Chen Chih-Hao, Chen Shih-Hsien, Kuo Chang-Yi, Li Meng-Lun, Chen Jyh-Ping
Department of Chemical and Materials Engineering, Chang Gung University, Taoyuan 33302, Taiwan.
Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital, Kwei-San, Taoyuan 33305, Taiwan.
Nanomaterials (Basel). 2017 Aug 11;7(8):219. doi: 10.3390/nano7080219.
Silk fibroin (SF) and fiber alignment were introduced into polycaprolactone (PCL)-based electrospun nanofibers as chemical and physical cues for tendon tissue engineering applications. The physicochemical properties of random PCL (RP) nanofibers, random PCL/SF (RPSF) nanofibers and aligned PCL/SF (APSF) nanofibers were characterized for fiber orientation and SF blending effects. An in vitro cell culture with rabbit dermal fibroblasts (RDFBs) on nanofibers indicated that SF promotes cell proliferation to a higher extent than fiber alignment. Cells aligned in the direction of fiber axes could be confirmed through scanning electron microscopy (SEM) observation and cytoskeleton staining. The quantitative real-time polymerase chain reaction (qRT-PCR) experiments indicated up-regulated gene expression of tendon marker proteins (type I collagen (Col I), fibronectin and biglycan) on APSF nanofibers and tendon reconstruction was confirmed from Col III gene expression. Animal experiments with Achilles tendon defect repairs in rabbits were carried out with RPSF and APSF scaffolds. The beneficial effects of fiber alignment were verified from histological and immunohistochemical staining, where cell migration and extracellular matrix protein deposition tend to stretch in a parallel direction along the axial direction of APSF nanofibers with enhanced Col I and tenascin C production. Biomechanical testing indicated the tensile stiffness and maximum load of cell-seeded APSF scaffolds were 60.2 and 81.3% of normal tendon values, respectively, which are significantly higher than cell-seeded RPSF or acellular APSF and RPSF scaffolds. These results suggest that APSF nanofiber scaffolds combined with RDFBs have the potential to repair the gap defects of Achilles tendons in vivo and to effectively restore the function and structure of tendons.
丝素蛋白（SF）和纤维排列被引入基于聚己内酯（PCL）的电纺纳米纤维中，作为肌腱组织工程应用的化学和物理线索。对随机PCL（RP）纳米纤维、随机PCL/SF（RPSF）纳米纤维和排列PCL/SF（APSF）纳米纤维的物理化学性质进行了表征，以研究纤维取向和SF混合效果。在纳米纤维上用兔真皮成纤维细胞（RDFB）进行的体外细胞培养表明，SF比纤维排列更能促进细胞增殖。通过扫描电子显微镜（SEM）观察和细胞骨架染色可以确认细胞沿纤维轴方向排列。定量实时聚合酶链反应（qRT-PCR）实验表明，APSF纳米纤维上肌腱标记蛋白（I型胶原（Col I）、纤连蛋白和双糖链蛋白聚糖）的基因表达上调，并且从Col III基因表达证实了肌腱重建。用RPSF和APSF支架对兔跟腱缺损修复进行了动物实验。从组织学和免疫组织化学染色验证了纤维排列的有益效果，其中细胞迁移和细胞外基质蛋白沉积倾向于沿APSF纳米纤维轴向平行方向伸展，同时Col I和腱生蛋白C的产生增加。生物力学测试表明，接种细胞的APSF支架的拉伸刚度和最大负荷分别为正常肌腱值的60.2%和81.3%，显著高于接种细胞的RPSF或无细胞APSF和RPSF支架。这些结果表明，APSF纳米纤维支架与RDFB结合有潜力在体内修复跟腱的间隙缺损，并有效恢复肌腱的功能和结构。
