BACKGROUNDVarious tissue engineering strategies have been developed to facilitate axonal regeneration after spinal cord injury. This study aimed to investigate whether neural stem cells (NSCs) could survive in poly(L-lactic-co-glycolic acid) (PLGA) scaffolds and, when cografted with Schwann cells (SCs), could be induced to differentiate towards neurons which form synaptic connection and eventually facilitate axonal regeneration and myelination and motor function.

METHODSNSCs and SCs which were seeded within the directional PLGA scaffolds were implanted in hemisected adult rat spinal cord. Control rats were similarly injured and implanted of scaffolds with or without NSCs. Survival, migration, differentiation, synaptic formation of NSCs, axonal regeneration and myelination and motor function were analyzed. Student's t test was used to determine differences in surviving percentage of NSCs. One-way analysis of variance (ANOVA) was used to determine the differences in the number of axons myelinated in the scaffolds, the mean latency and amplitude of cortical motor evoked potentials (CMEPs) and Basso, Beattie & Bresnahan locomotor rating scale (BBB) score. The χ(2) test was used to determine the differences in recovery percentage of CMEPs.

RESULTSNSCs survived, but the majority migrated into adjacent host cord and died mostly. Survival rate of NSCs with SCs was higher than that of NSCs without SCs ((1.7831 ± 0.0402)% vs. (1.4911 ± 0.0313)%, P < 0.001). Cografted with SCs, NSCs were induced to differentiate towards neurons and might form synaptic connection. The mean number of myelinated axons in PLGA + NSCs + SCs group was more than that in PLGA + NSCs group and in PLGA group ((110.25 ± 30.46) vs. (18.25 ± 3.30) and (11.25 ± 5.54), P < 0.01). The percentage of CMEPs recovery in PLGA + NSCs + SCs group was higher than in the other groups (84.8% vs. 50.0% and 37.5%, P < 0.05). The amplitude of CMEPs in PLGA + NSCs + SCs group was higher than in the other groups ((1452.63 ± 331.70) µV vs. (428.84 ± 193.01) µV and (117.33 ± 14.40) µV, P < 0.05). Ipsilateral retransection resulted in disappearance again and functional loss of CMEPs for a few days. But contralateral retransection completely damaged the bilateral motor function.

CONCLUSIONSNSCs can survive in PLGA scaffolds, and SCs promote NSCs to survive and differentiate towards neurons in vivo which even might form synaptic connection. The scaffolds seeded with cells facilitate axonal regeneration and myelination and motor function recovery. But regenerating axons have limited contribution to motor function recovery.

Animals ; Axons ; physiology ; Cells, Cultured ; Electrophysiology ; Female ; Fluorescent Antibody Technique ; Lactic Acid ; chemistry ; Nerve Regeneration ; physiology ; Neural Stem Cells ; cytology ; Polyglycolic Acid ; chemistry ; Pregnancy ; Rats ; Rats, Wistar ; Schwann Cells ; cytology ; Spinal Cord Injuries ; therapy ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

Olfactory ensheathing cells (OECs) are a unique type of glia with common properties of astrocyte and Schwann cells. Cultured OECs have two morphological phenotypes, astrocyte-like OECs and Schwann cell-like OECs. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of these reversible changes is still unknown. The aim of this paper is to establish a method for the morphology plasticity of cultured OECs, and investigate the underlying mechanism. Using the primary culture of OECs and immunocytochemistry, the morphology of OECs was observed under serum, serum free media or dB-cAMP drug treatment. Statistical analysis was performed to test differences among the percentages of OEC subtypes under these conditions. The results showed that under serum free media, (95.2±3.7)% of OECs showed Schwann cell-like morphology, and (4.8±3.7)% of OECs showed astrocyte-like morphology; however, under 10% serum media, (42.5±10.4)% of OECs exhibited Schwann cell-like morphology, and (57.5±10.4)% of OECs exhibited astrocyte-like morphology. When media was changed back to serum free media for 24 h, (94.8±5.0)% of OECs showed Schwann cell-like morphology, and (5.2±5.0)% of OECs showed astrocyte-like morphology. Furthermore, culture condition with or without serum did not affect the expression of OEC cell marker, p-75 and S-100. Finally, dB-cAMP, an analog of cAMP, through inhibiting the formation of F-actin stress fibers and focal adhesion, induced the morphology switch from astrocyte-like to Schwann cell-like morphology under serum condition, promoted the branches and the growth of processes. These results suggest that serum induces the morphology plasticity of cultured OECs, which is mediated by cytoplasmic cAMP level through regulating the formation of F-actin stress fibers and focal adhesion.
Animals ; Astrocytes ; cytology ; physiology ; Cells, Cultured ; Culture Media ; pharmacology ; Cyclic AMP ; physiology ; Male ; Neuroglia ; cytology ; physiology ; Olfactory Bulb ; cytology ; physiology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; physiology ; Serum ; physiology

BACKGROUNDPeripheral nerve regeneration across large gaps is clinically challenging. Scaffold design plays a pivotal role in nerve tissue engineering. Recently, nanofibrous scaffolds have proven a suitable environment for cell attachment and proliferation due to similarities of their physical properties to natural extracellular matrix. Poly(propylene carbonate) (PPC) nanofibrous scaffolds have been investigated for vascular tissue engineering. However, no reports exist of PPC nanofibrous scaffolds for nerve tissue engineering. This study aimed to evaluate the potential role of aligned and random PPC nanofibrous scaffolds as substrates for peripheral nerve tissue and cells in nerve tissue engineering.

METHODSAligned and random PPC nanofibrous scaffolds were fabricated by electrospinning and their chemical characterization were carried out using scanning electron microscopy (SEM). Dorsal root ganglia (DRG) from Sprague-Dawley rats were cultured on the nanofibrous substrates for 7 days. Neurite outgrowth and Schwann-cell migration from DRG were observed and quantified using immunocytochemistry and SEM. Schwann cells derived from rat sciatic nerves were cultured in electrospun PPC scaffold-extract fluid for 24, 48, 72 hours and 7 days. The viability of Schwann cells was evaluated by 3-[4,5-dimethyl(thiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT) assay.

RESULTSThe diameter of aligned and random fibers ranged between 800 nm and 1200 nm, and the thickness of the films was approximately 10 - 20 µm. Quantification of aligned fiber films revealed approximately 90% alignment of all fibers along the longitudinal axis. However, with random fiber films, the alignment of fibers was random through all angle bins. Rat DRG explants were grown on PPC nanofiber films for up to 1 week. On the aligned fiber films, the majority of neurite outgrowth and Schwann cell migration from the DRG extended unidirectionally, parallel to the aligned fibers. However, on the random fiber films, neurite outgrowth and Schwann cell migration were randomly distributed. A comparison of cumulative neurite lengths from cultured DRGs indicated that neurites grew faster on aligned PPC films ((2537.6 ± 987.3) µm) than randomly-distributed fibers ((493.5 ± 50.6) µm). The average distance of Schwann cell migration on aligned PPC nanofibrous films ((2803.5 ± 943.6) µm) were significantly greater than those on random fibers ((625.3 ± 47.8) µm). The viability of Schwann cells cultured in aligned PPC scaffold extract fluid was not significantly different from that in the plain DMEM/F12 medium at all time points after seeding.

CONCLUSIONSThe aligned PPC nanofibrous film, but not the randomly-oriented fibers, significantly enhanced peripheral nerve regeneration in vitro, indicating the substantial role of topographical cues in stimulating endogenous nerve repair mechanisms. Aligned PPC nanofibrous scaffolds may be a promising biomaterial for nerve regeneration.

Animals ; Biocompatible Materials ; chemistry ; Cells, Cultured ; Ganglia, Spinal ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Nanofibers ; chemistry ; Nerve Regeneration ; physiology ; Nerve Tissue ; cytology ; metabolism ; ultrastructure ; Polymers ; chemistry ; Propane ; analogs & derivatives ; chemistry ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; metabolism ; ultrastructure ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo investigate the phenotypic, molecular and biological characteristics of adipose tissue-derived stromal cells (ADSCs) differentiated alonely a Schwann cells (SCs) lineage and to provide a new cells' seed source for nerve tissue engineering or cell therapy.

METHODSCultured ADSCs were isolated from SD rats and the undifferentiated ADSCs were confirmed by detection of MSC-specific cell-surface markers. The ADSCs were differentiated along a glial cell lineage using an established cocktail of growth factors. Following differention, we used immunofluorescene staining and RT-PCR to evaluate the characteristics of differentiated WJMSCs.

RESULTSADSCs were successfully isolated from the rats' fat tissue. The isolated ADSCs expressed CD29, CD90 but not CD34, CD44 nor CD45. Osteogenic differentiation was detected by Alizarin red staining and adipogenic differentiation was comfirmed by Oil-red O staining. ADSCs treated with a mixture of glial growth factors adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and RT-PCR analysis revealed that the treated cells expressed the glial markers S100, P75 and glial fibrillary acidic protein indicative of differentiation.

CONCLUSIONADSCs can be differentiated into cells that are Schwann-like in terms of morphologic features and phenotype and could be suitable Schwann-cell substitutes for nerve repair in clinical applications.

Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology

OBJECTIVETo investigate the effect of mitogen-activated protein kinase (MEK) kinase cascade, extracellular signal-regulated kinase (ERK1/2) signal pathway on Schwann cells proliferation promoted by Pyrroloquinoline Quinine (PQQ) and its molecular mechanisms.

METHODSSchwann cells were cultured and purified in vitro. The purity was identified by S-100. Different time and concentration of PQQ was added into culture medium. The expression of ERK1/2 and phosphorylated-ERK1/2 was detected by western blot. The expression of p-ERK1/2 after blocking of MEK signal pathway by specific inhibitor PD98059 was detected by western blot.

RESULTSMorphological change was observed in PQQ treated Schwann cells. 1-500 nmol/L PQQ could up-regulate the expression of p-ERK1/2, and 1000 nmol/L had no effects, while 10 000 nmol/L exhibited inhibitory effect (P < 0.05). p-ERK1/2 increased to peak 1 h after PQQ added, and this up-regulation of p-ERK1/2 was inhibited by PD98059 (P < 0.05).

CONCLUSIONSPQQ could affect morphology of Schwann cells and activation of ERK1/2. MEK inhibitor PD98059 could, block this activation. It suggests that MEK/ERK signal pathway should be involved in Schwann cells proliferation promoted by PQQ.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; physiology ; Pyrroles ; pharmacology ; Quinolines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; drug effects ; Signal Transduction

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals ; Axotomy ; Cell Movement ; Cell Proliferation ; Male ; Membrane Glycoproteins/*physiology ; Membrane Proteins/pharmacology ; *Nerve Regeneration ; Nerve Tissue Proteins/pharmacology ; Neurites/drug effects/*physiology/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/pharmacology ; Schwann Cells/cytology/*physiology ; Sensory Receptor Cells/*physiology

The purpose of this investigation was to evaluate the effect of transforming growth factor-beta (TGF-beta) on the efficacy of Schwann cell (SC) and on the repair of peripheral nerve defect. 50 ng x ml(-1) TGF-beta was shown to promote the proliferation of SC by MTT and flow cytometry (FCM) assay, and NGF synthesis in SC culture media was noted to be of significantly higher concentration by ELISA method (P<0.05). SCs mixed with bovine acellular matrix (BAM), fetal bovine serum and media based on definite ratio were injected into polylactideco-glycolide acid (PLGA)guide. 30 SD rats, each had a man-made sciatic nerve defect 15 mm long, were randomly divided into 3 groups: experiment group (PLGA conduit+SC+TGF-beta), control group(PLGA conduit+SC), and autograft group. After 16 weeks, it was demonstrated that the effect of the test group was not significantly different from that of the autograft group, but it was better than that of the control group by means of electrophysiological test and sciatic nerve function index (SFI). TGF-beta can promote not only the proliferation, but also the NGF synthesis of SC obviously. The use of exogenous TGF-beta in the repair of peripheral nerve defect may produce better curative effect.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Female ; Guided Tissue Regeneration ; Lactic Acid ; chemistry ; Nerve Growth Factor ; biosynthesis ; Nerve Regeneration ; drug effects ; Polyglycolic Acid ; chemistry ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; Sciatic Nerve ; injuries ; physiology ; Tissue Engineering ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the synergistic effect of Schwann cells (SCs) and retinoic acid (RA) on differentiation and synaptogenesis of neural stem cells (NSCs) derived from hippocampus of neonatal rats.

METHODSThe classical method for 2x2 factorial analysis experiment was used to assess synergistic action of SCs and RA. NSCs were treated with RA, SCs, and SCs + RA in DMEM/F12 with 0.5% fetal bovine serum for six days, respectively. Double immunofluorescent staining was used to detect the differentiation of NSCs including nestin, glial fibrillary acidic protein (GFAP) and Map2. The expression of PSD95 was used to demonstrate synaptogenesis.

RESULTSAfter NSCs were treated with RA or SCs, the expression of nestin and GFAP was significantly decreased while the expression of Map2 and PSD95 was significantly increased in comparison with the control. Factorial ANOVA showed that interactions between SCs and RA could induce the expression of Map2 and PSD95.

CONCLUSIONSCs and RA could promote synergistically the neuronal differentiation and synaptogenesis of hippocampal neural stem cells in vitro while they decreased the astrocytes and nestin positive NSCs.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Drug Synergism ; Fluorescent Antibody Technique ; Glial Fibrillary Acidic Protein ; metabolism ; Hippocampus ; cytology ; drug effects ; ultrastructure ; Intermediate Filament Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; metabolism ; Stem Cells ; cytology ; drug effects ; ultrastructure ; Synapses ; drug effects ; physiology ; Tretinoin ; pharmacology

OBJECTIVETo test a nerve bridge substitute for peripheral nerve repair by tissue-engineering approach.

METHODSAn artificial nerve fabricated with a scroll of amnion derivative (ZQ membrane) and cultured autogenous Schwann cell was sutured to bridge sciatic nerve defect of 2.5 cm in length in rats. The specimens were assessed with tracking study, histology, electrophysiological technique, NF200, and synaptophysin-38 (SYP) immuno histochemical staining 3 months postoperatively.

RESULTSThe regenerated nerve sprouted 3 months after the operation. The regenerated nerve fibers were plentiful and could grow into the recipient nerve and target muscle's motor end plate (MEP) areas to reinnervate target muscle, and reconstruct function of nerve-muscle junction. Functional recovery could reach to 40%-60% of normal control. Nerve-muscle conduction velocity (N-MCV) arrived at 21.77 +/- 1.15 m/s.

CONCLUSIONSA tissue engineering material fabricated with a scroll of ZQ membrane and cultured autologous Schwann cell may be a useful substitute for nerve repair.

Amnion ; cytology ; Animals ; Cells, Cultured ; Female ; Male ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; Sciatic Nerve ; injuries ; surgery ; Tissue Engineering ; methods

OBJECTIVETo investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells.

METHODSBone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry.

RESULTSMononuclear cells separated by Percoll's were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6x10(7) times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%-0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein (GFAP) positive cells were identified; S100 expression was detected among 0.1%-0.2% cells.

CONCLUSIONSBone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; Humans ; Immunohistochemistry ; Intermediate Filament Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurofilament Proteins ; metabolism ; Phosphopyruvate Hydratase ; metabolism ; S100 Proteins ; metabolism ; Schwann Cells ; cytology