Quantification of the mechanical properties of living tissue equivalents (LTEs) is essential for assessing their ultimate functionality as tissue substitutes, yet their delicate nature makes failure testing problematic. For this study, we evaluated the validity of using an inflation device for quantifying the biaxial tensile failure properties of extremely delicate fibroblast-populated collagen gels (CGs) and fibrin gels (FGs). Small samples were circularly clamped and then inflated until rupture. Each sample assumed an approximately spherical shape and burst at its center indicating effective clamping. After two weeks in culture, all LTEs tested were fragile, but the FGs were significantly stronger and more extensible than the CGs (ultimate tensile strength 6.0 kPa +/- 2.0 kPa vs. 2.8 kPa +/- 0.7 kPa; failure strain 3.5 +/- 0.9 vs. 0.26 +/- 0.05, n = 4). After an additional 11 days of culture, the strength of the FGs increased significantly (26.5 kPa +/- 12.7 kPa), and the extensibility decreased (1.9 +/- 0.8, n = 3). This study demonstrates that subtle differences in the properties of LTEs can be measured using inflation methods with minimal sample handling and without having to grow the tissues into anchors or cut the specimens.

Transforming growth factor-beta1 (TGF-beta1) is commonly used to promote matrix production for engineered tissues in vitro, yet it also enhances fibroblast contractility. For applications where contraction is undesirable, we hypothesized that epidermal growth factor (EGF) would yield equivalent mechanical properties without enhancing contractility. In this study, the response of human dermal fibroblasts to EGF (5 ng/mL) and TGF-beta1 (5 ng/mL) was determined within hemispheric fibrin-based gels by assessing matrix compaction and strength, cell number, collagen production, and contractility. After 3 weeks, both cytokines enhanced compaction relative to controls, and EGF roughly doubled matrix strength over controls and TGF-beta1-treated samples. TGF-beta1 induced alpha-smooth muscle actin (alphaSMA) expression whereas EGF did not. TGF-beta1 also increased retraction following substrate release while EGF reduced retraction. Treatment with cytochalasin D revealed that, regardless of growth factor, approximately 10% of the total retraction was due to residual matrix stress accumulated during cell-mediated remodeling. EGF increased the cell number by 17%, whereas TGF-beta1 decreased the cell number by 63% relative to controls. EGF and TGF-beta1 stimulated greater collagen content than controls by 49% and 33%, respectively. These data suggest that EGF may be an attractive alternative to TGF-beta1 for engineering fibrin-based connective tissue substitutes with adequate strength and minimal tissue contractility.

Extracellular matrices without animal components and with high mechanical strength are needed for the development of the next generation of viable skin replacements. The goal of this study was to determine the optimal concentration of epidermal growth factor (EGF) to maximize the strength and collagen content of cell-derived matrix (CDM) produced by fibroblasts in vitro in serum-free media. Scaffold-free CDM samples were produced by human dermal fibroblasts in the presence of 0-50 ng/mL EGF in chemically defined media. After 21 days of culture, a membrane inflation system was used to measure the biaxial tensile strength, failure stretch ratio, and thickness of each treatment group. The fibroblasts treated with 5 ng/mL EGF produced the thickest matrix (270 microm). A thinner (130 microm) matrix, produced when the fibroblasts were treated with 0.5 ng/mL, had an ultimate tensile strength (895 kPa), greater than two times that of the other treatment groups. The fibroblasts treated with 0.5 ng/mL also had the highest collagen density (23.5 mg/cm(3)). Fibroblasts stimulated with the lowest (0.05 ng/mL) and highest (50 ng/mL) concentrations of EGF produced significantly weaker matrices and lower collagen densities. There was no significant correlation between UTS and collagen density suggesting that mechanisms other than density contribute to the strength of the matrix. Taken together, these data indicate that the optimal EGF concentration depends upon the relative importance of matrix strength and volume in a given application and that 0.5-5.0 ng/mL EGF promotes production of a robust extracellular matrix in only 3 weeks.