Tissue-engineered arteries based on entrapment of human dermal fibroblasts in fibrin gel yield completely biological vascular grafts that possess circumferential alignment characteristic of native arteries and essential to their mechanical properties. A bioreactor was developed to condition six grafts in the same culture medium while being subjected to similar cyclic distension and transmural flow resulting from pulsed flow distributed among the graft lumens via a manifold. The lumenal pressure and circumferential stretch were noninvasively monitored and used to calculate stiffness in the range of 80-120 mmHg and then to successfully predict graft burst strength. The length of the graft was incrementally shortened during bioreactor culture to maintain circumferential alignment and achieve mechanical anisotropy comparable to native arteries. After 7-9 weeks of bioreactor culture, the fibrin-based grafts were extensively remodeled by the fibroblasts into circumferentially-aligned tubes of collagen and other extracellular matrix with burst pressures in the range of 1400-1600 mmHg and compliance comparable to native arteries. The tissue suture retention force was also suitable for implantation in the rat model and, with poly(lactic acid) sewing rings entrapped at both ends of the graft, also in the ovine model. The strength achieved with a biological scaffold in such a short duration is unprecedented for an engineered artery.

Tissue engineering provides a means to create functional living tissue replacements. Here, we examine the effects of 3 weeks of cyclic distension (CD) on fibrin-based tubular tissue constructs seeded with porcine valve interstitial cells. CD with circumferential strain amplitude ranging from 2.5% to 20% was applied to evaluate the effects of CD on fibrin remodeling into tissue. We hypothesized that during long-term CD cells adapt to cyclic strain of constant strain amplitude (constant CD), diminishing tissue growth. We thus also subjected constructs to CD with strain amplitude that was incremented from 5% to 15% over the 3 weeks of CD [incremental CD (ICD)]. For constant CD, improvement occurred in construct mechanical properties and composition, peaking at 15% strain: ultimate tensile strength (UTS) and tensile modulus increased 47% and 45%, respectively, over statically incubated controls (to 1.1 and 4.7 MPa, respectively); collagen density increased 29% compared with controls (to 27 mg/ml). ICD further improved outcomes. UTS increased 98% and modulus increased 62% compared with the largest values with constant CD, and collagen density increased 34%. Only in the case of ICD was the ratio of collagen content to cell number greater (70%) than controls, consistent with increased collagen deposition per cell. Studies with human dermal fibroblasts showed similar improvements, generalizing the findings, and revealed a 255% increase in extracellular signal-regulated kinase signaling for ICD vs. constant CD. These results suggest cell adaptation may limit conventional strategies of stretching with constant strain amplitude and that new approaches might optimize bioreactor operation.ERK signal ͉ tissue engineering ͉ valve interstitial cells ͉ dermal fibroblast ͉ mechanical loading ͉ conditioning

Current pediatric heart valve replacement options are suboptimal because they are incapable of somatic growth. Thus, children typically have multiple surgeries to replace outgrown valves. In this study, we present the in vivo function and growth potential of our tissue-engineered pediatric tubular valve. The valves were fabricated by sewing two decellularized engineered tissue tubes together in a prescribed pattern using degradable sutures and subsequently implanted into the main pulmonary artery of growing lambs. Valve function was monitored using periodic ultrasounds after implantation throughout the duration of the study. The valves functioned well up to eight weeks, four weeks beyond the suture strength half-life, after which their insufficiency index worsened. Histology from the explanted valves revealed extensive host cell invasion occurred within the engineered root and commenced from the leaflet surfaces. These cells expressed multiple phenotypes, including endothelial, and deposited elastin and collagen IV. Although the tubes fused together along the degradable suture line, as designed, the leaflets shortened compared to their original height. This shortening is hypothesized to result from inadequate fusion at the commissures prior to suture degradation. With appropriate commissure reinforcement, this novel heart valve may provide the somatic growth potential desired for a pediatric valve replacement.

The performance of completely biological, decellularized engineered allografts in a sheep model was evaluated to establish clinical potential of these unique arterial allografts. The 4-mm-diameter, 2-3-cm-long grafts were fabricated from fibrin gel remodeled into an aligned tissue tube in vitro by ovine dermal fibroblasts. Decellularization and subsequent storage had little effect on graft properties, with burst pressure exceeding 4000 mmHg and the same compliance as the ovine femoral artery. Grafts were implanted interpositionally in the femoral artery of six sheep (n = 9), with contralateral sham controls (n = 3). At 8 weeks (n = 5) and 24 weeks (n = 4), all grafts were patent and showed no evidence of dilatation or mineralization. Mid-graft lumen diameter was unchanged. Extensive recellularization occurred, with most cells expressing aSMA. Endothelialization was complete by 24 weeks with elastin deposition evident. These completely biological grafts possessed circumferential alignment/mechanical anisotropy characteristic of native arteries and were cultured only 5 weeks prior to decellularization and storage as ''off-the-shelf'' grafts.

Diseased aortic valves often require replacement, with over 30% of the current aortic valve surgeries performed in patients who will outlive a bioprosthetic valve. While many promising tissue-engineered valves have been created in the lab using the cell-seeded polymeric scaffold paradigm, none have been successfully tested long-term in the aortic position of pre-clinical model. The high pressure gradients and dynamic flow across the aortic valve leaflets require engineering a tissue that has the strength and compliance to withstand high mechanical demand without compromising normal hemodynamics. A long-term preclinical evaluation of an off-the-shelf tissue-engineered aortic valve in sheep model is presented here. The valves were made from a tube of decellularized cell-produced matrix mounted on a frame. The engineered tissue matrix is primarily composed of collagen, with strength and organization comparable to native valve leaflets. In vitro testing showed excellent hemodynamic performance with low regurgitation, low systolic pressure, and large orifice area. The implanted valves showed large-scale leaflet motion and maintained effective orifice area throughout the duration of the 6-month implant, with no calcification. After 24 weeks implantation (over 17 million cycles), the valves showed no change in tensile mechanical properties or collagen content. In addition, histology and DNA quantitation showed repopulation of the engineered matrix with interstitial-like cells and endothelialization. New extracellular matrix deposition, including elastin, further demonstrates positive tissue remodeling in addition to recellularization and valve function. Long-term implantation in the sheep model resulted in functionality, matrix remodeling, and recellularization, unprecedented results for a tissue-engineered aortic valve.