Kafi Md Abdul, Aktar Khudishta, Todo Mitsugu, Dahiya Ravinder
BEST Group, School of Engineering, University of Glasgow, Glasgow G12 8QQ, UK.
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
Regen Biomater. 2020 Mar;7(2):141-151. doi: 10.1093/rb/rbz034. Epub 2019 Sep 30.
Scaffold engineering has attracted significant attention for three-dimensional (3D) growth, proliferation and differentiation of stem cells . Currently available scaffolds suffer from issues such as poor ability for cell adhesion, migration and proliferation. This paper addresses these issues with 3D porous chitosan scaffold, fabricated and functionalized with cysteine-terminated Arg-Gly-Asp (Cys-RGD) tri-peptide on their walls. The study reveals that the compressive moduli of the scaffold is independent to RGD functionalization but shows dependence on the applied freezing temperature (TM) during the fabrication process. The low freezing TM (-80°C) produces scaffold with high compressive moduli (14.64 ± 1.38 kPa) and high TM (-30°C) produces scaffold with low compressive moduli (5.6 ± 0.38 kPa). The Cys-RGD functionalized scaffolds lead to significant improvements in adhesion (150%) and proliferation (300%) of human mesenchymal stem cell (hMSC). The RGD-integrin coupling activates the focal adhesion signaling (Paxillin-FAK-ERK) pathways, as confirmed by the expression of p-Paxillin, p-FAK and p-ERK protein, and results in the observed improvement of cell adhesion and proliferation. The proliferation of hMSC on RGD functionalized surface was evaluated with scanning electron microscopy imaging and distribution though pore was confirmed by histochemistry of transversely sectioned scaffold. The hMSC adhesion and proliferation in scaffold with high compressive moduli showed a constant enhancement (with a slope value 9.97) of compressive strength throughout the experimental period of 28 days. The improved cell adhesion and proliferation with RGD functionalized chitosan scaffold, together with their mechanical stability, will enable new interesting avenues for 3D cell growth and differentiation in numerous applications including regenerative tissue implants.
支架工程在干细胞的三维（3D）生长、增殖和分化方面引起了广泛关注。目前可用的支架存在细胞黏附、迁移和增殖能力差等问题。本文采用3D多孔壳聚糖支架解决这些问题，该支架在其壁上用半胱氨酸末端的精氨酸-甘氨酸-天冬氨酸（Cys-RGD）三肽进行了制备和功能化。研究表明，支架的压缩模量与RGD功能化无关，但取决于制备过程中施加的冷冻温度（TM）。低冷冻温度TM（-80°C）产生具有高压缩模量（14.64±1.38kPa）的支架，而高TM（-30°C）产生具有低压缩模量（5.6±0.38kPa）的支架。Cys-RGD功能化的支架显著改善了人间充质干细胞（hMSC）的黏附（150%）和增殖（300%）。RGD-整合素偶联激活了粘着斑信号通路（桩蛋白-FAK-ERK），p-桩蛋白、p-FAK和p-ERK蛋白的表达证实了这一点，并导致观察到的细胞黏附和增殖改善。通过扫描电子显微镜成像评估了hMSC在RGD功能化表面上的增殖，并通过对横向切片支架的组织化学证实了其在孔中的分布。在28天的实验期内，具有高压缩模量的支架中hMSC的黏附和增殖显示出压缩强度的持续增强（斜率值为9.97）。RGD功能化壳聚糖支架改善的细胞黏附和增殖以及它们的机械稳定性，将为包括再生组织植入物在内的众多应用中的3D细胞生长和分化开辟新的有趣途径。
