Objective To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroincollagen and to investigate its cytocompatibil ity in vitro. Methods Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckl ing rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 × 104 cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold. Results SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 ± 9) nm and (205 ± 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% ± 1.1% and 85.3% ± 1.3%, respectively; the water absorption rate was 2 647% ± 172% and 2 593% ± 161%, respectively; the tensile strength was (0.32 ± 0.03) MPa and (0.28 ± 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% ± 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P lt; 0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P gt; 0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells prol iferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM. Conclusion The 3D porous nanofiber scaffoldof PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibil ity and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.
Objective To study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lacitic acid) (PDLLA) catheters. Methods SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres andECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissueengineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was appl ied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Eelectrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively. Results All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P lt; 0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P lt; 0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P lt; 0.05), and compound ampl itude (AMP) as well as action potential area (AREA） of group E were bigger than those of groups B, C and D (P lt; 0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (Plt; 0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myel inated fibers in group E were significantly differents from those in groups A, B and C (Plt; 0.05). The density and diameter of myel inated fibers in group E were smaller than those in group A (Plt; 0.05), but bigger than those in groups B, C and D (P lt; 0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P lt; 0.05); the number of myel inated fibers in group E was significantly different from those in groups A, B, C (P lt; 0.05); and the density and diameter of myel inated fibers in group E were bigger than those in groups B and C (Plt; 0.05). Conclusion The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeables PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.
Objective To investigate the velvet antler polypeptide (VAP) on sciatic nerve regeneration in rats through local administration and VAP-PLGA compound membrane. Methods The 3, 15 mg/g of VAP-PLGA compound membranewere prepared by compounding VAP and PLGA, respectively. Seventy-two Wistar rats, male or female, aged 3-6 months and weighing (250 ± 50) g, were selected to make the model of sciatic nerve section. Then, all rats were randomized into 4 groups (n=18): group A in which nothing was given after anatomosis, group B in which 1 mL of VAP at the concentration of 10 mg/L was injected into the gastrocnemius muscle medial for every other day, group C in which 3 mg/g of VAP-PLGA compound membrane was given to the nerve anastomotic stoma and group D in which 15 mg/g of VAP-PLGA compound membrane was given to the nerve anastomotic stoma. The sciatic adhesion degree observation, electrophysiological examination, immunohistochemical staining and hemi-quantity calculation and horseradish peroxidase (HRP) retrograde tracing were conducted 2, 4 and 6 weeks after operation, respectively. Results All rats survived to the end of the experiment, without foot ulcer or neuroma. Severer nervous adherence was observed in group A, mild adherence in group B, and no adherence in groups C and D 2, 4 and 6 weeks after operation, respectively. The recovery rate of the evoked potential of triceps surae in groups B, C and D was better than that in group A (P lt; 0.01), group D was superior to groups B and C (P lt; 0.05) at each time point. No significant difference between group B and group C (P gt; 0.05) 2, 4 weeks after operation was detected, but group C was superior to group B (P lt; 0.05) 6 weeksafter operation. For the regenerative fiber axon and the expression of myelin sheath TGF-β1 and IGF antigen, the staining intensity in groups B, C and D was higher than that in group A at each time point (P lt; 0.05), and there were significant differences between group D and groups B and C 6 weeks after operation (P lt; 0.05), but no difference between groups B and C (P gt; 0.05). The HRP retrograde tracing showed that the myelinated nerve fiber stained by HRP gradually increased as time passed by and myelinated nerve fiber stained by HRP in groups B, C and D was much more than that in group A, and group D was superior to the other groups. No significant difference between group B and group C was detected. Conclusion To apply VAP through either local administration or VAP-PLGA compound membrane around the attached site of nerve anastomosis is capable of promoting nerve regeneration, which has an obvious dose-effect relationship with the dose of VAP. Meanwhile, VAP-PLGA compound membrane can prevent the nerve adhesion.
Objective To evaluate the effect of the plasma treated PLGA nerve conduits seeded BMSCs on repairing SD rat sciatic nerve defects. Methods BMSCs were acquired from 30 newborn SD rats. After ampl ified and passaged for 3 times, PLGA nerve conduits were prepared and some of them were treated with plasma. A 1-cm-length sciatic nerve defect wasmade in 30 4-week-old SD rats, then they were randomly divided into 3 groups for three different nerve defects reconstruction methods (n=10). In the experimental group, defect was repaired by plasma treatment and PGLA nerve conduits seeded with BMSCs; in the control group, by normal PLGA nerve conduits seeded with BMSCs; and in the autologous group, by autologous nerve. At 6 weeks after the surgery, the dynamic walking pattern was recorded and the sciatic function index (SFI) was calculated; the electrophysiological test was taken; the gastrocnemius wet weight recovery rate was calculated; and the image analysis of regenerated nerve was made. Results All rats survived after the surgery and l ived to the end of the experiment. At 6 weeks after the surgery, the dynamic walking pattern of the experimental group and autologous group was better than that of the control group. The SFI value of the experimental, control and autologous groups was —51.02 ± 6.54, —58.73 ± 7.87 and —48.73 ± 3.95, respectively, showing statistically significant differences among the experimental group, control group and autologous group (P lt; 0.05). The results of the motor nerve conduction velocity and wave ampl itude showed that there were statistically significant differences between the experimental group and the control group (P lt; 0.05), and between the control group and the autologous group (Plt; 0.01); but no significant difference between the experimental group and autologous group(Pgt; 0.05); The gastrocnemius wet weight recovery rate of the experimental, control and autologous groups was 56.13% ± 4.27%, 43.14% ± 6.52%, 59.47% ± 3.85%, respectively; showing statistically significant differences among experimental group, control group and autologous group (P lt; 0.05). The density, diameter of regenerated nerve fiber as well as neural sheath thickness of the experimental group were all higher than those of the control group (P lt; 0.05) and lower than those of the autologous nerve group (P lt; 0.05); there was significant difference between the control group and the autologous group (P lt; 0.01). Conclusion Plasma treated PLGA nerve conduits seeded with BMSCs can effectively repair sciatic nerve defects and provide a new strategy for the development of tissue engineered nerve to repair the peripheral nerve defects.
目的:探讨PLGA材料构建的纳米粒载体导入表皮生长因子受体(EGFR)反义寡核苷酸在头颈鳞癌基因治疗中的可行性,为头颈肿瘤基因治疗中载体的选择提供一个新的研究思路。方法:以PLGA为材料,采用油包水双乳化溶剂蒸发法制备载EGFR正义、反义寡核苷酸纳米颗粒;纳米颗粒转染SCCⅦ细胞株;MTT法了解纳米颗粒对细胞的毒性;通过实时荧光定量PCR检测转染后EGFR基因mRNA表达水平。结果:获得了制备载寡核苷酸PLGA纳米颗粒工艺流程,PLGA纳米颗粒平均粒径116nm±7.57nm。纳米颗粒体外转染SCCⅦ细胞,MTT结果显示纳米颗粒对细胞生长无明显抑制效应,同时具有明显抑制EGFR基因mRNA表达效应。结论:PLGA纳米颗粒可以有效地载入反义寡核苷酸,达到抑制靶基因的效果,同时无明显的细胞毒性。