The ultimate goal in vascular tissue engineering is the generation of bioartificial blood vessels that resemble the morphology and function of native vessels as accurately as possible. Previous studies have shown that the tunica intima and tunica media of native blood vessels can be resembled in bioartificial vessels by applying physiological mechanical stimulation using pulsatile perfusion in vitro. However, until today only very few studies have focused on the integration of a functional tunica adventitia including a vascular network known as vasa vasorum, which are of pivotal importance for graft integration and nutrition. For this, it is not only essential to integrate a complex microvascular network in the outer layer of the prosthesis, but also to resemble the physiological longitudinal alignment of capillary vasa vasorum of native vessels. Here, we investigated the effect of physiological in vitro perfusion on the alignment of vasa vasorum capillaries in the tunica adventitia of small-diameter fibrin-based bioartificial blood vessels.
Two-layered fibrin vessels were generated in a step-wise molding technique. First, acellular tunica media-equivalents were molded in a cylindrical mold. For this, a high-concentration fibrin matrix was generated using 25 mg/ml human fibrinogen resuspended in Medium 199. Polymerization was initiated by adding thrombin, factor XIII and Calcium chloride. After initial polymerization, compaction of the matrix was performed by centrifugation using a custom-built rotation device. The compacted tunica media-equivalent was then transferred into a second mold and the tunica adventitia-equivalent was molded around it. For cellularization of this layer, red fluorescent protein expressing human umbilical vein endothelial cells (EC) and adipogenous stem cells (ASC) were suspended in a low-concentration fibrin matrix. Consequently, segments were implemented in bioreactors and incubated for 72 h under longitudinal tension of 50% (tension alone), pulsatile perfusion with physiological cyclic stretch of 5% (pulse alone) at a frequency of 60 bpm, or both factors combined (tension+pulse). Control segments were incubated in cell culture tubes without mechanical stimulation (static).
Complex microvascular networks were generated by endothelial cell self-assembly in the tunica adventitia of every group. Both, longitudinal tension and pulsatile perfusion induced physiological longitudinal alignment of vasa vasorum capillaries parallel to the main vessel axis. This effect was even more pronounced when both stimuli were applied simultaneously. Opposed to that, statically incubated controls showed randomly organized capillary networks.
Integration of a pre-vascularized tunica adventitia in bioartificial blood vessels is a promising strategy to facilitate early graft integration and immediate cell nutrition throughout the vessel wall after implantation of bioartificial vascular grafts. With longitudinal tension and cyclic stretch, we identified two mechanical stimuli that influence capillary tube orientation in tunica adventitia equivalents of fibrin-based bioartificial blood vessels in vitro. Thus, mechanical stimulation represents an effective strategy to generate physiologically aligned vasa vasorum capillaries in cardiovascular tissue engineering."