bFGF-PLGA缓释微球生物活性组织工程神经的构建和效果评价研究

目的探讨应用SC、ECM凝胶、bFGF-PLGA缓释微球、PLGA纤维微丝整合至高渗透性聚乳酸poly(D,L-lacitic acid),PDLLA]导管中,构建组织工程神经的方法,并评价其修复周围神经缺损的效果。方法培养和纯化1d龄SD近交系乳鼠SC,取第1代细胞(1×107个/mL)与bFGF-PLGA缓释微球、ECM凝胶复合,注入内置PLGA纤维微丝的高渗透性PLLDA导管中,构建组织工程神经。取成年SD大鼠60只,制备坐骨神经15mm缺损模型后,根据移植物不同随机分为5组(n=12)。A组:采用自体神经移植修复;B组:PDLLA导管,管内注满PBS液;C组:含PLGA纤维微丝的PDLLA导管,管内注满30?M凝胶;D组:含PLGA纤维微丝的PDLLA导管,管内注满30?M凝胶与SC的混合物;E组:组织工程神经。术后观察大鼠一般情况,于术后12、16、20及24周行坐骨神经功能指数(sciatic function index,SFI)测定,术后12及24周神经电生理检测,并取材行大体观察和组织学观察。结果术后大鼠均存活至实验完成。术后12、16周,E组SFI与B组差异有统计学意义(P<0.05);术后20、24周,E组与B、C组差异有统计学意义(P<0.05)。术后12周,电生理检测E组坐骨神经传导速度(nerve conduct velocity,NCV)大于B、C组(P<0.05),复合动作电位振幅(compound amplitude,AMP)和动作电位面积分数(action potential area,AREA)大于B、C及D组(P<0.05);术后24周,E组NCV、AMP和AREA大于B、C组(P<0.05)。组织学观察术后12周,E组再生神经组织面积及有髓神经纤维数与A、B、C组比较,差异有统计学意义(P<0.05);有髓神经纤维密度和直径小于A组(P<0.05),大于B、C、D组(P<0.05)。术后24周,E组再生神经组织面积大于B组(P<0.05),有髓神经纤维数与A、B、C组比较有统计学意义(P<0.05);有髓神经纤维密度和直径大于B、C组(P<0.05)。结论SC、ECM凝胶、bFGF-PLGA缓释微球、PLGA纤维微丝与高渗透性PDLLA导管相互整合构建的组织工程神经修复神经缺损后,能促近神经再生,修复效果接近于自体神经移植。

CONSTRUCTION AND EVALUATION OF THE TISSUE ENGINEERED NERVE OF bFGF-PLGA SUSTAINED RELEASE MICROSPHERES

Objective To study the outcomes of nerve defect repair with the tissue engineered nerve,which is composed of the complex of SCs,30?M 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 and ECM gel,and then were injected into permeable PDLLA catheters with PLGA microfilaments inside.In this way,the tissue engineered 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?M gel inside,were used.In group D,PDLLA catheters,with PLGA microfilaments,SCs and 30?M gel inside,were used.In group E,the tissue engineered nerve was applied.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<0.05).At 20 and 24 weeks after the operation,group E was significantly different from groups B and C in SFI (P<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<0.05),and compound amplitude(AMP)as well as action potential area(AREA)of group E were bigger than those of groups B,C and D(P<0.05).At 24 weeks after the operation,NCV,AMP and AREA of group E were bigger than those of groups B and C(P<0.05).At 12 weeks after the operation,histological observation showed the area of regenerated nerves and the number of myelinated fibers in group E were significantly differents from those in groups A,B and C(P<0.05).The density and diameter of myelinated fibers in group E were smaller than those in group A(P< 0.05),but bigger than those in groups B,C and D(P<0.05).At 24 weeks after the operation,the area of regenerative nerves in group E is bigger than those in group B(P<0.05);the number of myelinated fibers in group E was significantly different from those in groups A,B,C(P<0.05);and the density and diameter of myelinated fibers in group E were bigger than those in groups B and C(P<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.