In order to observe the role of genetically modified Schwann cell (SC) with pSVP0Mcat in the regeneration of injured spinal cord, the cells were implanted into the spinal cord. Ninety SD rats were used to establish a model of hemi-transection of spinal cord at the level of T8, and were divided into three groups, randomly, that is, pSVP0Mcat modified SC implantation (Group A), SC implantation (Group B) and without cell implantation as control (Group C). After three months the presence of axonal regeneration of the injured spinal cord was examined by means of horseradish peroxidase (HRP) retrograde labelling technique and stereography. The results indicated that HRP labelled cells in Group A and B could be found in the superior region of injured spinal cord and the brain stem such as the red nuclei and oculomotor nuclei. The density of ventral hom neurons of the spinal cord and the number of myelinated axons in 100 microns of the white matter was A gt; B gt; C group. In brief, the pSVP0Mcat modified SC intraspinal implantation could promote regeneration of the injured spinal cord.
Objective To investigate an effect of differenttemperature cryopreservation of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat. Methods Eighty femaleSD rats were randomly divided into 8 groups of 10 rats each. One was the control group and 7 were the experimental groups. Two 2-cm-long sciatic nerve segments were respectively taken from both legs of each rat. In the control group, the sciatic nerve segments did not undergo the treatment of cryopreservation; however, in the 7 experimental groups, the sciatic nerve segments respectively underwent the different temperature cryopreservation of the twostep freezing method at -20℃, -30℃, -40℃, -50℃, -60℃, -70℃ and -80℃. The sciatic nerve segments were cryopreserved for 2 hours，and then placed into the liquid nigrtrogen at -196℃. After 48 hours of storage，the nerve segments werethawed quickly in the 37℃ water bath box for 1 minute. Then, the sciatic nerve segments each group were harvested. The cells of the sciatic nerve were incubated with Calcein-AM for 15 minutes. The average fluorescence intensity of the cells was measured by the flow cytometry. The nerve fibers were also incubated with Calcein-AM for 15 minutes. The fluorescence intensity of the cells was analyzed by the confocal fluorescence microscope. The Schwann cell biological activity intensity was measured. Results The fluorescence intensity in the -40℃ group was the best and the Schwann cell biological activity in this group was thebest among all the groups（P＜0.01）. The fluorescence intensity in the 8 groups measured by the flow cytometry was as follows：242.522 0±9.568 4 in the control group，168.677 0±10.207 0 in the -20 ℃ group，214.992 0±8.329 1 in the -30 ℃ group，235.526 0±9.280 5 in the -40 ℃ group，222.434 0±8.515 5 in the -50 ℃ group，217.409 0±9.515 7 inthe -60 ℃ group，132.376 0±13.459 7 in the -70 ℃ group， and 108.132 0±16.033 1 in the -80 ℃ group. The fluorescence intensity detected by the confocal fluorescence microscope was as follows：143.700 0±5.567 8 in the control group，119.700 0±5.161 5 in the -20 ℃ group，121.300 0±4.347 4 in the -30 ℃ group，700 0±5.012 2 in the -40 ℃ group，121.000 0±4.546 1 in the -50 ℃ group，118.400 0±4.9261 in the -60 ℃ group，81.200 0±5.116 4in the -70 ℃ group，and 79. 000 0±5.716 4 in the -80 ℃ group. Conclusion The Schwann cell biological activity treated by the two-step freezing methodcan be preserved and the activity is cryopreserved best at -40 ℃.
ObjectiveTo investigate the mechanism of muscle-derived cells (MDCs) in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.MethodsThe hind limb skeletal muscles of mice carrying enhanced green fluorescent protein (EGFP) was collected to extract and culture EGFP-MDCs to P1 generation for later experiments. Five-mm-long nerve defects were created in the right sciatic nerves of C57BL/6 mice to establish a peripheral nerve defect model. The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane (PUR) conduit. For the MDC group, EGFP-MDCs were injected into the PUR conduit. The PUR group without EGFP-MDCs was used as the negative control group. At 1 and 2 weeks after operation, the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve. Immunofluorescence staining of S100β, the marker of Schwann cells, was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells, and observe the source of the Schwann cells expressing S100β. Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2 (p-ErbB2) and phosphorylated focal adhesion kinase (p-FAK) in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.ResultsThe general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation, while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation. Immunofluorescence staining showed that the Schwann cells expressing S100β in proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation. At 2 weeks after operation, the Schwann cells expressing S100β in the two nerve stumps of the MDC group were connected, but not in the PUR group. At 2 weeks after operation, the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation, and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation, the differences were all significant (P<0.05). At 1 week after operation, the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group (P<0.05). There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation (t=0.327, P=0.747), while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group (t=4.470, P=0.000). At 1 and 2 weeks after operation, the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group (P<0.05). At 1 and 2 weeks after operation, part of Schwann cells expressing S100β, which were derived from EGFP-MDCs, could be observed in the regenerated nerves of MDC group.ConclusionMDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice, and promote the migration of Schwann cells. MDCs can be differentiated into cells expressing the Schwann cell marker S100β, or as other cellular components, to involve in the early repair of peripheral nerves.
Objective To investigate the outcome of repairing the peripheral nerve defects with the tissue engineered nerve constructed by Schwann cells and fibrin glue. Methods Wallerian degenerated sciatic nerve were harvested from the 4-week-old New Zealand rabbits for culture of Schwann cells. The Schwann cells were then separated, amplified and purified, and then were identified by the S-100 protein immunochemical staining. The cultured Schwann cells (1×106/ml) were mixed with fibrin glue to form the Schwann cell-fibrin glue compound, which was observed by the inverted phase contrastmicroscope. The compound filled some silicone tubes (Group A) and biomembrane (Group B) to fabricate the tissue engineered nerves with a purpose of repairing the 10-mm defects in the New Zealand rabbit tibia nerves. The autologous nerve grafting was performed in Group C. The electrophysiological examination and the histomorphological analysis were performed at 10 weeks after the transplantation. Results All the rabbits survived through the experiment. In Group A, all the rabbits developed an ulcer in the soles of their left feet at 3-4weeks after the transplantation, while less ulceration developed in Groups B and C. At 10 weeks after the transplantation, the electrophysiological examination was performed, the elective stimulation failed to pass through the nerve grafts, and no composed muscular action potential was found in all the rabbits in Group A; the elective stimulation could pass through all the nerve grafts in Groups B and C, and could evoke the composed muscular action potential; the composed muscular action potential and the nerve conduct velocity in the two groups were 4.21±0.82 mV and 3.40±5.40 m/s vs. 4.80±1.15 mV and 36.55±6.43 m/s(Pgt;0.05). In Group A, no regrown axon was found in the nerve grafts, but neuromawas found to have formed in the both ends of the silicon tube. In Groups B and C, there was no obvious neuroma formation but regrown axons could be found to have regenerated. The histomorphological analysis on the regrown axons showed thatthere was no statistically significant difference between Groups B and C. Conclusion The tissue engineered nerve fabricated with Schwann cells, fibrin glue, and biomembrane can promote the nerve regeneration, and its reparative effect is similar to that of the autologous nerves; therefore, the future of its clinical practice is brilliant.
Schwann cells (SC) play an important role in nerve regeneration. The cultures of both human and rabbit SC (gt;99%) were obtained, and were separately derived from the sciatic nerve of the human fetus and the rabbit respectively by "the method of reexplantation". In addition, the cryostore and resuscitation of SC were carried out, and the resuscitated cells could retain their growth properties.
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.
ObjectiveTo systemically review the efficacy and safety of Schwann cells (SCs) or activated Schwann cells (ASCs) transplantation in the treatment of traumatic spinal-cord injury (TSCI) in rats models.
MethodsRandomized controlled trials (RCTs) about the effects of SCs and ASCs transplantation for TSCI in rats were searched in PubMed, EMbase, The Cochrane Library (Issue 12, 2014), CBM, CNKI, WanFang Data and VIP from inception to December 2014. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data, and assessed the risk of bias of included studies. Then meta-analysis was performed using RevMan 5.3 software.
ResultsA total of 14 RCTs involving 510 rats were included. The results of meta-analysis showed that:compared with the control group, the Basso, Beattie and Bresnahan (BBB) scores in the SCs or ASCs transplantation group were superior in 4 weeks (SMD=2.31, 95%CI 1.48 to 3.13, P<0.000 01), 8 weeks (SMD=3.93, 95%CI 3.06 to 4.81, P<0.000 01) and 12 weeks (SMD=6.15, 95%CI 4.30 to 8.00, P<0.000 01) after surgery. The BBB scores in the SCs or ASCs transplantation combined with other therapies group were also better in 4 weeks (SMD=1.06, 95%CI 0.44 to 1.68, P=0.000 8), 8 weeks (SMD=2.26, 95%CI 1.57 to 2.96, P<0.000 01) and 12 weeks (SMD=1.49, 95%CI 0.72 to 2.25, P<0.000 01) after surgery. Compared with the SCs group, the BBB score in the ASCs transplantation group were superior in 4 weeks (SMD=4.31, 95%CI 3.50 to 5.13, P<0.000 01) and 12 weeks (SMD=5.44, 95%CI 3.99 to 6.89, P<0.000 01) after surgery. No significant difference was found in mortality between the transplantation group and the control group.
ConclusionCurrent evidence indicates that SCs and ASCs can promote the recovery of motor function in the rats with TSCI. More functional recoveries can be obtained in ASCs transplantation compared with SCs transplantation. Due to limited quality of the included studies, the above conclusion should be verified by conducting more large-scale, high quality RCTs.
The biomaterial, chitin, was used to create a nerve regeneration chamber for bridging healing experiment of sciatic nerve of rats having a defect of 12mm. The crude Schwann cells were introduced into the chambers in one group and the other group had no crude Schwann cells in the chamber and the results of the two groups were compared with those having the nerve defects bridged with skeletal muscles. The specimens were observed by macroscopic, microdissection. electrophysiologic testing, HRP retrograde labelling, histologic and electron microscopic examinations at 4, 8, and 12 weeks after the operation. The results showed that atthe 8th week, the regenerating nerve fibers from the cephalad ends had united with the fibers of the caudal ends of the divided nerves either the crude Schwanneclls were introduced or not, but the morphology of the regenerating nerve, the way of regeneration and the recovery of the function of the extremities were far superior in the group that no cruds Schwann cells had been introduced than those with crude Schwann cell introduced and those bridged by skeletal muscles.
Objective To review the research progress on the role of Schwann cells in regulating bone regeneration. MethodsThe domestic and foreign literature about the behavior of Schwann cells related to bone regeneration, multiple tissue repair ability, nutritional effects of their neurotrophic factor network, and their application in bone tissue engineering was extensively reviewed. ResultsAs a critical part of the peripheral nervous system, Schwann cells regulate the expression level of various neurotrophic factors and growth factors through the paracrine effect, and participates in the tissue regeneration and differentiation process of non-neural tissues such as blood vessels and bone, reflecting the nutritional effect of neural-vascular-bone integration. ConclusionTaking full advantage of the multipotent differentiation ability of Schwann cells in nerve, blood vessel, and bone tissue regeneration may provide novel insights for clinical application of tissue engineered bone.
Objective To explore the advance in physical materials,chemical matrix, and biological seed cells for fabricating artificial nerve. Methods Recent literature relevant to artificial nerve, especially the achievement in physical material, chemical matrix and biological seed cells for fabricating artificial nerve, were extensively reviewed. Results Polymers of polylactic acid or polyglycolic acid and their polymer, polymer of hyaluronic acid and glut-aldehyde, polymer of polyacrylonitrile and polyvinylchloride were artificial nerve materials with the properties of good biocompatibility and biodegradation. A conduit with multichannel and high percentage of pores was beneficial to the regeneration of nerve. The activated Schwann cells were excellent seeds of artificial nerve. A suitable chemical matrix, such as laminin and alginate, could promote the regeneration of nerve. Conclusion The successful fabrication of artificial nerve lies in the advance in the mechanism of nerve regeneration and physical material, chemical matrix and biological seed cells.
