Partial decellularization eliminates immunogenicity in tracheal allografts

Abstract There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long‐segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T‐cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.


| INTRODUCTION
Tracheal defects are rare but life-threatening.End-to-end anastomosis is not feasible beyond a certain length, warranting tissue replacement for reconstruction. 1Unfortunately, there is no clinical standard for tracheal replacement.Creation of a graft that can recapitulate the complex structure and function of the trachea is a priority within the field of regenerative medicine.
][8][9][10][11][12] Tracheal cartilage provides the primary structural support for the organ and efforts to decellularize chondrocytes from the dense extracellular matrix result in a loss of graft patency. 9,104][15] Advances in tracheal tissue engineering have relied upon partial decellularization or deepithelialization, resulting in removal of the cells within the tracheal epithelium with preservation of graft chondrocytes. 15,16ing a murine model of orthotopic tracheal transplant, we previously established that partially decellularized tracheal grafts (PDTG) are capable of tracheal neotissue formation with preservation of mechanical properties. 16,17Still, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of PDTG is a critical step toward clinical translation.We use our murine microsurgical model to determine if partial decellularization eliminates allograft immunogenicity, and assess the impact of immunogenicity.
Briefly, tracheas were rinsed with 1Â PBS with 1% penicillin/ streptomycin (P/S, Gibco, Thermo Fisher Scientific, Waltham, MA) and treated with 0.01% sodium dodecyl sulfate (SDS, Sigma-Aldrich, MO) and 0.9% sodium chloride (NaCl, Fisher Scientific, Fair Lawn, NJ) for 5 min.The tracheas were then subjected to consecutive 3 h graded SDS treatments of 0.01% and 0.1% SDS before soaking in 0.2% SDS and 0.1% SDS for 15 min each.They were then treated with 1% Triton X-100 in distilled water for 5 min to remove nucleic acids and underwent a final 0.9% NaCl wash for 15 min.All steps were performed on a shaker at the speed of 48 rounds/min.PDTG were cryopreserved at À80 C until use.

| Implantation of tracheal grafts
Syngeneic tracheal grafts (STG), PDTA, PDTS, and allografts (ATG) were implanted via previously published methods (N = 5/STG, N = 10/PDTA, PDTS, and ATG). 8,18A 5 mm tracheal segment was harvested and immersed in phosphate-buffered saline for implantation.At the time of implantation, 4 mm segment was resected and orthotopically implanted. 8Type of grafts implanted were randomized within blocks of "day of procedure" and "surgeon" to experimental groups and euthanized at various timepoints.Only female mice were used in this study to avoid sex-specific host responses to implanted grafts. 202][23] Animals were closely monitored for early (humane) euthanasia criteria including respiratory distress (labored breathing, stridor) and/or more than 20% weight loss compared to weight before surgery.At a planned or humane endpoint, animals were euthanized with Ketamine/Xylazine cocktail.Once euthanasia was confirmed, the entire trachea including the graft was harvested and placed in 10% Neutral buffered formalin.

| Epithelial height
Epithelialization was assessed with hematoxylin and eosin (H&E) and immunofluorescent staining of post-implantation tracheal sections.
Images of stained sections were captured using bright field and immunofluorescent microscopy (Zeiss, Oberkochen, Germany).Average epithelial height was measured by dividing the area of the graft epithelium by the length of the graft basement membrane.

| Epithelialization
Longitudinal sections were stained with Mouse anti-Acyl Alphatubulin (ACT) to identify ciliated epithelial cells.The extent of epithelial infiltration was measured by dividing percent coverage of the ACT-positive cells by the length of the graft. 18

| Submucosal thickness
Submucosal thickness was quantified using ImageJ software (U. S. National Institutes of Health, Bethesda, MD) and calculated by averaging five regularly spaced measurements of the submucosal height between the cartilage and basement membrane on each graft cartilage ring.

| Micro-computed tomography
Micro-computed tomography (microCT) imaging was performed on live animals to assess graft patency at 1 month and 3 months using a μPET/CT system (U-PET6CTHR, MILabs, Utrecht, The Netherlands).
The animals were anesthetized with inhalational isoflurane in room air at 1-3 L/min and positioned prone.The scan was set as full 360 rotation, x-ray tube of 0.33 mA and 55 kV, 0.750 per step, 1 projection per step, 1 Â 1 binning, and 40 ms exposure time.All microCT images were reconstructed using MILabs reconstruction software v12.0 (Utrecht, The Netherlands) with a 40 μm voxel grid, Hann projection filter, and Gaussian volume filter (160 μm).The area of airway lumen was quantified from each slice of graft scans and analyzed using ImageJ software.

| Statistical analysis
Normally distributed data were compared using Welch's t-test for data with non-equal variances and unpaired t-test and ANOVA for data with equal variances.Non-parametric tests (Mann Whitney-U) were used for data that were not distributed normally.Statistical tests were performed using the GraphPad Prism 8 software (GraphPad Software Inc., CA).Statistical difference was defined as p < 0.05.Experimental data were expressed as means ± standard deviations (SD).

| Orthotopic tracheal transplantation resulted in similar graft patency and survival among graft types
All graft types remained patent with no evidence of stenosis and had similar survival rates (Figure 1a-c).The animals tolerated orthotopic tracheal transplantation well and did not exhibit signs of respiratory distress at the time of euthanasia.At Day 10, allografts were found to have diffuse epithelial sloughing and eosinophilic cellular infiltrate within the lamina propria consistent with acute rejection.This process also resulted in an increase in epithelial height and the loss of ciliated cells (Figure 1d-l; Figure S1). 21,24,25There was no sign of epithelial injury or eosinophilic infiltrate in surgical control (STG), which maintained an epithelium morphologically identical to the native trachea (Figure S1).PDTG also lacked signs of injury and eosinophilic cell infiltration, exhibiting early graft epithelialization.Terminally differentiated ciliated cells were seen repopulating PDTG at Day 10 and recreated pseudostratified epithelium by 1-month (Figure 1i-l; Figure S2).Conversely, allografts demonstrated a blunted epithelium with less ciliation (p = 0.0149) (Figure 1j-l).There was no difference in epithelial morphology between allograft-derived (PDTA) and syngeneic-derived (PDTS) partially decellularized grafts (Figure 1d-l; Figures S2 and S3).

| Partial decellularization attenuates CD8+ T-cell mediated rejection
We measured T-cell infiltration during acute (Day 10) and chronic (1, 3 months) intervals to quantify the immunogenicity of PDTG.In allografts, there was both acute and chronic elevation of CD8+ T-cells within lamina propria (Figure 2a-f).Large amounts of CD8+ T-cells in allografts were associated with apoptotic cells, both of which were not found STG and PDTG (Figure 2g).CD8+ T-cells were found to be similar in PDTA and PDTS, suggesting that partial decellularization eliminated allograft immunogenicity.In both PDTG and allografts, CD4+ T-cells increased at 10 days and 1 month (Figure 2h-k).At 3 months, CD4+ T-cells in PDTG were found to be equivalent to control, while allograft CD4+ T-cells remained persistently elevated.

| DISCUSSION
Using a mouse microsurgical model, we determined that tracheal graft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium characterized by both epithelial height and ciliated cell coverage of the graft. 21,24,25Presence of CD4+ and CD8+ T-cells was not associated with stenosis.7][28] Furthermore, partial decellularization removed graft immunogenicity, with no differences between PDTA and PDTS neotissue formation, and CD8+ T-cell levels are similar to controls and baseline.Since PDTA is generated from an allogenic trachea, this suggests that partial decellularization does not result in acute or chronic rejection.
Our findings also suggest that neo-epithelialization is at least  regeneration. 29,30While the increased CD4+ T-cell presence in allografts could be attributed to rejection, the elevated CD4+ T-cell levels in PDTG at 1 month with a subsequent decrease at 3 months after ciliated epithelial regeneration, suggests that CD4+ presence is associated with neotissue formation.This is further supported by the baseline levels of CD4+ T-cells in STG, which serve as a control for tracheal replacement.
One limitation of this study is the inability to use the wellestablished method of flow cytometry to assess alloreactivity and Tcell phenotypes due to the prohibitive size of the mouse trachea.In summary, we have established the potential of partial decellularization to eliminate the immunogenicity of tracheal allografts while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

| CONCLUSION
We have established that partial decellularization creates grafts that are able to support epithelization while remaining patent in vivo with similar survival rates to surgical controls.Moreover, partial decellularization does not result in rejection, indicating its potential to eliminate the immunogenicity of tracheal allografts.

2 | METHODS 2 . 1 |
Animal care and ethics statement The Institutional Animal Care and Use Committee of the Abigail Wexner Research Institute at Nationwide Children's Hospital (Columbus, OH) reviewed and approved the protocol (AR15-00090).All animals received humane care by standards published by the Public Health Service, National Institutes of Health (Bethesda, MD) in the Care and Use of Laboratory Animals (2011), and US Department of Agriculture (USDA) regulations outlined in the Animal Welfare Act.

Formalin
-fixed STG, PDTA, PDTS, and allografts were decalcified in 15% EDTA at 4 C overnight before being paraffin-embedded.Longitudinal sections (4 μm) were then sectioned with microtome.The sections were then de-paraffinized with xylenes, rehydrated with decreasing concentrations of ethanol, and stained with hematoxylin (Sigma-Aldrich, MO) and counterstained with eosin.Collagen fibers of tracheal grafts implantation were stained with Masson's Trichrome.Apoptotic cells were identified using the Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL).

2. 4
.1 | T-cell infiltration T-cell infiltration was assessed via immunofluorescent staining of CD4 and CD8 T-cells.Briefly, the sections were stained with Rabbit Anti-CD4 (1:500 dilution) and Rabbit Anti-CD8 (1:250 dilution) and Anti-rabbit Alexa Fluor 594 as the secondary antibody.T-cell infiltration was assessed by quantifying the number of T-cells per submucosal area.