In Vitro Construction Of Tissue Engineered Nerves And Their Use For Sciatic Nerve Repair In Rats

Part I In vitro construction of tissue engineered nervesObjectiveWe aimed to prepare nerve equivalents by combining a co-culture of DRG explantsand Schwann cells with a silk fibroin (SF)-based scaffold in an attempt to develop atissue-engineered nerve that was significantly similar to a native nerve.Methods1. Schwann cells (SCs) were harvested from sciatic nerves of1d Sprague-Dawley(SD) rats and purified, followed by culture for3weeks.2. The L4-6DRGs were removed from SD rat embryos (E14.516.5), and thedissociated DRGs were plated onto the SF filaments inside the scaffold that had beenfabricated by injection-molding.3. A nerve equivalent was prepared by introducing a co-culture of DRG explants andSCs inside a silk fibroin-based scaffold.4. Immunohistochemistry was used to identify DRG-SC co-culture inside aSF-based scaffold after2weeks.5. Scanning and transmission electron microscopy was carried out to identifyDRG-SC co-culture inside a SF-based scaffold after designated times.Results1. We developed an in vitro co-culture system of SCs/DRGs inside a silkfibroin-based scaffold, and thus constructed successfully a novel tissue engineered nerve.2. Immunochemistry showed that S-100positive and NF200positive cells,identified as SCs and DRG neurites, adhered to and encircled around the inner wall of a SF-based scaffold.3. Scanning electron micrographs displayed that extracellular matrix (ECM)-liketissues appeared along longitudinal aligned SF filaments inside a scaffold, and SCs witha spindle or spherical shape encircled the axons of DRGs.4. Transmission electron microscopy clearly demonstrated the formation of myelinsheaths and the lamella structure after SCs wrapping DRG axons.ConclusionBy using co-culture of DRGs and SCs that was residing in a SF-based scaffold, wedeveloped a novel tissue-engineered nerve, which was confirmed to show nativenerve-like characteristics as featured by existence of nerve fibers and myelin sheath.Part II Use of tissue engineered nerves to repair sciaticnerve defect in ratsObjectiveWe aimed to evaluate the outcome of nerve regeneration and functional recoveryafter bridging sciatic nerve defects with our developed tissue engineered nerves.Methods1. The aboved constructed tissue-engineeered nerves, autografts, and SF-basednerve scaffolds alone were respectively used to bridge10mm sciatic nerve defects inrats.2. Twelve weeks after nerve grafting, the compound muscle amplitude potential(CMAP) value was measured and the motor nerve conduction velocity (MCV) wascaculated.3. After2,4,6,8,10and12weeks after nerve grafting, walking track analysis wasperformed to evaluate the sciatic nerve function index (SFI).4. Four and twelve weeks after nerve grafting, immunohistochemistry with anti-NFand anti-S100was performed to investigate the regeneration of myelinated nerve fibers. 5. Twelve weeks after nerve grafting, immunochemistry and transmission electronmicroscopy were used to detect the sheath thickness or myelin lamellae number.6. Twelve weeks after nerve grafting, the cross-sectional area of target muscles andaverage percentage of collagen were measured.7. Western blot was used to analysis the protein expression level of N-cadherin andPMP22.Results1. Twelve weeks after nerve grafting, there were no significant differences in theCMAP and MCV value between rats repaired by autografts and tissue-engineeerednerves, but the CAMP value was significantly larger in rats repaired by autografts ortissue-engineeered nerves than in rats repaired by SF-based nerve scaffolds (P<0.05).2. Twelve weeks after nerve grafting, the SFI value was significantly higher in ratsrepaired by autografts or tissue-engineeered nerves than in rats repaired by SF-basednerve scaffolds(P<0.05).3. Four weeks after nerve grafting, the myelinated nerve fibers were found in ratsrepaired by tissue-engineeered nerves, and at twelve weeks an increased number ofmyelinated nerve fibers appeared.4. Four and twelve weeks after nerve grafting, immunochemistry and transmissionelectron microscopy indicated that the sheath thickness or myelin lamellae number wassignificantly greater in rats repaired by autografts or tissue-engineeered nerves than inrats repaired by SF-based nerve scaffolds(P<0.05).5. Masson trichrome staining indicated that the gastrocnemius muscle atrophy wasmore serious in unrepaired rats that in repaired rats, and the alleviation in muscle atrophywas the greatest in rats repaired by autografts(P<0.05).6. According to Western blot analysis, the N-cadherin expression was significantlyhigher in rats repaired by tissue-engineeered nerves than in rats repaired by SF-basednerve scaffolds starting from4weeks after nerve grafting(P<0.05), while the PMP22expression was significantly higher in rats repaired by tissue-engineeered nerves than in rats repaired by SF-based nerve scaffolds starting from2weeks after nervegrafting(P<0.01).Conclusion1. After bridging10mm sciatic nerve defect in rats, nerve regeneration andfunctional recovery were significantly better in rats repaired by tissue-engineeered nervesthan in rats repaired by SF-based nerve scaffolds. This result showed that our developedtissue engineered nerves could achieve the satisfactory outcome of peripheral nevrerepair.2. The repairing capacity of tissue-engineeered nerves might be induced byincreased expressions of2nerve regeneration-related proteins in regenerated nervetissues.Part III Gene expression profiles and identification of keyregulatory factors in injured peripheral nervesObjectiveWe aimed to analyze the differential expressed genes in injured proximal nervesegments and identify the key regulatory factors by Signal-flow networks in an attemptto unveil the important factors mediating acute responses of injured peripheral nerves.Methods1. The differential expressed genes were analyzed by a random variance model(RVM) after the sciatic nerves of SD rats were transected at0,0.5,1,3,6and9h,respectively.2. Based on the gene ontology (GO) database, significance analysis of the genefunction significant expression tendency (STC)-GO was applied to analyze the mainfunction of the differential expression genes.3. Significance analysis of the differential gene pathway was performed for thedifferential genes and a significant pathway involving the differential genes was obtained.4. According to the significant pathway involving differential genes, theSignal-Flow among the genes in different time series was studied in a quantitative way,taking advantage of the regulatory relations among the genes and the expression of thegenes in the database.5. The identified regulators were identified by the following measurements:(1) qRT-PCR using mRNA isolated from proximal nerve segments;(2) immunohistochemistry and Western blot analysis to examine the expression andlocalization of the key regulators;(3) siRNA knockdown experiments to detect the effects of the key regulators on invitro Schwann cells.(4) the exploration of specific mechanisms of related key factors.Results1. The Random variance model (RVM) F-test was applied to filter the differentiallyexpressed genes of the control. A total of2850diferential genes wereidentified(P<0.01)．2. Based on the KEGG database, a total of9canonical pathways were identified(Fisher’s exact test, P value0.001), which were associated with cytokine-cytokinereceptor interaction, cell adhesion molecules, Toll-like receptor signaling pathway andapoptosis, etc.3. Signal-flow analysis indicated that anti-apoptosis, intercellular tight junction, cellpolarity and adhesion were the key elements in the gene regulatory network．Birc2, Birc3and Tnfrsfla, which were involved in anti-apoptosis, showed signicant weight values.4. TNF-α ELISA showed that TNF-α consistantly increased in the extracts of5mmproximal segments collected at0,0.5,1,3,6,9and12h after injury. qRT-PCRdemonstrated that the expression of Birc2, Birc3and Tnfrsf1a was accordinglyupregulated.5. Immunohistochemistry revealed that Birc2, Birc3and Tnfrsfla were co-localized with S100, suggesting that Schwann cells might be responsible for anti-apoptosis actions.6. Western blot showed that cleaved caspase-3was highly upregulated in SCs aftertreatment with40ng/ml TNF-α for12h or with10-40ng/ml for24h.7. Both TUNEL and flow cytometry indicated that siRNAs transfection increasedthe cell number of apoptotic Schwann cells. After transfection with Birc2-, Birc3-, andTnfrsf1a-siRNAs for48h, the cell number of apoptotic Schwann cells increased from11.18to25.87(P<0.05), to28.94(P<0.05), and to28.64%(P<0.01) of control cells,respectively. The apoptotic SC number increased from16.91to32.23(P<0.01),36.01(P<0.01), and37.52%(P<0.01) of control cells after transfection with Birc2-Birc3-andTnfrsf1a-siRNAs, respectively, for72h.8. Caspase3and caspase6colorimetric assays showed that both caspase3andcaspase6were upregulated after knockdown of Birc2, Birc3or Tnfrsf1a withtransfection of siRNAs for48or72h (P<0.05).9. EdU analysis indicated that treatment with2ng/ml TNF-α significantly enhancedcell proliferation of Schwann cells as compared to no treatment (P<0.05), whereastreatment with higher than10ng/ml TNF-α significantly inhibited the cell proliferation(P<0.01).10. The cell migration assay demonstrated that TNF-α treantment with higher than10ng/ml inhibited migration of Schwann cells.11. Transmission electron microscopy showed that TNF-α treantment with higherthan10ng/ml induced apoptosis of Schwann cells, characterized by the condensation ofnuclear chromatin, cytoplasmic blebbing and vacuolization, or the formation of a fewapoptotic bodies. A chemical inhibitor of TNF-α reduced cell apoptosis, compared totreatment with40ng/ml TNF-α (P<0.01). Accordingly, the expression of caspase-7,-8,and-9increased following treatment with40ng/ml TNF-α.12. Co-immunoprecipitation showed that either FADD or RIP was able to bind toTRADD and form a protein complex in Schwann cells. Western blot analysisdemonstrated that the protein level of FADD bound to TRADD was elevated with increase in the dosage of added TNF-α (P<0.01). In contrast, the protein level of RIPbound to TRADD reached a maximum value when cells were treated with2ng/ml ofTNF-α, and was then dramatically decreased when cells were treated with5-40ng/ml ofTNF-α (P<0.01).13. Western blot analysis revealed that the expression level of phosphorylated-(p-)JNK or p-NF-κB p65changed in a manner similar to that of FADD or RIP (P<0.01),respectively, in response to treatment with different dosages of TNF-α (P<0.01).Conclusion1. The injured peripheral nerves upregulated the key regulators Birc2, Birc3andTnfrsf1a for anti-apoptosis in reponse to the influx of inflammatory cytokines. TNF-αmay be the main regulatory cytokine and Schwann cells are mainly responsed cells.2. Low concentration of TNF-α (≤2ng/ml) promoted proliferation and migratin ofSchwann cells, wheras high concentration of TNF-α (≥10ng/ml) induced apoptosis.3. Low concentration of TNF-α (≤2ng/ml) promoted the interaction of RIP andTRADD to prevent from cell apoptosis, but high concentration of TNF-α (≥10ng/ml)promoted an interaction of FADD and TRADD to induce apoptosis.4. High concentration of TNF-α (≥10ng/ml) induced cell apoptosis by signalingthrough caspase-3, caspase-6, caspase-7, caspase-8and caspase-9.