Moreira Helena R, Raftery Rosanne M, da Silva Lucília P, Cerqueira Mariana T, Reis Rui L, Marques Alexandra P, O'Brien Fergal J
3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, Avepark, Barco, 4805-017 Guimarães, Portugal.
Biomater Sci. 2021 Mar 21;9(6):2067-2081. doi: 10.1039/d0bm01560a. Epub 2021 Jan 21.
Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth factors which are associated with high cost, bolus release and short half-life. As an alternative to growth factors, we hypothesized that delivering genes-encoding angiogenic growth factors to cells in a scaffold microenvironment would lead to a controlled release of angiogenic proteins promoting vascularization, simultaneously offering structural support for new matrix deposition. Two non-viral vectors, chitosan (Ch) and polyethyleneimine (PEI), were tested to deliver plasmids encoding for vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF2) to human dermal fibroblasts (hDFbs). hDFbs were successfully transfected with both Ch and PEI, without compromising the metabolic activity. Despite low transfection efficiency, superior VEGF and FGF-2 transgene expression was attained when pVEGF was delivered with PEI and when pFGF2 was delivered with Ch, impacting the formation of capillary-like structures by primary human dermal microvascular endothelial cells (hDMECs). Moreover, in a 3D microenvironment, when PEI-pVEGF and Ch-FGF2 were delivered to hDFbs, cells produced functional pro-angiogenic proteins which induced faster formation of capillary-like structures that were retained in vitro for longer time in a Matrigel assay. The dual combination of the plasmids resulted in a downregulation of the production of VEGF and an upregulation of FGF-2. The number of capillary-like segments obtained with this system was inferior to the delivery of plasmids individually but superior to what was observed with the non-transfected cells. This work confirmed that cell-laden scaffolds containing transfected cells offer a novel, selective and alternative approach to impact the vascularization during tissue regeneration. Moreover, this work provides a new platform for pathophysiology studies, models of disease, culture systems and drug screening.
血管化仍然是组织工程中的主要挑战之一。在组织再生的背景下，通常通过添加生长因子来触发毛细血管样结构的形成，而这些生长因子成本高昂、呈大剂量释放且半衰期短。作为生长因子的替代方案，我们推测在支架微环境中将编码血管生成生长因子的基因递送至细胞会导致血管生成蛋白的可控释放，从而促进血管化，同时为新的基质沉积提供结构支持。测试了两种非病毒载体，壳聚糖（Ch）和聚乙烯亚胺（PEI），以将编码血管内皮生长因子（pVEGF）和成纤维细胞生长因子-2（pFGF2）的质粒递送至人真皮成纤维细胞（hDFbs）。hDFbs用Ch和PEI均成功转染，且未损害代谢活性。尽管转染效率较低，但当用PEI递送pVEGF以及用Ch递送pFGF2时，可实现更高的VEGF和FGF-2转基因表达，影响原代人真皮微血管内皮细胞（hDMECs）形成毛细血管样结构。此外，在三维微环境中，当将PEI-pVEGF和Ch-FGF2递送至hDFbs时，细胞产生功能性促血管生成蛋白，这些蛋白诱导毛细血管样结构更快形成，并且在基质胶试验中在体外保留更长时间。质粒的双重组合导致VEGF产生下调以及FGF-2上调。用该系统获得的毛细血管样节段数量低于单独递送质粒，但优于未转染细胞的情况。这项工作证实，含有转染细胞的载细胞支架为影响组织再生过程中的血管化提供了一种新颖、选择性且替代的方法。此外，这项工作为病理生理学研究、疾病模型、培养系统和药物筛选提供了一个新平台。
