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.
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.
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 summarize the recent advance in the research of tissue engineered nerve grafts. Methods The cl inical and experimental research papers about tissue engineered nerve grafts were extensively reviewed and analyzed. Results The porosity, mechanical property and surface topography of a nerve scaffold, which was either made up of natural biodegradable polymers or synthetic polyesters, were pivotal factors that influenced the capacity of the scaffold in supporting nerve regeneration. Of various candidate supporting cells for nerve tissue engineering, the bone marrowmesenchymal stem cells had been paid more attention because of their advantages. Several model designs of drug del ivery systems for controlled release of growth factors had been attempted. In cl inical settings, short nerve gaps were demonstrated to be treatable with several nerve conduits which were commercially available, with functional recovery approximating tonerve autografting. Conclusion The field of nerve tissue engineering has witnessed a rapid development not only in experimental research but also in cl inical appl ication.
Objective To construct adenovirus expressing NGF (Ad-NGF) and to investigate its promotive effect on the reparation and regeneration of sciatic nerve injury in rats. Methods NGF gene sequence was cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK-293 cells, the recombinant adenoviruses-Ad-NGF underwent sequence identification. Thirty-two male SD rats weighing 180-200 g were randomly divided into 4 groups (n=8 rats per group). Sciatic nerve injury model was establ ished by disconnecting and direct suturing the right sciatic nerve in the rat. Theright gastrocnemius muscle of group A and C received Ad-NGF injection and adenovirus vector without NGF gene sequence injection, respectively, and 1 × 108 PFU/per time was given every other day for three times. Group B and D received NGF injection (200 U/d) and normal sal ine (100 ?L/d), respectively, for 3 weeks. The effect of various treatments on injured sciatic nerve was evaluated by performing sciatic nerve function index and nerve electrophysiology detections 31 days after operation. Meanwhile, the sciatic nerve in the anastomosis and at the site 1 cm distal to the anastomosis were obtained, and underwent RTPCR and Western blot analysis for detecting NGF mRNA and protein expression level in the injured sciatic nerve in the rats. Histology, immunohistochemistry, and transmission electron microscope observations were conducted. Results Ad-NGF carrying NGF gene sequence was constructed successfully and confirmed by sequence analysis. The sciatic nerve function index, nerve conduction velocity, evoked potential ampl itude, and latent period of group A was better than those of other groups (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). RT-PCR and Western blot detection: the expression levels of NGF mRNA and protein in group A were greater than those of group B, C, and D (P lt; 0.05), and no significant differences were noted among group B, C, and D (P gt; 0.05). Histology and immunohistochemistry observation showed that the regeneration of the sciatic nerve in group A was obvious superior to that of other groups. Transmission electron microscopy observation suggested there was significant difference between group A and groups B, C, and D in terms of axonal diameter of sciatic nerve cross-section, myel in sheath thickness and nerve fiber number (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). Conclusion Ad-NGF can effectively promote the repair of sciatic nerveinjury in rats, and is a new method for obtaining large amounts of NGF in the area of injured peripheral nerve.
OBJECTIVE In order to investigate the opportunity of repair and prognosis of recurrent laryngeal nerve injuries after thyroidectomy. METHODS Twelve cases with recurrent laryngeal nerve injuries after thyroidectomy were immediately and delayed operated on nerve repair and reinnervation. In immediate operation, 5 cases were repaired by direct recurrent laryngeal nerve suture, and 1 case was treated by transposition of the phrenic nerve to the recurrent laryngeal nerve and sutured the adductor branch to the branch of ansa cervicalis. In delayed operation, 3 cases were treated by anastomosis the main trunk of ansa cervicalis to the adductor branch of recurrent laryngeal nerve, and 3 cases were operated on neuromuscular pedicle to reinnervate posterior cricoarytenoid muscle. RESULTS Followed up 6 months, the effect was excellent in 1 case who was immediately operated by selective reinnervation of the abductor and adductor muscles of the larynx, better in 9 cases, and poor in 2 cases who were delayed operated over 12 months. CONCLUSION It can be concluded that the earlier reinnervation is performed, the better prognosis is.
ObjectiveTo summarize the research progress of autologous vein nerve conduit for the repair of peripheral nerve defect. MethodsThe recent domestic and foreign literature concerning autologous vein nerve conduit for repair of peripheral nerve defect was analyzed and summarized. ResultsA large number of basic researches and clinical applications show that the effect of autologous venous nerve conduit is close to that of autologous nerve transplantation in repairing short nerve defect, especially the compound nerve conduit has a variety of autologous nerve tissue, cells, and growth factors, etc. ConclusionAutologous vein nerve conduit for repair of non-nerve defect can be a good supplement of autologous nerve graft, improvement of autologous venous catheter to repair peripheral nerve defect is the research direction in the future.