Determining the Immunogenicity and Structural Integrity of Decellularized Tracheal Allografts

Purpose Tracheal reconstruction is required following large circumferential defects that are not amenable to direct anastomosis. The indications for such reconstructions are malignancy, traumatic injury and tracheal stenosis. Airway allotransplantation has recently been described as a solution to this problem but involves risks related to the use immunosuppression. Decellularization techniques have been used in many organs and allow for the use of functional matrices without the need for immunosuppression. The purpose of this study is to determine the immunogenicity and the structural integrity of decellularized tracheal allografts.

Methods Tracheas were harvested from Yorkshire pigs. These matrices were decellularized using multiple cycles of two different detergent-enzymatic reactions described in the literature: Sodium deoxycholate + DNAse I (Protocol A) and Triton X-100 + Pronase + CHAPS (Protocol B). Native tracheas and decellularized tracheas were placed in a device, sealed at both ends and exposed to increasing static negative pressures. CT Scan imaging of these tracheas at different negative pressures was used to plot the luminal area over the pressure to determine tracheal compliance. Following each cycle of decellularization, tracheal sections were paraffin-embedded for histological sections and stained with H&E, DAPI, MHCI and MHCII. Baseline in vitro lymphocyte proliferation assays were also performed in syngeneic and allogeneic pigs and following exposure to native and decellularized tracheal segments.

Results Native tracheas and decellularized tracheas using Protocol A and Protocol B were exposed to increasing static negative pressures. Luminal area, volume and compliance were measured using CT Imaging. Preliminary data shows that native tracheas collapsed completely at a pressure of -30mmHg. Following five cycles of decellularization, decellularized tracheas using Protocol A collapsed at a pressure of -12mmHg while tracheas using Protocol B collapsed at a pressure similar to that of native tracheas. Baseline immunological response via lymphocyte proliferation assays and immunostaining will also be presented.

Conclusions Decellularized tracheal matrices can be used for airway transplantation without the need for immunosuppression. Structural integrity appears to be maintained using protocols with neutral constituents (such as CHAPS). The correlation between maintenance of function and immunogenicity remains to be determined.

Future directions -Determine the structural integrity of decellularized tracheas following recellularization with mesenchymal stem cells and epithelial cells

-Determine the local and systemic immune response following heterotopic implantation of decellularized and recellularized tracheal grafts