Engineered Arteries Developed In a Multi-Graft Flow-Stretch Bioreactor with In Vivo Evaluation

Introduction: Tissue engineering provides a means to create arterial grafts that can maintain vascular function comparable to native vessels. To date, successful vascular grafts have been developed using the cell-sheet method as well as synthetic polymer-based approaches. Mechanical conditioning such as cyclic conditioning in bioreactor has been shown to improve collagen production and mechanical properties of engineered tissue (Syedain et al 2008). Nutrient transport also plays an important role in tissue development. Here we present a novel bioreactor designed to apply cyclic stretch combined with transmural flow (Pulse flow stretch (PFS) bioreactor) to arterial constructs. Further, arteries developed in the bioreactor were evaluated in vivo in a rat model.

 

Material and Methods: Fibrin-based engineered arteries were fabricated using 4 mg/mL fibrin and 1 million neonatal human dermal fibroblasts (nHDF) per mL. After 2 weeks, they were mounted in PFS bioreactor for 5 wk at 7% stretch. Grafts conditioned in bioreactor were tested for mechanical properties and collagen production. Further, 1.5 mm ID grafts were implanted interpositionally in the abdominal aorta in 10 wk old HSD:RH-Fox^rnu (nude) rats (Harlin) with end-to-end anastomosis using 10-0 nylon sutures. Rats were maintained post-operatively on an anti-coagulant regiment of Aspirin and Plavix.  The grafts were harvested at 12 and 24 weeks post-implantation.

Result and Discussion:  A bioreactor was developed to condition up to 6 grafts in a single manifold with applied cyclic flow leading to simultaneous circumferential stretching and transmural flow in order to promote tissue growth (Figure 1). The pressure and diameter were non-invasively monitored and used to calculate stiffness in the range of 80-120 mmHg.  Using pressure-diameter correlations, a method was validated to non-invasively predict burst strength during bioreactor culture.  The cell induced axial shortening of the construct was also accommodated to achieve circumferential alignment and mechanical anisotropy comparable to native arteries. Grafts implanted in Rat were evaluated at 12 and 24 weeks. At 12 weeks, grafts (n=2) were aneurysmal.  Several other grafts indicated clotting at 12 weeks.  At 24 weeks (n=1), a graft from a separate fabrication showed no dilation or substantial intimal hyperplasia (Fig. 2a,c). The elastin concentration increased dramatically, becoming comparable to the native artery (Fig. 2b). Histology showed organized elastin similar to the native artery as well as complete graft endothelialization (Fig. 2c).

 

Conclusion: Fibrin-based tissue engineering provides a means to create completely biological engineered arteries in short duration (7 weeks of incubation). Here, we present a bioreactor system to condition artery to achieve physiological compliance and burst strength. While successful implants have been accomplished using synthetic scaffolds and a cell sheet-based method, arterial implants have not been reported to date for a completely biological scaffold. To this end, we present preliminary findings of arterial implants showing function to 6 months in vivo is possible using completely biological tissue-engineered arteries fabricated from fibrin and human cells.

References:

Syedain et al. PNAS 2008: 105(18) p6537-6542.

Acknowledgment:

Funding from NIH R01 HL083880 (to R.T.T.)