A Multimodal Approach to Quantify Chondrocyte Viability for Airway Tissue Engineering

Abstract

Objectives/hypothesis: Partially decellularized tracheal scaffolds have emerged as a potential solution for long-segment tracheal defects. These grafts have exhibited regenerative capacity and the preservation of native mechanical properties resulting from the elimination of all highly immunogenic cell types while sparing weakly immunogenic cartilage. With partial decellularization, new considerations must be made about the viability of preserved chondrocytes. In this study, we propose a multimodal approach for quantifying chondrocyte viability for airway tissue engineering.

Methods: Tracheal segments (5 mm) were harvested from C57BL/6 mice, and immediately stored in phosphate-buffered saline at -20°C (PBS-20) or biobanked via cryopreservation. Stored and control (fresh) tracheal grafts were implanted as syngeneic tracheal grafts (STG) for 3 months. STG was scanned with micro-computed tomography (μCT) in vivo. STG subjected to different conditions (fresh, PBS-20, or biobanked) were characterized with live/dead assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and von Kossa staining.

Results: Live/dead assay detected higher chondrocyte viability in biobanked conditions compared to PBS-20. TUNEL staining indicated that storage conditions did not alter the proportion of apoptotic cells. Biobanking exhibited a lower calcification area than PBS-20 in 3-month post-implanted grafts. Higher radiographic density (Hounsfield units) measured by μCT correlated with more calcification within the tracheal cartilage.

Conclusions: We propose a strategy to assess chondrocyte viability that integrates with vivo imaging and histologic techniques, leveraging their respective strengths and weaknesses. These techniques will support the rational design of partially decellularized tracheal scaffolds.

Level of evidence: N/A Laryngoscope, 133:512-520, 2023.

Keywords: Regenerative medicine; biobanking; chondrocyte viability; tissue engineering; tracheal replacement.

Figure 1.. Study design

A. Chronologic workflow and characterization. B. Tracheal segments were explanted. C. Tracheal grafts were either implanted immediately as fresh tracheal segments (a), stored in PBS at −20° C (PBS-20) (b), or cryopreserved at −80° C (biobanked) (c). D. Tracheal grafts were implanted orthotopically in syngeneic recipients for 3 months.

Figure 2.. Characterization of pre-implanted tracheal grafts using live/dead assay.

A. Representative live/dead images of tracheal cross-sections of fresh STG (a), PBS-20 stored overnight (b), PBS-20 stored for 1 month (c), biobanked overnight (d), and biobanked for 1 month (e). B. Quantification of chondrocyte viability. * represents a significant difference between PBS-20 and biobanking at 1 month (p=0.0001) and overnight (p<0.0001). Red * denotes significant differences compared to fresh STG (1 mon PBS-20 p<0.0001, 1 mon biobanking p=0.0266, overnight PBS-20 p<0.0001, overnight biobanking p=0.0081).

Figure 3.. Histologic characterization of pre-and post-implanted tracheal grafts.

A. Representative H&E images. B. Representative images of TUNEL-stained chondrocytes. C. Quantification of chondrocyte viability detected by TUNEL assay. D. Chondrocyte population quantified by DAPI+ cells number/mm2. Red * denotes significant lower chondrocyte population in 3-month post-implanted grafts than pre-implanted grafts (p=0.0218 for fresh, p=0.0296, for PBS-20, p=0.0024 for biobanked grafts); # represent lower chondrocyte population in grafts than in host (p=0.0033 for fresh, p=0.0043 for PBS-20, p=0.0008 for biobanked grafts); * represents lower chondrocyte population in PBS-20 and biobanked tracheal grafts compared to fresh graft (p=0.0003, and p<0.0001), and lower chondrocyte population in biobanked host than fresh host (p=0.0215).

Figure 4.. In vivo Hounsfield units.

A. Representative sagittal view of fresh, PBS-20, and biobanking grafts at end time point using μCT. B. Average HU quantification. Red * denotes significant difference compared to host (p=0.0148, p=0.0002, p=0.0009); * represents significantly higher HU in PBS-20 STG and biobanked STG compared to fresh STG after in vivo for 3 months (p=0.0002, biobanked p=0.0118).

Figure 5.. Calcification correlation with HU.

A. Pre- and post-implant von Kossa stain. B. Proportion of cartilage area calcified in fresh, PBS-20, and biobanking pre- and post-implants. Red * represents 3-month post-implant is higher than pre-implant in PBS-20 and biobanking (p<0.0001, p=0.0002); red # represent host is lower than graft in PBS-20 and biobanking groups at 3 months (p<0.0001, p=0.0008); * represents PBS-20>biobanking>fresh grafts in 3-month post-implants (p=0.0003 PBS vs. biobanking, p<0.0001 PBS vs. fresh, p=0.0002 biobanking vs. fresh). C. Correlation between cartilage calcification area and HU (r=0.9193, p<0.0001).