Objective To investigate the origin of small saphenous vein of distally-based of sural nerve nutrient vessels flap and its clinical application. Methods The origins of nutrient vessels of small saphenousvein and communicating branches of superficial-deep vein were observed on specimens of 30 adult cadaveric low limbs by perfusing red gelatin to dissect the artery. Results The nutrient vessels of small saphenous vein originated from the heel lateral artery, the terminal perforator branches of peroneal artery and intermuscular septum perforating branches of peroneal artery. There were 2 to 5 branches ofsuch distally-based perforating branches whose diameters ranged from 0.6 to 1.0 mm. Those perforating branches included fascia branches, cutaneous branches nerve and vein nutrient branches. Those nutrient vessels formed a longitudinalvessel chain of sural nerve shaft, vessel chain of vein side and vessel networkof deep superficial fascia. The small saphenous vein had 1 to 2 communicating branches of superficial-deep vein whose diameter was 1.7±0.5 mm， 3.4±0.9 cm to the level of cusp of lateral malleolus, and converged into the fibular vein. Conclusion Distally-based sural nerve, small saphenous vein, and nutrient vessles of fascia skin have the same region. The communicating branches of superficial-deep vein is 3 to 4 cm to the level of cusp lateral malleolus. These communicating branches could improve the venousdrainage of the flap.
Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
Objective To study the development, investigation, and application of the artificial vertebral body so as to provide an essential reference for the future research and clinical application. Methods The recent articles on materials, types, and clinical applications of the artificial vertebral body were reviewed.Results The materials used for the artificial vertebral body were porcelain, alloy, variant bone, and composite. But each of them had its own advantages and disadvantages. The types of the artificial vertebral body were grouped as expandable and non-expandable ones; however, the expandable type was much better. The artificial vertebral body had been applied to the treatments of spinal tumor, tuberculosis, fracture, and infection, with better effects. Conclusion The artificial vertebral body can beextensively applied. However, the materials and types need to be improved.
Objective To assess the curative effect of the subtalararthrodesis on the serious subtalar joint with the posterior tibial tendon dysfunction.Methods From October 2000 to February 2006, 31 patients (18 males, 13 females; age 23-62 years, averaged 36.4years) with serious subtalar joint osteoarthrisis and stage Ⅱ posterior tibial tendon dysfunction were treated by the subtalar arthrodesis. The tibial tendon dysfunction involved 15 right and 16 left lower extremities, which were caused by retrograde osteoarthritis in 14 patients,sequel of an injury in 8 patients, infection in 7 patients, and anatomic structural abnormity in 2 patients. The treatment course averaged 9.5 months (range, 6-30 months). Before the subtalar arthrodesis, the injured tendons were repaired, and then the bone grafting was performed in the tarsus sinus. All of the patients were assessed before and after operation according to the Hindfoot scores system (American Orthopedics Foot and Ankle Society, AOFAS). Results Among the patients, 28 were followed up on an average of 23.6 months (range, 8-61 months). The AOFAS scores ranged from 45.30±1.08 before operation to 79.60±2.14 afteroperation. The pain indexes ranged from 15.40±2.23 before operation to 38.50±2.61 after operation. The functional indexes of the foot and ankle joint ranged from averaged 21.60±3.01 before operation to averaged 37.40±2.83 after operation. The statistical analysis of the t-test on all the above data showed that there was a significant difference between beforeoperation and after operation (P＜0.01). The angles between the longitudinal line of the talar and the calcaneal bone were 43.70±1.06° before operation and 29.40±0.98° after operation, and the deviation angles between the calcanealline and the talus were 48.20±0.85° before operation and 39.40±1.02° after operation. There was a significant difference between before operation and after operation (P＜0.01). Conclusion The subtalar arthrodesis combined with the bone grafting in the tarsus sinus and the repair of the injured tendons can effectivelycorrect the deformity of the deformity of the metapodium, relieve the pain, retin the adjacent joint motion ability, and this method can be recommended for the adult patient who suffers from serious subtalar osteoarthritis and stage Ⅱ osterior tibial tendon dysfunction.
Objective To establish an effective way to cryopreserveprecartilaginous stem cells(PSCs) of neonate rat. Methods PSCs ［fibroblast growth factor-3(FGFR-3) positive cells］ were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction(RT-PCR), immunohistochemistry, and immunofluorescence. Results Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be dectected by RT-PCR in cryopreserved PSCs.Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo.Conclusion Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.
ObjectiveTo determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides(ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs(2.5，5.0，7.5，10.0 and 12.5 μl) were put into 100 μl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50，100，150，200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution ofSP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.Results The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a ber fluorescence intensitywith a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12±3.92 U/L in the 72hgroup, which was significantly higher those that in the 26h group（49.61±17.13 U/L）and the 48h group（120.26±8.42 U/L）(Plt;0.01). At 26 h after the transfection, there were no statistical differences at the above LDHlevels in the different-concentration groups（Pgt;0.05）. Conclusion Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
Objective To evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repairof tibial defects. Methods Twelve goats were made 2.1 cm segmental defects in he tibial diaphysis and divided into 2 groups. AdvhBMP2 transfected marrow mesenchymal stem cells(MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n=7) and untransfected group (n=5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213±1.278) and the untransfected group(－0.310±0.147，Plt;0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544±0.957) and the untransfected group (3.104±0.644，Pgt;0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359±0226, 2.297±0.200, 2.214±0.215 and 2.297±0.210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0.874±0.524 in untransfected group, being significant differences betweentwo groups(Plt;0.05). Conclusion Adenovirus mediated BMP-2gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.
Objective To resolve the tough problem of how to observe the growing cells in an opaque vector. Methods The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethralepithelial cells were cultured in the collagen chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetateacetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. Results The urethral epithelial cells grew and proliferated very well in the collagen chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were(1.09±0.13)×10.8 and (2.04±0.13)×10.8, respectively. However, after 14and 21 days, the fluorescent density amount of the surviving cells was (0.55± 0.09)×10.8 and (0.47±0.03)×108, respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days（P＜0.05）.Conclusion Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidlymeasured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.
Objective To establish the artificial bladder reflex arc by the normal body reflex pathway above the horizon of spinal cord injury to reinnervate the flaccid bladder and restore bladder micturition function. Methods An intradural microanastomosis was performed on the L6 ventral root tothe S2 ventral root. After axonal regeneration,the “patellar ligament-spinal cord center-bladder” reflex pathway was reestablished. A longterm function of the reflex arc was observed in the nerve electrophysiological experiment, detrusor electromyography experiment, and urodynamic testing 8 months after anastomosis. Results Trains of the stimuli(200 μV,5 ms) in the left L6 dorsal root and the nerve at the anastomosizedsite resulted in motor evoked potential from the disal to the anastomosized site before and after the spinal cord was destroyed horizontally between S1 and S4 segment levels in 2 Beegle dogs.The figure and amplitude of the evoked potential were similar to those of the control and general stability which showed anoninterventional wave. The urodynamic test revealed a rapid increase of the bladder pressure and a minor increase in the abdominal pressure. This showed that the bladder detrusor mainly resulted in the pressure increase.The bladder pressure increased to 60% of the normal on average compared with the controls when resulted in the left L6 dorsal root and the nerve anastomosized site were stinulated. Conclusion The long-term observation by the nerveelectrophysiological experiment, detrusor electromyography experiment, and urodynamic test indicate that the new artificial reflex arc can be established successfully. The somatic motor axons can regenerate into the parasympathetic endoneurial tubes of the autonomic nerve.
Objective To investigate the effects of ectomesenchymalstem cells on hematopoiesis after total body irradiation in rats. Methods The primary ectomesenchymal stem cells were isolated from E11.5 SD fetal mandibular processes by 25g/L trypsin and cultured with DMEM/F12. The morphology and growthrate were observed by inverted microscope. Eighty SD male rats randomly dividedinto ectomesenchymal stem cells group (n=20), fibroblast group(n=20), saline group(n=20) and control group(n=20), the first three groups were irradiated with 60Co γ rays at 6.0 Gy. The number of their bone marrow nucleated cells was counted after 4 weeks; the forming ability of colony-forming unit-granulocyte macrophage(CFU-GM) and histopathology of bone marrow were also observed. Results The cultured cells displayed monolayer growth and fibroblast-like with 2-4 processes. The ectomesenchymal stem cells could increase the number of bone marrow nucleated cells and peripheral blood white cell count, and improve the forming ability of CFU-GM. After 4 weeks of transplantation, the number of the peripheral blood white cells in group A was more than that in groups B and C(Plt;0.05), the contents of Hb in groups A and D was significantly higher than those in groups B and C(Plt;0.0). After 4 weeks, the bone morrow nucleated cells in group A were significant more than those in groups B and C(Plt;001); CFU-GM in groups A and D was higher than that in groups B and C(Plt;0.01). Conclusion Ectomesenchymal stem cells have characteristics of stem cells. It may improve hematopoiesis recovery of irradiated rats.