载柚皮苷复合支架对兔骨软骨缺损修复的实验研究
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摘要
目的探讨载柚皮苷复合支架的性能及其对兔骨软骨缺损修复的效果。方法利用W/O/W方法制备载柚皮苷和无载柚皮苷缓释微球;以凹凸棒石和Ⅰ型胶原蛋白为材料,通过"3层夹心法"分别构建载柚皮苷、无载柚皮苷和载TGF-β_1复合支架。分别利用体外缓释、扫描电镜和细胞计数试剂盒8法评价载柚皮苷微球的缓释效果、支架的形貌和细胞相容性。取40只日本大耳白兔随机分为A、B、C、D4组,每组10只。于兔双侧股骨髁间窝处制备直径4.5 mm、深4 mm的骨软骨缺损模型,A组为缺损组(空白对照),B、C、D组分别于骨软骨缺损处植入无载柚皮苷复合支架(阴性对照组)、载柚皮苷复合支架(实验组)及载TGF-β_1复合支架(阳性对照组)。分别于术后3、6个月时取材,行大体、HE染色、甲苯胺蓝染色,分别观察骨软骨缺损修复效果;Western blot检测新生软骨Ⅱ型胶原蛋白表达水平。结果载柚皮苷微球具有良好的缓释效果;构建的骨软骨复合支架有较好的孔隙;载柚皮苷软骨层支架细胞的增殖率与无载柚皮苷支架比较明显增加,差异有统计学意义(P<0.05)。兔体内植入实验大体观察示,术后3个月C、D组缺损范围与A、B组相比明显缩小;术后6个月C组缺损处被新生软骨所覆盖,D组新生软骨与周围正常软骨整合良好。组织学染色示,术后3个月A、B组缺损处被少量纤维组织填充,C、D组可见少量软骨生成;术后6个月C、D组新生骨软骨组织与正常骨软骨类似,A、B组缺损处以大量纤维组织为主。Western blot检测示,术后3、6个月C、D组缺损处新生组织中Ⅱ型胶原蛋白表达量均显著高于A、B组,差异有统计学意义(P<0.05);C、D组间比较差异无统计学意义(P>0.05)。结论载柚皮苷复合支架具有良好的组织相容性,并对兔关节骨软骨缺损有较好的修复效果。
Objective To investigate the performance of loading naringin composite scaffolds and its effects on repair of osteochondral defects.Methods The loading naringin and unloading naringin sustained release microspheres were prepared by W/O/W method;with the materials of the attpulgite and the collagen type Ⅰ,the loading naringin,unloading naringin,and loading transforming growth factor β_1(TGF-β_1) osteochondral composite scaffolds were constructed respectively by "3 layers sandwich method".The effect of sustained-release of loading naringin microspheres,the morphology of the composite scaffolds,and the biocompatibility were evaluated respectively by releasing in vitro,scanning electron microscope,and cell counting kit 8.Forty Japanese white rabbits were randomly divided into groups A,B,C,and D,10 rabbits each group.After a osleochondral defect of 4.5 mm in diameter and 4 mm in depth was made in the intercondylar fossa of two femurs.Defect was not repaired in group A(blank control),and defect was repaired with unloading naringin composite scaffolds(negative control group),loading naringin composite scaffolds(experimental group),and loading TGF-β_1 composite scaffolds(positive control group) in groups B,C,and D respectively.At 3 and 6 months after repair,the intercondylar fossa was harvested for the general,HE staining,and toluidine blue staining to observe the repair effect.Western blot was used to detect the expression of collagen type II in the new cartilage.Results Loading naringin microspheres had good effect of sustained-release;the osteochondral composite scaffolds had good porosity;the cell proliferation rate on loading naringin composite scaffold was increased significantly when compared with unloading naringin scaffold(P<0.05).General observation revealed that defect range of groups C and D was reduced significantly when compared with groups A and B at 3 months after repair;at 6 months after repair,defects of group C were covered by new cartilage,and new cartilage well integrated with the adjacent cartilage in group D.The results of histological staining revealed that defects were filled with a small amount of fibrous tissue in groups A and B,and a small amount of new cartilage in groups C and D at 3 months after repair;new cartilage of groups C and D was similar to normal cartilage,but defects were filled with a large amount of fibrous tissue in groups A and B at 6 months after repair.The expression of collagen type Ⅱ in groups C and D was significantly higher than that in groups A and B(P<0.05),but no significant difference was found between groups C and D(P>0.05).Conclusion Loading naringin composite scaffolds have good biocompatibility and effect in repair of rabbit articular osteochondral defects.
Objective To investigate the performance of loading naringin composite scaffolds and its effects on repair of osteochondral defects.Methods The loading naringin and unloading naringin sustained release microspheres were prepared by W/O/W method;with the materials of the attpulgite and the collagen type Ⅰ,the loading naringin,unloading naringin,and loading transforming growth factor β_1(TGF-β_1) osteochondral composite scaffolds were constructed respectively by "3 layers sandwich method".The effect of sustained-release of loading naringin microspheres,the morphology of the composite scaffolds,and the biocompatibility were evaluated respectively by releasing in vitro,scanning electron microscope,and cell counting kit 8.Forty Japanese white rabbits were randomly divided into groups A,B,C,and D,10 rabbits each group.After a osleochondral defect of 4.5 mm in diameter and 4 mm in depth was made in the intercondylar fossa of two femurs.Defect was not repaired in group A(blank control),and defect was repaired with unloading naringin composite scaffolds(negative control group),loading naringin composite scaffolds(experimental group),and loading TGF-β_1 composite scaffolds(positive control group) in groups B,C,and D respectively.At 3 and 6 months after repair,the intercondylar fossa was harvested for the general,HE staining,and toluidine blue staining to observe the repair effect.Western blot was used to detect the expression of collagen type II in the new cartilage.Results Loading naringin microspheres had good effect of sustained-release;the osteochondral composite scaffolds had good porosity;the cell proliferation rate on loading naringin composite scaffold was increased significantly when compared with unloading naringin scaffold(P<0.05).General observation revealed that defect range of groups C and D was reduced significantly when compared with groups A and B at 3 months after repair;at 6 months after repair,defects of group C were covered by new cartilage,and new cartilage well integrated with the adjacent cartilage in group D.The results of histological staining revealed that defects were filled with a small amount of fibrous tissue in groups A and B,and a small amount of new cartilage in groups C and D at 3 months after repair;new cartilage of groups C and D was similar to normal cartilage,but defects were filled with a large amount of fibrous tissue in groups A and B at 6 months after repair.The expression of collagen type Ⅱ in groups C and D was significantly higher than that in groups A and B(P<0.05),but no significant difference was found between groups C and D(P>0.05).Conclusion Loading naringin composite scaffolds have good biocompatibility and effect in repair of rabbit articular osteochondral defects.
引文
1 Cui W,Wang Q,Chen G,et al.Repair of articular cartilage defects with tissue-engineered osteochondral composites in pigs.J BiosciBioeng,2011,111(4):493-500.
2王鲁云,郭林,田丰德.兔自体松质骨颗粒修复关节软骨损伤.临床骨科杂志,2016,19(4):500-503,507.
3赵家宁,徐宝山,杨强.组织工程骨软骨复合组织构建研究进展.中国矫形外科杂志,2014,22(23):2156-2160.
4 Reyes R,Delgado A,Solis R,et al.Cartilage repair by local delivery of transforming growth factor-β_1 or bone morphogenetic protein-2from a novel,segmented polyurethane/polylactic-co-glycolic bilayered scaffold.J Biomed Mater Res A,2014,102(4):1110-1120.
5 Li B,Yang J,Ma L,et al.Influence of the molecular weight of poly(lactide-co-glycolide)on the in vivo cartilage repair by a construct of poly(lactide-co-glycolide)/fibrin gel/mesenchymal stem cells/transforming growth factor-β_1.Tissue Eng Part A,2014,20(1-2):1-11.
6 Yin F,Cai J,Zen W,et al.Cartilage Regeneration of AdiposeDerived Stem Cells in the TGF-β_1-Immobilized PLGA-Gelatin Scaffold.Stem Cell Rev,2015,11(3):453-459.
7李运涛,程科,胡德志,等.柚皮苷对经TNF-α诱导体外培养兔关节软骨细胞增殖及SOD、NOS影响.辽宁中医药大学学报,2008,10(5):175-177.
8苏友新,陈宝军,王艺茹,等.壮骨健膝方含药血清中药物成分骨碎补柚皮苷对IL-1β诱导兔退变软骨细胞“caveolin-p38MAPK”信号通路的影响.中国中西医结合杂志,2014,34(12):1492-1498.
9童华,刘婷婷,杨范莉,等.柚皮苷口腔黏附片的制备及体外释放度研究.西北药学杂志,2014,29(5):500-502.
10王慎东.自体及异体骨软骨移植与骨缺损修复.中国组织工程研究与临床康复,2008,12(40):7905-7908.
11张佼佼,王冬青,王志强,等.组织工程软骨支架材料的应用选择.中国组织工程研究与临床康复,2011,15(29):5467-5470.
12 Muller WE,Neufurth M,Wang S,et al.Morphogenetically active scaffold for osteochondral repair(polyphosphate/alginate/N,Ocarboxymethyl chitosan).Eur Cell Mater,2016,31:174-190.
13林建华,赖建明,林文平,等.胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测.中国修复重建外科杂志,2012,26(8):1001-1006.
14 Da H,Jia SJ,Meng GL,et al.The impact of compact layer in biphasic scaffold on osteochondral tissue engineering.PLoS One,2013,8(1):e54838.
15 Li W,Wang C,Peng J,et al.Naringin inhibits TNF-a induced oxidative stress and inflammatory response in HUVECs via Nox4/NF-κB and PI3K/Akt pathways.Curr Pharm Biotechnol,2014,15(12):1173-1182.
16 Zhao Y,Li Z,Wang W,et al.Naringin Protects Against Cartilage Destruction in Osteoarthritis Through Repression of NF-κB Signaling Pathway.Inflammation,2016,39(1):385-392.
17徐世希,欧阳丽娜,丁劲松.凹土棒石黏土及其应用进展.中南药学,2009,7(6):441-445.
18张晓敏,王世勇,李根,等.Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究.中国生物工程杂志,2016,36(5):27-33.
19张晓敏,宋学文,王维,等.凹凸棒石/Ⅰ型胶原/聚己内酯复合修复兔骨缺损的实验研究.中国修复重建外科杂志,2016,30(5):626-633.
20王长昇,林建华,吴朝阳.TGF-β_1和IGF-1对人骨髓基质干细胞体外增殖和分化的影响.临床和实验医学杂志,2008,7(1):3-4.
21 Fang J,Xu L,Li Y,et al.Roles of TGF-beta 1 signaling in the development of osteoarthritis.Histol Histopathol,2016,31(11):1161-1167.
2王鲁云,郭林,田丰德.兔自体松质骨颗粒修复关节软骨损伤.临床骨科杂志,2016,19(4):500-503,507.
3赵家宁,徐宝山,杨强.组织工程骨软骨复合组织构建研究进展.中国矫形外科杂志,2014,22(23):2156-2160.
4 Reyes R,Delgado A,Solis R,et al.Cartilage repair by local delivery of transforming growth factor-β_1 or bone morphogenetic protein-2from a novel,segmented polyurethane/polylactic-co-glycolic bilayered scaffold.J Biomed Mater Res A,2014,102(4):1110-1120.
5 Li B,Yang J,Ma L,et al.Influence of the molecular weight of poly(lactide-co-glycolide)on the in vivo cartilage repair by a construct of poly(lactide-co-glycolide)/fibrin gel/mesenchymal stem cells/transforming growth factor-β_1.Tissue Eng Part A,2014,20(1-2):1-11.
6 Yin F,Cai J,Zen W,et al.Cartilage Regeneration of AdiposeDerived Stem Cells in the TGF-β_1-Immobilized PLGA-Gelatin Scaffold.Stem Cell Rev,2015,11(3):453-459.
7李运涛,程科,胡德志,等.柚皮苷对经TNF-α诱导体外培养兔关节软骨细胞增殖及SOD、NOS影响.辽宁中医药大学学报,2008,10(5):175-177.
8苏友新,陈宝军,王艺茹,等.壮骨健膝方含药血清中药物成分骨碎补柚皮苷对IL-1β诱导兔退变软骨细胞“caveolin-p38MAPK”信号通路的影响.中国中西医结合杂志,2014,34(12):1492-1498.
9童华,刘婷婷,杨范莉,等.柚皮苷口腔黏附片的制备及体外释放度研究.西北药学杂志,2014,29(5):500-502.
10王慎东.自体及异体骨软骨移植与骨缺损修复.中国组织工程研究与临床康复,2008,12(40):7905-7908.
11张佼佼,王冬青,王志强,等.组织工程软骨支架材料的应用选择.中国组织工程研究与临床康复,2011,15(29):5467-5470.
12 Muller WE,Neufurth M,Wang S,et al.Morphogenetically active scaffold for osteochondral repair(polyphosphate/alginate/N,Ocarboxymethyl chitosan).Eur Cell Mater,2016,31:174-190.
13林建华,赖建明,林文平,等.胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测.中国修复重建外科杂志,2012,26(8):1001-1006.
14 Da H,Jia SJ,Meng GL,et al.The impact of compact layer in biphasic scaffold on osteochondral tissue engineering.PLoS One,2013,8(1):e54838.
15 Li W,Wang C,Peng J,et al.Naringin inhibits TNF-a induced oxidative stress and inflammatory response in HUVECs via Nox4/NF-κB and PI3K/Akt pathways.Curr Pharm Biotechnol,2014,15(12):1173-1182.
16 Zhao Y,Li Z,Wang W,et al.Naringin Protects Against Cartilage Destruction in Osteoarthritis Through Repression of NF-κB Signaling Pathway.Inflammation,2016,39(1):385-392.
17徐世希,欧阳丽娜,丁劲松.凹土棒石黏土及其应用进展.中南药学,2009,7(6):441-445.
18张晓敏,王世勇,李根,等.Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究.中国生物工程杂志,2016,36(5):27-33.
19张晓敏,宋学文,王维,等.凹凸棒石/Ⅰ型胶原/聚己内酯复合修复兔骨缺损的实验研究.中国修复重建外科杂志,2016,30(5):626-633.
20王长昇,林建华,吴朝阳.TGF-β_1和IGF-1对人骨髓基质干细胞体外增殖和分化的影响.临床和实验医学杂志,2008,7(1):3-4.
21 Fang J,Xu L,Li Y,et al.Roles of TGF-beta 1 signaling in the development of osteoarthritis.Histol Histopathol,2016,31(11):1161-1167.