The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Biofabrication. 2014 Jun;6(2):024102. doi: 10.1088/1758-5082/6/2/024102. Epub 2014 Jan 24.
Pre-vascularization is important for the reconstruction of dense and metabolically active myocardial tissue and its integration with the host myocardium after implantation. Herein, we demonstrate that the fabrication of micro-channels in alginate scaffold combined with the presentation of adhesion peptides and an angiogenic growth factor promote vessel-like networks in the construct, both in vitro and in vivo. Using a CO2 laser engraving system, 200 µm diameter channels were formed from top to bottom of the 2 mm thick alginate scaffold, with a channel-to-channel distance of 400 µm. Cells were seeded in a sequential manner onto the scaffolds: first, human umbilical vascular endothelial cells (HUVECs) were seeded and cultured for three days, then neonatal rat cardiomyocytes (CMs) and cardiofibroblasts were added at a final cell ratio of 50:35:15, respectively, and the constructs were cultivated for an additional seven days. A vessel-like network was formed within the cell constructs, wherein HUVECs were organized around the channels in a multilayer manner, while the CMs were located in-between the channels and exhibited the characteristic morphological features of a mature cardiac fiber. Acellular scaffolds with the affinity-bound basic fibroblast growth factor were implanted subcutaneously in mice. Increased cell penetration into the channeled scaffold and greater vessel density were found in comparison with the nonchanneled scaffolds. Our results thus point to the importance of micro-channels as a major structural promoter of vascularization in scaffolds, in conjunction with the sequential preculture of ECs and angiogenic factor presentation.
血管预形成对于致密且代谢活跃的心肌组织的重建及其在植入后的与宿主心肌的整合至关重要。在此，我们证明了在海藻酸钠支架中制造微通道，结合黏附肽和血管生成生长因子的呈现，可促进构建体中的类似血管的网络的形成，无论是在体外还是体内。使用 CO2 激光雕刻系统，从 2 毫米厚的海藻酸钠支架的顶部到底部形成 200 µm 直径的通道，通道之间的距离为 400 µm。细胞以顺序方式接种到支架上：首先，接种人脐静脉内皮细胞（HUVEC）并培养三天，然后加入新生大鼠心肌细胞（CMs）和心成纤维细胞，最终细胞比例分别为 50:35:15，再培养另外七天。在细胞构建体中形成了类似血管的网络，其中 HUVEC 以多层方式围绕通道排列，而 CMs 位于通道之间，表现出成熟心肌纤维的特征形态特征。将具有亲和结合的碱性成纤维细胞生长因子的无细胞支架皮下植入小鼠体内。与无通道支架相比，发现细胞更深入地渗透到有通道的支架中，并且血管密度更高。因此，我们的结果表明，微通道作为支架中血管生成的主要结构促进剂，与 EC 顺序预培养和血管生成因子呈现相结合非常重要。
