Evaluation of a Physical-Chemical Protocol for Porcine Tracheal Decellularization

Abstract

Introduction/ObjectiveTracheal resection with primary reconstruction is the definitive treatment for many tracheal benign and malignant diseases. When primary resection is not deemed feasible as a result of the length of the stenosis, airway transplantation may become a solution. Tissue engineering offers an alternative way for creating tracheal substitutes. The development of tracheal allograft transplantation includes the decellularized tracheal scaffolds made of extracellular matrix that are seeded with the receptor's cells. Many protocols are used to obtain a decellularized scaffold. Most of them consist of cyclical physical-chemical steps with enzymes. This study proposes a protocol for decellularization based only in physical-chemical steps. MethodsDecellularization of pig tracheal segments was carried out using a standardized protocol consisting of freezing and thawing, 10 cycles of agitation, exposure to sodium deoxycholate, and washing. The degree of decellularization was determined by quantifying residual DNA. We also analyzed the morphology under hematoxylin and eosin staining. ResultsFourteen porcine tracheal segments were decellularized. All scaffolds obtained showed less than 2% of residual DNA (mean 20 ± 8 ng/mg) when compared to the fresh samples (mean 850 ± 123 ng/mg), P = .001. Morphological analysis showed that the epithelium and mixed glands were completely removed. It was possible to identify residual nuclei inside the cartilaginous rings (73.7 ± 12 × 26 ± 8 nuclei/field, P < .001). ConclusionThe protocol tested was able to provide effective decellularization of porcine tracheas.