Multimodal Imaging and Theranostics Laboratory, School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal 711103, India.
ACS Appl Bio Mater. 2021 May 17;4(5):4328-4344. doi: 10.1021/acsabm.1c00115. Epub 2021 Apr 12.
We report a significant improvement of adipose-derived mesenchymal stem cells' (ADMSCs) biocompatibility and proliferation on hierarchically patterned porous honey-incorporated silk fibroin scaffolds fabricated using a combination of soft lithography and freeze-drying techniques. Parametric variations show enhanced surface roughness, swelling, and degradation rate with good pore interconnectivity, porosity, and mechanical strength for soft-lithographically fabricated biomimetic microdome arrays on the 2% honey silk fibroin scaffold (PHSF2) as compared to its other variants, which eventually made PHSF2 more comparable to the native environment required for stem cell adhesion and proliferation. PHSF2 also exhibits sustained honey release with remarkable antibacterial efficacy against methicillin-resistant (MRSA). Honey incorporation (biochemical cue) influences microdome structural features, that is, biophysical cues (height, width, and periodicity), which further allows ADMSCs pseudopods (filopodia) to grasp the microdomes for efficient cell-cell communication and cell-matrix interaction and regulates ADMSCs behavior by altering their cytoskeletal rearrangement and thereby increases the cellular spreading area and cell sheet formation. The synergistic effect of biochemical (honey) and biophysical (patterns) cues on ADMSCs studied by the nitro blue tetrazolium assay and DCFDA fluorescence spectroscopy reveals limited free radical generation within cells. Molecular expression studies show a decrease in p53 and p21 expressions validating ADMSCs senescence inhibition, which is further correlated with a decrease in cellular senescence-associated β galactosidase activity. We also show that an increase in CDH1 and CK19 molecular expressions along with an increase in SOX9, RUNX2, and PPARγ molecular expressions supported by PHSF2 justify the substrate's efficacy of underpinning mesenchymal to epithelial transition and multilineage trans-differentiation. This work highlights the fabrication of a naturally healing nutraceutical (honey)-embedded patterned porous stand-alone tool with the potential to be used as smart stem cells delivering regenerative healing implant.
我们报告了一种显著提高脂肪间充质干细胞（ADMSCs）生物相容性和增殖的方法，即在使用软光刻和冷冻干燥技术相结合的方法制造的分层多孔蜂蜜复合丝素纤维支架上。参数变化表明，与其他变体相比，具有良好的孔连通性、孔隙率和机械强度的 2%蜂蜜丝素纤维支架（PHSF2）的表面粗糙度、溶胀和降解速率得到增强，其仿生微穹顶阵列通过软光刻制造，最终使 PHSF2 更类似于干细胞黏附和增殖所需的天然环境。PHSF2 还表现出持续释放蜂蜜的特性，对耐甲氧西林金黄色葡萄球菌（MRSA）具有显著的抗菌功效。蜂蜜的加入（生化信号）影响微穹顶的结构特征，即生物物理信号（高度、宽度和周期性），这进一步允许 ADMSCs 的伪足（丝状伪足）抓住微穹顶，实现有效的细胞间通讯和细胞-基质相互作用，并通过改变细胞骨架重排来调节 ADMSCs 的行为，从而增加细胞的扩展面积和细胞片形成。通过氮蓝四唑试验和 DCFDA 荧光光谱研究 ADMSCs 上的生化（蜂蜜）和生物物理（图案）信号的协同作用表明，细胞内自由基的生成受到限制。分子表达研究表明，p53 和 p21 的表达减少，证实了 ADMSCs 衰老的抑制，这与细胞衰老相关的β-半乳糖苷酶活性的降低有关。我们还表明，随着 CDH1 和 CK19 分子表达的增加，以及 SOX9、RUNX2 和 PPARγ 分子表达的增加，PHSF2 支持间充质向上皮转化和多谱系转分化，证明了该基质的有效性。这项工作强调了制造具有天然愈合能力的营养保健品（蜂蜜）嵌入式图案多孔独立工具的潜力，可作为智能干细胞传递再生愈合植入物。
