Intrinsic pulmonary sealing, its mechanisms and impact on validity and translational value of lung sealant studies: a pooled analysis of animal studies

Introduction

Prolonged pulmonary air leak (pPAL) occurs in up to 30% of lung resections, causing increased morbidity (empyema and post-operative complications), reinterventions (4.8%), readmissions [odds ratio (OR) =2] and mortality (OR =1.9) (1-7). Preclinical animal studies with lung sealants have shown promising results, and 74% of surgeons use sealants in their high-risk patients (8-10). Results of lung sealants in preventing pPAL in clinical studies, however, are mixed and guidelines do not recommend their routine use (11-16). Recently, in a European Society of Thoracic Surgeons (ESTS) survey, the majority of the 258 responding thoracic surgeons affirmed the lack of sufficient evidence for lung sealants, and an unmet clinical need for more effective lung sealants was described (17). The marked discrepancy between the more positive preclinical studies and often unsatisfactory clinical results indicate a potential translational gap.

For effective clinical use, the performance of lung sealants should be investigated during their development in properly validated animal models. For a valid model, pulmonary air leakage (PAL) needs to be present of sufficient magnitude and without the capacity to resolve spontaneously for the study duration, ensuring accurate assessment of both acute and prolonged aerostatic efficacy of applied lung sealants. Furthermore, in contrast to patients undergoing lung resections, animals used in lung sealing experiments are healthy in 92% of cases and may poses enhanced intrinsic sealing and regenerative capacities, which can possibly invalidate positive study findings if unaccounted for (18-20). In the present literature, no standardized animal model exists that guarantees clinically significant pPAL, and negative control groups were only used in 18.7% of preclinical studies (20,21). Therefore, the validity of many previously tested lesions for pPAL in animal models is unknown (20,22,23).

Several enhancements to animal models for lung surgery research have been described, that might reduce the translational gap. First of all, the type of lesion might impact PAL, as larger lesions and lesions involving bronchioles seem more likely to result in pPAL (21,24). Secondly, disease models have been described, including models for emphysema and heparinized models (25,26). Although these disease models increase the risk of pPAL, these models may come at the cost of increased variation and decreased animal welfare. In the present study, we assessed the impact of different types of lesions on PAL from the results of negative controls (untreated lesions) in three animal experiments. These results provide insight in the impact of intrinsic sealing mechanisms on the validity and translational value of animal models for lung sealant research. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-180/rc).

Methods

Study setup

The intrinsic sealing capacities of both superficial parenchymal lesions and lesions involving bronchioles are investigated in in vivo and post-mortem ovine models, by measuring air leakage (AL) and characterizing the responsible mechanisms histopathologically. To simulate a real post-operative scenario, an ovine acute aerostasis model with both mechanical ventilation and spontaneous breathing was used, based on previous models (21,24). A bilateral thoracotomy was performed sequentially and varying standardized lung lesions were created in an explorative study design, after which AL was measured on both lungs after chest closure with a digital drainage system.

The sheep included in the analysis were used in three experiments, one post-mortem study and two in vivo studies. First, superficial parenchymal lung lesions were tested in mechanically ventilated post-mortem sheep (N=2 cadavers), to test the hypothesis that pleural apposition diminishes AL for superficial lesions after chest closure (27,28). Then, such lesions were created in a live sheep model (in vivo), testing their natural healing course in the presence of intact coagulation (N=3 sheep). Finally, based on the previous results, more extensive lesions involving macroscopically visible bronchioles were tested (N=7 sheep). Part of the lesions made in the in vivo studies were treated with a sealant. For the present study, we only pooled results for untreated lesions, as the focus of this investigation was to study the mechanisms of intrinsic sealing.

Lung lesions

Lesions were made to simulate the clinical problem of an alveolar-pleural fistula, defined as PAL arising distal to the segmental bronchus (29). Three different lung lesions were tested: superficial parenchymal, deep bowl-shaped and sequential lung amputation lesions. Superficial parenchymal lesions were made by creating n × n perpendicular cuts in a 25 mm × 25 mm square with a scalpel limited to 1–3 mm depth (Figure 1A,1B). Deep bowl-shaped lesions were made by cutting a ⌀25 mm bowl lesion using biopsy punches and scissors (Figure 1C,1D). Sequential lung amputations were made by cutting away tissue perpendicularly to the expected bronchiole branching pattern, at a width of 3–5 cm in steps of 1 cm until either bronchioles of >⌀1.5 or ⌀0.5 mm were encountered, on the ventral tips of the right middle lobe (RML) and right lower lobe (RLL) on the right, plus the left upper lobe (LUL) and the left lower lobe (LLL) on the left side (Figure 1E,1F). This method for inducing lesions involving bronchioles was found to be the most repeatable in our ex vivo investigations [Supplementary file (Appendix 1)] and is based on the model of Ranger (21). All lesions were made on a static lung with a positive end-expiratory pressure (PEEP) of 10 cmH₂O. Precise lesion types per experiment are explained in Table 1.

Figure 1 Lung lesions. (A,B) Superficial parenchymal lesion creation in vivo; (C,D) deep bowl-shaped lesion creation in vivo; (E,F) sequential lung amputation lesion in vivo. White arrow points at a macroscopically visible bronchiole (marked with a suture for recognition at obduction).

Outcome measures

In all experiments, after lesion creation and chest closure, AL was measured using a digital chest drainage system (Thopaz^®, Medela, Baar, Switzerland). The time point since start of drainage when AL was <20 mL/min, was noted as time until intrinsic sealing. For lesions involving bronchioles, the AL was measured using a mechanical ventilator (SERVO-I^®, Getinge, Gotenburg, Sweden), based on the inspiratory (TVi) and expiratory (TVe) tidal volumes, right before and after lesion induction. The minimal leaking pressure (MLP) was determined for these lesions by dialing down the PEEP in steps of 1 cmH₂O until leakage disappeared or increasing in steps of 1 cmH₂O until leakage appears. In the in vivo series, the post-mortem measurements of MLP were performed either ex-situ after lung explantation, or in-situ using selective intubation (in E4–E6, Table 1). Bronchial diameters (lumen, ⌀) were measured using a ruler with markings every 0.5 mm (Aesculap AA804R), and approximated in 0.5 mm increments. Finally, macroscopy was recorded descriptively, paying attention to mechanisms of sealing, hemostasis and atelectasis.

Animal procedures

All experiments were performed under a project license (No. AVD10300202114869) granted by national authorities (‘Centrale Commissie Dierproeven’, CCD) after review by an independent ethics board (‘Dierexperimentencommissie’, DEC) in the Netherlands, in compliance with institutional guidelines for the care and use of animals. Humane care and anesthesia were provided throughout the experiments. Experimental protocols were approved by the animal welfare body and registered internally at our institute (Nos. 2021-0012-001 and 2021-0012-002).

Adult mixed-breed female sheep (N=2) which were previously utilized for the production of antibodies in an unrelated project were euthanized using an overdose of pentobarbital, and the cadavers were directly re-used in the post-mortem pilot model. Healthy adult mixed-breed female sheep (N=10) were used in the in vivo animal model. Anesthetic protocols in vivo involved deep surgical anesthesia during surgery and lighter sedation during a spontaneous ventilation observation period [detailed in Supplementary file (Appendix 1)]. Mechanical ventilation was guided by the visual presence of atelectasis on lung surfaces and ventilation and oxygenation requirements in the live animals. Reduction of ruminal tympany was ensured throughout the procedure.

A thoracotomy was performed on both sides of in the fifth intercostal space sequentially, always beginning with the right lung. After lesion creation, hemostasis was performed if required using gauzes and diathermy, and time until hemostasis was recorded. If necessary, fibrin plugs were removed using a small tweezers from the bronchiole lesions following hemostasis. Following all measurements, a silicone drainage tube (size Ch30) was placed apically and exited the thorax ventrally on both sides. The thoracotomy was closed in layers to ensure air-tightness for accurate AL measurements. After an observation period in a back position (post-mortem model, minimum of one hour) or abdominal position (in vivo model, minimum of three hours) under mechanical or spontaneous ventilation, the live animals were euthanized using pentobarbital. The lungs were explanted through a median sternotomy for inspection, post-mortem measurements and histology.

Histology

For histological analysis, samples were taken from the created defects after MLP determination and stored in 4% formaldehyde. Subsequently, they were embedded in paraffin and 4 μm thick sections were cut and stained with hematoxylin-eosin staining. These coupes were digitalized and assessed by an experienced pathologist (Vos S).

Statistical analysis

ThopEasy + software (Medela, Baar, Switzerland) was used to import AL data, giving mean AL values (mL/min) every 10 minutes. For all lesions (post-mortem and in vivo), the mean AL was calculated over the first 80 minutes of drainage (minimum drainage time across all groups). In vivo, mean AL was calculated over the first 180 minutes of drainage, and in 30 minute intervals for the first 5 hours of drainage. For statistical comparison, drainage AL data was normalized as: ALnormalized=ln(ALNleaks), were N_leaks denotes the number of effective ALs on the drained side. Normalized values were then compared using an analysis of variance (ANOVA) with Bonferroni-Holm post-hoc test (α=0.05/3). Intraoperative AL (mL/min) based on the mechanical ventilator was calculated as: AL=(∑k=15(TVik−TVek)5)×RR, were RR denotes respiratory rate. This AL was corrected for AL measured before the lesion was created as: ALcorrected=ALlesion−ALbaseline. In case this calculation resulted in a negative AL, an AL of 0 mL/min was noted. MLP, time until hemostasis and AL data were compared between lesions involving bronchioles (⌀0.5 mm vs. >⌀1.5 mm lesions) using a Mann-Whitney U test (two-tailed α=0.05). IBM SPSS Statistics 27 (IBM Corp., Armonk, New York, USA) was used for statistical testing.

Results

Summary of experiment characteristics

In twelve animals, 25 untreated lesions were created and analyzed (N=4 superficial parenchyma post-mortem, N=5 superficial parenchyma in vivo, N=16 lesions involving bronchioles, N=4 ⌀0.5 mm and N=11 >⌀1.5 mm bronchioles, N=1 missing diameter). One animal in the in vivo group (P2) did not regain spontaneous ventilation and was kept on the mechanical ventilator during the observation period, another (P6) died due to acute cardiac arrest right after closure of the left thoracotomy, resulting in missing left lung drainage data and a shorter follow-up period. All experiment characteristics are displayed in Table 1.

Air leak characteristics for lesion subtypes

75% of superficial parenchymal lesions resulted in AL after thorax closure in the post-mortem model [mean ± standard deviation (SD): 760±693 mL/min, N=4]. One lesion did not exhibit AL, likely due to extensive atelectasis of the affected lobe seen at obduction. For the in vivo superficial parenchymal lesions, only minimal and rapidly decreasing post-operative AL was observed (mean ± SD: 42±33 mL/min, N=5, Figure 2A). All of these lesions stopped leaking within a median time of 20 minutes [interquartile range (IQR), 10–75 minutes, N=5]. The average AL at 80 minutes showed a trend towards statistical significance between the post-mortem and in vivo groups (P=0.055).

Figure 2 AL results for parenchymal and bronchiole lesions. (A) Average AL from first 80 minutes of thoracic drainage; (B) average AL from the first 180 minutes of thoracic drainage; (C) average AL values per 30 minutes from in vivo experiments. Dashed lines show individual drain measurements. *, absolute AL values for left or right drains, uncorrected for number of leaks per side (Table 1); **, statistical testing performed using ANOVA (α=0.05) on log-normalized AL values and Bonferroni-Holm correction for post-hoc testing (only significant comparisons shown). min., minutes; h, hours; AL, air leakage; ANOVA, analysis of variance.

In comparison to superficial parenchymal lesions in vivo, all lesions involving bronchioles (pooled ⌀0.5 mm and >⌀1.5 mm lesions) led to significantly higher AL post-operatively (normalized flow mean ± SD: 459±221 mL/min, P=0.003, N=9, Figure 2A,2B). Despite relevant AL initially, the magnitude of the AL still decreased over the observation period (Figure 2C). At termination of the experiment, 5/9 (55.6%) of these lungs were still leaking (median drain time: 273 minutes, IQR, 207–435 minutes, N=5), and intrinsic sealing for the remaining lungs occurred within a median of 115 minutes (IQR, 52–245 minutes, N=4). The shortest time until intrinsic sealing was observed for the lung affected with a ⌀0.5 mm bronchiole lesion (50 minutes, N=1).

Bronchiole diameter and leakage capabilities

Comparing both sizes of bronchioles, >⌀1.5 mm lesions (N=11) were found to require longer time until hemostasis then ⌀0.5 mm lesions (N=4) (mean ± SD: 6.0±2.7 vs. 2.0±0 minutes, P=0.012). However, no significant difference was found between >⌀1.5 mm/⌀0.5 mm lesions for MLP (median ± IQR, 5±2 vs. 7±5 cmH₂O, P=0.226) nor mechanical ventilator AL (median ± IQR, 581±1,012 vs. 140±305 mL/min, P=0.104) during the live observation. Furthermore, post-mortem MLP did not differ significantly between >⌀1.5 mm/⌀0.5 mm (median ± IQR, 6±13 vs. 10±41 cmH₂O, P=0.199) A trend was seen for higher AL alive and lower MLP post-mortem for the >⌀1.5 mm lesions. Of note, N=2 lesions were excluded from MLP analysis in the >⌀1.5 mm subgroup due to blood contact during obduction, which possibly influenced the MLP (15 and 45 cmH₂O).

Macroscopy and histology

A rapid intrinsic sealing of the in vivo superficial parenchymal lesions was observed intraoperatively (illustrated in Video 1). At obduction, all superficial parenchymal lesions of the in vivo model were covered with a coagulated fibrin sealing layer (Figure 3A,3B). For the lesions involving bronchioles, the bronchioles appeared contracted or were no longer macroscopically visible (Figure 3C,3D).

Figure 3 Macroscopic findings. (A) Intrinsically sealed superficial parenchymal lesion, with a coagulated fibrin layer over the lesion. (B) Small bronchiole (white arrow) as air leak focus of a deep bowl-shaped lesion with some secretions. Note the intrinsic sealing layer over the lung parenchyma. (C) Macroscopic aspect of sequential amputation in vivo and (D) the same lesion at obduction. Note that the bronchiole (white arrow) is no longer visible at obduction and that a coagulated sealing layer is present on top of the parenchyma.

Histology for the superficial parenchymal lesions (N=2 lesions after <12 h) revealed an area of alveolar collapse surrounding the injured sites, extending beyond the coagulated parenchymal incisions. Within this area of alveolar collapse, intra-alveolar hemorrhage was observed. Going further proximally, the normal air contents of the parenchyma gradually returned. Minimal influx of immune cells was seen, but no notable immune response was noted (Figure 4). The histological response for the lesions involving bronchioles was similar to the parenchymal lesions in vivo [Supplementary file (Appendix 1), Figure S3].

Figure 4 Histology of intrinsically sealed lung parenchyma. (A) Aspect of a 3 mm deep parenchymal incision (5× magnification). Three areas can be distinguished in the intrinsic sealing layer: area I shows coagulation in the incision itself; area II shows alveolar collapse and intra-alveolar hemorrhages extending beyond the actual incision site (detailed at 40× magnification in B) and area III shows the return of normal air containing alveoli. (C) Uninjured lung site shows normal air containing parenchyma (40× magnification). Hematoxylin-eosin staining.

Discussion

This study revealed that superficial parenchymal defects are unsuited for lung sealant testing in a healthy ovine model of PAL, due to rapid intrinsic sealing. Sequential amputation lesions involving bronchioles do result in AL in 56% of cases (median observation time: 4:33 h), but with 44% of cases sealing intrinsically in a median time of 1:55 h. Bronchioles of >⌀1.5 mm are preferable over ⌀0.5 mm, due to a trend towards a longer time to sealing. These lesions can be created in a reproducible manner and therefore seem valid for acute lung sealant testing. Nevertheless, the time to sealing is still too short for analysis of pPAL. These observed intrinsic sealing mechanisms could explain a translational gap in lung sealant research, especially in studies lacking negative control groups to demonstrate pPAL.

Comparison to literature

Presently, many clinical studies have been performed, testing numerous different sealant products. Some sealants were shown to be effective, but there were fluctuating results and no clear evidence based recommendations (16). As we suggest, disappointing results in clinical studies may be a consequence of a translational gap when negative controls are not used in the preclinical phase to ensure significant PAL. For example, effectiveness of fibrin glue was seen in an animal study that compared bursting pressures of fibrin glue with sutures in a rabbit model, without a negative control group (i.e., lesion with no treatment) (30). In contrast, the animal study by McCarthy et al., which included a negative control group to demonstrate significant PAL, showed no real effect of fibrin glue. Fifty percent of the animals in their study were free of AL after 24 h in both the fibrin glue and negative control groups (i.e. similar effectiveness to natural healing) (24). This study is in line with clinical literature, as fibrin glue has not been found significantly effective for reducing the length of hospital stay in the clinical trials by Fleisher, Wong and Mouritzen (31-33).

Our study confirms the hypothesis that it is only possible to induce PAL in healthy animal lungs when creating lesions with adequate depth, lacerating terminal bronchiole (21). Previous studies with negative control groups had similar findings, as amputations of lung lobes or deep incisions, likely resulting in bronchiolar leaks, resulted in AL complications [Supplementary file (Appendix 1), Table S1] (21,34-36). As an example, Ranger et al. produced similar bronchiolar leaks of >⌀1.5 mm and found higher AL (mean 1.4 L/min, range 0.6–3.5 L/min) in small dogs with similar drainage settings, as compared to our finding. These differences may be explained by sampling variation or lobe/species effects (21). In contrast, various superficial lesions did not lead to AL problems (18,37-39). Also in larger parenchymal lesions, AL may persist, although in small dogs and pigs (17–35 kg) it is unclear whether this is due to some degree of bronchiolar laceration at depths of ~5 mm (24,40).

Intrinsic sealing mechanisms

Pathologically, the intrinsic sealing layer is hypothesized to be more than just superficial coagulation, an extended area of alveolar collapse might be an explanation for the strong and rapid intrinsic sealing seen in this study. Similar mechanisms have previously been described, including compressed or partially aerated alveoli, subpleural alveolar edema, intra-alveolar bleeding and intra-alveolar deposition of fibrin strands (19,41-43). Based on these studies and the findings in our study, the following mechanisms are proposed to be principally responsible: first, alveolar collapse is physically initiated by air escaping from the subpleural alveolar layer, by direct impression of the surgical instrument and a lower resistance to flow of this distal AL path (41). Simultaneously, hemorrhage spreads through the pores of Kohn and channels of Lambert (43). Blood proteins and lipids are known to cause an inhibition of surfactant function, increasing alveolar surface tension (Laplace’s law, P=2Tr) and thereby attenuating alveolar collapse biophysically (44-48). This results in a reduction of the common radius of the subpleural alveoli, increasing the resistance and reducing the AL by Poiseuille’s law. This attenuates coagulation mechanisms analogous to vasoconstriction in a bleeding wound (41,43). Inflammatory mechanisms were not seen in our sample (due to the short follow-up period), but may further enhance sealing through alveolar compression, due to capillary enlargement and edema as previously described (19,42,43). Pleural apposition may also reduce AL, but we hypothesize this to play a small role based on our post-mortem observations and rapid sealing seen intra-operatively (27).

These micromechanics could also provide a biophysical explanation for the lower intrinsic sealing capabilities in diseased human lungs. Pulmonary emphysema is a major risk factor for pPAL, and emphysema scores have shown to be predictive of pPAL (1,49-51). Conceptually, in emphysematous lungs, lower pressures are required to prevent alveolar collapse, due to the larger radius of emphysematous alveoli (Laplace’s law). Therefore, it is hypothesized that the proposed biophysical intrinsic sealing mechanisms will not induce the same degree of intrinsic sealing by alveolar collapse as seen in the healthy, non-emphysematous ovine lungs. Further study of intrinsic sealing mechanisms in emphysematous lungs might help us understand mechanisms of pPAL, which may improve treatment of these lesions.

The reduction in bronchiole diameter which was observed macroscopically post-mortem, may be due to sympathetic nervous system activation or bronchiole collapse due to absence of hyaline cartilage supporting structures. Especially bronchioles <1 mm lack supporting hyaline cartilage, which was also seen histologically in our study [Supplementary file (Appendix 1), Figure S3] (52). In this case, as surrounding alveolar tissues collapse due to a steal phenomenon from the leaking orifice, especially bronchioles without cartilage support could collapse and seal intrinsically (as shown in Figure 3D). A bronchiole of >⌀1.5 mm appears preferable over ⌀0.5 mm to prevent rapid and strong intrinsic sealing in the acute aerostasis model from this perspective (21).

There might be species specific considerations for sheep, which explain the strong and rapid intrinsic sealing. These include intra-vascular macrophages and an increased white blood cell count, which may cause immune mediated attenuation of intrinsic sealing mechanisms (53-55). Coagulation times are comparable to those of humans (55). High doses of intravenous propofol (dissolved in lipids) were sometimes required during surgical anesthesia, and it was hypothesized that this might result in hyperlipidemia and subsequently increased blood viscosity and coagulability. However, previous in vitro and in vivo experiments have shown no effect on blood viscosity, and even describe a reduced platelet aggregation (56).

Translational value of air leak models

The face validity of a bronchiolar AL model is reduced, as clinical pPAL mostly arises from the alveoli in superficial parenchymal injury, such as from the dissection of fissures or pleural adhesions. As an acute aerostasis model, the construct validity remains preserved. PAL >500 mL/min intraoperatively was found to predict pPAL, and AL sizes of 150–400 mL/min have been used for sealant application in a clinical trial, with positive results (57,58). Thus, the bronchiole AL seems sufficient for acute sealant testing. The model allows testing on an actively leaking lung, after thoracic closure, at least before intrinsic sealing of specific bronchiole lesion occurs. Thereby, other important mechanisms such as coagulation, immune response, pleural mechanisms and physiological breathing can be replicated, which is presently not feasible in animal-free alternatives (59,60).

Disease models may be suitable to induce pPAL, but it remains unclear how long PAL should last in a model for sealant testing. Based on the hypothesized micromechanics, emphysema may result in longer pPAL, while heparinization seems a less effective disease model, as this would not inhibit alveolar collapse, and might even enhance spreading of blood through the alveoli. One study found significantly lower bursting pressures for sealants applied to emphysematous lungs, but did not measure PAL postoperatively (26). The lower bursting pressures could be biochemically ascribed to a lower crosslink density on emphysematous lungs, which may be another explanation for a translational gap (26). When developing disease models, disease induction might be associated with an increased baseline variation, higher sample size requirement and decreased animal welfare, which needs to be weighed against the potential added benefit of these models.

Limitations

These experiments offer a valuable addition to the present preclinical literature, by confirming the possibility of a translational gap and offering novel insights into intrinsic sealing mechanisms. However, based on these experiments, it is not possible to define a global standardized model for pPAL and lung sealant research. For this, further investigations are needed. First of all, it needs to be studied how long PAL needs to be present in a model for sealant testing to ensure accurate translation. Formulated differently, the time until intrinsic sealing of human lungs when a sealant is applied needs to be studied. Disease models are hypothesized to be suitable options for developing longer leaking PAL, but remain entirely unconfirmed, and come with added costs (higher sample size, decreased animal welfare). Another option might be to create leaks in large bronchi (e.g., segmental bronchi), but this further decreases the face validity. Finally, due to small sample size and heterogenous methods in the present study, all findings described here should be interpreted with appropriate caution and need to be confirmed in a-priori statistically powered experiments.

Recommendations for further research

Clinicians and experimental researchers should be aware of the intrinsic sealing mechanisms of healthy animal lungs. Success of sealants in animal studies should therefore be interpreted cautiously. In new experimental design, negative control groups should always be considered to measure the actual treatment effect. PAL may be created in animal models by creating especially large defects or lacerating bronchioles of >⌀1.5 mm (21,24). For development of a global standardized pPAL model, further research is required into the requirements of valid pPAL models (e.g., duration of PAL), and methods for inducing longer PAL (e.g., emphysema or heparinization) (25,26). Such disease models could also be used to study mechanisms of PAL in clinical scenario’s, such PAL arising from stapler lines. The understanding of PAL sealing mechanisms is still incomplete, and the proposed mechanisms should be further investigated. With a thorough understanding hereof, a clinical solution for the problem of pPAL might be discovered, for example by making the surgical treatment better synergize with the underlying mechanisms of the specific lesion type. The conduction of animal systematic reviews and adherence to ARRIVE guidelines should be encouraged to further improve scientific rigorousness of animal studies (20,61).

Conclusions

Superficial parenchymal lesions exhibit an intrinsic sealing mechanism in a healthy ovine lung model, explained pathologically by an extended area of alveolar collapse attenuating coagulation mechanisms. These mechanisms may reduce model validity and contribute to a translational gap in lung sealant research, especially when negative control groups are not used. Experimental researchers should account for these mechanisms when designing experiments, to improve clinical applicability. One such approach for acute lung sealant testing in an ovine model, is to create deep parenchymal defects involving at least bronchioles of >⌀1.5 mm. Further study into PAL models is required to develop and validate a universal standardized acute aerostasis model.

Acknowledgments

The authors would like to thank Nicole Calon, Jort Evers, Alex Hanssen, Stefanie Schönfeld, Maikel School and Pim van Sambeeck (all are affiliated with the Radboud University Medical Center in Nijmegen, The Netherlands) for their help during the experiments, Pieter Verbost and Manon van Hulzen for their help with the experimental protocols and licenses and all other supporting personnel in the animal laboratory for their great caretaking of all laboratory animals. We want to thank the Radboudumc Department of Statistics for its help with the statistical analysis and the Department of Pathology for helping with processing the histological samples. Part of this work has previously been presented at the SEOHS 2022 conference in the Netherlands. Finally, we thank Willem de Boode for help with conceptual understanding of the intrinsic sealing mechanism.

Funding: This work was supported by GATT Technologies B.V. (Nijmegen, The Netherlands).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-180/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-180/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-180/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-180/coif). EAR is an employee of GATT Technologies B.V. and HvG was a scientific advisor for GATT Technologies B.V. until 31 December 2021, but not in relation to lung sealing technology. BPH received funding and study materials through the institution for conduction of the study from GATT Technologies B.V. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All experiments were performed under a project license (No. AVD10300202114869) granted by national authorities (‘Centrale Commissie Dierproeven’, CCD) after review by an independent ethics board (‘Dierexperimentencommissie’, DEC) in the Netherlands, in compliance with institutional guidelines for the care and use of animals. Humane care and anesthesia were provided throughout the experiments. Experimental protocols were approved by the animal welfare body and registered internally at our institute (Nos. 2021-0012-001 and 2021-0012-002).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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