ObjectiveTo investigate the in vivo degradation and histocompatibility of modified chitosan based on conductive composite nerve conduit, so as to provide a new scaffold material for the construction of tissue engineered nerve.MethodsThe nano polypyrrole (PPy) was synthesized by microemulsion polymerization, blended with chitosan, and then formed conduit by injecting the mixed solution into a customized conduit formation model. After freeze-drying and deacidification, the nano PPy/chitosan composite conduit (CP conduit) was prepared. Then the CP conduits with different acetyl degree were resulted undergoing varying acetylation for 30, 60, and 90 minutes (CAP1, CAP2, CAP3 conduits). Fourier infrared absorption spectrum and scanning electron microscopy (SEM) were used to identify the conduits. And the conductivity was measured by four-probe conductometer. The above conduits were implanted after the subcutaneous fascial tunnels were made symmetrically on both sides of the back of 30 female Sprague Dawley rats. At 2, 4, 6, 8, 10, and 12 weeks after operation, the morphology, the microstructure, and the degradation rate were observed and measured to assess the in vivo degradation of conduits. HE staining and anti-macrophage immunofluorescence staining were performed to observe the histocompatibility in vivo.ResultsThe characteristic peaks of the amide Ⅱ band around 1 562 cm−1 appeared after being acetylated, indicating that the acetylation modification of chitosan was successful. There was no significant difference in conductivity between conduits (P>0.05). SEM observation showed that the surfaces of the conduits in all groups were similar with relatively smooth surface and compact structure. After the conduits were implanted into the rats, with the extension of time, all conduits were collapsed, especially on the CAP3 conduit. All conduits had different degrees of mass loss, and the higher the degree of acetylation, the greater the mass change (P<0.05). SEM observation showed that there were more pores at 12 weeks after implantation, and the pores showed an increasing trend as the degree of acetylation increased. Histological observation showed that there were more macrophages and lymphocytes infiltration in each group at the early stage. With the extension of implantation time, lymphocytes decreased, fibroblasts increased, and collagen fibers proliferated significantly. ConclusionThe modified chitosan basedon conductive composite nerve conduit made of nano-PPy/chitosan composite with different acetylation degrees has good biocompatibility, conductivity, and biodegradability correlated with acetylation degree in vivo, which provide a new scaffold material for the construction of tissue engineered nerve.
Objective To investigate the time l imit of repairing old sciatic nerve defect in rats and observe the repair effect of autogenous nerve transplantation on old sciatic nerve defect in rats. Methods Thirty-six SD rats of clean grade wererandomized into 6 groups (n=6 per group). The animal model of nerve defect was made by transecting left sciatic nerve at the mid-thigh level. For groups A1, B1 and C1, defects were repaired by the contralateral autogenous nerve transplantation 1, 3 or 6 months after nerve damage and for the control groups of A2, B2 and C2, defects were not repaired. After operation, the gait, toe skin and leg muscle were examined weekly. Three months after autograft, a combination of electrophysiology examination, fluoro gold (FG) retrograde tracing and histological assessment including l ight microscopy, TEM was util ized to investigate the nerve functional recovery. Results Lameness and foot skin ulcers were observed in each group after nerve damage. At 2 months after autograft, such denervation symptoms were only improved in groups A1 and B1. At 3 months after autograft, the motor conduction velocity was (21.84 ± 6.74), (20.02 ± 4.17) and (16.09 ± 8.21) m/s in groups A1, B1 and C1, respectively, showing no statistically significant difference between them (P gt; 0.05). The ampl itude of compound muscle action potential (CAMP) was (12.68 ± 4.38), (9.20 ± 3.43) and (1.22 ± 0.39) mV in groups A1, B1 and C1, respectively, indicating significant differences between groups A1, B1 and group C1 (P lt; 0.05). No CAMP was evident in groups A2, B2 and C2. FG retrograde tracing conducted 3 months after autograft showed that the positive cells were most common in group A1 with big soma, mild in group B1 and lest in group C1 with smallest soma. Gastrocnemius Masson staining showed that the fiber morphology of gastrocnemius in groups A1 and B1 was close to normal, while the rest 4 groups had an obvious atrophy of muscle fiber. The fiber cross-section area was (340.73 ± 118.46), (299.88 ± 119.75), (54.33 ± 53.43), (78.60 ± 51.38), (65.62 ± 25.36), and (40.93 ± 28.22) μm2 in groups A1, B1, C1, A2, B2 and C2, respectively, indicating a significant difference between groups A1, B1 and groups C1, A2, B2 (P lt; 0.05). Neurohistology observation showed that more regenerated nerve fibers were observed in group A1 and B1, but less in group C1. The myel in sheath was thick in groups A1 and B1, while it was thin in group C1. Only SCs and hyperplastic collagen fiber were found in groups A2, B2 and C2. Conclusion Autogenous nerve transplantation is capable of repairing 1- and 3- month sciatic nerve defect to some degree in rat, but repair effect is not obvious on 6-month sciatic nerve defect in rats.
Objective To explore the effect of short-term low-frequency electrical stimulation (SLES) during operation on nerve regeneration in delayed peripheral nerve injury with long gap. Methods Thirty female adult Sprague Dawley rats, weighing 160-180 g, were used to prepare 13-mm defect model by trimming the nerve stumps. Then all rats were randomly divided into 2 groups, 15 rats in each group. After nerve defect was bridged by the contralateral normal sciatic nerve, SLES was applied in the experimental group, but was not in the control group. The spinal cords and dorsal root ganglions (DRGs) were harvested to carry out immunofluorescence histochemistry double staining for growth-associated proteins 43 (GAP-43) and brain-derived neurotrophic factor (BDNF) at 1, 2, and 7 days after repair. Fluorogold (FG) retrograde tracing was performed at 3 months after repair. The mid-portion regenerated segments were harvested to perform Meyer’s trichrome staining, immunofluorescence double staining for neurofilament (NF) and soluble protein 100 (S-100) on the transversely or longitudinal sections at 3 months after repair. The segment of the distal sciatic nerve trunk was harvested for electron microscopy and morphometric analyses to measure the diameter of the myelinated axons, thickness of myelin sheaths, the G ratio, and the density of the myelinated nerve fibers. The gastrocnemius muscles of the operated sides were harvested to measure the relative wet weight ratios. Karnovsky-Root cholinesterase staining of the motor endplate was carried out. Results In the experimental group, the expressions of GAP-43 and BDNF were higher than those in the control group at 1 and 2 days after repair. The number of labeled neurons in the anterior horn of gray matter in the spinal cord and DRGs at the operated side from the experimental group was more than that from the control group. Meyer’s trichrome staining, immunofluorescence double staining, and the electron microscopy observation showed that the regenerated nerves were observed to develop better in the experimental group than the control group. The relative wet weight ratio of experimental group was significantly higher than that of the control group (t=4.633,P=0.000). The size and the shape of the motor endplates in the experimental group were better than those in the control group. Conclusion SLES can promote the regeneration ability of the short-term (1 month) delayed nerve injury with long gap to a certain extent.
Objective To prepare nano polypyrrole (PPy)/chitin composite membrane and observe their biocompatibility. Methods The nano PPy was synthesized by microemulsion polymerization, blended with chitosan and then formed membranes. The membranes were then modified by acetylation to get the experimental membranes (nano PPy/chitin composite membranes, group A). The chitosan membranes (group B) and chitin ones (group C) modified by acetylation acted as control. Scanning electron microscopy and FT-IR spectra were used to identify the nano PPy and the membranes of each group. And the conductivity of membranes of each group was measured. Schwann cells were co-cultured in vitro with each group membranes to observe the biocompatibility by inverted microscope observing, living cell staining, cell counting, and immunofluorescence staining. The lysozyme solution was used to evaluate the degradation of the membranes in vitro. Results The FT-IR spectra showed that the characteristic vibrational absorption peaks of C=C from nano PPy appeared at 1 543.4 cm–1 and 1 458.4 cm–1. Scanning electron microscopy observation revealed that the size of nano PPy particles was about 100-200 nm. The nano PPy particles were synthesized. It was successful to turn chitosan to chitin by the acetylation, which was investigated by FT-IR analysis of membranes in groups A and C. The characteristic peaks of the amide Ⅱ band around 1 562 cm–1 appeared after acetylated modification. Conductivity test showed that the conductivity of membranes in group A was about (1.259 2±0.005 7)×10–3 S/cm, while the conductivity of the membranes in groups B and C was not detected. The nano PPy particles uniformly distributed on the surface of membranes in group A were observed by scanning electron microscope; the membranes in control groups were smooth. As a result, the nano PPy/chitin composite membranes with electrical conductivity were obtained. The cultured Schwann cells were found to survive with good function by fluorescein diacetate live cell staining, soluble protein-100 immunofluorescence staining, and inverted microscope observing. The cell counting showed that the proliferation of Schwann cells after 2 days and 4 days of group A was more than that of the two control groups, and the differences were significant (P<0.05). It indicated that the nano PPy/chitin composite membranes had better ability of adhesion and proliferation than those of chitosan and chitin membranes. The degradation of membranesin vitro showed that the degradation rates of membranes in groups A and C were significantly higher than those in group B at all time points (P<0.05). In a word, the degradation performance of the membranes modified by acetylation was better than that of chitosan membranes under the same condition. Conclusion The nano PPy and chitosan can be blended and modified by acetylation successfully. Nano PPy/chitin composite membranes had electrical conductivity, degradability, and good biocompatibility in vitro.