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Published in final edited form as: Laryngoscope. 2022 May 25;133(3):512–520. doi: 10.1002/lary.30206

A Multimodal Approach to Quantify Chondrocyte Viability for Airway Tissue Engineering

Coreena Chan 1, Lumei Liu 2, Sayali Dharmadhikari 3,4, Kimberly M Shontz 5, Zheng Hong Tan 6, Maxwell Bergman 7, Terri Shaffer 8, Nguyen K Tram 9, Christopher K Breuer 10,11, Mitchel R Stacy 12, Tendy Chiang 13,14
PMCID: PMC9691794  NIHMSID: NIHMS1805029  PMID: 35612419

Abstract

Objectives

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 were 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 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 in vivo imaging and histologic techniques, leveraging their respective strengths and weaknesses. These techniques will support the rational design of partially decellularized tracheal scaffolds.

Keywords: Regenerative medicine, tracheal replacement, biobanking, chondrocyte viability, tissue engineering

Introduction

There is no surgical solution for the management of long-segment tracheal defects1–3. The lack of a tracheal replacement stems from a paucity of autologous tissue. Tissue engineering has the potential to create a viable tracheal replacement capable of renewal, regeneration, and repair3–8. However, trials have revealed that synthetic scaffolds and fully decellularized grafts lack the biological or mechanical cues to support regeneration9,10. Recent innovations using partially decellularized tracheal allografts demonstrate the ability to remove highly antigenic cell types with the preservation of weakly immunogenic cartilage. Partially decellularized tracheal allografts are capable of supporting epithelial regeneration, endothelialization, and chondrocyte viability in vivo in the absence of immunosuppression11–15. However, novel approaches for partial decellularization introduce new considerations for graft cell viability. In vivo and ex vivo strategies to quantify graft viability would provide critical input for graft development.

Beyond partial decellularization, other factors have been shown to impact graft viability of a donor tracheal graft, including ischemia time and graft storage16–19. Quantification of chondrocyte viability can be determined with functional assays and detection of membrane or DNA damage. However, each assay carries inherent limitations. We present an integrated approach to quantify chondrocyte viability in tracheal grafts from different storage conditions using in vivo and in vitro methods. A combination of histological and radiographic techniques permits both pre- and post-implant assessment, as well as longitudinal surveillance of chondrocyte viability. We studied this strategy of quantifying chondrocyte viability with the use of a mouse model of orthotopic tracheal replacement.

Material and methods

Acquisition of segmental tracheal grafts

Tracheal segments were harvested from 6 to 8-week-old female C57BL/6 mice as previously described20,21. The number of animals assigned for experimental and control groups was determined based on anticipated drop from previous surgical morbidity and mortality11,20. Proximal tracheas were dissected free and 5 mm segmental tracheal grafts were explanted from donors. Native tracheal grafts were directly immersed in 1 ml 1X phosphate-buffered saline (PBS) in 1.5 ml tubes and then stored at −20° C immediately. Another group of tracheal grafts was biobanked. Briefly, the native tracheal grafts were directly immersed in 1 ml cryopreservation solution (Dulbecco’s Modified Eagle Medium (DMEM, ATCC, Manassas, VA) with 10% fetal bovine serum, 1% Penicillin/streptomycin, and 5% Dimethyl sulfoxide (DMSO, ATCC)). Then the tubes were inserted in a cryopreservation container (Mr. Frosty™ Freezing Container, ThermosFisher Scientific, Waltham, MA), and stored at −80° C. The tracheal grafts were stored in PBS at −20° C (PBS-20) and biobanked overnight and for 1 month. Fresh native tracheal grafts were not stored and directly processed following explantation (control group). Tracheal grafts in PBS-20 or in biobanked conditions were thawed at 37° C incubator before implantation; fresh tracheal grafts were immediately implanted in the recipient following explantation.

Tracheal graft implantation

Native tracheal grafts were implanted in syngeneic female C57BL/6 mice (6 to 8-week-old) as syngeneic tracheal grafts (STG, N=4 for fresh STG, N=10 for PBS-20 STG, and N=10 for biobanked STG) following previous protocols11,20,21. First, animals were anesthetized and sedated, followed by an aseptic midline incision from the sternum to the hyoid bone. The strap muscles were divided, and the trachea was dissected circumferentially. The trachea was separated away from the recurrent laryngeal nerves and esophagus. The distal end of the tracheal segment was secured to the sternal notch to create a temporary tracheostomy site. The graft was implanted to the superior native airway with 9–0 sterile nylon suture. The tracheostomy site was released and a 3~4mm (three rings) segment of native trachea was excised, representing a long segment tracheal defect in the mouse model. A segment of native trachea was resected followed by the distal anastomosis. One operator performed all of the animal procedures. Animals were carefully observed and euthanized if humane endpoint criteria were met (respiratory distress, loss of >20% pre-implantation weight). The remaining surviving animals were euthanized 3 months post-implantation. The study design is shown in Figure 1. The STG at 3 months (STG-3mon) were harvested and fixed in formalin and embedded in paraffin for subsequent histology.

Figure 1. Study design.

Figure 1.

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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.

Histology

Native trachea and STG-3mon were longitudinally sectioned for pre- and post-implant characterization. Grafts were stained with Hematoxylin and Eosin (H&E) (Sigma-Aldrich, St. Louis, MO, USA) after decalcification to evaluate chondrocyte population (cell number per mm2). Chondrocyte viability was evaluated using the live/dead assay and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Live/dead cytotoxicity kit (Invitrogen™, Thermo Fisher Scientific) was used to stain an isolated tracheal graft with 0.6 μl/ml Calcein-AM and 13 μl/ml ethidium homodimer-1 in PBS (300μl/sample). After incubation for 15 minutes, transverse sections of the cartilage were imaged with a confocal microscope (Zeiss LSM 700, Oberkochen, Germany). Live cells were defined as green-fluorescent calcein-AM representing intracellular esterase activity. Damaged/dead cells were defined as red-fluorescent ethidium homodimer-1 representing diminished plasma membrane integrity. Cellular viability was defined as the percentage of living cells out of total cells [Live % = 100 × (Nlive cells / Ntotal cells) %]. Apoptotic chondrocytes defined by DNA fragmentation were quantified using the TUNEL assay (Sigma-Aldrich). Longitudinal sections were stained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI, Invitrogen) counterstain. Viability was calculated as [Live % = 100 × (Nnuclei +TUNEL+ / Nnuclei+) %]. Images were captured using a Zeiss Imager.M2 fluorescent microscope equipped with an AxiocamHRc black and white digital camera. Live cells were defined as DAPI+ nuclei while dead cells were defined as the overlap of TUNEL+ and DAPI+ nuclei22. A von Kossa stain was used to evaluate the extent of calcification of STG cartilage before decalcification. The amount of calcification was defined as the area of calcification out of the total area of cartilage [Calcification % = 100 × (AreaCalcification / AreaTotal) %] detected using ImageJ software (National Institutes of Health).

Micro-computed tomography (μCT) imaging

μCT imaging was performed using a μPET/CT system (U-PET6CTHR, MILabs, Utrecht, The Netherlands) to assess graft patency and cartilage Hounsfield Units (HU) in host and STG in vivo at 90 days. The animals were anesthetized with inhalational isoflurane in room air at 1–3L/min and positioned prone. The scan settings were as follows: full 360° rotation, X-ray tube settings of 0.33 mA and 55 kV, 0.750° degree per step, 1 projection per step, 1 × 1 binning, and 40 ms exposure time. All μCT images were reconstructed using MILabs reconstruction software v12.0 with an 40 μm voxel grid, Hann projection filter, and Gaussian volume filter (160 μm). Images of the airway lumen were analyzed using ImageJ software (National Institutes of Health). A fixed attenuation threshold of 500 HU was used to segment the cartilage23,24.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., CA, USA). The Shapiro-Wilk test was used to assess the normality of the data. Data with normal distribution were compared using Welch’s t-test for data with significantly different variances or using an unpaired t-test for data with equal variances. The Mann–Whitney non-parametric test was used for data with non-normal distribution. Paired t-test was used to compare data between graft and host in each animal. Two groups were used for each comparison. Pearson’s correlation was performed to analyze the correlation between graft HU and cartilage calcification. Statistical difference was considered significant at p-value <0.05. All experimental data were conveyed as mean ± standard deviation (SD).

Results

Live/dead assay detected higher chondrocyte viability of biobanked trachea compared to PBS-20

We first created segmental tracheal grafts with different storage conditions including fresh, frozen in PBS-20, or biobanked overnight or for 1 month (Figure 2). We used live/dead assay to characterize graft chondrocytes before orthotopic implantation into a syngeneic recipient. Live/dead assay showed that biobanked trachea preserved higher chondrocyte viability than PBS-20 both overnight and at 1 month (p<0.0001, p=0.0001) (Figure 2B). All storage conditions showed decreased chondrocyte viability compared to fresh (1 month PBS-20 p<0.0001, 1 month biobanking p=0.0266, overnight PBS-20 p<0.0001, overnight biobanking p=0.0081). Biobanking was able to better preserve chondrocyte viability than PBS-20. There was no difference in viability between 1-month and overnight storage conditions in either PBS-20 or biobanking, suggesting that chondrocyte viability is not affected by storage duration for up to 1 month.

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

Figure 2.

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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).

Storage conditions do not alter the proportion of apoptotic cells

We used H&E and TUNEL staining to characterize epithelium and chondrocytes in grafts before and after 3 months of implantation (Figure 3). At 3 months, the survival rates of STG animals were 100% (4 out of 4) for fresh group, 70% (7/10) for biobanked group, 60% (6/10) for PBS-20 group. Overall tracheal morphology of cartilage rings and epithelium in pre- and post-implanted fresh, PBS-20, and biobanked grafts are visualized in Figure 3A. The pre-implanted tracheal grafts stored at PBS-20 displayed sloughing epithelium, while the biobanked tracheal grafts showed similar epithelium morphology to fresh. Following 3 months implantation, both PBS-20 stored and biobanked tracheal grafts exhibited intact epithelium identical to control.

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

Figure 3.

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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).

TUNEL staining is used to assess cell apoptosis indicated by DNA fragmentation. Visualization of chondrocytes from TUNEL and DAPI staining revealed minimal purple-fluorescent appearing nuclei throughout the pre- and post-implants in addition to notably fewer central nuclei in the cartilage rings of PBS-20 and biobanked grafts (Figure 3B). The TUNEL assay showed that viability was not different between pre-implants and post-implanted grafts or host, nor between storage conditions (Figure 3C, all p>0.05). These data suggest that storage condition does not result in chondrocyte DNA damage-induced cell death. DAPI was further used to quantify the chondrocyte population, defined as the number of chondrocyte nuclei per mm2 (Figure 3D). Fresh STG at 3 months had a larger chondrocyte population than both PBS-20 (*, p=0.0003) and biobanked (*, p<0.0001) grafts at 3 months (Figure 3D). Fresh, PBS-20, and biobanked grafts at 3 months demonstrated fewer chondrocyte population than both pre-implants (red *, p=0.0218, p=0.0296, p=0.0024, respectively) and host (#, p=0.0033, p=0.0043, p=0.0008, respectively) at 3 months. At 3 months, the fresh graft had more chondrocytes than both biobanking and PBS-20 grafts. The fresh host showed more chondrocytes than biobanked (p=0.0215) but not PBS-20. These data suggest that storage in PBS-20 or biobanking did not damage DNA but did reduce graft chondrocytes at 3 months.

Increased attenuation in the graft on μCT correlates with cartilage calcification

Before graft explantation, μCT was performed to monitor graft patency and quantify Hounsfield units (HU) of fresh, PBS-20, and biobanked grafts compared to the host. All grafts were patent in vivo at the end time point. Increased attenuation (HU) was revealed in PBS-20 (p=0.0002) and biobanked grafts (p=0.0118) compared to fresh STG (Figure 4, *). All STG regardless of storage condition had increased HU compared to the host at 3 months (Red *, p=0.0148, p=0.0002, p=0.0009). After explantation, von Kossa stain was used to assess the amount of calcification in the tracheal cartilage rings.

Figure 4. In vivo Hounsfield units.

Figure 4.

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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).

Von Kossa demonstrates more calcification of cartilage rings in PBS-20 and biobanked grafts at 3 months compared to pre-implant (Figure 5A). Calcification area quantification (Figure 5B) demonstrated increased calcification in PBS-20 STG at 3 months compared to pre-implant PBS-20 (p<0.0001), fresh at 3 months (p<0.0001), and host at 3 months (p<0.0001). Biobanked STG at 3 months showed decreased calcification compared to PBS-20 STG at 3 months (p=0.0003). Biobanked STG at 3 months showed increased calcification compared to pre-implant biobanked tracheal grafts (p=0.0002), fresh at 3 months (p=0.0002), and host at 3 months (p=0.0008). Overall, PBS-20 at 3 months had the greatest calcification area, in agreement with the μCT results. Significant correlation (Pearson) between HU and cartilage calcification area from von Kossa stain (p<0.0001) suggests that micro CT can be reasonably used to longitudinally measure cartilage calcification in vivo (Figure 5C).

Figure 5. Calcification correlation with HU.

Figure 5.

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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).

Discussion

Long-segment tracheal defects represent a significant source of morbidity and mortality as there is no existing surgical cure1–3. Previous studies using autologous, biologic, and synthetic constructs have not been successful, but tissue engineering shows promise for the creation of a viable tracheal replacement3–6,20,25. Although decellularized tracheal grafts have represented a major advance in regenerative medicine, attempts at transplantation of decellularized tracheal grafts have been limited by recurrent collapse due to impaired mechanical properties7,8,26,27. Due to the immunopriviliged nature of cartilage, partial decellularization is emerging as a method to create non-immunogenic allograft sources for tracheal grafts11–15,28. Graft chondrocyte viability is now a new and important consideration. Chondrocytes are responsible for the maintenance of the graft matrisome; viability of these cells could prove to play a critical role in graft performance29–31. Beyond decellularization, graft viability is related to ischemia time and storage conditions; these factors can limit the creation of a donor tissue bank. Well-established approaches of cryopreservation methods to biobank organs have been developed to enhance viability32,33.

Previous attempts at determining the viability of chondrocytes have included histologic means, though they have been complicated by unique challenges often derived from uncertainty regarding measurement validity and difficulty defining the term viability. For instance, one of the earliest attempts at quantifying tracheal cartilage viability by Kushibe, K., et al. intended to measure chondrocyte viability through Na235SO4 incorporation. However, this method only indirectly assessed viability by measuring glycosaminoglycan synthesis17,34. Live/dead cytotoxicity assay is another demonstrated method for quantifying chondrocyte viability. Live/dead assay has been used in articular cartilage and measures cell membrane damage-induced apoptosis that can occur in the absence of DNA fragmentation11,35–38. Depending on the extent of injury, some of the damaged cells stained positive by EthD-1 in live/dead assay can be reversible due to cell repair39,40. In contrast, the TUNEL assay has been widely used to quantify chondrocyte viability in articular cartilage and animal trachea detecting chondrocyte apoptosis defined by DNA strand breaks but no other indicators41–43. TUNEL stains cells that have undergone irreversible cell death and exhibit the biochemical hallmark of apoptosis—internucleosomal DNA fragmentation44–50. These methods have inherent disadvantages for use in detecting chondrocyte viability. Live/dead can be tissue consumable and time-intensive due to whole-mount imaging, while TUNEL can only detect late-stage cell apoptosis when there is DNA damage (Table 1).

Table 1.

Summary of chondrocyte viability detection methods.

Live/Dead assay TUNEL assay μCT
Principle of the method Membrane injury DNA fragmentation Calcification—increased HU
Advantages

  • Sensitive to membrane damage defined cell death

  • Quick

  • Sensitive to DNA damage defined cell death

  • Conserves tissue through use of paraffin embedded tissue sections

  • Allows immunofluorescent co-staining

  • Real time, non-destructive, and longitudinal detection in vivo

  • Fast scanning (5min/animal)

  • Can provide time sensitive viability change within the same animal
Disadvantages

  • Not in real time

  • Tissue consumable and time intensive due to whole mount imaging

  • Unable to perform co-stain

  • Not sensitive to early cell death or membrane damage

  • Not in real time

  • Requires histological section

  • Time-consuming

  • Indirect measurement of chondrocyte death
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Our previous work has shown that STG provides an idealized version of the tracheal construct as it is biocompatible, capable of re-epithelialization, and not limited by the extent of defect or poor vascularization20. In this study, we used our previous STG model as a control and implanted STG of different storage conditions (PBS-20 and biobanking) to assess the optimal approach to quantify chondrocyte viability. Biobanking is superior to PBS-20 to maintain chondrocyte viability, indicating biobanking could serve as an approach to store donor grafts for clinical and preclinical use. Establishing the strengths and weaknesses of these methods of quantifying chondrocyte viability methods in STG first provides a path for translation into our partially decellulaized tracheal graft. Storage of trachea in PBS is a common protocol in the production of fully decellularized trachea27,51–54. However, it has been shown that storage of cells at −20° C, or sub-zero temperatures in general, can cause cold shock-induced cell membrane damage resulting in cell mortality16. In addition, storage of trachea using cryopreservation has been suggested to preserve graft viability and cartilage integrity, as well as reduce allogenicity17–19. These different storage conditions did not affect epithelium regeneration and allow us modulate the chondrocyte viability of STG. Then we characterized chondrocyte viability in pre- and post-implanted tracheal grafts.

We quantified chondrocyte viability before implantation in grafts using live/dead assay and TUNEL staining. We also present a novel method using μCT to quantify in vivo chondrocyte viability indicated by calcification. μCT has been used in diagnosing the damage in articular cartilage and bone24,55,56. We demonstrated the tracheal HU detected by μCT had a positive correlation with cartilage calcification detected by von Kossa staining, which detects calcium salt formation57,58. Pathologic calcification of articular cartilage has been correlated to both increased age and decreased cartilage cellularity, which is in accordance with the low cartilage cellularity (Figure 3) and high HU and calcification (Figure 4, 5) in our results; This is possibly explained by chondrocyte-derived apoptotic bodies depositing calcium and forming crystals59,60. Vesiculating apoptotic chondrocytes form matrix vesicles that are involved in the calcification process . Hypertrophic chondrocytes are able to synthesize apoptotic bodies or matrix vesicles resulting in matrix mineralization61. Using μCT, we were able to detect cartilage calcification in vivo and longitudinally, thus quantifying real-time chondrocyte apoptosis. A limitation of this method is that, as an indirect measurement of chondrocyte viability, it can only provide relative values compared to host or control, but cannot provide absolute value based on viable chondrocyte number (Table 1).

Based on our results, we recognize that there is no ideal method for quantifying chondrocyte viability, as each of these techniques attempts to measure a varied definition of cell viability. We conclude that a combination of radiologic and histologic techniques is the optimal strategy to quantify chondrocyte viability, rather than an individual technique. In application, knowledge of the pros and cons of each method may be relevant depending on the research question or goal, such as use in cartilaginous tissues outside of the trachea or in a study with limited sample size. Ultimately, clinical performance of any tissue engineered tracheal replacement is expected to rely on longitudinal measures, including radiographic and bronchoscopic procedures. In evaluating the value of each method for chondrocyte viability quantification, feasibility of clinical adoption in humans must be considered. Accordingly, μCT may be a critical answer for longitudinal assessment of cartilage quality and chondrocyte viability in the trachea.

Our study does have limitations. Despite evidence that chondrocytes contribute to the mechanical properties of trachea, these properties were not tested in this study. Due to the small size of mouse trachea, there is no commercial mechanical testing system that can be used to measure the anterior-posterior compression force of our samples. Therefore, the effect of storage condition on the role of chondrocyte viability in mechanical properties was not able be illustrated. Although, μCT was used to visualize the graft patency to indirectly indicate the similarity in graft dimensions between tracheal grafts stored in different conditions. This study did also use histological slides for characterization. Due to the staining and imaging limitations of whole mount tissue, histological staining on slides is currently a standard approach to visualizing cells in tissue. Finally, histological and μCT measurements may be subject to bias and random error. To minimize the impact of these limitations, all quantification of chondrocyte viability was completed in a blinded fashion.

Conclusion

Quantification of chondrocyte viability for a living tracheal graft needs comprehensive approaches. We predict that a strategy involving selection of radiologic and histologic techniques based on their respective strengths and weaknesses can be used in combination to assess chondrocyte viability. In the future, these techniques may prove useful in determining the merit of partially decellularized tracheal allograft.

Acknowledgements

This work was supported in part by a The Ohio State University College of Medicine (Roessler) research scholarship (C.C.) and the National Institutes of Health (NIH NHLBI K08HL138460, NIH NHLBI R01HL157039 (T.C.)).

Footnotes

Conflict of Interest Statement

We have no conflicts of interest to disclose.

Meeting

Triological Society Combined Sections Meeting, Coronado, California, USA, January 20–22, 2022

Contributor Information

Coreena Chan, College of Medicine, The Ohio State University, Columbus, OH, USA.

Lumei Liu, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Sayali Dharmadhikari, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA; Department of Pediatric Otolaryngology, Nationwide Children’s Hospital, Columbus, OH, USA.

Kimberly M Shontz, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Zheng Hong Tan, College of Medicine, The Ohio State University, Columbus, OH, USA.

Maxwell Bergman, Department of Otolaryngology-Head & Neck Surgery, The Ohio State University Medical Center, Columbus, OH, USA.

Terri Shaffer, Small Animal Imaging Facility, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Nguyen K Tram, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Christopher K Breuer, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA; Department of Pediatric Surgery, Nationwide Children’s Hospital, Columbus, OH, USA.

Mitchel R Stacy, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Tendy Chiang, Center for Regenerative Medicine, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA; Department of Pediatric Otolaryngology, Nationwide Children’s Hospital, Columbus, OH, USA.

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