Abstract】 Objective To measure the changes of bone mineral density and bone micro-architecture of thefemoral head that harvested from the three-foot bearing ethanol destroyed canine model for osteonecrosis of femoral head, and discuss the influences of local injection of ethanol and biomechanical loading to the structural properties of the femoral head. Methods Twenty-four Beagles were divided randomly into four-foot bearing canines and three-foot bearing canines. One fore-l imb was fixed randomly in three-foot bearing canines. Osteonecrosis was induced in all experimental animals by local injection of 5 mL pure ethanol into one side of the femoral head. The hind l imbs injected with NS were acted as control group, that of three-foot canines injected with ethanol were acted as three-foot canine group, and that of four-foot canines injected with ethanol were acted as four-foot canine group. The contralateral femoral head was injected into equal amount of NS. Animals were sacrificed at the time intervals of 1, 3, 6, and 12 weeks after the injection of ethanol. Quantitative microcomputedtomography was used to characterize changes in bone micro-architecture and bone mineral density of femoralhead. Results The clear three-dimensional model of trabecular bone of necrotic femoral head were obtained. There were no significant differences among 3 groups according to the time l ine by 1 week after ethanol injection(P gt; 0.05). At 3 weeks after injection of ethanol, in three-foot canine group and four-foot canine group, the volume of BMC, BMD, BVF, and BS/BV increased gradually as the distance to the drill ing canal increased. There were significant differences between 3 regions (P lt; 0.05). At 6 weeks, in three-foot canine group and four-foot canine group, the volume of BMC, BMD,BVF, and Tb.N of region I and II decreased significantly compared with region III (P lt; 0.05). At 12 weeks, there are no differences among 3 groups (P gt; 0.05). There were significant decreases in BMD values, BVF, BS/BV, Tb.N, Tb.Sp and Tb.Th after the injection of pure ethanol. And, the changes were more and more obvious by the time l ine. These changes were differentiable at 3 weeks after injection of ethanol, and became obvious at 6 weeks. These changes were more obvious at the part that near the injection canal. The changes in threefoot canine group were more obvious than that in four-foot canine group. Conclusion Resorption of necrotic compact bone trabecular may weaken the structural properties of the femoral head. Moreover, remodel ing and repairing process of necrotic bone trabecular may be hampered by constant biomechanical loading that presented in three-foot bearing canines, and thereby further weaken the structural properties of the femoral head. Biomechanical loading may be one of the critical reasons that lead to the collapse of femoral head.
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 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 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.
Objective
To analyze the application of rigid intra-oral tooth borne distraction device in dento-alveolar distraction osteogenesis.
Methods
Six patients who underwent orthodontic treatment for maxillary and/or mandibular canine tooth from January to December 2016 in Hanzhong Central Hospital were collected. The bilateral canine tooth was retracted after the first premolar extraction by using the conventional method, and were distracted by the rigid intra-oral tooth borne distraction device, which was made of stainless steel. The tooth movement distance and time, pain and adverse reaction of patients in the process of orthodontics were investigated.
Results
The number of orthodontic tooth of each patient was 2–4, and the movement range of canine retraction was 6.5–8.0 mm. The time required for canine tooth moving to the second premolar was 13–17 days, and the canine tooth of all the patients were moved, inclined and buccal expanded after three weeks of enhanced fusion. Two patients felt pain and discomfort, one patient experienced buccal mucosa ulcer, and none of the six patients suffered from dysmasesia, dysphagia, periodontitis or tooth enamel loss.
Conclusion
As an effective tool for orthodontic treatment, the new rigid intra-oral tooth borne distraction device could accelerate the speed of canine movement, and shorten the orthodontic time with few adverse reactions.
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.
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.
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 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 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.
