Proteomic Analysis of Decellularized Tissue Engineered Laryngeal-Tracheal Scaffolds

Abstract

Objective: The clinical success of a fully tissue-engineered tracheal transplant has indicated that there may be more to using decellularized tissue as a scaffold construct. It has been hypothesized that remnant polypeptides reside in these matrices. We sought to determine the proteome profile of a decellularized laryngeal-tracheal scaffold construct.

Method: A laryngeal-tracheal construct was fabricated using standard decellularization protocols and underwent immunohistochemistry, electron microscopy, biomechanical analysis, and qPCR for total DNA content. Samples from anatomical subsites were prepared for proteomic analysis. Tandem mass-spectrometry (LC-MS/MS) was used to identify peptide sequences and validated using multicolor immunohistochemistry and western blotting.

Results: Decellularization of laryngea-tracheal scaffold airway construct effectively removed cells, MHC components, and DNA while maintaining similar biomechanical properties to native (nondecellularized) tissue. We identified variable expression of polypeptides in the anatomical subsites of the airway scaffold construct. A minimum core set of polypeptides was identified with a subset of proteins with putative roles beyond structural integrity. Of particular interest were small molecular weight polypeptides known to have significant roles in epithelialization and angiogenesis.

Conclusion: We demonstrate that mass spectrometry can be used as a tool to accurately identify residual proteins in a laryngeal-tracheal scaffold. The identification of signaling proteins may be of crucial importance in designing the next generation of “intelligent” off-the-shelf scaffolds.