Tissue engineering of implantable small-diameter blood vessels using fibrin gel as scaffold
In this work, first we optimize the conditions of fabricating and culturing fibrin gel seeded with smooth muscle cells (SMC) for tissue-engineered blood vessel (TEBV). The microtopology of fibrin matrix is influenced by the concentration of fibrinogen and calcium, while fibrinolysis and matrix remodeling are affected by the presence of the fibrinolytic inhibitor, aprotinin. Here we report the effects of these components on two key properties of TEBVs, namely mechanical strength and vasoreactivity. We found that high concentrations of fibrinogen or calcium decreased both strength and reactivity, significantly. Surprisingly, aprotinin increased mechanical strength but decreased vascular reactivity in a dose dependent manner. TGF-[beta]1 and insulin had a moderate effect on mechanical strength but enhanced reactivity, through receptor and non-receptor mediated pathways significantly. In addition, the combination of TGF-[beta]1, insulin and aprotinin resulted in significant improvement of both properties. Our data suggest that the microtopology of fibrin matrix and the rates of fibrinolysis and extracellular matrix synthesis may affect the properties of TEBVs significantly. They also indicate that biomaterial and culture parameters may have differential effects on mechanical properties vs. vascular reactivity and therefore, engineering blood vessels under conditions that maximize tissue strength may not always result in optimal function. Instead, strength and reactivity must be used in concert for more accurate evaluation of tissue engineered vascular constructs. To improve the mechanical properties, we propose a novel method to engineer TEBVs with improved by sequential application of layers each one serving a different function. To this end, we prepared TEBVs that were composed of two layers: one cellular layer containing smooth muscle cells (SMC) embedded in fibrin hydrogel to provide vascular reactivity and matrix remodeling and a second cell-free fibrin layer composed of high concentration fibrinogen to provide mechanical strength. The time of addition of the second layer was critical in maintaining cohesion of the two layers. The break force of bi-layered TEBVs increased with FBG concentration in the cell-free layer in a dose dependent manner. Bi-layered TEBVs exhibited burst pressure that was 10-fold higher than single-layered tissues and significantly higher than arterial pressure. Interestingly, vascular reactivity, the ability of blood vessels to constrict or dilate, remained high even though the cells were constricting an additional tissue layer. Notably, SMCs in the cellular layer aligned circumferentially in the absence of pulsatile pressure, possibly due to the passive force applied by the mandrel and the acellular layer. These results demonstrate a scalable methodology for engineering implantable TEBVs with improved matrix remodeling, cellular orientation and mechanical properties.