ObjectiveTo evaluate the effect of using Schwann-like cells derived from human umbilical cord blood mesenchymal stem cells (hUCBMSCs) as the seed cells to repair large sciatic nerve defect in rats so as to provide the experimental evidence for clinical application of hUCBMSCs. MethodsFourty-five male Sprague Dawley (SD) rats in SPF grade, weighing 200-250 g, were selected. The hUCBMSCs were harvested and cultured from umbilical cord blood using lymphocyte separating and high molecular weight hydroxyethyl starch, and then was identified. The hUCBMSCs of 3rd generation were induced to Schwann-like cells, and then was identified by chemical derivatization combined with cytokine. The acellular nerve basal membrane conduit was prepared as scaffold material by the sciatic nerve of SD rats through repeated freezing, thawing, and washing. The tissue engineered nerve was prepared after 7 days of culturing Schwann-like cells (1×107 cells/mL) on the acellular nerve basal membrane conduit using the multi-point injection. The 15 mm sciatic nerve defect model was established in 30 male SD rats, which were randomly divided into 3 groups (10 rats each group). Defect was repaired with tissue engineered nerve in group A, with acellular nerve basal membrane conduit in group B, and with autologous sciatic nerve in group C. The nerve repair was evaluated through general observation, sciatic function index (SFI), nerve electrophysiology, weight of gastrocnemius muscle, and Masson staining after operation. ResultsThe hUCBMSCs showed higher expression of surface markers of mesenchymal stem cells, and Schwann-like cells showed positive expression of glia cell specific markers such as S100b, glial fibrillary acidic protein, and P75. At 8 weeks after operation, the acellular nerve basal membrane conduit had no necrosis and liquefaction, with mild adhesion, soft texture, and good continuity at nerve anastomosis site in group A; group B had similar appearance to group A; adhesion of group C was milder than that of groups A and B, with smooth anastomotic stoma and no enlargement, and the color was similar to that of normal nerve. SFI were gradually decreased, group C was significantly greater than groups A and B, group A was significantly greater than group B (P<0.05). The compound action potential could be detected in anastomotic site of 3 groups, group C was significantly greater than groups A and B, and group A was significantly greater than group B in amplitude and conduction velocity (P<0.05). Atrophy was observed in the gastrocnemius of 3 groups; wet weight's recovery rate of the gastrocnemius of group C was significantly greater than that of groups A and B, and group A was significantly greater than group B (P<0.05). Masson staining showed that large nerve fibers regeneration was found in group A, which had dense and neat arrangement with similar fiber diameter. The density and diameter of medullated fibers, thickness of myelinated axon, and axon diameter of group C were significantly greater than those of groups A and B, and group A was significantly greater than group B (P<0.05). ConclusionTissue engineered nerves from hUCBMSCs-derived Schwann-like cells can effectively repair large defects of the sciatic nerve. hUCBMSCs-derived Schwann-like cells can be used as a source of seed cells in nerve tissue engineering.
【摘要】 目的 探讨高原地区桡神经损伤的治疗效果，并总结影响疗效的因素。 方法 回顾性分析2005年6月-2010年6月收治的桡神经损伤并有完整随访资料的54例患者，其中男40例，女14例；年龄8～69岁，平均32.6岁。开放性损伤5例，闭合性损伤49例；左侧26例，右侧28例。受伤原因:刀伤5例，医源性损伤（手术牵拉伤、被钢板挤压伤）10例，肱骨干骨折合并桡神经损伤39例。神经损伤类型:桡神经完全断裂12例；大部分断裂15例；挫伤27例，挫伤长度1.5~4.5 cm。所有患者均有典型的感觉及运动功能障。采用神经吻合修复27 例，神经松解减压27例。骨折均用钢板内固定。 结果 所有患者手术均顺利，术后切口均I期愈合，无手术相关并发症发生。54例均获随访16~24个月，平均18个月。骨折于术后8～14个月达临床愈合。末次随访时根据中华医学会手外科上肢周围神经功能评定标准，神经吻合的27例中，获优14例，良8例，差5例；神经松解减压术治疗的27例均获优。总优良率为91%。 结论 上臂桡神经损伤宜早期手术修复，神经吻合的疗效较神经松解减压术差。【Abstract】 Objective To explore the therapeutic effect on radial nerve injuries in plateau area, and to analyze the influencing factors. Methods The clinical data of 54 patients with radial nerve injuries who were treated between June 2005 and June 2010 were retrospectively analyzed. The patients included 40 males and 14 females and aged 8-69 years (averaged 32.6 years old). Of these 54 patients, 5 were open injuries, 49 were closed injuries; 26 were on the left side, and 28 were on the right sides. Causes of injuries included: 5 direct cut injuries, 10 iatrogenic injuries (including traction injuries and crush injuries by steel plates), and 39 humeral shaft fracture and radial nerve injuries. Types of nerve injuries included: 12 complete radial neurotmesis, 15 partial radial neurotmesis, and 27 radial contusions (with contusion length ranged 1.5-4.5 cm). All patients had radial nerve injuries experienced significant motor dysfunctions. Among these patients, 27 underwent nerve anastomosis, the remaining 27 were treated by nerve decompression; all fractures were treated with internal fixation with steel plates. Results During the average follow-up of 18 months (16-24 months), all 54 patients completely recovered from radial nerve injuries without any complications. The time for fracture healing ranged 8-14 months. According to the evaluation standards for radial nerve functional recovery, developed by the Chinese Medical Association, among the 27 cases treated by nerve anastomosis, 14 were “optimal”, 8 were “fair”, and 5 were “bad”; and all 27 cases treated by nerve decompression were “optimal”. Conclusion It is suggested to have early surgical treatment for the upper arm radical nerve injuries. The nerve decompression had better curative effects than the nerve anastomosis does.
Objective To obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.
Objective To analyze the therapy and effectiveness of ulnar styloid fracture complicated with wrist dorsal branch of ulnar nerve injury. Methods Between October 2005 and October 2012, 16 cases of ulnar styloid fracture complicated with wrist dorsal branch of ulnar nerve injury were treated. There were 14 males and 2 females with an average age of 42 years (range, 22-58 years). Fracture was caused by traffic accident in 8 cases, by mechanical crush in 5 cases, and by falling in 3 cases. According to the anatomical features of the ulnar styloid and imaging findings, ulnar styloid fractures were classified as type I (ulnar styloid tip fracture) in 1 case and type II (ulnar styloid base fracture) in 15 cases. The skin sensation of ulnar wrist was S0 in 5 cases, S1 in 1 case, S2 in 7 cases, and S3 in 3 cases according to the criteria of the British Medical Research Council in 1954 for the sensory functions of the ulnar wrist. The time from injury to operation was 6-72 hours (mean, 18 hours). Fracture was treated by operative fixation, and nerve was repaired by epineurium neurolysis in 13 cases of nerve contusion and by sural nerve graft in 3 cases of complete nerve rupture. Results All incisions healed by first intention. Sixteen patients were followed up for an average time of 14 months (range, 6-24 months). The X-ray films showed that all of them achieved bone union at 4-10 weeks after operation (mean, 6 weeks). No patient had complications such as ulnar wrist chronic pain and an inability to rotate. According to Green-O’Brien wrist scoring system, the results were excellent in 13 cases and good in 3 cases; according to the criteria of the British Medical Research Council in 1954 for the sensory functions of the ulnar wrist, the results were excellent in all cases, including 11 cases of S4 and 5 cases of S3+. Two-point discrimination of the ulnar wrist was 5-9 mm (mean, 6.6 mm). Conclusion For patients with ulnar styloid fracture complicated with wrist dorsal branch of ulnar nerve injury, internal fixation and nerve repair should be performed. It can prevent ulnar wrist pain and promote sensory recovery.
Objective To review the possible mechanisms of the mammal ian target of rapamycin (mTOR) in theneuronal restoration process after nervous system injury. Methods The related l iterature on mTOR in the restoration ofnervous system injury was extensively reviewed and comprehensively analyzed. Results mTOR can integrate signals fromextracellular stress and then plays a critical role in the regulation of various cell biological processes, thus contributes to therestoration of nervous system injury. Conclusion Regulating the activity of mTOR signaling pathway in different aspects cancontribute to the restoration of nervous system injury via different mechanisms, especially in the stress-induced brain injury.mTOR may be a potential target for neuronal restoration mechanism after nervous system injury.
ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
ObjectiveTo investigate the feasibility of the 3rd-6th intercostal nerve transfer to the suprascapular nerve for reconstruction of shoulder abduction. MethodsFifteen thoracic walls (30 sides) were collected from human cadavers. The 3rd-6th intercostal nerve length which can be dissected between the midaxillary line and midclavicular line, and the transfer distance between the midaxillary line and midpoint of the clavicular bone (prepared point for neurotization) were measured. ResultsIn 30 sides of specimens, the 3rd and 4th intercostal nerves could be obtained between the midaxillary line and midclavicular line, the available length of which was significantly greater than the transfer distance (P lt; 0.01). Six sides of the 5th intercostal nerve and 16 sides of 6th intercostal nerve were covered by the costal cartilage before reaching the midclavicular line. The available length of the 5th intercostal nerve was similar to the transfer distance (P gt; 0.01), while the available length of the 6th intercostal nerve was significantly less than transfer distance (P lt; 0.01). The suprascapular nerve could be dissociated and turned to the clavicular bone of more than 2 cm. The whole length of the available 5th intercostal nerve length and the turning length (2 cm) of suprascapular nerve was significantly greater than the transfer distance (P lt; 0.01), but for the 6th intercostal nerve, the whole length was still less than transfer distance (P lt; 0.01). ConclusionIt could be an alternative method to use the 3rd, 4th, and 5th intercostal nerve transfer to the suprascapular nerve for reconstruction of shoulder abduction. And for the 6th intercostal nerve, longer dissociated length may be required for direct coaptation or using a graft for nerve repair.
Objective To investigate the effects of 3 methods (suture removal, suture removal with epineurium neurolysis, and l igated femoral nerve resection with end-end suture) in repairing femoral nerve injury after l igation in different periods so as to provide a reference for cl inical use of repairing iatrogenic l igation injury of the peri pheral nerve. Methods A total of 120 adult female Sprague Dawley rats, weighing (200 ± 20) g, were used to prepare the animal models of left femoralnerve l igation, and were divided into groups A (n=40), B (n=40), and C (n=40) according different repairing methods. Atimmediate, 1, 3, and 5 months (10 rats each time point) after l igation, suture removal was performed in group A, suture removal with epineurium neurolysis in group B, and l igated femoral nerve resection with end-end suture in group C. At 3 months after operation, the foot-base angle (FBA) and the heels-tail angle (HTA), action potential and conduction velocity of femoral nerve, and wet weight of quadriceps femoris muscle (QFM) were measured; the samples of quadriceps femoris and femoral nerve were harvested for histological observation, muscle fiber count, and nerve fiber passing rate measuring. Results The FBA in group A was significant smaller than that in group C at immediate, 1, 3, and 5 months (P lt; 0.05), but there was no significant difference between groups A and B (P gt; 0.05). The HTA in group A was significantly smaller than that in group C at immediate, 1, 3, and 5 months (P lt; 0.05), and the THA in group B was significantly smaller than that in group C at 1, 3, and 5 months (P lt; 0.05). The wet weight of QFM in group B was significantly higher than that in group C at immediate, 3, and 5 months (P lt; 0.05), and the wet weight of QFM in group A was significantly higher than that in group C at immediate and 3 months (P lt; 0.05), but no significant difference was found between groups A and B at immediate, 1, and 3 months (P gt; 0.05). There was significant difference in the action potential of femoral nerve between group A and groups B and C at immediate and 1 month (P lt; 0.05), but there was no significant difference between other groups at 3 and 5 months (P gt; 0.05) except between groups A and C at 5 months (P lt; 0.05). The conduction velocity of femoral nerve in group A was significantly faster than that in group C at immediate, 1, and 5 months (P lt; 0.05), and it was significantly faster in group A than in group B at immediate and 1 month (P lt; 0.05), but no significant difference was found between groups A and B at 3 and 5 months (P gt; 0.05), between groups B and C at other time points (P gt; 0.05) except at immediate (P lt; 0.05). The count of muscle fibre of the quadriceps femoris was significantly more in groups A and B than in group C at immediate (P lt; 0.05); it was significantly more in group A than in group B at 5 months (P lt; 0.05). The passing rate of the femoral nerve fiber was significantly higher in group A than in groups B and C at 3 months (P lt; 0.05), but no significant difference was found between the other groups (P gt; 0.05). Conclusion After femoral nerve l igation, suture removal method has the best effect at early term, the next is epineurium neurolysis method, and the worst is the l igation femoral nerve resection with end-end suture repair.
Objective To review the basic researches and the cl inical appl ication of the nano-neural tissue engineering materials, especially the electrically conductive carbon nanotubes (CNT). Methods The l iterature concerning the basic and cl inical researches of the conductive materials of nano-neural tissue engineering, especially the electrically conductive CNT were reviewed. Results The researches of conductive materials of nano-neural tissue engineering have made some progress, the electrically conductive CNT can not only promote Schwan cells’ adhension, migration, and prol iferation, but also mimic the function of electric conductivity of neural myel in and enhance neurite growth and regeneration. So the electrically conductive CNT make great sense in stimulating and directing the growth of neurite and the regeneration of axons. Conclusion Because of these unique properties, the electrically conductive CNT have great advantages in peripheral nerve repair and function reconstruction, and are promising to provide a novel method for cl inical peri pheral nerve repair and function reconstruction after injury.
Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) embedded in fibrin glue around chemical extracted acellular nerve allograft (CEANA) on the peripheral nerve regeneration. Methods Twenty-oneadult male C57 mice (weighing 25-30 g) and 15 adult male Balb/c mice (weighing 25-30 g) were selected. The sciatic nerves were harvested from the Balb/c mice to prepare CEANA. The BMSCs were isolated from 3 C57 mice and were cultured; BMSCs embedded in fibrin glue were cultured for 3, 7, 14, and 21 days. Then the supernatant was obtained and co-cultured with PC12 cells for 2 days to observe the PC12 cell growth in vitro. The other 18 C57 mice were used to establ ish the left sciatic nerve defect models of 10 mm and divided into 3 groups: autogenous nerve graft with fibrin glue (group A, n=6), CEANA graft with BMSCs (5 × 106) embedded in fibrin glue (group B, n=6), and CEANA graft with fibrin glue (group C, n=6). The right sciatic nerves were exposed as the controls. At 2, 4, 6, and 8 weeks, the mouse static sciatic index (SSI) was measured. The histomorphometric assessment of triceps surae muscles and nerve grafts were evaluated by Masson staining, toluidine blue staining, and transmission electron microscope (TEM) observationat 8 weeks after operation. Results BMSCs were uniform distribution in fibrin glue, which were spherical in shape, and the cells began to grow apophysis at 3 days. PC12 cells differentiated into neuron-l ike cells after addition supernatant co-cultured after 2 days. Incisions healed well in each group. At 2, 4, 6, and 8 weeks, the SSI increased gradually in 3 groups. SSI in group A was higher than that in groups B and C at 4, 6, and 8 weeks after operation (P lt; 0.05). SSI in group B was sl ightly higher than that in group C, but had no significant difference (P gt; 0.05). At 8 weeks, the wet weight recovery rate of triceps surae muscles and fibers number of myel inated nerve were better in group B than in group C, but worse in group B than in group A, showing significant differences (P lt; 0.05). The triceps surae muscle fibers area and myel in sheath thickness had significant differences between group B and group C (P lt; 0.01), but there was no significant difference between group A and group B (P gt; 0.05). Conclusion BMSCs embedded in fibrin glue around CEANA can improve functional recovery of peripheral nerve injury.