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.

Most cross-linking methods utilize chemistry or physical processes that are detrimental to cells and tissue development. Those that are not as harmful often do not provide a level of strength that ultimately meets the required application. The purpose of this work was to investigate the use of a ruthenium-sodium persulfate cross-linking system to form dityrosine in fibrin-based engineered tissue. By utilizing the tyrosine residues inherent to fibrin and cell-deposited proteins, at least 3-fold mechanical strength increases and 10-fold stiffness increases were achieved after cross-linking. This strengthening and stiffening effect was found to increase with culture duration prior to cross-linking such that physiologically relevant properties were obtained. Fibrin was not required for this effect as demonstrated by testing with collagen-based engineered tissue. Cross-linked tissues were implanted subcutaneously and shown to have minimal inflammation after 30 days, similar to non-cross-linked controls. Overall, the method employed is rapid, non-toxic, minimally inflammatory, and is capable of increasing strength and stiffness of engineered tissues to physiological levels.

Tissue engineering utilizing fibrin gel as a scaffold has the advantage of creating a completely biological replacement. Cells seeded in a fibrin gel can induce fibril alignment by traction forces when subjected to appropriate mechanical constraints. While gel compaction is key to successful tissue fabrication, excessive compaction can result due to low gel stiffness. This study investigated using ruthenium-catalyzed photo-cross-linking as a method to increase gel stiffness in order to minimize over-compaction. Cross-links between the abundant tyrosine molecules that comprise fibrin were created upon exposure to blue light. Cross-linking was effective in increasing the stiffness of the fibrin gel by 93% with no adverse effects on cell viability. Long-term culture of cross-linked tubular constructs revealed no detrimental effects on cell proliferation or collagen deposition due to cross-linking. After 4 weeks of cyclic distension, the cross-linked samples were more than twice as long as non-cross-linked controls, with similar cell and collagen contents. However, the cross-linked samples required a longer incubation period to achieve a UTS and modulus comparable to controls. This study shows that photo-cross-linking is an attractive option to stiffen the initial fibrin gel and thereby reduce cell-induced compaction, which can allow for longer incubation periods and thus more tissue growth without compaction below a useful size.

Nutrient transport limitation remains a fundamental issue for in vitro culture of engineered tissues. In this study, perfusion bioreactor configurations were investigated to provide uniform delivery of oxygen to media equivalents (MEs) being developed as the basis for tissue-engineered arteries. Bioreactor configurations were developed to evaluate oxygen delivery associated with complete transmural flow (through the wall of the ME), complete axial flow (through the lumen), and a combination of these flows. In addition, transport models of the different flow configurations were analyzed to determine the most uniform oxygen profile throughout the tissue, incorporating direct measurements of tissue hydraulic conductivity, cellular O 2 consumption kinetics, and cell density along with ME physical dimensions. Model results indicate that dissolved oxygen (DO) uniformity is improved when a combination of transmural and axial flow is implemented; however, detrimental effects could occur due to lumenal pressure exceeding the burst pressure or damaging interstitial shear stress imparted by excessive transmural flow rates or decreasing hydraulic conductivity due to ME compaction. The model was verified by comparing predicted with measured outlet DO concentrations. Based on these results, the combination of a controlled transmural flow coupled with axial flow presents an attractive means to increase the transport of nutrients to cells within the cultured tissue to improve growth (increased cell and extracellular matrix concentrations) as well as uniformity.

We examined the effect of insulin supplementation and hypoxic culture (2% vs. 20% oxygen tension) on collagen deposition and mechanical properties of fibrin-based tubular tissue constructs seeded with neonatal human dermal fibroblasts. The results presented here demonstrate that constructs cultured under hypoxic conditions with insulin supplementation increased in collagen density by approximately five-fold and both the ultimate tensile strength (UTS) and modulus by more than three-fold compared with normoxic (20% oxygen tension), noninsulin supplemented controls. In addition, collagen deposited on a per-cell basis increased by approximately four-fold. Interaction was demonstrated for hypoxia and insulin in combination in terms of UTS and collagen production on a per-cell basis. This interaction resulted from two distinct processes involved in collagen fibril formation. Western blot analysis showed that insulin supplementation alone increased Akt phosphorylation and the combined treatment increased collagen prolyl-4-hydroxylase. These molecules are distinct regulators of collagen deposition, having an impact at both the transcriptional and posttranslational modification stages of collagen fibril formation that, in turn, increase collagen density in the tissue constructs. These findings highlight the potential of utilizing insulin supplementation and hypoxic culture in combination to increase the mechanical strength and stiffness of fibrin-based engineered tissues.