Abstract
Purpose
Preclinical aerosol studies using animals are essential for evaluating toxic or therapeutic effects on human respiratory tract.
Macaques are relevant animal models for respiratory studies, but they are sensitive, expensive and difficult-to-access.
Methods
In the context of preliminary studies before animal experiments, we set up an alternative in vitro anatomical model of macaque airways to reduce, refine and replace (3Rs) the animals. We printed an in vitro anatomical cast until the third bronchial division from X-ray computed tomography data of a healthy cynomolgus macaque. This in vitro model was then connected to a respiratory pump to mimic macaque’s breathing. We assessed the relevance of this in vitro model, by comparing aerosol deposition patterns obtained with the anatomical model and in three macaques using planar gamma camera imaging. DTPA-99mTechnetium aerosols were produced using three jet nebulizers, generating three different particle sizes: 13.1, 3.2 and 0.93 µm in terms of the mass median aerodynamic diameter (MMAD).
Results
The data showed no statistical differences between the animal and anatomical in vitro models in terms of total aerosol deposited in the airways. However, the distribution of the deposition in the airways showed a higher deposited fraction in the upper respiratory tract in the animals than the in vitro model for all particle sizes.
Conclusions
The anatomical printed model appears to be a relevant in vitro tool to predict total aerosol deposition in macaque airways.
Keywords: aerosol, deposition, gamma camera, inhalation, macaque
Introduction
Aerosol inhalation is a long-standing practice, mainly used in the treatment of upper respiratory tract/ URT (nose and nasal cavity, pharynx, and larynx) diseases, such as sinus disorders [1–4], or lower respiratory tract/ LRT (trachea, bronchi, bronchioles, and alveoli)pulmonary diseases [1–3], such as asthma [4], cystic fibrosis, some infectious diseases, and chronic obstructive pulmonary disease [5–8].
Their efficacy and dispersion in the airways depend on the size of the generated particles used to target the URT and/or LRT, how the aerosols are administered and the interface between the host and aerosol delivery device (mask for example).Thus, in 2003, US-FDA authorities confirmed that it is essential to assess and quantify the amount of aerosols deposited inside nasal airways [9] and to determine, by extrapolation, the amount deposited in the LRT. Furthermore, different types of nebulizers generating the same size aerosols can result in different deposition sites inside the airways [10]. Consequently, aerosol size is not the only critical parameter for predicting regional deposition inside the body.
Thus, studies targeting the different parts of the airways are essential for predicting aerosol deposition. Furthermore, inhalation studies are necessary for inhaled-drug marketing authorization and the transposition of preclinical results to clinical application [11].
Preclinical studies are also necessary for the testing of toxic aerosols (biological infectious agents, toxins, environmental compounds) [12, 13], and the development or evaluation of medical countermeasures [14].
Relevant animal models for inhalation studies are non-human primates (NHPs) and pigs[15] due to their similarity to humans in terms of anatomy and lung lobation. NHPs are the only animal model that depicts the complexity of the anatomical respiratory structures and air transmission inside the lungs of humans.
Nevertheless, even for regulatory, purposes (drug or vaccine development for example) NHP models are difficult to access (availability and cost) and require specific housing combined with dedicated animal welfare procedures.
It is therefore crucial, in accordance with the reinforcement of the 3Rs (replace, refine, and reuse), rule, to favor alternative NHP aerosol models [16] before experimentation on living NHPs, such as macaques. One attractive possibility is to use an in vitro replica of the NHP respiratory system. Such an in vitro tool would allow a better understanding of aerosol deposition, predict pharmacokinetics/dynamics of any aerosolized substance, and reduce the use of NHPs. Using such a tool during the pre-characterization step (before using animals) would allow the set-up of all technological elements required for aerosols delivery before moving on to animals, which, ultimately, would allow extrapolation to humans.
Such printed in vitro 3D models are already available for humans and have been recently validated for the study of aerosol deposition in the upper airways [10, 17–19]. Furthermore, it is undeniable that, preclinical experimentation on animals is facing a re-evaluation, given the increasing of restrictions and demanding procedures required to justify the use of animals. Ehrmann et al.[20], confirmed that many alternative models are already available in the clinical domain, from ex vivo cell models to others diverse models of respiratory tracts. Thus, deposition studies have been performed with human airways cast from cadavers [21] or that have been 3D printed [18, 22].
We aimed to develop, describe, and evaluate an alternative in vitro anatomical model of the macaque respiratory tract and compare it to that of animals and assess the deposition aerosols of different size.
The resulting 3D cast was designed using a X-ray scan of the respiratory tract of a living macaque. We assessed the relevance of this model, by comparing the aerosol deposition in the 3D in-vitro model to that in three female macaques. The aerosol deposition studies were performed using three different nebulizers and measured by gamma camera imaging using radiolabelled aerosols.
Materials and Methods
In Vitro Cast Printed Model
Two highly similar models were printed using 3D- printing technology from the whole head until the first bronchia divisions of a healthy living male cynomolgus macaque at IDMIT (Infectious Diseases Models for Innovative Therapies, CEA, Fontenay-aux-Roses, France). The casts were generated using the 3D printing stereolithography technique from raw computed tomography CT data of the macaque. The chosen macaque was large (6.6 kg), with large airways, a recommended condition for the printing technique that was used.
CT Equipment and Whole-Body Scanning of Macaques: First Step for the Printing of the In Vitro 3D Cast
A clinical CT imaging system (Vereos-Ingenuity, Philips Healthcare, Best, Netherlands) was used with a 64 × 0.6 mm CT detector collimation using a tube voltage of 120 kV and an intensity of approximately 120–130 mAs. CT images were reconstructed with a slice thickness of 1.25 mm and an interval of 0.25 mm. The clinical CT equipment was used to produce the scans needed for the 3D printing of the in vitro cast.
The selected macaque (6.6 kg body weight) underwent a whole-body high-resolution X-ray CT scan after being anesthetized with an intramuscular injection of ketamine (0.03 mg/kg, Centravet, Dinan, France) and Domitor® (0.22 µg/kg, Viatris,Meyzieu, France). During the scans, the animal was lying on its back in a dorsal decubitus position and was spontaneously breathing. CT acquisition lasted approximately about 15 s. The obtained DICOM raw images were converted into STL files using Materialise (Materialise® NV, Leuven, Belgium) Mimics 23 software (Materialise® Mimics Innovation Suite, Leuven, Belgium). We printed the trachea and bronchial trees with major branches according to the capabilities of the printer, with a layer thickness of ≥ to 2 mm (stereolithography).
Description of the In Vitro 3D Printed Cast Model
The final printed model hereafter called the in vitro 3D cast model was made of a rigid transparent washable tusk® material (tusk XC2700T, Materialise, NV, Leuven, Belgium). The printed cast model (described in Fig. 1) is composed of two parts attached to each other:
The first part of the cast model (URT) consists of the whole head, segmented into four longitudinal parts, allowing visualization of the aerosol and deposition inside the transparent waterproof nasal cavities. The head was printed from the exterior details (ears, forehead, and complete mouth, with the teeth etc.) to the internal details (sinuses and, ostia of the naso-pharynx).
The second part of the model, which constitutes the LRT, is connected to the first part. It includes the trachea and bronchial tree with six bronchial divisions.
Fig. 1.
In vitro experimental set-up. A pediatric human facemask (DTF Medical, Saint Etienne, France) was used with the three types of nebulizers. The facemask covers the nose and mouth when placed on the face. The total duration of nebulization (for each of the three nebulizer experiments) was 10 min.
Holes were printed at the extremity of the six bronchi, allowing connection to filters (PARI® filter 041B0523, Starnberg, Germany) and a respiratory pump (Harvard Apparatus, Massachusetts, USA; tidal volume = 26 mL, respiratory rate = 33 cycles /minute, I/E (inspiratory/expiratory) = 40/60) to simulate the spontaneous physiological breathing of a healthy macaque.
The experimental setup is presented in Fig. 1.
Nebulizers
Three medical jet nebulizers were used to generate submicrometric and micrometric aerosols: Microcirrus® (Intersurgical, Berkshire, UK), Sidestream® (Philips Healthcare Andover, USA) and a prototype NL20® (DTF Medical, Saint-Etienne, France). The Microcirrus® and Sidestream® nebulizers were loaded with a 150 MBq (3 mL) solution of diethylenetriamine penta-acetic acid (99mTc-DTPA), (Curium Life Forward, Gif-sur-Yvette, France). The NL20® prototype nebulizer was filled with a 150 MBq (5 mL) solution of 99mTc-DTPA. The Sidestream® and Microcirrus® nebulizers were connected to an air source operating at 8 L/minute to produce aerosols. The NL20® prototype operates at an airflow source rate of 4 L/minute; air was added at 4 L/minute in a second opening of the nebulizer, making a total of 8 L /minute, to have the same aerosol velocity for the three nebulizers.
Animals and In Vivo Experimental Design
Three female cynomolgus macaques (Macaca fascicularis), bred in Mauritius were used to compare aerosol deposition with that of 3D cast model.
The experimental animal protocol was conducted in accordance with the European regulations for animal experimentation committee, under ethics committee agreement number 29462#2021020212179142V2.
Following acclimation, the three macaques (3.3 kg, 4.3 kg, and 4.5 kg) received veterinary check-ups to ascertain their global health, in particular, their respiratory capacity, before inclusion in the study. The integrity of the lungs was evaluated before the aerosol deposition studies using a thoracic scanner (CT, IRIS-CL, Inviscan, Strasbourg, France). A two-bed CT scan was performed consisting of 576 projections over 360 degrees, at 80 kV and 0.9 mA, with an exposure time of 90 ms, leading to a total acquisition time of 104 s. The three-dimensional image was reconstructed using beam hardening correction, ring artifact pre-correction, resulting in 1190 slices with a voxel size of 160 × 160 × 160 μm.
Each macaque in our experimental setup randomly received aerosols generated from each of the three selected nebulizers two times (n = 6). For inhalation of the aerosols, animals were sedated in a unique contention chair. Acclimatization and training of the macaque in the chair and to breath spontaneously through a facemask were performed, for each animal one week before each experiment, to limit stress.
The animals received aerosols after being anesthetized by intramuscular injection of ketamine (3 mg/kg), (Imalgene® 1000, Centravet, Dinan, France) and Sededorm® (40 µg/kg) (Medetomidine 1 mg/Kg).
A gel (Ocry-gel®) was used to lubricate and close the eyes of the animals to avoid eye exposure and dryness. Once the mask was in place on the face, the nebulizer was connected, and the nebulization started simultaneously with a timer (to achieve a 10 min nebulization process) and a saturometer placed on their fingers to control vital parameters (blood saturation and cardiac rate).
Once back in their cage, the macaques were slowly awakened using an intramuscular injection of Alzane® (Atipamezole, 200 µg/kg).
Particle- Size Measurements
Particles-size distributions of the 99mTc-DTPA aerosols generated by each of the three jet nebulizers were measured three times for each nebulizer using cascade impactors. A new generation impactor, (NGI, Copley Scientific Ltd, Nottingham, United Kingdom) operating at 15L/min was used for the NL20® and Sidestream® nebulizers and a DEKATI® low-pressure Impactor (DLPI) operating at 10L/min for the Microcirrus® nebulizer. At the end of nebulization process, the quantity of particles deposited for each stage of either the DLPI or NGI was determined by scintigraphy imaging using a gamma camera (Single Head Ecam Siemens healthcare, Erlangen, Germany). Thus, the median mass aerodynamic diameter (MMAD) was calculated for the polydisperse aerosols of the three nebulizers. We also calculated the particle fractions for diameters smaller than 0.5 µm, 1 µm, 2 µm and 5 µm. These fractions were calculated using the European and French guideline (EN13544-1) [23, 24] for characteristics and performance of inhalation systems. Providing particles fractions, helps to predict the fraction of aerosols likely to be deposited in the URT and LRT [25] as function of aerosol size [23].
Deposition Measurements: In vivo and in vitro Image Acquisition
Comparative CT Scans of the In Vitro vsIn Vivo Models
Comparative X-Ray CT scans (Vereos, Philips, Netherlands) were performed to compare the geometry of the in vitro 3D cast model to that of the initial animal model. We compared scans of the 3D cast model with those of the real macaque model by specifically measuring various defined distances: sinuses, ostia, and trachea. Measurements on the in vitro and in vivo scans were performed using IntelliSpacePortal 8.0 software (Philips Healthcare, Netherlands). We measured the diagonal diameter and height of the sphenoid sinuses, named a and b and a’ and b’ respectively, for the in vivo and in vitro CT scans. We also measured the diameter of the left and right maxillary ostia of the sinuses, named c and c’ respectively in vivo and in vitro. Finally, we measured the beginning of the tracheal lumen, named d and d’. The measured distances are presented in Fig. 2.
Fig. 2.
Comparison of CT scans and measurements of the printed in vitro 3D cast model and in vivo macaque model. (A) Comparison of the whole body and head until the third bronchial division of a 6.6 kg male fascicularis macaque (in vivo) and the in vitro 3D model printed using a stereolithography manufacturing process (A’) until the third bronchial divisions, lateral view. Comparison of the internal face of the macaque (C) and in vitro 3D cast model (C’), frontal view and of the tracheal lumen, in vivo (D) and in vitro (D’), dorsal view.
Assessment of Aerosol Deposition and Acquisition by Gamma Scintigraphy Imaging
After 10 min of nebulization, aerosol deposition was measured by gamma camera scintigraphy imaging (Single Head Ecam, Siemens healthcare, Erlangen, Germany) for the in vitro and in vivo models under the same experimental and analytical conditions for both models (in vitro vs in vivo). Images were recorded using a Siemens planar gamma camera (Single Head ECAM, Siemens Healthineers, Erlangen, Germany) with a resolution matrix of 128 × 128 using a single planar detector.
Before starting nebulization, a 1-min acquisition was performed for each nebulizer to assess the initial radioactive scintigraphy signal. Then, after nebulization, the gamma scintigraphy images of the animals or 3D cast model were acquired for 5 min.
Image Analysis: Gamma Scintigraphy Measurement
To quantify the amountof aerosol deposition by gamma camera imaging, the regions of interest (ROI) were separated into two regions for the in vivo and in vitro models: the URT and LRT. We applied three different corrections to estimate aerosol deposition: the radioactive decay of 99mTc, tissues attenuation, and the acquisition time.
Tissue Attenuation for Scintigraphy Image Analysis
We used a 2 min scintigraphy image acquisition time after internal or external radioactivity exposure to determine the attenuation coefficient for each tissue.
Attenuation was calculated for four tissues. The same calculation method for the attenuation coefficients was applied to the in vitro model and the animal, except for the stomach and lungs, as the in vitro 3D cast model does not have these elements.
Lung tissue attenuation: an intravenous bolus injection of 1 mL/37 MBq of radiolabelled macroaggregated albumin (LyoMAA-99mTc, Pulmocis, France) was performed on each macaque. 99 m Tc- LyoMAA particles were biodistributed solely in the pulmonary capillaries by introduction through the saphenous vein to visualize them by scintigraphy imaging (perfusion scintigraphy) of the lung. Due to the limited printing of the in vitro model lower airways, lung attenuation consisted of bronchial attenuation (6): the same volume of radioactivity (99mTc- DTPA) was introduced to each of the six (6) divisions and then measured by scintigraphy imaging.
Stomach tissue attenuation: a gastric catheter was introduced by oropharyngeal route down to the stomach. The stomach was then filled with 10 mL 0.9% NaCl - 99mTc-DTPA.
Nasal cavity attenuation: each model (animal and 3D cast) received approximately150 µL /18.5 MBq per nostril of a radioactivity through a thin adapted intranasal atomization syringe device (MAD 30, Teleflex®, Morrisville, USA).
Oral cavity attenuation: a tube filled with 1 mL/37 MBq of radioactivity was placed inside the oral cavity of the animal or the nasal cast.
We used the average of the nasal and oral cavity attenuation to determine the attenuation of each, as the nasal attenuation calculations were similar to those for oral attenuation and we could not distinguish between the two cavities by gamma camera imaging.
Quantification of Deposited Aerosolized 99 m-Tc-DTPA
The ROIs were then adapted to each aerosols image to estimate the in vivo and in vitro radioactivity deposited in the URT and LRT. All calculations considered the background radiation, the radioactive decay of the 99 m Tc (radioactive period of 6 h) and previously calculated tissue attenuation coefficient. The lungs were delimited manually using the perfusion scintigraphy ROIs, describe above for the lung attenuation coefficient with LyoMAA. Results are expressed in terms of total aerosol mass deposited (URT + LRT) in the airways based on the nebulizer load counted by a gamma camera and relationship between liquid and mass (density = 1). Results are also expressed in terms of the distribution of deposition between the URT and LRT using a normalization calculation.
Statistical Analysis
Data are presented as the mean ± standard deviation (SD). GraphPad® software (GraphPad® Prism 9 version 9.2.0, San-Diego California, USA) was used for graphical results. Statistical analyses were performed using StatXact® software (StatXact-3 3.0.2, Cytel Software Corporation, France). Non-parametric stratified permutation tests were used to compare the in vitro and in vivo aerosol deposition in terms of the total aerosol mass deposited (URT + LRT) and the distribution of deposition between the URT and LRT. P-values (p) ≤ 0.05 were considered statistically significant and p > 0.1 were not.
Results
Aerosol Particle Size Distribution
The jet nebulizers used in this study produced different polydisperse aerosols.
The results for the particles characterized by their MMAD and their % distribution (particle fraction) are summarized in Table I.
Table I.
Characteristics of the Three Jet Nebulizers: MMAD (Mass Median Aerodynamic Diameter)
| Jet nebulizer (Q = 8 L / minute) |
MMAD (µm): NGI / DLPI (Microcirrus®) and gamma camera |
Particle fraction < 5 µm (FP) |
Particle fraction < 2.5 µm |
Particle fraction < 1 µm |
Particle fraction < 0.5 µm |
|---|---|---|---|---|---|
| NL20®Prototype | 13.1 ± 9.70 | 21.6 ± 3.9% | 10.1 ± 3.3% | 2.4 ± 1.4% | 1.9 ± 1.0% |
| Sidestream® | 3.2 ± 1.05 | 69.5 ± 12.1% | 38.6 ± 2.7% | 10.1 ± 2.0% | 6.6 ± 1.3% |
| Microcirrus® | 0.93 ± 0.19 | 76.3 ± 7.0% | 75.5% ± 5.7% | 53.9 ± 8.2% | 19.5 ± 10.4% |
The MMAD obtained ranged from 13.1 µm for the NL20® prototype nebulizer to 3.2 µm for the Sidestream® and 0.93 µm for the Microcirrus® nebulizers.
The NL20® prototype nebulizer produced the largest aerosols (13.1 ± 9.7 µm) among the three nebulizers tested. The percentage of the fine particle fraction was: 21.6 ± 3.9% for particles < 5 µm and 2.4 ± 1.4% for particle < 1 µm for this device.
The Microcirrus® nebulizer produced the smallest aerosol particles among the three nebulizers tested. Indeed, 76.3 ± 7.0% of the particles generated were < 5 µm and 53.9 ± 8.2% were < 1 µm.
The percentage of particles < 0.5 µm was 19.5% for the Mircocirrus®, 6.6% for the Sidestream® and 1.9% for the NL20®.
Among the three nebulizers, the NL20® prototype produced the smallest (21.6%) percentage of particles < 5 µm, whereas, the Sidestream® and Microcirrus® produced 69.5 and 76.3% respectively.
This suggests that the NL20® nebulizer generated aerosols in a size range > 5 µm, likely to be deposited predominantly in the URT.
In Vitro 3D Printed Cast Model Validation
We compared the CT scans and anatomical measurements of the in vivo and in vitro models to evaluate whether the in vitro 3D cast model was anatomically and accurately printed up to the third bronchial divisions. The height of the frontal sinuses for the in vivo and in vitro models were 17.8 (a) and 17.6 (a’) mm respectively (Fig. 2B & B’). The diagonal distances of the frontal sinuses in vivo and in vitro were 48.8 (b) and 48.4 (b’) mm respectively. The measured distances from the left to right sinuses were 37.5 mm for both models (Fig. 2C & C’). Finally, the tracheal lumen diameters were 8.8 (d) and 9.0 mm (d’) respectively, (Fig. 2D & D’).
The distances and diameters measured in the scans of both models’ (vivo and vitro) were similar. The CT scan measurements confirm the high preservations of the anatomy.
The printed in vitro model accurately reproduces the in vivo model in terms of anatomy.
Comparison of In vivo and In vitro Deposition
Data of qualitative scintigraphy images showed three deposition patterns of aerosols depending on the three different sizes of the aerosols generated and tested (Fig. 3). Images show two spots in the URT in vivo results likely corresponding to hair in the animals’ jowls. This was not the case of the in vitro model, as it lacks fur. The LRT deposition of the in vitro 3D cast model showed three spots, corresponding to the three of the six filters of the bronchial divisions.
Fig. 3.
In vivo (top images) and in vitro (bottom images) comparative scintigraphy images of the URT and LRT of aerosol deposition experiments. Images were recorded after 10 min 99mTc-DTPA aerosols inhalation of particles of large to small MMAD with the three jet nebulizers. All images are from the same macaque and same in vitro 3D cast model of aerosolization. The blue and red colours indicate a high concentration of radioactivity, whereas orange color represents a low concentration of radioactivity.
In vivo and in vitro deposition data obtained for the three size ranges studied are presented in Fig. 4. The total aerosol deposition in the URT and LRT, showed no statistically significant, (p = 0.4) differences (ns) between in vitro and in vivo total airway deposition. These data show that, the macaque in vitro 3D cast model is predictive of global aerosol airway deposition in defined conditions of aerosolization. The difference in total deposition (expressed as the quantity (g) of the aerosol) between the types of nebulizers can be explained by the difference of nebulizer output.
Fig. 4.
Comparative results of the total amount (URT + LRT) of aerosol mass (mean ± SD) deposited in the respiratory airways depending on aerosol size (13.1, 3.2, 0.93 µm) for the in vitro 3D- cast model (tiled columns) and macaque model (grey column).
There was a statistically significant difference (p < 0.0001*) between the in vivo and in vitro models in terms of the distribution of deposition (URT/LRT).
Globally, fewer particles were deposited in the LRT versus the URT for the three particle sizes in vitro and in vivo (Fig 5).
Fig. 5.
Distribution of deposition in the URT versus the LRT (mean), for aerosols from 0.93 to 13.1 µm.
LRT (grey colour) deposition was consistently higher in the in vitro 3D-cast model than in the in vivo model for all the three sizes tested:
17.0 ± 1.8% (in vitro) vs 4.5 ± 1.2% (in vivo) for 13.1 µm; 62 ± 6.5% (in vitro) vs 14.0 ± 6.9% (in vivo) for 3.2 µm, and; 51.0 ± 26.2% (in vitro) vs 22.6 ± 8.1% (in vivo) for 0.93 µm.
Overall, the in vitro 3D cast model overestimates LRT deposition relative to the in vivo macaque.
Discussion
No other study has compared aerosol deposition between a macaque in vitro 3D-cast model and living macaques [26, 27].
Our in-vivo 99mTc-DTPA aerosol deposition data, can be compared to the in vivo data of Albuquerque-Silva et al. [25] measured by scintigraphy imaging in three healthy living baboons. They quantified the deposition in what they called extra-thoracic (ET) and thoracic (TH) regions of interest (ROIs), which are similar to what we defined as our URT and LRT ROIs. Interestingly, their largest particles (2.8 µm) and our intermediate sized particles (3.2 µm) showed similar deposition values for the URT deposition in baboons and macaques: 72 ± 17% vs. 86 ± 7%, respectively.
Among tested nebulizers, only jet nebulizers have provided clear-cut values for small-, intermediate-, and large-sized particles, whereas the mesh nebulizers tested were not able to generate droplet sizes with an MMAD < 1 µm. In a comparative clinical study, Vecellio et al. [22] determined the percentage of URT deposition of 5.6-µm 99mTc aerosols, in terms of volume mean diameter (VMD), using a nasal jet nebulizer in seven healthy human male volunteers by gamma camera imaging. Their values of 73 ± 10% obtained in human beings are close to ours (86.0 ± 6.9%) obtained in macaques for a comparable particle size [22, 28].
In terms of in vitro cast models, our results share several similarities with those of two similar studies of aerosol deposition using 3D cast models of NHP. Kelly et al. [26] compared nasal airway deposition inside an acrylic transparent mould nasal cast replica of a male monkey cadaver and human in-vitro mould nasal airway replicas manufactured by stereolithography. Kesavan et al. [27] conducted an in vitro study of aerosol deposition using 3D cast models from living macaque (four females and one male) CT scans and compared it to that in humans. To simulate natural mucus, they coated their cast with Mazola corn oil®.
These authors showed that aerosol deposition is not influenced by the gender of the monkeys but rather by the physical parameters that characterize the behaviour of aerosols (Stokes and Reynolds number). Of note, using a NHP cadaver to develop a cast model was shown to introduce geometric differences relative to humans [24]. Thus, it is likely more appropriate to use living NHPs to produce a macaque in vitro cast model.
Another important element is the printing technique. We used the same stereolithography printing technique based on CT scans of a healthy macaque as Kesavan et al. [26]. However, we printed a realistic trachea and bronchial tree, whereas they used "plastic tubing" to mimic the LRT. In addition, they modified the original macaque printed 3D-cast model by closing the mouth. In doing so, they did not accurately reproduce the physiological characteristics of macaque nose and mouth-breathing [12].
Of note, our in vitro and in vivo deposition data show that the macaque in vitro cast is predictive of total aerosol deposition. However, it underestimates URT deposition and consequently overestimates that in the LRT.
One possible explanation for the URT underestimation is that, despite macaques being nose and mouth breathers [29, 30], it is not possible to exclude that they breathed more by the nose during the aerosolization process. On the contrary, the 3D-cast model imposes simultaneous nose and mouth “breathing”. In addition, the fact that the nose, contrary to the mouth, is an anatomical filtration zone could explain the higher URT deposition in the in vivo model than the in vitro model. Another possibility is that although we showed anatomical similarities between the in vitro and in vivo models, the actual in vitro 3D cast models do not accurately depict the exact texture of all the internal structures that exist in vivo [10]. Thus, it cannot be excluded that the synthetic material used to print the in vitro models could have influenced the deposition in the upper cavities, possibly explaining the differences between in vivo and in vitro aerosol deposition. Another explanation could be that we applied the same method for the URT attenuation coefficients, whereas the 3D cast is made of plastic and the airways of the macaque are composed of physiological tissues. In addition, because gamma camera imaging is unable to precisely distinguish anatomical structures, what is called the URT is, in fact, the entire face of the animals, with aerosols that penetrate to the URT. Indeed, the aerosol droplets can stick to the fur of the animal in the in vivo model, whereas fur was not an issue in the in vitro model. Overall, these factors could explain why the quantified URT deposition was underestimated by the in vitro model relative to the in vivo model.
The in vivo LRT aerosol deposition was consistently less than in vitro. This may be related to the fact that only fine to ultra-fine particles target the LRT, particularly the lungs, and depend on various parameters, such as the animals’ breathing parameters and the effects of the anaesthesia. A second possibility is that the 3D cast model was printed until the first bronchial division, which was then connected to artificial tubes and filters. This setup is obviously not entirely physiological. Consequently, as filters were used, it is likely that all of the aerosol crossing the filter was retained in the filter and not exhaled, as occurs in the real lung. In addition, the overestimation of aerosol deposition in the LRT airways in vitro could be explained by anatomical differences between the in vitro and in vivo models. In particular, deep deposition (LRT) is expected to be higher for a 6.6 kg animal (in vitro model) than in the animals used (in vivo assays), which had a weight ranging from 3 to 4 kg. The flow velocities in the ducts were necessarily different for the same volume/minute setting between the different tracheal/bronchial diameters.
Finally, the observed differences could have been influenced by the lack of mucus and mucocilliary clearance in the 3D cast. Indeed, the thickness of the mucus and mucocilliary clearance condition the deposition and behaviour (rheology characteristics, such as surface tension and viscosity) of deposited particles [31], especially in the URT. Williams and Suman [32] also described that the lack of mucosal surface and ciliary clearance constitutes a limitation for nasal anatomical models. The moisture content and temperature in the airways could also modify the particle size, affecting the deposition along the airways [33]. However, despite a similar lack of mucus, moisture, and body temperature (37 °C) in the airways model, a human 3D cast model was shown to provide results comparable to those obtained with in vivo human CT scans [10]. Thus, the use of the stereolithography technique based on accurate healthy macaque CT scans appears to provide researchers with valuable and accurate in vitro models, as shown by MRI images [34] or cadavers [11, 18, 26, 35, 36].
Conclusion
The in vitro 3D cast model we developed from a macaque scan printed until the third bronchial divisions provides a good overall predictive model of the total amount of aerosols deposition inside the respiratory tracts for aerosols ranging from 0.93 to 13.1 µm in term of MMAD.
Despite the observed overestimation of URT deposition, this is a helpful tool for preliminary experiments prior to animal studies.
Further studies concerning nasal cast development should improve the relevance of this tool to predict aerosol deposition.
Although macaques are still necessary for aerosol studies, using such an in vitro model will help to reduce the number of macaques included in aerosol studies, in accordance with the requirements of the 3Rs rule.
Acknowledgements
We thank all collaborators from the CEPR of the University of Tours for their expertise in aerosolization and their help in the animal experimentation.
Special thanks are addressed to Deborah Le PENNEC for her practical help concerningthe development of the 3D cast model and the use of nebulizers and their characterization process.
We particularly acknowledge PST-A platform for its important support for the in vivo experimentation, from the regulatory aspects (Jerome MONTHARU) to the technical aspects of the macaque experimentation and housing conditions (Lucas GARANGER).
Concerning the macaque whole-body check-up CT scans before the beginning of the study, we thank Laurent GALINEAU and Sophie SERRIERE from the “Département d'Imagerie Préclinique, Plateforme Scientifique et Technique Analyse des Systèmes Biologiques”, and “UMR 1253, IBrain, Équipe Imagerie, Biomarqueurs et Thérapie”, University of Tours, Inserm, Tours, France.
We also thank Sandrine Le GUELLEC from DTF medical for providing the NL20® Prototype and her expertise.
We finally acknowledge Quentin PASCAL (DVM) from the CEA of Fontenay-aux-Roses for his clinical expertise in validating the 3D cast model.
Funding
This project was funded by the French Agency Innovation Defence (Former General Direction of Army), DGA, for their NRBCE program and by the CEA of Fontenay-aux-Roses.
Justina CREPPY thanks the same Institutions for her PhD Fellowship.
Data Availability
The data associated with this work will be made available on request.
Declarations
Conflict of Interest
Laurent Vecellio was employed by DTF Medical from 2001 to 2018 and by Nemera from 2018 to 2020.
Footnotes
Publisher's Note
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Associated Data
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Data Availability Statement
The data associated with this work will be made available on request.