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An acellular xenogeneic scaffold derived from the bovine vocal fold lamina propria has shown some promise for in vitro vocal fold tissue engineering. To further explore the potential of the scaffold for cellular attachment, migration, and infiltration, as well as the transport of oxygen, proteins, and nutrients in vivo, this study examined the architecture of pores in the scaffold in terms of several key parameters. Porosity was determined using a standard fluid replacement method with a pycnometer. Average pore size and the pore size distribution were assessed using digital image analysis of scanning electron micrographs. The intrinsic permeability to water was measured using a custom-built hydrostatic pressure apparatus as an estimation of the overall porous nature of the acellular scaffold. The results indicated that the bovine acellular scaffold has a reasonably high porosity (90.49 +/- 4.33%), a proper pore size distribution (60% of the pores with equivalent diametersor =10 microm and100 microm) that could facilitate cellular attachment and infiltration, as well as a relatively high intrinsic permeability (0.21-3.21 darcy) for the transport of soluble factors. These findings offered preliminary support of the potential of the scaffold for facilitating functional extracellular matrix remodeling in vocal fold reconstruction.

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.