A fibrin-based arterial media equivalent.

We report here, with respect to collagen production and the mechanical properties of a fibrin-based media equivalent (ME), on our efforts to optimize the culture conditions of neonatal SMCs entrapped in tubular fibrin gels. We examined several factors, including the concentration of fibrinolysis inhibitor, the cell source and initial number, the addition of TGF-beta and insulin to the culture medium, and the time in culture. We found that varying the concentration of epsilon-aminocaproic acid (ACA), an inhibitor of fibrinolysis, did not affect the collagen production, but that lower concentrations resulted in a compromised physical integrity of the ME. While use of neonatal SMCs yielded superior results over adult SMCs, a higher initial cell number did not improve results. The addition of 1 ng/mL of TGF-beta to the medium increased the collagen content fourfold and the ultimate tensile strength (UTS) and modulus approximately tenfold after 3 weeks, while the addition of both TGF-beta and insulin improved collagen content sixfold and UTS and modulus almost 20-fold. Additional TGF-beta (5 ng/mL) did not improve any of the properties measured, but additional time in culture did. Samples incubated for 6 weeks with TGF-beta and insulin contained about seven times the amount of collagen and had a three-times higher UTS and modulus than did samples incubated for only 3 weeks. When compared to collagen MEs, the fibrin MEs compacted to a greater extent and were both stronger and stiffer when cultured under the same conditions, having after 6 weeks a tensile modulus and ultimate tensile strength similar to those of rat abdominal aorta.

Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent.

We report here on studies examining the use of fibrin as an alternative to collagen for the entrapment of neonatal aortic rat smooth muscle cells (SMCs) in the fabrication of media equivalents. The studies show increased collagen production by fibroblasts entrapped in fibrin, which suggests that fibrin may be used in the fabrication of tissue equivalents to promote increased protein synthesis and remodeling. However, one of the challenges of working with fibrin is the rapid degradation by SMCs. This degradation was effectively inhibited with the addition of epsilon-aminocaproic acid (EACA) to the culture medium in concentrations ranging from 0.25 to 1 mg/mL. We also present results showing that fibrin stimulates collagen production by SMCs. SMCs in fibrin produced 3.2 and 4.9 times the amount of collagen produced by SMCs in collagen when supplemented with 1 and 0.25 mg/mL EACA, respectively. More than half of the collagen produced appeared in the medium rather than the matrix. The collagen in the medium appeared to be processed beyond the proform and may be in an aggregate form. In addition, the presence of type-III collagen or a type-I trimer was indicated by the results of an analysis of the medium by autoradiography.

Mechanisms of stiffening and strengthening in media-equivalents fabricated using glycation.

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.