This work is part of a comprehensive inhalation toxicology study on e-vapor aerosols compared with CS exposure; the study also includes assessment of systemic, respiratory, and cardiovascular effects in the ApoE–/– mouse model. Here, we summarize the main procedures relevant to the investigation of the effects of exposure on bone in this study; for further details and other endpoints, the reader is referred to the other topical reports on this study (Szostak et al. 2020), as well as to the corresponding data sets on INTERVALS (https://www.intervals.science/studies/#/apoe_p4).
All procedures performed in this study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted (a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and licensed by the Agri-Food & Veterinary Authority of Singapore, with approval from an Institutional Animal Care and Use Committee (IACUC, protocol #15044)) and in compliance with the National Advisory Committee for Laboratory Animal Research Guidelines on the Care and Use of Animals for Scientific Purposes (Naclar 2004). All applicable international, national, and institutional guidelines for the care and use of animals were followed.
Female ApoE–/– mice (B6.129P2-Apoetm1/Unc N11) bred under specific pathogen-free conditions were obtained from Taconic Biosciences (Rensselaer, NY, USA). The health status of the animals was verified using the health check certificate provided by the breeder. Mice were maintained and exposed under specific hygienic conditions as described previously, with filtered conditioned fresh air at 22 ℃ ± 2 ℃ and 55% ± 15% humidity. Additional details of animal housing, randomization, and acclimatization have been published previously (Boue et al. 2012; Lietz et al. 2013; Phillips et al. 2016a).
Animal groups and exposure
At the age of 8 weeks, mice were randomly allocated using the body weights to five exposure groups and subjected for up to 6 months of whole-body exposure (Table 1): Sham (exposure to fresh, conditioned air); diluted mainstream CS from the 3R4F reference cigarette; or three groups of e-vapor aerosol exposure (CARRIER, BASE, and TEST). The CARRIER e-vapor liquid contained the humectants propylene glycol (PG) and vegetable glycerin (VG) alone; BASE contained the humectants and nicotine; and TEST contained the humectants, 4% nicotine, and flavors (Table 2). For the BASE and TEST formulations containing nicotine (4%), mixtures of acids (1%) were added, with the resulting pH of ~8. The BASE and TEST group exposure atmospheres were configured to deliver a nicotine concentration of 35 µg/L (corresponding to the nicotine level of 560 µg/L total particulate matter (TPM) from 3R4F cigarettes). Schematic in Fig. 1 gives an overview of the general study design (Fig. 1).
Mice were exposed for 3 h per day, 5 days per week, for up to 6 months. Intermittent exposure to fresh filtered air for 30 min after the first hour of exposure and for 60 min after the second hour of exposure was provided to avoid accumulation of excessive carboxyhemoglobin (COHb) in the 3R4F group.
3R4F reference cigarettes were purchased from the University of Kentucky (College of Agriculture 2019). Mainstream CS from 3R4F cigarettes was generated on 30-port rotary smoking machines in accordance with the Health Canada intense smoking protocol (Health_Canada (1999)), which is based on ISO standard 3308 (ISO3308 1991.): 55-mL puff volume, one puff per 30 s, and 100% blockage of ventilation holes (Government 2000); the 3R4F puff count was 10–11 per cigarette (average, 10.4 ± 0.3). For whole-body exposure, the concentration of 3R4F CS was 562 ± 65 (mean ± standard deviation) µg TPM per liter (600 µg /L target concentration) and 35.2 ± 4.8 (mean ± standard deviation) µg nicotine/L.
E-vapor aerosols for the CARRIER, BASE, and TEST groups were prepared by adding each component and diluting to the final mass composition (Table 2). The prepared mix was stored away from light, at a controlled temperature of 2–8 ℃ under uncontrolled humidity conditions. The CARRIER, BASE, and TEST aerosols were generated using a capillary aerosol generator developed by Philip Morris International Inc. and further refined by Virginia Commonwealth University (Gupta et al. 2003; Howell and Sweeney 1998). This aerosol generator was previously shown to deliver aerosols in a consistent manner and at similar particle size distribution and concentrations as a prototype e-cig device (Werley et al. 2016). The temperature of the capillary aerosol generator was set at 250–275 °C to match the temperature of the heated coil during puffing of the e-cig device (Geiss et al. 2016). The generator was fitted with a diffuser and compressed air to prevent aerosol backflow. Condensation aerosol was created when the output from the generator was diluted with air at ambient temperature. A portion of the formed aerosol from the generator was further diluted with filtered air to achieve the target concentrations in the test atmosphere and delivered via glass tubing to the exposure chamber.
For whole-body exposure, the CARRIER aerosol contained PG at a concentration of 179.4 ± 21.3 µg/L and VG at a concentration of 576.9 ± 65.6 µg/L (without nicotine). The BASE aerosol was nicotine-matched to 3R4F and PG/VG-matched to the CARRIER (35.5 ± 4.9 µg nicotine/L, 171.3 ± 16.7 µg PG/L, and 543.5 ± 68.4 µg VG/L); the TEST aerosol was also nicotine-matched to 3R4F and PG/VG-matched to the CARRIER (35.7 ± 5.6 µg nicotine/L, 173.0 ± 19.4 µg PG/L, and 546.2 ± 74.2 µg VG/L).
Analysis of biomarkers of exposure
Blood COHb concentrations were determined as described previously (Phillips et al. 2016a; Phillips et al. 2015). Blood was collected from the facial vein under anesthesia within 15 min after exposure (at months 2 and 5). For plasma collection, blood was placed on ice after collection and processed. Aliquoted plasma was transferred to storage at ≤ − 70 °C. Plasma PG, nicotine, and cotinine concentrations were measured by ABF GmbH (Planegg, Germany). Urine was collected during exposure and for approximately 18 h post exposure in individual metabolic cages. Urine collected during exposure, urine from the 18 h overnight collection, and water collected during rinsing of the cage (with approximately 100 µL of water) were pooled per animal, aliquoted, and stored at ≤ − 70 °C. Nicotine metabolites (trans-3′-hydroxycotinine, norcotinine, cotinine, nicotine-N′-oxide, and nornicotine) in urine were analyzed by LC-MS/MS after 1,3-diethyl-2-thiobarbituric acid derivatization at ABF.
The bone phenotype of female ApoE–/– mice exposed to various smoke/aerosol conditions was determined by micro-computed tomography (µCT), biomechanical testing by three-point bending, and histological analysis of intact tibiae. On the scheduled necropsy date and approximately 16–24 h after the last exposure, mice were anesthetized with 100 mg/kg pentobarbital before exsanguination and perfusion with 0.9% saline. Both tibiae of each animal were harvested by disarticulation of the hip joint to separate the lower limb from the mouse torso as described before (Reumann et al. 2011a, b). The patellar tendon was cut horizontally, and the tibia was separated from the femur. Subsequently, all surrounding muscle was removed from both bones. The fibula was removed along with the soft tissue. As some tibiae got damaged during preparation, only intact bones were further analyzed and served for general characteristics (Sham: N = 10; 3R4F: N = 16; CARRIER: N = 13; BASE: N = 11; TEST: N = 14). For bone length analysis, X-ray images were acquired using an X-ray Bucky table (BuckyDiagnost CS Optimus, Philips, Hamburg, Germany) and standard cassettes (IP Cassette Type CC, FCR standard cassette 18x24cm, Fujifilm, Germany). Settings for the index finger (50 kV; 2.5 mAs; free exposure with small focal spot of 0.6; and a focus-detector distance of 1.05 m) were used to achieve high spatial resolution for the small bones. X-ray images were analyzed using an electronic PACS system (IMPAX 184.108.40.2063 Version 2014, AGFA HealthCare N.V., Mortsel, Belgium). Bone weight analysis was performed for all intact tibiae using the Kern ABJ scale (Kern & Sohn GmbH, Balingen, Germany).
Characterization of bone architecture
The overall 3D morphology of total bone compartment of the whole intact right tibiae (N = 7/group) was assessed using a Scanco µCT 80 system (Scanco Medical, Bassersdorf, Switzerland). Additionally, a detailed analysis of the midshaft cortical area was performed. The midshaft cortical region of interest was defined in the middle area of the long bone. Each bone was measured in length and the exact midpoint was used as landmark. From there, a total of 50 sections, 25 sections distal and proximal from landmark, were defined for further midshaft analysis for each bone. All bones were scanned in 4% formalin. Parameters of 20 µm voxel size, 70 KVp, a 200 ms exposure and one frame per view were used for the scans. The Scanco µCT software (µCT evaluation program V6.0, Scanco Medical, Switzerland) was used for 3D reconstruction, evaluation and viewing of images. After 3D reconstruction, the volumes of interest were segmented and analyzed for whole bone and for midshaft cortical bone. Directly measured bone volume fraction (BV/TV), mean/density TV and mean/density were calculated for all whole bone (Table 3). BV/TV, total area (TA), bone area (BA) and bone total area ratio (BA/TA) of the cortex, polar (pMOI), maximum (Imax) and minimum (Imin) moments of inertia were calculated for the midshaft cortical areas of the bones (Table 4).
Characterization of bone strength
The mechanical properties of intact left tibiae were evaluated by a three-point bending test (Sham: N = 6; 3R4F: N = 8; CARRIER: N = 6; TEST: N = 8; BASE: N = 7). The tests were conducted at room temperature on a precision load frame (Zwicki Z2.5 TN, Zwick Roell, Ulm, Germany). The tibiae were tested with their posterior side loaded in compression and the anterior side in tension. The anterior surface was placed on the two lower supports, which were set 10 mm (60% of mean tibial length) apart for all tibiae of all groups. Load was applied at 0.025 mm/s, with a preload of 1 N, until failure. Structural mechanical properties dependent on geometry were measured using the testXpert II V3.3 program (Zwick Roell, Ulm, Germany). Parameters included ultimate load (N), stiffness (N/mm), work to fracture (Nmm), and post-yield displacement (PYD; mm). The setup and parameters were based on those reported by Jepsen et al. (Jepsen et al. 2015).
Characterization of bone morphology
All dissected tibia samples (Sham: N = 4; 3R4F: N = 2; CARRIER: N = 3; BASE: N = 3; TEST: N = 3) were fixed in 4% formalin at room temperature for 24 h. All samples were embedded in paraffin after decalcification in ethylenediaminetetraacetate (10% EDTA, pH 7.4) for 48 h at 37 °C. For each sample, serial longitudinal sections (2.5 µm thickness) were prepared, deparaffinized in xylene, and rehydrated in an ethanol gradient. The sections were then stained with hematoxylin and eosin (H&E), Alcian blue, Masson trichrome stain, and Van Gieson stain using standard procedures. Digital bright field images from slides were captured using a Mirax Scan or Axio 2 imaging system (Carl Zeiss MicroImaging GmbH, Jena, Germany).
Statistical analysis for the bone-related endpoints was performed using GraphPad Prism 5.0 (La Jolla, CA, USA). Differences between the groups were evaluated by analysis of variance, the Kruskal–Wallis H-Test followed by Dunn’s multiple comparison test. Significance was defined as p < 0.05. All data are shown as box-and-whisker (Tukey) plots, indicating the median and interquartile range (IQR). Whiskers show the 1.5 IQR. Individually plotted values are outliners beyond the 1.5 IQR.
More details on the exposure characterization data and statistical analysis are available in Szostak et al. (Szostak et al. 2020). Data are expressed as mean ± standard error of the mean. Pairwise comparisons between groups were performed, and unadjusted p values are reported. For continuous variables, if the data of the two groups being compared did not exhibit strong deviation from the normal distribution (as assessed by a Shapiro-Wilk test at the 5% level on the standardized residuals of both groups), a two-sample t-test accounting for variance heterogeneity was performed. Otherwise, an exact Mann–Whitney–Wilcoxon two-sample test was used (Monte Carlo estimates of the exact p values were used). Results were considered significantly different for a specific comparison if p<0.05.