OBJECTIVE To investigate the methods to fabricate repair materials of tissue engineered peripheral nerve with bioactivity of Schwann cells (SC). METHODS 1. The materials were made by dry-wet spinning process to fabricate PLA hollow fiber canal with external diameter of 2.3 mm, internal diameter of 1.9 mm, thickness of 0.4 mm, pore size of 20 to 40 microns, pore ratio of 70% and non-spinning fiber net with pore size of 100 to 200 microns, pore ratio of 85%. 2. SC were implanted into excellular matrix (ECM) gel to observe the growth of SC. 3. SC/ECM complex were implanted into non-spinning PLA fiber net to observe the growth of SC. 4. SC, SC/ECM and SC/ECM/PLA were implanted into PLA hollow fiber canal to bridge 10 mm defect of rat sciatic nerve. RESULTS 1. SC were recovered bipolar shape at 1 day after implantation, and could be survived 14 days in ECM gel. 2. After SC/ECM complex was implanted into PLA net, most of SC were retained in the pore of PLA net with the formation of ECM gel. SC could be adhered and grown on PLA fiber. 3. Most of SC in ECM gel could be survived to 21 days after transplantation. Survival cell numbers of SC/ECM and SC/ECM/PLA groups were obviously higher than SC suspension group. CONCLUSION Non-spinning PLA porous biodegradable materials with ECM is benefit for SC to be adhered and grown.
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 study the migration of Schwann cells from the nerve autograft in the acellular nerve allograft of the rats in vivo. Mehtods The sciatic nerves (20 mm long) of the SD rats were harvested and prepared for the acellular nerve grafts by the chemical extraction. Then, they were observed by the gross view, HE staining, and Antilamininstaining, respectively. Another 32 female SD rats weighing 250-300 g were obtained for the study. A 2-mm-long nerve autograft was interposed between the two 10-mm-long nerve allografts to form a 22-mm-long composite. Then, the composite was placed in the muscle space, together with a sole 22-mm-long nerve allograftas a control. They were harvested at 5，10，15 and 20 days, respectively, and were then given the HE staining and the S-100 staining. Results The acellular nerve graft was semitransparent under the gross view. HE staining showed that no cell was observed within the nerve graft. Anti-laminin staining showed that the basal membrane was partially interrupted, with a positive result (dark brown). All the nerve grafts in both the groups exhibited the existenceof the cells. The S-100 positive cells were observed from the 15th day at the far ends of the two allografts of the composite; however, there were no suchcells observed within the sole nerve allograft. Conclusion Schwann cells from the sciatic nerves (2 mm- long) of the rats can migrate in the acellular nerve allograft to the far ends of the neighboring 10-mm-long nerve allografts at 15 days after operation, which offers the theoretical basis forthe repair of the longrange nerve defect by the composite of the acellular nerve allografts with the interposed nerve autograft.
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 ℃.
Objective To evaluate the effect of Schwann cell (SC) on the proliferation of marrow mesenchymal stem cells (MSCs) and provide evidence for application of SC in construction of the tissue engineered vessels.Methods SC and MSCs were harvested from SD rats(weight 40 g). SC were verified immunohstochemically by the S-100 staining, and MSCs were verified by CD 44, CD 105, CD 34 and CD 45. The 3rd passages of both the cells were cocultured in the Transwell system and were amounted by the 3H-TDR integration technique at 1, 3, 5 and 7 days,respectively. The results were expressed by the CPM(counts per minute, CPM) values. However, MSCs on both the layers were served as the controls. The Westernblot was performed to assess the expression of the vascular endothelial growth factor (VEGF), its receptor Flk-1, and the associated receptor neuropilin 1(NRP-1) in SC, the trial cells, and the controls. Results SC had a spindle shape in the flasks, and more than 90% of SC had a positive reaction for the S-100 staining.MSCs expressed CD44 and CD105, and had a negativesignal in CD 34 and CD 45. The CPM values of MSCs in the trial groups were 2 411.00±270.84，3 016.17±241.57，6 570.83±2 848.27 and 6 375.8±1 431.28at 1, 3, 5 and 7 days, respectively. They were significantly higher in their values than the control group (2 142.17±531.63，2 603.33±389.64，2 707.50±328.55，2 389.00±908.01), especially at 5 days （P＜0.05）. The Western blot indicated that VEGF was expressedobviously in both the SC group and the cocultured MSCs grou,p and was less visible in the control cells. The expressions of Flk-1 and NRP-1 inthe cocultured MSCs were much ber than in the controls. Conclusion SC can significantly promote the proliferation of MSCs when they are cocultured. The peak time of the proliferation effect appeared at 5 days. This effect may be triggered by the up-regulation of VEGF in MSCs, which also leads to the upregulation of Flk-1 and NRP-1 .
Objective Bone marrow mesenchymal stem cells (BMSCs) are multi potent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and ol igodendrocytes in rats at different ages in vitro. Methods SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myel in basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregul in 1 (NRG1) was detected by ELISA method. Results SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or ol igodendrocytes-l ike morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% ± 2.86%, 12.89% ± 1.78%, and 2.69% ± 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% ± 2.39%, 6.33% ± 1.40%, and 0.92% ± 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P lt; 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P lt; 0.05). Conclusion The microenvironment formed by SCs can promote BMSCs differentiation into neurons and ol igodendrocytes, but the differentiation capabil ity of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is l ikely important factors that induce the differentiation of BMSCs into neurons and ol igodendrocytes.
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.
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 research the protective effects of different allogeneic cells injected into denervated muscles on ventricornual motor neuron. Methods Thirty-six adult female SD rats, weighting 120-150 g, were individed into four groups randomly and each group had nine. Left ischiadic nerves of all the SD rats, which were cut down on germfree conditions,were operated by primary suture of epineurium. Different cells were injected into the triceps muscles of calf in each group after operation with once a week for 4 weeks:1 ml Schwann cells (1×106/ml) in group A, 1 ml mixed cells ofSchwann cells and myoblast cells (1∶1,1×106/ml) in group B, 1 ml extract from the mixed cells of Schwann cells, myoblast cells and endotheliocytes (1∶1∶1,1×106/ml)in group C,and 1 ml culture medium without FCS as control group(group D). The observation of enzymohistochemistry and C-Jun expression in the ventricornual motor neuron was made after three months of operation. Results After 3 months of operation, the expressions of C-Jun in groups A, B and C were superiorto that in group D; the number of neuron was more than that of group D. The expressions of C-Jun in the ventricornual motor neuron were as follows: 128.591±0.766 in group A, 116.729±0.778 in group B, 100.071±2.017 in group C and 144.648±2.083 in group D; showing statistically significant difference between groupsA, B, C and D(P＜0.01). Enzymohistochemistry showed the well outlined and wellstacked cell body of neuron in groups A, B and C, and illdefined boundary of cytoplasm and nucleus. There was statistically significant defference in enzyme activity of the ventricornual motor neuron between groups(P＜0.01). Conclusion All of the Schwann cells,mixed cells of Schwann cells with myoblast cells,and the extract from Schwann cells, myoblast cells and endotheliocytes can protect the ventricornual motor neuron. And the protectiveeffect of the extract from Schwann cells, myoblast cells and endotheliocytes is superior to that of Schwann cells and mixed cells.
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.