Panda Asish Kumar, K Ravikumar, Gebrekrstos Amanuel, Bose Suryasarathi, Markandeya Yogananda S, Mehta Bhupesh, Basu Bikramjit
Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore 560012, India.
Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India.
ACS Appl Mater Interfaces. 2021 Jan 13;13(1):164-185. doi: 10.1021/acsami.0c17257. Epub 2020 Dec 24.
Engineering cellular microenvironment on a functional platform using various biophysical cues to modulate stem cell fate has been the central theme in regenerative engineering. Among the various biophysical cues to direct stem cell differentiation, the critical role of physiologically relevant electric field (EF) stimulation was established in the recent past. The present study is the first to report the strategy to switch EF-mediated differentiation of human mesenchymal stem cells (hMSCs) between neuronal and glial pathways, using tailored functional properties of the biomaterial substrate. We have examined the combinatorial effect of substrate functionalities (conductivity, electroactivity, and topography) on the EF-mediated stem cell differentiation on polyvinylidene-difluoride (PVDF) nanocomposites , without any biochemical inducers. The functionalities of PVDF have been tailored using conducting nanofiller (multiwall-carbon nanotube, MWNT) and piezoceramic (BaTiO, BT) by an optimized processing approach (melt mixing-compression molding-rolling). The DC conductivity of PVDF nanocomposites was tuned from ∼10 to ∼10 S/cm and the dielectric constant from ∼10 to ∼300. The phenotypical changes and genotypical expression of hMSCs revealed the signatures of early differentiation toward neuronal pathway on rolled-PVDF/MWNT and late differentiation toward glial lineage on rolled-PVDF/BT/MWNT. Moreover, we were able to distinguish the physiological properties of differentiated neuron-like and glial-like cells using membrane depolarization and mechanical stimulation. The excitability of the EF-stimulated hMSCs was also determined using whole-cell patch-clamp recordings. Mechanistically, the roles of intracellular reactive oxygen species (ROS), Ca oscillations, and synaptic and gap junction proteins in directing the cellular fate have been established. Therefore, the present work critically unveils complex yet synergistic interaction of substrate functional properties to direct EF-mediated differentiation toward neuron-like and glial-like cells, with distinguishable electrophysiological responses.
利用各种生物物理线索在功能平台上构建细胞微环境以调控干细胞命运，一直是再生工程的核心主题。在引导干细胞分化的各种生物物理线索中，生理相关电场（EF）刺激的关键作用在最近得以确立。本研究首次报道了一种策略，即利用生物材料底物的定制功能特性，在神经元和神经胶质途径之间切换EF介导的人间充质干细胞（hMSCs）分化。我们研究了底物功能（导电性、电活性和拓扑结构）对聚偏二氟乙烯（PVDF）纳米复合材料上EF介导的干细胞分化的组合效应，且未使用任何生化诱导剂。通过优化的加工方法（熔融混合 - 压缩成型 - 轧制），使用导电纳米填料（多壁碳纳米管，MWNT）和压电陶瓷（钛酸钡，BT）对PVDF的功能进行了定制。PVDF纳米复合材料的直流电导率从10⁻⁹ S/cm调节到10⁻⁴ S/cm，介电常数从10调节到300。hMSCs的表型变化和基因表达揭示了在轧制的PVDF/MWNT上向神经元途径早期分化以及在轧制的PVDF/BT/MWNT上向神经胶质谱系晚期分化的特征。此外，我们能够利用膜去极化和机械刺激区分分化的神经元样和神经胶质样细胞的生理特性。还使用全细胞膜片钳记录确定了EF刺激的hMSCs的兴奋性。从机制上讲，已经确定了细胞内活性氧（ROS）、钙振荡以及突触和缝隙连接蛋白在引导细胞命运中的作用。因此，本工作批判性地揭示了底物功能特性之间复杂而协同的相互作用，以引导EF介导的向神经元样和神经胶质样细胞的分化，并具有可区分的电生理反应。
