3B's Research Group, I3Bs-Research Institute on Biomaterials, Biodegradables and Biomimetics , University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine , AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra , Barco, 4805-017 Guimarães , Portugal.
ICVS/3B's-PT Government Associate Laboratory , Braga/Guimarães , Portugal.
ACS Appl Mater Interfaces. 2019 Jun 5;11(22):19830-19840. doi: 10.1021/acsami.9b04616. Epub 2019 May 24.
Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan, and cellulose nanocrystals to recreate the inherent tendon hierarchy from a nano-to-macro scale. These were then coated with tropoelastin (TROPO) through polydopamine (PDA) linking, to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs toward the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin, and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM.
肌腱组织工程策略，通过重建生物物理和生物化学的天然微环境，具有更大的潜力来实现再生。在这里，我们使用具有机械能力的聚己内酯、壳聚糖和纤维素纳米晶体纱线开发了肌腱仿生支架，从纳米到宏观尺度重建了固有肌腱层次结构。然后，通过聚多巴胺 (PDA) 连接将原弹性蛋白 (TROPO) 涂覆在这些支架上，以模拟天然细胞外基质 (ECM) 的组成和弹性。PDA 和 TROPO 涂层都降低了表面硬度，而不会掩盖底层基质。我们发现，接种在这些 TROPO 仿生支架上的人脂肪来源干细胞 (hASC) 更快地获得了它们的纺锤形形态和高长宽比特征，这是肌腱细胞的特征。免疫细胞化学显示，PDA 和 TROPO 涂层表面促进了 hASC 向肌腱谱系的分化，肌腱相关标志物 Scleraxis 和 Tenomodulin 的表达可持续至培养的 21 天。此外，这些表面支持肌腱样细胞外基质的沉积，这得益于 I 型和 III 型胶原蛋白、腱生蛋白和饰胶蛋白的表达。基因表达分析显示，与对照组相比，TROPO 存在时下调了成骨和成纤维标志物，表明适当的细胞外基质沉积。值得注意的是，与 PDA 涂层和未涂层条件相比，暴露于 TROPO 的分化细胞由于明显的弹性蛋白合成和沉积而获得了弹性蛋白特性，有助于形成更具模拟性的基质。总之，我们的仿生基质结合生物物理和生物学线索调节干细胞行为，增强其长期的肌腱形成能力和富含弹性蛋白的细胞外基质的产生。
