Max Bergmann Center of Biomaterials, Technische Universität Dresden , Budapester Straße 27, 01069 Dresden, Germany.
Centre for Nano Science and Engineering, Indian Institute of Science , Bangalore 560012, India.
ACS Appl Mater Interfaces. 2015 Oct 21;7(41):23015-28. doi: 10.1021/acsami.5b06390. Epub 2015 Oct 7.
The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.
物理刺激（如表面弹性、形貌和化学性质）对间充质干细胞增殖和分化的影响已经得到了很好的研究。在这方面，我们采用了一种完全不同的方法，证明了固有基底导电性、细胞微环境的定义化学成分以及间歇性电脉冲的传递在驱动间充质干细胞向成骨分化方面的相互作用。为此，我们用聚吡咯烷酮（PANI）基底涂覆单独的胶原蛋白 I （Coll）或与硫酸软骨素（sHya）结合形成人工细胞外基质（aECM），模拟骨组织的天然微环境。此外，骨髓来源的人骨髓间充质干细胞（hMSCs）在这些中等导电性（10（-4）-10（-3）S/cm）的涂覆有 PANI 的 aECM 基底上培养，并通过变压器样耦合（TLC）方法间歇性地暴露于脉冲电场（PEF）中，持续 28 天。基于对一系列终点的分析，推断 Coll/sHya 涂覆的 PANI（PANI/Coll/sHya）基底在培养的 28 天内增加了 hMSCs 的增殖能力，即使在没有 PEF 刺激的情况下也是如此。相反，所采用的 PEF 刺激方案（7ms 矩形脉冲，3.6mV/cm，10Hz）被证明可以增强 hMSCs 的成骨分化潜力。此外，PEF 刺激的 hMSCs 也表现出与非刺激细胞不同的形态特征。同时，在 PANI/Coll/sHya 基底上也观察到碱性磷酸酶活性的早期开始，并导致更多的钙沉积。此外，实时聚合酶链反应结果表明碱性磷酸酶和骨钙素的 mRNA 水平较高，而其他成骨标志物如 runt 相关转录因子 2、Col1A 和骨桥蛋白的表达呈现出与在成骨培养基中培养的对照细胞相似的动态模式。综上所述，我们的实验结果说明了多种参数（如基底导电性、电场刺激和 aECM 涂覆）在体外调节 hMSC 增殖和分化中的相互作用。
