Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital at Keelung, Keelung 20401, Taiwan.
Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital at Linkou, Collage of Medicine, Chang Gung University, Taoyuan 33305, Taiwan.
Int J Mol Sci. 2021 Oct 18;22(20):11215. doi: 10.3390/ijms222011215.
To recreate the in vivo niche for tendon tissue engineering in vitro, the characteristics of tendon tissue underlines the use of biochemical and biophysical cues during tenocyte culture. Herein, we prepare core-sheath nanofibers with polycaprolactone (PCL) sheath for mechanical support and hyaluronic acid (HA)/platelet-rich plasma (PRP) core for growth factor delivery. Three types of core-sheath nanofiber membrane scaffolds (CSNMS), consisting of random HA-PCL nanofibers (Random), random HA/PRP-PCL nanofibers (Random+) or aligned HA/PRP-PCL (Align) nanofibers, were used to study response of rabbit tenocytes to biochemical (PRP) and biophysical (fiber alignment) stimulation. The core-sheath structures as well as other pertinent properties of CSNMS have been characterized, with Align showing the best mechanical properties. The unidirectional growth of tenocytes, as induced by aligned fiber topography, was confirmed from cell morphology and cytoskeleton expression. The combined effects of PRP and fiber alignment in Align CSNMS lead to enhanced cell proliferation rates, as well as upregulated gene expression and marker protein synthesis. Another biophysical cue on tenocytes was introduced by dynamic culture of tenocyte-seeded Align in a bioreactor with cyclic tension stimulation. Augmented by this biophysical beacon from mechanical loading, dynamic cell culture could shorten the time for tendon maturation in vitro, with improved cell proliferation rates and tenogenic phenotype maintenance, compared to static culture. Therefore, we successfully demonstrate how combined use of biochemical/topographical cues as well as mechanical stimulation could ameliorate cellular response of tenocytes in CSNMS, which can provide a functional in vitro environmental niche for tendon tissue engineering.
为了在体外重现肌腱组织工程的体内微环境，肌腱组织的特性强调了在肌腱细胞培养过程中生物化学和生物物理线索的使用。在此，我们制备了具有聚己内酯（PCL）鞘的核壳纳米纤维，用于机械支撑，以及具有透明质酸（HA）/富含血小板的血浆（PRP）核的用于生长因子传递。使用三种类型的核壳纳米纤维膜支架（CSNMS）来研究兔肌腱细胞对生物化学（PRP）和生物物理（纤维排列）刺激的反应，这三种 CSNMS 由随机 HA-PCL 纳米纤维（Random）、随机 HA/PRP-PCL 纳米纤维（Random+）或定向 HA/PRP-PCL 纳米纤维（Align）组成。CSNMS 的核壳结构以及其他相关特性已得到表征，其中 Align 表现出最佳的机械性能。从细胞形态和细胞骨架表达证实了定向纤维形貌诱导的肌腱细胞的单向生长。PRP 和纤维排列的组合效应在 Align CSNMS 中导致细胞增殖率提高，以及基因表达和标记蛋白合成上调。通过在具有循环张力刺激的生物反应器中对肌腱细胞-接种的 Align 进行动态培养，向肌腱细胞引入了另一个生物物理线索。通过机械加载的生物物理信标增强后，与静态培养相比，动态细胞培养可以缩短肌腱在体外成熟的时间，同时提高细胞增殖率和肌腱形成表型的维持。因此，我们成功地证明了如何联合使用生物化学/形貌线索以及机械刺激可以改善 CSNMS 中肌腱细胞的细胞反应，这可以为肌腱组织工程提供功能性的体外微环境。
