(638f) 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.)