电纺明胶聚己内酯纳米纤维气凝胶结合软骨细胞外基质修复兔软骨损伤

Electrospun gelatin polycaprolactone nanofiber aerogel combined with cartilage extracellular matrix for repair of cartilage injury in rabbits

目的探讨电纺明胶聚己内酯(GT/PCL)纳米纤维气凝胶(NFA)结合软骨细胞外基质(ECM)对兔软骨损伤的修复作用。方法静电纺丝法制备GT/PCL电纺膜,高速研磨后制成短纤维溶液,取新鲜牛关节软骨制备ECM,将短纤维溶液与ECM溶液(1:10)混匀后冻干,制备GT/PCL/ECM(NFA)三维支架。采用扫描电镜、傅立叶红外(FUR)光谱仪对GT/PCL、ECM、GT/PCL/ECM支架的组成成分、微观结构、溶胀率、孔隙率、抗压强度及降解率进行表征,将支架与软骨细胞共培养进行生物相容性检测。选择15只雄性新西兰大白兔,按随机数字表法分为空白对照组(A组,5只)和ECM组(B组,5只)和复合支架(GT/PCL/ECM)组(C组,5只),建立软骨损伤模型后B、C组植入支架材料。术后3周时对三组动物采用半定量整体MRI成像评分系统(WORMS)评分评估膝关节软骨修复情况;处死动物后对各组膝关节采用国际软骨修复协会组织学评分标准(ICRS)行大体评分,HE染色及番红O-固绿染色行ICRS组织学评分。结果三种支架扫描电镜下均呈现多孔三维结构,FIRT光谱显示GT、PCL已成功引入,电纺GT/PCL NAF的结构松散无法进行材料学表征。GT/PCL/ECM支架的溶胀率为(1092.0±32.2)%,高于ECM支架[(933.6±16.3)%](P<0.01);GT/PCL/ECM支架孔隙率为(92.3±2.9)%,高于ECM支架[(85.9±2.2)%](P<0.05);ECM支架抗压强度[(2.7±0.1)kPa]与GT/PCL/ECM支架抗压强度[(2.4±0.1)kPa]差异无统计学意义(P>0.05);ECM支架降解率高于GT/PCL/ECM支架,但差异无统计学意义(P>0.05)。GT/PCL/ECM支架细胞毒性评级为Ⅰ级,生物相容性较好。3周时C组WORMS评分为(49.0±11.4)分,较B组[(40.0±6.7)分]、A组[(24.0±6.5)分]明显升高(P<0.05或0.01);ICRS大体评分C组为(7.4±1.1)分,较B组[(4.6±1.1)分]、A组[(3.0±1.2)分]明显升高(P<0.01);ICRS组织学评分C组、B组分别为(6.8±0.8)分、(4.2±0.8)分,较入组[(2.8±0.8)分]均明显升高(P<0.05或0.01)。结论T/PCL/ECM(NFA)支架具有与天然软骨相似的组织结构,并在物理特性和生物相容性方面优于传统ECM支架,为软骨细胞的黏附和生长提供了稳定的环境,促进了胶原再生,从而加速软骨损伤的修复。

Objective To investigate the repair effect of electrospun gelatin polycaprolactone(GT/PCL)nanofiber aerogels(NFA)combined with cartilage extracellular matrix(ECM)for treatment of cartilage injuries in rabbits.Methods Firstly,the GT/PCL electrospun membrane was prepared by electrospinning and was ground into the short fiber at high speed.ECM was extracted and separated from fresh bovine articular cartilage,which mixed with the short fiber solution(10:1).Subsequently,it was used to prepared GT/PCL/ECM(NFA)three-dimensional scaffold.Finally,the physical characteristics of the three different scaffolds(GT/PCL,ECM and GT/PCL/ECM)were detected by scanning electron microscope and Fourier-transform infrared(FTIR)spectrometer,including the composition,microstructure,swelling rate,porosity,compressive strength and degradation rates.And the hiocompatibility research was getting on by co-culturing the scaffold with chondrocytes.Fifteen male New Zealand white rabbits were divided into blank control group(Group A,n=5),ECM group(group B,n=5)and composite scaffold(GT/PCL/ECM)group(Group C,n=5)according to the random number table.An injury model was established and three types of bio-scaffold materials were implanted into different groups.At 3 weeks,the cartilage repair was evaluated among groups by semi quantitative global MRI scoring system(WORMS).After the animals were killed,the knee joints of each group were scored by the international society for cartilage repair histological score(ICRs);the ICRs histological score was performedby HE staining and safranine green staining.Results Three scaffolds showed a porous three-dimensional structure under the scanning electron microscope.FIRT showed that GT and PCL were introduced into the scaffolds successfully.The GT/PCL NAF was loose and unable to be characterized by materials science.The swelling rate of GT/PCL/ECM scaffold[(1,092.0±32.2)%]was higher than that of ECM scaffold[(933.6±16.3)%](P<0.01).The porosity of GT/PCL/ECM scaffold[(92.3±2.3)%]was higher than that of ECM scaffold[(85.9±2.2)%](P<0.05).The compressive strength of ECM scaffold[(2.7±0.1)kPa]and of GT/PCL/ECM scaffold[(2.4±0.1)kPa]showed no statistical difference(P>0.05).The degradation rate of ECM scaffold was higher than that of GT/PCL/ECM scaffold,but the difference was not statistically significant(P>0.05).The cytotoxicity rating of GT/PCL/ECM scaffold was grade Ⅰ,indicating that its hiocompatibility was better.At 3 weeks,the MRI WORMS score in Group C[(49.0±11.4)points]was significantly higher than that in Group B[(40.0±6.7)points]and that in Group A[(24.0±6.5)points](P<0.05 or 0.01);the general ICRS score of group C was[(7.4±1.1)points],which was significantly higher than that of group B[(4.6±1.1)points]and group A[(3.0±1.2)points](P<0.01);The ICRS histological scores of group C and group B were f(6.8±0.8)points]and[(4.2±0.8)points]respectively compared with group A[(2.8±0.8)points]were significantly higher(P<0.05 or 0.01).Conclusion GT/PCL/ECM(NFA)scaffold has similar tissue structure to natural cartilage and is superior to traditional ECM scaffold in physical properties and biocompatibility,which provides a stable environment for chondrocyte adhesion and grow th,promotes collagen regeneration,and thus accelerates the repair of cartilage injury.