OBJECTIVE: To purify and study Schwann cells cytoplasmic neurotrophic protein. METHODS: The dissociated SC taken from 300 newborn rats sciatic nerves were cultured, collected, ultrasonicated and ultraspeed centrifuged. The supernates were ultrafiltrated and concentrated by using ultrafiltration units with PM10, PM30, PM50 ultrafiltration membranes. The ultrafiltrated-concentrated solution with the protein molecular weight 10-30 ku, 30-50 ku and gt; 50 ku were collected respectively. The dissociated spinal cord motoneurons of 14 days embryonic rats were cultured with serum-free conditional medium and the additional SC cytoplasmic proteins were added into the medium. The results showed that the 10-30 ku and gt; 50 ku SC cytoplasmic proteins were able to maintain the survival of motoneurons for 24 hours. Then the 26 ku and 58 ku proteins were further extracted and purified from SC cytoplasm by high pressure liquid chromatography, and their neurobiological activities were studied. RESULTS: The 26 ku and 58 ku Schwann cell’s cytoplasmic proteins were able to maintain the survival of motoneurons cultured in the serum-free medium for 48 hours. The highest biological activity concentration is 20 ng per well. CONCLUSION: Schwann cells cytoplasm contains motoneuron neurotrophic proteins with molecular weight 26 ku and 58 ku.
Objective
Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps.
Methods
hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining.
Results
The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation.
Conclusion
The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
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.
ObjectiveTo study the effect of Schwann cells (SCs) promoting the function of nitric oxide (NO) secretion of bone marrow mesenchymal stem cells (BMSCs) derived endothelial cells so as to lay the experimental foundation for research of the effect of nerves on vessels during the process of tissue engineering bone formation.
MethodsSCs were collected from 1-day-old Sprague Dawley (SD) rats,and identified through S100 immunohistochemistry (IHC).BMSCs were collected from 2-week-old SD rats and induced into endothelial cells (IECs),which were identified through von Willebrand factor (vWF) and CD31 immunofluorescence (IF).Transwell system was used for co-culture of SCs and IECs without contact as the experimental group,and simple culture of IECs served as the control group.The NO concentration in the medium was measured at 1,3,5,and 7 days after culture; the mRNA expressions of nitric oxide synthetase 2 (NOS2) and NOS3 were detected by real-time fluorescence quantitative PCR (RT-qPCR) at 1,3,7,and 10 days.
ResultsSCs and IECs were identified through morphology and immunology indexes of S100 IHC,vWF and CD31 IF.Significant differences were found in the NO concentration among different time points in 2 groups (P<0.05); the NO concentration of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 3 days.NOS2 mRNA expression of the experimental group was significantly higher than that of the control group (P<0.05); difference was significant in the NOS2 mRNA expression among different time points in 2 groups (P<0.05).NOS3 mRNA expression of the experimental group was significantly higher than that of the control group at the other time points (P<0.05) except at 10 days.No significant difference was found in NOS3 mRNA expression among different time points in the experimental group (F=6.673,P=0.062),but it showed significant differences in the control group (F=36.581,P=0.000).
ConclusionSCs can promote NO secretion of BMSCs derived endothelial cells,which is due to promoting the activity of NOS.
Objective
To explore the construction and biocompatibility in vitro evaluation of the electrospun-graphene (Gr)/silk fibroin (SF) nanofilms.
Methods
The electrostatic spinning solution was prepared by dissolving SF and different mass ratio (0, 5%, 10%, 15%, and 20%) of Gr in formic acid solution. The hydrophilia and hydrophobic was analyzed by testing the static contact angle of electrostatic spinning solution of different mass ratio of Gr. Gr-SF nanofilms with different mass ratio (0, 5%, 10%, 15%, and 20%, as groups A, B, C, D, and E, respectively) were constructed by electrospinning technology. The structure of nanofilms were observed by optical microscope and scanning electron microscope; electrochemical performance of nanofilms were detected by cyclic voltammetry at electrochemical workstation; the porosity of nanofilms were measured by n-hexane substitution method, and the permeability were observed; L929 cells were used to evaluate the cytotoxicity of nanofilms in vitro at 1, 4, and 7 days after culture. The primary Sprague Dawley rats’ Schwann cells were co-cultured with different Gr-SF nanofilms of 5 groups for 3 days, the morphology and distribution of Schwann cells were identified by toluidine blue staining, the cell adhesion of Schwann cells were determined by cell counting kit 8 (CCK-8) method, the proliferation of Schwann cells were detected by EdU/Hoechst33342 staining.
Results
The static contact angle measurement confirmed that the hydrophilia of Gr-SF electrospinning solution was decreased by increasing the mass ratio of Gr. Light microscope and scanning electron microscopy showed that Gr-SF nanofilms had nanofiber structure, Gr particles could be dispersed uniformly in the membrane, and the increasing of mass ratio of Gr could lead to the aggregation of particles. The porosity measurement showed that the Gr-SF nanofilms had high porosity (>65%). With the increasing of mass ratio of Gr, the porosity and conductivity of Gr-SF nanofilm increased gradually, the value in the group A was significantly lower than those in groups C, D, and E (P<0.05). In vitro L929 cells cytotoxicity test showed that all the Gr-SF nanofilms had good biocompatibility. Toluidine blue staining, CCK-8 assay, and EdU/Hoechst33342 staining showed that Gr-SF nanofilms with mass ratio of Gr less than 10% could support the survival and proliferation of co-cultured Schwann cells.
Conclusion
The Gr-SF nanofilm with mass ratio of Gr less than 10% have proper hydrophilia, conductivity, porosity, and other physical and chemical properties, and have good biocompatibility in vitro. They can be used in tissue engineered nerve preparation.
Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.
ObjectiveTo summarize the applications of Schwann cells (SCs), stem cells, and genetically modified cells (GMCs) in repair of peripheral nerve defects.
MethodsThe literature of original experimental study and clinical research related with SCs, stem cells, and GMCs was reviewed and analyzed.
ResultsSCs play a key role in repair of peripheral nerve defects; the stem cells can be induced to differentiate into SCs, which can be implanted into nerve conduits to promote the repair of peripheral nerve defect; genetically modified technology can enhance the function of SCs and different stem cells, which has been regarded as a new option for tissue engineered nerve.
ConclusionAlthough great progress has been made in tissue engineered nerve recently, mostly limited to the experimental stage. The research of seed cells in application of tissue engineered nerve need be studied deeply.
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.
Objective
To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves.
Methods
The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis.
Results
At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B.
Conclusion
BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.
Objective To observe the effects of co-transfection of Nogo extracellular peptide residues 1-40 (NEP1-40) and neurotrophin 3 (NT-3) genes with Schwann cell-derived exosomes (SCDEs) on the survival and differentiation of neural stem cells (NSCs), and lay the foundation for the in vivo experiments of SCDE and NSC co-transplantation. Methods The NEP1-40 and NT-3 genes were transfected into Schwann cells by lentiviral vector, and SCDEs were collected for identification. The NSCs that have been passaged for 3 times were selected and inoculated into the inoculation plate, and they were divided into conventional culture group, simple exosome culture group (adding empty vector plasmid to modify SCDE for culture) and two genes exosome culture group (adding two genes modified SCDE for culture). The activity of cells in each group was detected. The survival and differentiation of NSCs were evaluated by immunofluorescence detection of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and galactosylceramidase (GALC) positive cells. Results After transfection of these two genes, the fluorescence intensity was higher and the cell state was better. The relative expression levels of messenger RNA and protein of NEP1-40 and NT-3 in the two gene groups were higher than those in the empty plasmid group (P<0.05). The relative expression levels of NEP1-40 and NT-3 proteins in SCDE of the two gene groups were higher than those of the empty vector group (P<0.05). There was no significant difference in the relative expression level of CD63 protein in SCDE between the two groups (P>0.05). In terms of cell activity, the cell activity of the two genes exosome culture group was the strongest, followed by the simple exosome culture group, and the conventional culture group was the weakest. The differences between any two groups were statistically significant (1.28±0.04 vs. 0.72±0.09 vs. 0.41±0.04, P<0.05). In terms of cell survival, NeuN-positive cells (5.23±0.22 vs. 2.36±0.09 vs. 1.00±0.01) and GALC-positive cells (2.29±0.06 vs. 1.75±0.02 vs. 1.00±0.04) of the two genes exosome culture group were the best, followed by the simple exosome culture group, and the conventional culture group were the weakest. The differences between any two groups were statistically significant (P<0.05). In terms of cell differentiation, NeuN-positive cells (0.44±0.02 vs. 0.29±0.01 vs. 0.16±0.01) and GALC-positive cells (0.38±0.07 vs. 0.23±0.02 vs. 0.12±0.01) of the two genes exosome culture group were the best, followed by the simple exosome culture group, and the conventional culture group were the weakest. The differences between any two groups were statistically significant (P<0.05). The differentiation of GFAP-positive cells in the conventional culture group was the best, followed by the simple exosome culture group, and the two genes exosome culture group was the worst (0.52±0.05 vs. 0.42±0.03 vs. 0.30±0.09). The differences between any two groups were statistically significant (P<0.05). Conclusion NEP1-40 and NT-3 genes can be successfully transfected into Schwann cells by lentiviral vector, which can effectively increase the content of related proteins in SCDE, and the exosomes can effectively promote the survival and differentiation of NSCs in vitro.
