Naskar Sharmistha, Panda Asish Kumar, Kumaran Viswanathan, Mehta Bhupesh, Basu Bikramjit
Centre for Biosystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India.
Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, India.
ACS Appl Bio Mater. 2018 Aug 20;1(2):414-435. doi: 10.1021/acsabm.8b00147. Epub 2018 Jul 31.
The combinatorial influence of a biophysical cue (substrate stiffness) and biomechanical cue (shear flow) on the osteogenesis modulation of human mesenchymal stem cells (hMSCs) is studied for bone regenerative applications. In this work, we report stem cell differentiation on an ultra high molecular weight polyethylene (UHMWPE)-based hybrid nanobiocomposite [reinforced with a multiwalled carbon nanotube (MWCNT) and/or nanohydroxyapatite (nHA)] under a physiologically relevant shear flow (1 Pa) in a custom-built microfluidic device. Using a genotypic assessment with qRT-PCR and phenotypic assessment through analysis of cytoskeletal remodelling and marker proteins, the role of shear on the progression of osteogenesis modulation has been quantitatively established with statistically significant differences between nHA-reinforced and MWCNT-reinforced UHMWPE. Early-stage (alkaline phosphatase activity at day 8), middle-stage (matrix collagenation at day 14), and late-stage (matrix calcification at day 20) events were analyzed using mRNA expression changes of a limited cell volume after microfluidic culture experiments. The conventional Petri dish culture (static) exhibited an increased osteogenesis for nanoparticle-reinforced UHMWPE, irrespective of the type of nanoparticle. The shear-mediated culture experiments resulted in noticeable differences in the degree of osteogenesis with MWCNT being more effective than nHA reinforcement. The shear-mediated osteogenesis has been attributed to the skewed cellular morphology with a higher cell adhesion (vinculin expression) on UHMWPE and nHA than that of UHMWPE and MWCNT. The signatures of the cytoskeletal changes are reflected in terms of left-to-right (L-R) chirality as well as alignment and pattern of actin fibers. Moreover, stemness (vimentin expression) was found to be decreased because of differentiation. The electrophysiological analysis using patch clamp experiments also revealed a higher inward calcium current and intracellular calcium activity for the cells grown on the UHMWPE and nHA nanobiocomposite under shear. Overall, the present study conclusively establishes the synergistic role of substrate stiffness and shear on osteogenesis of hMSCs, in vitro.
为了骨再生应用，研究了生物物理线索（基质硬度）和生物力学线索（剪切流）对人间充质干细胞（hMSCs）成骨调节的组合影响。在这项工作中，我们报告了在定制的微流控装置中，在生理相关的剪切流（1 Pa）下，超高分子量聚乙烯（UHMWPE）基混合纳米生物复合材料[用多壁碳纳米管（MWCNT）和/或纳米羟基磷灰石（nHA）增强]上的干细胞分化情况。通过qRT-PCR进行基因分型评估，并通过分析细胞骨架重塑和标记蛋白进行表型评估，已定量确定了剪切在成骨调节进展中的作用，nHA增强的UHMWPE和MWCNT增强的UHMWPE之间存在统计学上的显著差异。在微流控培养实验后，使用有限细胞体积的mRNA表达变化分析了早期（第8天碱性磷酸酶活性）、中期（第14天基质胶原化）和晚期（第20天基质钙化）事件。传统的培养皿培养（静态）显示，无论纳米颗粒类型如何，纳米颗粒增强的UHMWPE的成骨作用均增强。剪切介导的培养实验导致成骨程度存在明显差异，MWCNT比nHA增强更有效。剪切介导的成骨作用归因于细胞形态的倾斜，在UHMWPE和nHA上的细胞粘附（纽蛋白表达）高于UHMWPE和MWCNT。细胞骨架变化的特征通过从左到右（L-R）的手性以及肌动蛋白纤维的排列和模式得以体现。此外，由于分化，干性（波形蛋白表达）降低。使用膜片钳实验的电生理分析还显示，在剪切作用下，生长在UHMWPE和nHA纳米生物复合材料上的细胞具有更高的内向钙电流和细胞内钙活性。总体而言，本研究最终确定了基质硬度和剪切对hMSCs体外成骨的协同作用。
