Objective To make a comparison for the change of maximum tensile intensity and stiffness of a whole implant that is placed into bone tunnel with various lengths tendon, by using beagle dog’s autogenous flexor tendons to reconstruct anterior cruciate l igament (ACL). Methods Sixty male beagle dogs were included in the experiment (weighting 13-16 kg). Three dogs were used for intact flexor tendon of both knees (normal control group), 3 dogs for the intact ACL andfemur-graft-tibia complex (auto control group) and 54 dogs (108 knees) for models of reconstructed ACL (6 experimentalgroups according to different lengths of tendon: 5, 9, 13, 17, 21 and 25 mm in the bone tunnel). The tensile intensity and stiffness were measured after 45, 90 and 180 days separately after operation. Results In the normal control group, the maximum tensile intensity of the intact flexor tendon was (564.15 ± 36.18) N, the stiffness was (59.89 ± 4.28) N/ mm. In the auto control group, the maximum tensile intensity of the intact ACL was (684.75 ± 48.10) N, the stiffness was (74.34 ± 6.99) N/ mm, all ruptured through the intra-articular portion of the graft. The maximum tensile intensity of femur-graft-tibia complex in the auto control group was (301.92 ± 15.04) N, the stiffness was (31.35 ± 1.97) N/mm. After 45 days of operation, all failure occurred at the tibial or femoral insertion site. After 90 days of operation, 24 of the breakpoints were scattered in tendon-bone junction, 12 (3 in 17 mm group, 5 in 21 mm group, 4 in 25 mm group) ruptured through the intra-articular portion. After 180 days of the operation, all breakpoints were distributed inside joint of the implant. The maximum tensile intensity and the stiffness were ber in 17, 21 and 25 mm groups than in 5, 9 and 13 mm groups after operation (P lt; 0.05). Conclusion Tendon with 17 mm length, which will be implanted into bone tunnel, is an appl icable index, in reconstruction of ACL by autogenous tendons.
ObjectiveTo investigate the situation of internal antibody positive rate of Chinese who exposed to rabies virus and received full procedure of rabies vaccination, and to provide the basis for the adjustment of rabies vaccination procedure and policy. MethodsWe electronically searched databases including WanFang Data, VIP, CNKI, PubMed and EMbase from inception to May 2015 to collect studies about Chinese exposed to rabies virus and received full procedure rabies vaccination. Two reviewers independently screened literature, extracted data, and assessed the methodological quality of included studies. Then, meta-analysis was performed using R software (R3.2.1). ResultsA total of 33 studies were included. The combined antibody positive rate was 93.99% with 95%CI 92.02% to 95.70%. Antibody positive rate among male was 93.73% with 95%CI 91.65% to 95.54%, while among female was 94.33% with 95%CI 92.35% to 96.04%, and there was no significant difference between male and female (P>0.05). The antibody positive rate of hamster kidney cell rabies vaccine was 89.94% with 95%CI 86.09% to 95%, while the antibody positive rate of vero cell rabies vaccine was 96.65% with 95%CI 94.99% to 94.99%, and there was significant difference between both groups (P<0.05). There was no statistical difference in antibody positive rates among different years of rabies vaccine (P>0.05). However, the antibody positive rate of rabies vaccine had a tendency to reduce with the increasing age ConclusionAntibody positive rate of vero cell rabies vaccine is higher than that of hamster kidney cell rabies vaccine. Older people have lower antibody positive rate after receiving rabies vaccination. We suggest using vero cell rabies vaccine when giving rabies vaccination; elderly people should receive booster vaccination after basic vaccination.
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 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 investigate the effect of canine decellularized tendon slices (DTSs) on tendon-bone healing in repairing rotator cuff injury of rabbit. Methods Canine DTSs were prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours from the adult Beagles Achilles tendons. Histological observation and cytocompatibility evaluation for the canine DTSs were performed in vitro. Twenty-four mature male New Zealand white rabbits, weighing 2.5-3.0 kg, were randomly selected. U-shaped defect of more than 50% of normal tendon in width and 8 mm in length was made in infraspinatus tendons of unilateral limb as the experimental group; the canine DTSs were used to repair defect, and the insertion of infraspinatus tendon on greater tuberosity of humerus was reconstructed in the experimental group. No treatment was done on the contralateral limb as the control group. At 4, 8, and 12 weeks after operation, the specimens were harvested for histological observation and biomechanical test. Results Histological examination showed that collagen fibers of canine DTSs were well preserved, without residual cells. The cytocompatibility examination showed that fibroblasts attached well to canine DTSs. Biomechanical test showed that the maximum load and stiffness increased significantly with time, and the maximum load and stiffness at 12 weeks were significantly higher than those at 4 and 8 weeks (P lt; 0.05). The maximum load and stiffness of the experimental group at 4 and 8 weeks were significantly lower than those of the control group (P lt; 0.05). The stiffness of the experimental group at 12 weeks was significantly lower than that of the control group (t= — 5.679, P=0.000), but no significant difference was found in the maximum load at 12 weeks between 2 groups (t=0.969, P=0.361). Histological observation showed that the control group displayed a 4-layer structure of the tendon-bone insertion. In the experimental group at 4 weeks, the tendon-bone interface was filled with granulation tissue, and a small amount of Sharpey’s fibers-like connected the tendon to bone; granulation tissue disappeared, and fibroblasts, Sharpey’s fiber, new cartilage, and chondrocytes significantly increased with time; tendon-bone interface became mature, but the tide line was not observed between the unmineralized fibrocartilage and mineralized fibrocartilage. Conclusion Canine DTSs prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours, can enhance the healing of host tendon-bone and improve the biomechanical characteristics of the rabbit infraspinatus tendon.
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 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 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.
Objective To investigate the effect of imbedding chemotherapy of sustained release of 5-fluorouracil on the healing of colonic stoma in dog. Methods Twenty-eight adult hybrid dogs were randomly divided into chemotherapy group （n=22) and control group (n=6). The canine sigmoid colon were firstly detached and then anastomosed via median abdominal incision, 200 mg sustained release of 5-fluorouracil was imbedded in the mesentery 1.0-1.5 cm away from colonic stoma in chemotherapy group, whereas the control substance was injected into the dogs in control group. Tissue samples were collected from mesentery and stomas on 3, 5, 7, 10 and 15 days after operation, respectively, in order to observe the healing of stoma. The drug concentrations in the stoma and in the tissues that were 0, 1, 3, 5, 7, 10 and 15 cm away from the imbedding point were also measured by high performance liquid chromatographymethod at different phases. Results The tissues from colonic stoma only showed inflammatory reaction at early stage, with no necrosis and cellular degeneration. It was observed that the stoma healed basically on the tenth day after operation. The drug concentrations in the tissues gradually decreased at the range of 0-15 cm over time, but all of which were higher than the anti-tumor effective concentration （0.10 μg/g）. Conclusion The imbedding chemotherapy of sustained release of 5-fluorouracil in mesentery has little effect on the healing of stoma, and it could remain an effective anti-tumor concentration in a period of time.
Abstract: Objective To observe the changes in morphology, structure, and ventricular function of infarct heart after bone marrow mononuclear cells (BMMNC) implantation. Methods Twenty-four dogs were divided into four groups with random number table, acute myocardial infarction (AM I) control group , AM I-BMMNC group , old myocardial infarct ion (OMI) control group and OM I-BMMNC group , 6 dogs each group. Autologous BMMNC were injected into infarct and peri-infarct myocardium fo r transplantation in AM I-BMMNC group and OM I-BMMNC group. The same volume of no-cells phosphate buffered solution (PBS) was injected into the myocardium in AM Icontrol group and OM I-control group. Before and at six weeks of cell t ransplantation, ult rasonic cardiography (UCG) were performed to observe the change of heart morphology and function, then the heart was harvested for morphological and histological study. Results U CG showed that left ventricular end diastolic dimension (LV EDD) , left ventricular end diastolic volume (LVEDV ) , the thickness of left ventricular postwall (LVPW ) in AM I-BMMNC group were significantly less than those in AM I-control group (32. 5±5. 1mm vs. 36. 6±3. 4mm , 46. 7±12. 1m l vs. 57. 5±10. 1m l, 6. 2±0. 6mm vs. 6. 9±0. 9mm; P lt; 0. 05). LVEDD, LVEDV , LVPW in OM I-BMMNC group were significantly less than those in OM I-control group (32. 8±4. 2 mm vs. 36. 8±4. 4mm , 48. 2±12. 9m l vs. 60.6±16.5m l, 7. 0±0. 4mm vs. 7. 3±0. 5mm; P lt; 0. 05). The value of eject fraction (EF) in OM I-BMMNC group were significantly higher than that in OM I-control group (53. 3% ±10. 3% vs. 44. 7%±10. 1% ). Compared with their control group in morphological measurement, the increase of infarct region thickness (7. 0 ± 1. 9mm vs. 5. 0 ±2.0mm , 6.0±0. 6mm vs. 4. 0±0. 5mm; P lt; 0. 05) and the reduction of infarct region length (25. 5±5. 2mm vs. 32. 1±612mm , 33. 6±5. 5mm vs. 39. 0±3. 2mm , P lt; 0. 05) were observed after transplantation in AM I-BMMNC group and OM I-BMMNC group, no ventricular aneurysm was found in AM I-BMMNC group, and the ratio between long axis and minor axis circumference of left ventricle increased in OM I-BMMNC group (0. 581±0. 013 vs. 0. 566±0.015; P lt; 0. 05). Both in AM I-BMMNC group and OM I-BMMNC group, fluorescence expressed in transplantation region was observed, the morphology of most nuclei with fluorescencew as irregular, and the differentiated cardiocyte with fluorescence was not found in myocardium after transplantation. The histological examination showed more neovascularization after transp lantation both in AMI and in OM I, and significant lymphocyte infiltration in AM I-BMMNC group. Conclusion BMMNC implantation into infarct myocardium both in AMI and OMI have a beneficial effect, which can attenuate deleterious ventricular remodeling in morphology and st ructure, and improve neovascularization in histology, and improve the heart function.