仿生可降解PCL-PLGA纤维支架负载人脐带间充质干细胞构建组织工程纤维环
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摘要
目的以聚己内酯(PCL)和聚乳酸聚乙醇酸共聚物(PLGA)为原料构建仿生可降解纤维支架,评估其作为组织工程纤维环支架的可行性。方法以PCL、PLGA混合物为原料,通过熔融纺丝法制备PCL-PLGA混合支架作为实验组,纯PCL支架作为对照组。采用扫描电子显微镜(SEM)观察成品支架微观结构(纤维直径和孔径),测量孔隙率;使用力学加载装置测量支架的弹性模量;体外监测支架降解情况;对支架接种人脐带沃顿胶间充质干细胞(HWJ-MSCs)后采用CCK-8法和细胞Live/dead染色检测支架的生物相容性。结果实验组和对照组支架的纤维直径、孔径和孔隙率组间差异无统计学意义(P>0.05);SEM镜下可见支架纤维取向性好,纤维成角60°;混合支架的压缩弹性模量为(1.42±0.11)MPa,拉伸弹性模量(5.47±0.23)MPa;纯PCL支架的压缩弹性模量为(2.36±0.19)MPa,拉伸弹性模量(8.95±0.22)MPa;体外降解检测结果表明混合支架的降解周期和纤维环自我修复过程相适应;CCK-8检测和细胞Live/dead染色结果显示HWJ-MSCs在支架上有良好的增殖能力和活性。结论采用熔融纺丝法制备的PCL-PLGA纤维支架能够模拟天然纤维环的微观结构,具有生物可降解性、良好的生物相容性和力学性能,是构建组织工程椎间盘合适的支架载体。
Objective To construct a biomimetic degradable fiber scaffold with polycaprolactone(PCL) and poly lactic-co-glycolic acid(PLGA) as raw materials, and to evaluate its feasibility as a tissue engineering annulus fibrosus(AF)scaffold.MethodsThe PCL-PLGA hybrid scaffold was prepared by melt spinning method using PCL and PLGA mixture as the experimental group. The pure PCL scaffold was used as the control group. The microstructure(fiber diameter and pore diameter) of the finished scaffold was observed by scanning electron microscopy(SEM), and the porosity was measured. The elastic modulus of the scaffold was measured using a mechanical loading device. The degradation of the scaffold was monitored in vitro. The biocompatibility of the scaffold was detected by CCK-8 method and cell Live/dead staining after seeding Human Wharton's jelly-derived mesenchymal stem cells(HWJ-MSCs).ResultsThere were no significant differences in fiber diameter, pore size and porosity between the experimental group and the control group under SEM(P>0.05). The orientation of the scaffold fiber was good under the microscope, and the fiber angle was 60°. The compression elastic modulus of the hybrid scaffold was(1.42±0.11) MPa, and tensile elastic modulus was(5.47±0.23) MPa. The compressive elastic modulus of the pure PCL scaffold was(2.36±0.19) MPa, and tensile elastic modulus was(8.95±0.22)MPa. The results of in vitro degradation assay indicated that the degradation cycle of the hybrid scaffold was compatible with the self-repair process of the annulus fibrosus. CCK-8 assay and Live/dead staining showed that HWJ-MSCs had good proliferative capacity and activity on the scaffold.ConclusionThe PCL-PLGA fiber scaffold prepared by melt spinning method can simulate the natural AF microstructure, which has biodegradability, good biocompatibility and mechanical properties. It is a suitable scaffold carrier for constructing tissue engineering intervertebral disc.
Objective To construct a biomimetic degradable fiber scaffold with polycaprolactone(PCL) and poly lactic-co-glycolic acid(PLGA) as raw materials, and to evaluate its feasibility as a tissue engineering annulus fibrosus(AF)scaffold.MethodsThe PCL-PLGA hybrid scaffold was prepared by melt spinning method using PCL and PLGA mixture as the experimental group. The pure PCL scaffold was used as the control group. The microstructure(fiber diameter and pore diameter) of the finished scaffold was observed by scanning electron microscopy(SEM), and the porosity was measured. The elastic modulus of the scaffold was measured using a mechanical loading device. The degradation of the scaffold was monitored in vitro. The biocompatibility of the scaffold was detected by CCK-8 method and cell Live/dead staining after seeding Human Wharton's jelly-derived mesenchymal stem cells(HWJ-MSCs).ResultsThere were no significant differences in fiber diameter, pore size and porosity between the experimental group and the control group under SEM(P>0.05). The orientation of the scaffold fiber was good under the microscope, and the fiber angle was 60°. The compression elastic modulus of the hybrid scaffold was(1.42±0.11) MPa, and tensile elastic modulus was(5.47±0.23) MPa. The compressive elastic modulus of the pure PCL scaffold was(2.36±0.19) MPa, and tensile elastic modulus was(8.95±0.22)MPa. The results of in vitro degradation assay indicated that the degradation cycle of the hybrid scaffold was compatible with the self-repair process of the annulus fibrosus. CCK-8 assay and Live/dead staining showed that HWJ-MSCs had good proliferative capacity and activity on the scaffold.ConclusionThe PCL-PLGA fiber scaffold prepared by melt spinning method can simulate the natural AF microstructure, which has biodegradability, good biocompatibility and mechanical properties. It is a suitable scaffold carrier for constructing tissue engineering intervertebral disc.
引文
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[16]Cui N,Qian J,Wang J,et al.Preparation,physicochemical properties and biocompatibility of PBLG/PLGA/bioglass composite scaffolds[J].Mater Sci Eng C Mater Biol Appl,2017,71:118-124.doi:10.1016/j.msec.2016.09.085.
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[2]Xu J,Liu S,Wang S,et al.Decellularised nucleus pulposus as a potential biologic scaffold for disc tissue engineering[J].Mater Sci Eng C Mater Biol Appl,2019,99:1213-1225.doi:10.1016/j.msec.2019.02.045.
[3]Long RG,Burki A,Zysset P,et al.Mechanical restoration and failure analyses of a hydrogel and scaffold composite strategy for annulus fibrosus repair[J].Acta Biomater,2016,30:116-125.doi:10.1016/j.actbio.2015.11.015.
[4]Agrawal CM,Ray RB.Biodegradable polymeric scaffolds for musculoskeletal tissue engineering[J].J Biomed Mater Res,2001,55(2):141-150.
[5]Kelnar I,Zhigunov A,Kapralkova L,et al.Facile preparation of biocompatible poly(lactic acid)-reinforced poly(ε-caprolactone)fibers via graphite nanoplatelets-aided melt spinning[J].J Mech Behav Biomed Mater,2018,84:108-115.doi:10.1016/j.jmbbm.2018.05.013.
[6]Hoyer B,Bernhardt A,Heinemann S,et al.Biomimetically mineralized salmon collagen scaffolds for application in bone tissue engineering[J].Biomacromolecules,2012,13(4):1059-1066.doi:10.1021/bm201776r.
[7]Pan Y,Zhou X,Wei Y,et al.Small-diameter hybrid vascular grafts composed of polycaprolactone and polydioxanone fibers[J].Sci Rep,2017,7(1):3615.doi:10.1038/s41598-017-03851-1.
[8]Sundaram B,Cherian AG,Kumar S.3D decellularized native extracellular matrix scaffold for in vitro culture expansion of human wharton′s jelly-derived mesenchymal stem cells(hWJ MSCs)[J].Methods Mol Biol,2018,1577:35-53.doi:10.1007/7651_2017_71.
[9]Nerurkar NL,Baker BM,Sen S,et al.Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus[J].Nat Mater,2009,8(12):986-992.doi:10.1038/nmat2558.
[10]Alvim Valente C,Cesar Chagastelles P,Fontana Nicoletti N,et al.Design and optimization of biocompatible polycaprolactone/poly(llactic-co-glycolic acid)scaffolds with and without microgrooves for tissue engineering applications[J].J Biomed Mater Res A,2018,106(6):1522-1534.doi:10.1002/jbm.a.36355.
[11]杜立龙,徐宝山,杨强,等.仿生圆周取向微米纤维支架构建组织工程椎间盘纤维环[J].天津医药,2018,46(11):1161-1166.Du LL,Xu BS,Yang Q,et al.Biomimetic circumferential oriented polycaprolactone microfiber scaffold for annulus fibrosus tissue engineering[J].Tianjin Med J,2018,46(11):1161-1166.doi:10.11958/20181572.
[12]杨强,丁晓明,徐宝山,等.取向性丝素蛋白仿生软骨支架的制备及其生物相容性评估[J].天津医药,2015,43(7):709-712.Yang Q,Ding XM,Xu BS,et al.Fabrication and biocompatibility assessment of a silk fibroin scaffold with biomimetic oriented microstructure for cartilage tissue engineering[J].Tianjin Med J,2015,43(7):709-712.doi:10.11958/j.issn.0253-9896.2015.07.002.
[13]Brown TD,Slotosch A,Thibaudeau L,et al.Design and fabrication of tubular scaffolds via direct writing in a melt electrospinning mode[J].Biointerphases,2012,7(1/2/3/4):13.doi:10.1007/s13758-011-0013-7.
[14]Borem R,Madeline A,Walters J,et al.Angle-ply biomaterial scaffold for annulus fibrosus repair replicates native tissue mechanical properties,restores spinal kinematics,and supports cell viability[J].Acta Biomater,2017,58:254-268.doi:10.1016/j.actbio.2017.06.006.
[15]Zhang Y,Chee A,Thonar EJ,et al.Intervertebral disk repair by protein,gene,or cell injection:a framework for rehabilitationfocused biologics in the spine[J].PM R,2011,3(6 Suppl 1):S88-94.doi:10.1016/j.pmrj.2011.04.020.
[16]Cui N,Qian J,Wang J,et al.Preparation,physicochemical properties and biocompatibility of PBLG/PLGA/bioglass composite scaffolds[J].Mater Sci Eng C Mater Biol Appl,2017,71:118-124.doi:10.1016/j.msec.2016.09.085.
[17]Ouyang HW,Goh JCH,Mo XM,et al.Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films[J].Materials Science and Engineering:C,2002,20(1):63-69.doi:10.1016/S0928-4931(02)00014-0.