犬BMSCs复合3D打印支架制备组织工程半月板及体外成软骨诱导时间对其分化的影响
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摘要
为了制备3D打印犬组织工程半月板支架并评价其性能,探索体外成软骨诱导时间对犬骨髓间充质干细胞(BMSCs)-支架复合物生长分化的影响,本试验使用3D打印技术制备聚己内酯(PCL)半月板支架,肉眼和扫描电镜观察支架形态及微观结构,生物力学试验测定支架压缩模量,使用CCK-8细胞毒性试验评价支架与细胞的相容性,并接种犬BMSCs在体外分别进行成软骨诱导7、14、21、28 d,通过倒置相差显微镜观察细胞在支架上的生长情况,通过测定不同诱导时间糖胺聚糖(GAG)含量和Ⅱ型胶原的表达量,分析和比较不同体外诱导时间对其基质合成的影响。结果显示,3D打印制备出的PCL支架对天然半月板解剖形状的还原度较高,孔隙均匀且孔间连通性好,有一定的力学抗压性能和缓慢的降解速度,接种其上的细胞数量呈递增趋势;在BMSCs-支架复合物的体外诱导过程中,细胞不断增殖,GAG和Ⅱ型胶原合成量都随诱导时间的延长而增加,诱导21 d时的合成量显著高于其他诱导时间的合成量(P<0.05)。结果说明,3D打印制备的PCL半月板支架具有良好的理化性能和细胞相容性,有望作为半月板组织工程支架,且体外诱导时间对BMSCs-支架复合物向软骨分化具有重要影响。
The goal of the present study was to prepare 3 D printed canine tissue engineering meniscus scaffold and evaluate its performance, explore the effect of in vitro chondrogenic induction time on the synthesis of extracellular matrix of canine bone marrow mesenchymal stem cells(BMSCs)-scaffold construction. After preparation of polycaprolactone(PCL) meniscus scaffold using 3 D printing technology, the morphology and microstructure of the scaffold were observed by gross and scanning electron microscopy. The compressive modulus of the scaffold was determined by biomechanical test. The compatibility of scaffold and cell was evaluated by CCK-8 cytotoxicity experiment. Canine BMSCs were seeded into scaffold and induced by cartilage in vitro for 7, 14, 21 and 28 d, respectively. The growth of the cells on the scaffold was observed by inverted phase contrast microscopy. The effects of different induction time in vitro on matrix synthesis were analyzed and compared by measuring the glycosaminoglycan(GAG) content and the expression of type Ⅱ collagen at different induction time. The results showed that the PCL scaffold prepared by 3 D printing had high degree of reduction of the natural anatomical shape of the meniscus, uniform pores and good inter-well connectivity, certain mechanical compressive properties and slow degradation rate,the number of cells increased. During the in vitro induction of BMSCs-scaffold construction, the cells proliferated continuously. The synthesis of GAG and type Ⅱ collagen increased with the induction time. The synthesis amount at 21 d was significantly higher than other induction time(P<0.05). The results proved that the PCL meniscus scaffold prepared by 3 D printing has good physical and chemical properties and cell compatibility. It is expected to be used as a meniscus tissue engineering scaffold, and in vitro induction time has an important effect on the differentiation of BMSCs-scaffold construction into cartilage.
The goal of the present study was to prepare 3 D printed canine tissue engineering meniscus scaffold and evaluate its performance, explore the effect of in vitro chondrogenic induction time on the synthesis of extracellular matrix of canine bone marrow mesenchymal stem cells(BMSCs)-scaffold construction. After preparation of polycaprolactone(PCL) meniscus scaffold using 3 D printing technology, the morphology and microstructure of the scaffold were observed by gross and scanning electron microscopy. The compressive modulus of the scaffold was determined by biomechanical test. The compatibility of scaffold and cell was evaluated by CCK-8 cytotoxicity experiment. Canine BMSCs were seeded into scaffold and induced by cartilage in vitro for 7, 14, 21 and 28 d, respectively. The growth of the cells on the scaffold was observed by inverted phase contrast microscopy. The effects of different induction time in vitro on matrix synthesis were analyzed and compared by measuring the glycosaminoglycan(GAG) content and the expression of type Ⅱ collagen at different induction time. The results showed that the PCL scaffold prepared by 3 D printing had high degree of reduction of the natural anatomical shape of the meniscus, uniform pores and good inter-well connectivity, certain mechanical compressive properties and slow degradation rate,the number of cells increased. During the in vitro induction of BMSCs-scaffold construction, the cells proliferated continuously. The synthesis of GAG and type Ⅱ collagen increased with the induction time. The synthesis amount at 21 d was significantly higher than other induction time(P<0.05). The results proved that the PCL meniscus scaffold prepared by 3 D printing has good physical and chemical properties and cell compatibility. It is expected to be used as a meniscus tissue engineering scaffold, and in vitro induction time has an important effect on the differentiation of BMSCs-scaffold construction into cartilage.
引文
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[2] PROFFEN B L,MCELFRESH M,FLEMING B C,et al.A comparative anatomical study of the human knee and six animal species[J].Knee,2012,19(4):493-499.
[3] FOX A J,WANIVENHAUS F,BURGE A J,et al.The human meniscus:a review of anatomy,function,injury,and advances in treatment[J].Clin Anat,2015,28(2):269-287.
[4] BRUCKER P U,VON CAMPE A,MEYER D C,et al.Clinical and radiological results 21 years following successful,isolated,open meniscal repair in stable knee joints[J].Knee,2011,18(6):396-401.
[5] ZHANG Z Z,JIANG D,WANG S J,et al.Scaffolds drive meniscus tissue engineering[J].RSC Adv,2015,5(123):102096.
[6] CHOI Y J,YI H G,KIM S W,et al.3D cell printed tissue analogues:a new platform for theranostics[J].Theranostics,2017,7(12):3118-3137.
[7] DING Z,HUANG H.Mesenchymal stem cells in rabbit meniscus and bone marrow exhibit a similar feature but a heterogeneous multi-differentiation potential:superiority of meniscus as a cell source for meniscus repair[J].BMC Musculoskelet Disord,2015,16:65.
[8] MAKRIS E A,GOMOLL A H,MALIZOS K N,et al.Repair and tissue engineering techniques for articular cartilage[J].Nat Rev Rheumatol,2015,11(1):21-34.
[9] SZOJKA A,LALH K,ANDREWS S H J,et al.Biomimetic 3D printed scaffolds for meniscus tissue engineering[J].Bioprinting,2017,8:1-7.
[10] ENGLUND M,HAUGEN I K,GUERMAZI A,et al.Evidence that meniscus damage may be a component of osteoarthritis:the Framingham study[J].Osteoarthr Cartilage,2016,24(2):270-273.
[11] LU L,ZHANG Q W,WOOTTON D M,et al.Mechanical study of polycaprolactone-hydroxyapatite porous scaffolds created by porogen-based solid freeform fabrication method[J].J Appl Biomater Funct Mater,2014,12(3):145-154.
[12] RONGEN J J,VAN TIENEN T G,VAN BOCHOVE B,et al.Biomaterials in search of a meniscus substitute[J].Biomaterials,2014,35(11):3527-3540.
[13] GROGAN S P,CHUNG P H,SOMAN P,et al.Digital micromirror device projection printing system for meniscus tissue engineering[J].Acta Biomater,2013,9(7):7218-7226.
[14] JUNG J P,BHUIYAN D B,OGLE B M.Solid organ fabrication:comparison of decellularization to 3D bioprinting[J].Biomater Res,2016,20:27.
[15] LI P,FENG X L,JIA X L,et al.Influences of tensile load on in vitro degradation of an electrospun poly(L-lactide-co-glycolide) scaffold[J].Acta Biomater,2010,6(8):2991-2996.
[16] BHARDWAJ N,KUNDU S C.Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends[J].Biomaterials,2012,33(10):2848-2857.
[17] HARRIS G M,PIROLI M E,JABBARZADEH E.Deconstructing the effects of matrix elasticity and geometry in mesenchymal stem cell lineage commitment[J].Adv Funct Mater,2014,24(16):2396-2403.
[18] CENGIZ I F,PEREIRA H,PêGO J M,et al.Segmental and regional quantification of 3D cellular density of human meniscus from osteoarthritic knee[J].J Tissue Eng Regen Med,2017,11(6):1844-1852.
[19] HORáK M,HANDL M,POD■,et al.Comparison of the cellular composition of two different chondrocyte-seeded biomaterials and the results of their transplantation in humans[J].Folia Biol (Praha),2014,60(1):1-9.
[20] ZEIFANG F,OBERLE D,NIERHOFF C,et al.Autologous chondrocyte implantation using the original periosteum-cover technique versus matrix-associated autologous chondrocyte implantation:a randomized clinical trial[J].Am J Sports Med,2010,38(5):924-933.
[21] FISCHER J,DICKHUT A,RICKERT M,et al.Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis[J].Arthritis Rheum,2014,62(9):2696-2706.