Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.

中文翻译：

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血流灌注下双层血管移植物中的层特异性细胞分化。

生物工程移植物具有克服自体和不可吸收的合成血管作为血管替代物的局限性的潜力。然而，创建这些活体移植物的挑战之一是利用仿生组织诱导和维持多种细胞表型。我们具有异型设计的仿生移植物通过将分层的细胞和组织组织引导为天然结构，有望实现功能性新血管再生。在这项研究中，为使这些血管移植物进一步成熟而设计了可灌注的两室式生物反应器腔室，使移植物的腔和外层在细胞特异性培养基中间隔暴露。我们将系统用于血管移植物中的内皮集落形成细胞和多能间充质基质细胞（MSC）的共培养，通过将静电纺丝和熔融电写结合在一起生产。结果表明，在灌注过程中可以诱导并维持靶向细胞表型（即内皮细胞（EC）和血管平滑肌细胞（vSMC））。ECs的融合腔腔层显示出流动响应性，如COX-2，KLF2和eNOS的上调以及应力纤维重塑和细胞伸长所表明。在外层，可以通过使用TGFβ成功地将MSC的周向定向多层结构分化为vSM样细胞，如αSMA，钙蛋白，胶原蛋白IV和（原代）弹性蛋白的上调所表明的那样，而不会影响内皮单层。细胞层抑制了外部和内部介质储层之间的扩散。这意味着将细胞层紧密密封在构建体中，从而形成真正分离的生物反应器隔室，确保内内皮和外平滑肌样层暴露于细胞特异性培养基。总之，使用此系统，我们成功地诱导了具有天然细胞样的组织的特定层细胞分化。这种共培养系统能够创建仿生新血管，因此可以用来研究和改进生物工程化的血管移植物。