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Tracheal decellularization using a combination of chemical, physical and bioreactor methods

Abstract

Introduction

The purpose of this study was to compare different decellularization protocols with the conventional detergent enzymatic method (DEM) using continuous agitation.

Methods

The first experiment compared conventional DEM with sonication and lyophilization+freeze-thaw cycles. A second experiment was carried out to compare time-adjusted DEM (2-hour instead of 4-hour incubations with 4% deoxycholate) to decellularization in a bioreactor. Cellularity was determined by DNA-quantitation, H&E-staining and immunostaining for major histocompatibility complex-1 (MHC-1).

Results

Compared to untreated trachea, DNA content significantly decreased after 2 cycles in all groups in the first experiment and dropped below the minimal criteria for efficient decellularization (<50 ng dsDNA/mg dry weight) after 4 cycles. However, nuclei were seen in the cartilage and MCH-1 staining was detected in some submucosal areas, indicating presence of chondrocytes and cellular residues that may render the scaffold immunogenic. In the second experiment DNA content significantly decreased after 1 cycle in both groups; however, even after 4 cycles, DNA content was above the minimal criteria for efficient decellularization. While collagen-levels remained stable, glycosaminoglycans diminished significantly after the initial cycles.

Conclusions

Efficient decellularization can be achieved after only 4 cycles of DEM compared to the 17 cycles previously reported. The use of a bioreactor can preserve the integrity of the extracellular matrix.

Post author correction

Article Type: ORIGINAL RESEARCH ARTICLE

DOI:10.5301/ijao.5000648

Authors

Yourka D. Tchoukalova, Justin M. Hintze, Richard E. Hayden, David G. Lott

Article History

Disclosures

Financial support: The research was supported by the Center for Regenerative Medicine, Mayo Clinic.
Conflict of interest: None of the authors has financial interest related to this study to disclose.

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Authors

Affiliations

  • Head and Neck Regeneration Program, Center for Regenerative Medicine, Mayo Clinic, Phoenix, AZ - USA
  • Department of Otolaryngology - Head and Neck Surgery, Mayo Clinic, Phoenix, AZ - USA

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