The generation of cellularized bioartificial blood vessels resembling all three layers of the natural vessel wall with physiological morphology and cell alignment is a long pursued goal in vascular tissue engineering. Simultaneous culture of all three layers under physiological mechanical conditions requires highly sophisticated perfusion techniques and still today remains a key challenge. Here, three-layered bioartificial vessels based on fibrin matrices were generated using a stepwise molding technique. Adipose-derived stem cells (ASC) were differentiated to smooth muscle cells (SMC) and integrated in a compacted tubular fibrin matrix to resemble the tunica media. The tunica adventitia-equivalent containing human umbilical vein endothelial cells (HUVEC) and ASC in a low concentration fibrin matrix was molded around it. Luminal seeding with HUVEC resembled the tunica intima. Subsequently, constructs were exposed to physiological mechanical stimulation in a pulsatile bioreactor for 72 h. Compared to statically incubated controls, mechanical stimulation induced physiological cell alignment in each layer: Luminal endothelial cells showed longitudinal alignment, cells in the media-layer were aligned circumferentially and expressed characteristic SMC marker proteins. HUVEC in the adventitia-layer formed longitudinally aligned microvascular tubes resembling vasa vasorum capillaries. Thus, physiologically organized three-layered bioartificial vessels were successfully manufactured by stepwise fibrin molding with subsequent mechanical stimulation.

细胞化生物人工血管的生成类似于天然血管壁的所有三层，具有生理形态和细胞排列，是血管组织工程中长期追求的目标。在生理机械条件下同时培养所有三层需要高度复杂的灌注技术，今天仍然是一个关键挑战。在这里，使用逐步成型技术生成基于纤维蛋白基质的三层生物人工血管。脂肪来源的干细胞 (ASC) 分化为平滑肌细胞 (SMC) 并整合到一个压缩的管状纤维蛋白基质中以类似于中膜。外膜_-在其周围模制在低浓度纤维蛋白基质中含有人脐静脉内皮细胞（HUVEC）和ASC的等效物。HUVEC 的腔内播种类似于内膜。随后，构建体在脉动生物反应器中暴露于生理机械刺激 72 小时。与静态孵育的对照相比，机械刺激在每一层中诱导生理细胞排列：管腔内皮细胞显示纵向排列，介质层中的细胞沿圆周排列并表达特征性SMC标记蛋白。外膜层中的 HUVEC形成了类似滋养管的纵向排列的微血管毛细血管。因此，通过逐步纤维蛋白成型和随后的机械刺激成功地制造了具有生理组织的三层生物人工血管。