Department of Materials Science and Engineering, Monash University, Wellington Road, Clayton, VIC 3800, Australia.
Department of Materials Science and Engineering, Monash University, Wellington Road, Clayton, VIC 3800, Australia.
Acta Biomater. 2020 Jan 1;101:249-261. doi: 10.1016/j.actbio.2019.11.016. Epub 2019 Nov 10.
Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.
水凝胶是组织工程中极具吸引力的候选材料，而对间充质干细胞（MSCs）进行包封和随后的分化是一种很有前途的策略，可用于修复和再生骨骼和软骨。然而，MSCs 对机械线索（尤其是基质硬度）非常敏感，因此水凝胶的固有柔软性与促进成骨的机械线索不匹配。克服这一限制的一种方法是利用机械转导信号通路并覆盖细胞从其环境中接收到的信号。先前的报告表明，机械敏感性 miRNA（miR-100-5p 和 miR-143-3p）可以增强 MSC 的成骨作用，使用复杂的多步程序转染、包封和分化细胞。在这项研究中，我们开发并表征了一种简便的方法，用于对包封在光交联明胶-PEG 水凝胶中的 MSC 进行原位转染。通过比较不同转染剂和水凝胶组成的影响，我们表明颗粒大小、电荷和水凝胶力学性能都会影响嵌入转染剂复合物的扩散。通过将 MSC 和转染剂都纳入水凝胶中，我们证明了对包封的 MSC 进行成功的原位转染。比较 miR-100-5p/miR-143-3p 的预转染和原位转染对水凝胶包封 MSC 的成骨能力的影响，我们的数据表明，原位转染后矿化和成骨基因表达更好。总的来说，我们展示了一种简单的、一锅法的用于 miRNA 的原位转染系统，以增强 MSC 的成骨潜力，从而显著提高 MSC 分化在用于骨组织工程应用的水凝胶中的效率。
间充质基质细胞（MSCs）对其周围微环境的线索敏感。在较硬基质上生长的 MSC 成骨能力增强，但在用于骨组织工程的水凝胶中受到限制。用机械敏感性 microRNAs（miRNAs）调节促成骨基因代表了克服这一挑战的潜在工具。在这里，我们报告了一种水凝胶平台，用于将 miRNA 递送到包封的 MSC 中。我们对水凝胶组成和转染剂类型对其迁移率和转染效率的影响进行了表征，证明了 MSC 的成功原位转染，并表明 miRNA 可以显著增强成骨矿物质沉积和标记基因表达。与封装前的传统 2D 转染相比，该系统更简单、更有效，因此有望提高骨组织工程中 MSC 的分化。
