Comparison of human fibroblast ECM-related gene expression on elastic three-dimensional substrates relative to two-dimensional films of the same material

Three-dimensional elastic substrates were fabricated from a commercially available polyurethane with an internal porosity of approximately 70% and elastic modulus of 27.4±2.76 KPa and examined for suitability in vocal fold tissue engineering. Using immunohistochemistry, biomechanical testing, and RT-PCR; we examined human fibroblast viability, distribution and extracellular matrix related gene expression within substrates for periods up to 4 weeks. We found that cells were capable of colonizing the entire volume of a 5 mm wide×3 mm deep×20 mm long substrate at high viability. Histological cross-sections showed extensive extracellular matrix deposited around the cells and throughout the pore structure of the substrates, which consisted of fibronectin and type I collagen. Cell seeded substrates displayed a significantly higher elastic modulus than unseeded controls similar to native tissue. The transfer of cell growth from two-dimensional to three-dimensional culture resulted in changes in ECM-related gene expression consistent with decreasing cell migration and increasing tissue formation. We found that fibroblasts cultured in three-dimensional substrates expressed significantly higher levels of mRNA for elastin and fibromodulin, while expressing significantly lower levels of mRNA for MMP-1 and hyaluronidase relative to two-dimensional substrates of the same material. The results suggest that three-dimensionally porous, Tecoflex-derived elastic biomaterials may be suitable substrates for engineering vocal fold tissue.