INTRODUCTION
An estimated 11.7% of the U.S. population smokes cigarettes despite its correlation with numerous health risk factors and significant socioeconomic burdens.1,2 Cigarette smoking detrimentally affects the respiratory and cardiovascular systems and contributes to the development of various cancers.3–5 Recent studies have also linked smoking with orthopedic conditions, such as degenerative disc disease. Specifically, clinical research using surveys and multivariate analysis has identified an association between patient smoking history and the development of intervertebral disc (IVD) degeneration and herniation.6–10 Medical imaging has indicated increased disc degeneration grades,6,11 decreased disc height,7,12–14 and Modic changes adjacent to the endplate in patients who smoke.9,15
To further elucidate IVD pathophysiology in the context of cigarette smoking, in vitro culture models and in vivo animal models have been developed to simulate cigarette smoking conditions.16–23 In vitro models have introduced either nicotine or cigarette smoke extract (CSE) into the cellular environment to examine the downstream molecular mechanisms on different IVD cell populations.16–18 These include the downregulation of anabolic genes in annulus fibrosis (AF) and nucleus pulposus (NP) cells,17,18 upregulation of catabolic genes in AF cells,17 decreased NP cell count,18 and reduced biosynthesis in AF and NP cells.16–18 CSE differs from nicotine in that it contains other water-soluble compounds found in cigarette smoke, particularly free radicals and other reactive oxygen species (ROS) that can lead to protein, DNA, and mitochondrial DNA damage.24–26 Although nicotine is recognized as a major cigarette constituent, the additional compounds found in CSE could have a more pronounced impact on cellular gene regulation, metabolism, and biosynthesis.17
In vivo animal models have examined the systemic effects of either intravenous nicotine injections or second-hand smoke exposure on IVD pathophysiology.18–20,22,23,27–30 Both of these treatments have led to detrimental cellular changes similar to those observed in vitro.18,21,22,31,32 Biochemical assays, employed only during smoke exposure studies, measured glycosaminoglycan content to decrease in the NP region.21,32 Histological analyses have confirmed morphologic changes in the IVD, including AF collagen fiber disorganization,19,23,27,31 NP hyalinization and fibrosis,19,21,27,31 and decreased NP cellularity.19,21,27 Furthermore, in vivo models have revealed chondrocyte apoptosis in the cartilage endplate (CEP),20 as well as a tendency toward endplate ossification.21,29 Notably, to our knowledge, only one in vivo study has examined smoke cessation (2 months) after a period of cigarette smoke exposure (2 months). That study observed that collagen fiber misalignment in the AF seemed irreversible after smoke exposure and that both the AF and NP tended to show extra fibrotic remodeling after the cessation period.19 The pathophysiology of the IVD, however, remains unclear, particularly regarding the spatial–temporal remodeling progression in the context of cigarette smoke exposure. Additionally, the effects of smoke cessation on IVD remodeling have not been quantitatively characterized.
Solute diffusion has been shown as crucial in depicting how three-dimensional structure and composition affect the integrity of avascular tissues, thereby defining their biomechanical function.33 It enables direct and quantitative comparison of the disc tissue remodeling, identifying which regions experience alterations in their structure and material properties due to smoke exposure, and further evaluating the outcomes of smoke cessation. Diffusion chambers have enabled the measurement of solute diffusivities in cartilaginous tissues ex vivo,34–37 but they are limited to unidirectional analyses, and require a substantial volume of intact tissue (5 mm in diameter).34,36,37 Alternatively, a fluorescent recovery after photobleaching (FRAP) microscopy technique seems promising for smaller regions [e.g., 387.5 μm × 387.5 μm (128 × 128 pixels) in this study], offering a much higher spatial resolution and the ability to capture diffusion rates in multiple directions simultaneously.33,38–41
Based on the previously discovered CEP chondrocyte apoptosis and endplate ossification tendency,20,29 we hypothesize that 2 months of cigarette smoke exposure will decrease solute diffusivity in the IVD, particularly in the CEP interface. Furthermore, we predict a continued reduction in diffusivities in all disc regions during a subsequent period of smoke cessation. The objective of this study is to establish a Sprague–Dawley (SD) rat model using a custom cigarette smoke exposure apparatus to assess the spatial–temporal IVD remodeling patterns. To test this hypothesis, we aim to quantify the solute diffusivity in the AF, NP, and CEP after smoke exposure and after smoke cessation using the FRAP technique. Additionally, this study characterizes changes in tissue porosity and collagen fiber structure, using a buoyancy method and label-free multiphoton excitation (MPE) imaging, respectively.34,42,43 This work helps to fill a current knowledge gap in IVD pathophysiology by quantifying the spatial–temporal alterations of diffusion properties and structural composition in the context of cigarette smoke exposure and smoke cessation.
METHODS
Twenty-four, 6-month-old (skeletally mature44–46) male SD rats were obtained following IACUC approval at the Medical University of South Carolina, and randomly assigned to two treatment groups: control and smoke exposure (Figure 1). Rats in the smoke exposure group underwent cigarette smoke exposure daily, for 2 h on weekdays and 1 h on weekends for 2 months. Research cigarettes (3R4F, University of Kentucky Tobacco Research & Development Center, Lexington, KY) were used for smoke exposure to mitigate irregularities in cigarette composition. Smoke exposure was performed using a programmable, custom-built smoke exposure apparatus (TE-10, Teague Enterprises, Woodland, CA) (Figure S1). Cigarettes were burned within the cigarette puffing chamber of the apparatus. A pressure differential created between the puffing chamber and the rat exposure chambers allowed the passage of smoke through the butt of the cigarette and into the rat exposure chambers to simulate the inhalation of cigarette smoke. Additionally, second-hand smoke generated within the puffing chamber was pumped into the rat exposure chambers. Total particulate matter (200.6 ± 73 mg/m3) was measured on an exhaust membrane filter (Pallflex® EMFAB TX40H120-WW, Pall laboratories, Westborough, MA) downstream from the rat exposure chamber (Figure S2), to ensure consistency with heavy chronic smoking in humans (approximately one pack of cigarettes per day).47,48 When not actively exposed to smoke, the animals were housed under normal conditions free from smoke contamination with adequate food, water, and bedding.
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After 2 months of smoke exposure, six animals in each group were randomly selected and euthanized using isoflurane (PHR2874, Sigma-Aldrich, Darmstadt, Germany), while the remaining six rats in each group were allowed to go through a five-month period of cessation. At the end of the cessation period, all remaining animals were euthanized using isoflurane, creating four treatment groups (n = 6/group): control (2 months of no smoke exposure), smoke exposure (2 months of smoke exposure), cessation control (7 months of no smoke exposure), and smoke cessation (2 months smoke exposure + 5 months of no smoke exposure). After euthanasia, the spine was dissected from the rat, individually wrapped in plastic wrap and gauze soaked in phosphate-buffered saline (PBS), and stored at −4°C to prevent tissue dehydration.43,49
Sample preparation
Two adjacent thoracic motion segments (T6-T7 and T7-T8) were removed from the vertebral column of each of the 24 rats and separated into bone-disc-bone complexes using a surgical scalpel (Figure 2). The motion segment complexes were embedded in optimal cutting temperature (OCT) fluid (23–730-571, Thermo Fisher Scientific, Waltham, MA), and trimmed using a freezing stage microtome (SM2400, Leica Biosystems, Deer Park, IL) to carefully remove the vertebral body on either side of the disc. The trimmed complexes were again embedded in OCT fluid and frozen in a plastic mold. Thin (~120 um) slices of disc tissue were sectioned using a cryostat (CM1800, Leica Biosystems, Deer Park, IL). The motion segments were sectioned either transversely from the midplane of the disc (consisting of AF and NP tissue; n = 24), or sagittally from the midplane of the motion segment (consisting of NP, AF, and CEP tissue; n = 24) (Figure 2). Three slices were obtained from each of the 48 motion segments (n = 144 slices total). The slices were immediately transferred to a microscope slide, and a 120 μm sticky well (S24737, Thermo Fisher Scientific, Waltham, MA) was adhered around the tissue. This well was then filled with a 0.2 mM fluorescein solution (332 Da, λex: 490 nm; λem: 514 nm, Fluka-Sigma-Aldrich, St. Louis, MO), and sealed with a glass coverslip to prevent evaporation of the fluorescein solution and minimize tissue swelling. The slides were stored at 4°C with no light exposure for a minimum of 16 h to allow equilibration of the dye solution within the tissue.
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Solute diffusivity measurement
The 2D FRAP tests were conducted in both the transverse and sagittal planes of the IVDs following previously established protocols.38,40,41,50 These experiments were performed on individually prepared tissue slices at room temperature (20°C) using a confocal microscope (DM6000 B, Leica Microsystems, Deerfield, IL). The dye solution within these slices was photobleached using an Ar-488 laser to create a circular bleach spot with a diameter of 48 μm, approximately 7 μm below the tissue surface. Prior to bleach spot creation, a multi-layer bleaching protocol was used to prevent diffusion along the optical axis, ensuring 2D recovery.38 A total of 250 images (128 × 128 pix, 387.5 × 387.5 μm2) were collected at a frame rate of either 0.355 or 0.669 s during the recovery period. Prior to bleaching, 5 images were collected, averaged together, and then subtracted from the recovery period images to minimize background fluorescent emission.51 A total of 2–3 FRAP tests were performed on each disc region per tissue slice. Recovery images were analyzed in MATLAB (MATLAB 2023a, MathWorks Inc., Natick, MA) using a custom code thatperformed spatial Fourier analysis on the images, enabling calculation of the principal fluorescein molecule diffusion rates (radial [Dradial], axial [Daxial], and circumferential [Dcircumferential]), and subsequently the average solute diffusivity [Daverage = (Dradial + Daxial + Dcircumferential)/3].39–41
Porosity measurements and MPE imaging
Three caudal motion segments (C1-C2, C2-C3, C3-C4) were dissected from the rat tails, and AF, NP, and CEP tissues were isolated carefully using a surgical scalpel under a dissection microscope. Tissue samples were weighed in air using an analytical balance and again while submerged in PBS using a density determination kit (Sartorius, Germany). After 1 week of lyophilization, dry samples were weighed, and porosity was calculated according to prior studies.34,49,52
MPE imaging was conducted to examine the regional IVD tissue structure. Lumbar motion segments (L1-L2, L3-L4) were fixed in formalin for 2 days and cut with a freezing stage microtome to expose the disc in either the transverse or sagittal plane. Prepared samples were then placed in a custom 3D-printed chamber, where they were immersed completely in PBS and prevented from swelling. The IVDs were imaged using a custom-built upright two-photon confocal microscope with a water immersion objective (MRP07220, Nikon Instruments Inc., Melville, NY). An ultrafast laser (Chameleon Ultra, Coherent Corp., Saxonburg, PA, λex = 740 nm) was used to image collagen fiber bundles, capturing second harmonic generation (SHG) emitted light (λem = 370 ± 36 nm). Simultaneously, ECM structure and cell morphology were imaged with two fluorescent emission channels (λem = 442 ± 42 nm, 607 ± 70 nm).42 Stacks of images (512 × 512 pixels, 547 × 547 μm2) were acquired from the surface of the tissue to a depth of 50 μm in increments of 2 μm. Imaging was repeated in a snake pattern over the whole surface of the transverse or sagittal midplane. Images were processed in ImageJ53 and stitched using Adobe Photoshop 2024 (Adobe, San Jose, CA).
The ImageJ plugin OrientationJ54 was used to calculate the coherency of the AF fiber bundles in the transverse and sagittal planes. Six regions of interest (0.0625 mm2) were selected on each image to encompass the entirety of the anterior AF. Coherency values were averaged for each specimen. Collagen bundle diameter was also obtained for each specimen using ImageJ. For each bundle shown in the sagittal images, the diameter was measured at three distinct locations (superior, inferior, and midpoint) and averaged.
Statistical analysis
AF and NP diffusivities (approximately 330 FRAP tests total in each disc region) were measured in the radial, axial, and circumferential directions, while the CEP (n ~ approximately 170 tests) was quantified only in the radial and axial directions (due to its thin structure preventing tissue preparation in the transverse plane). Transversely prepared slices enabled diffusivity measurement in the radial and circumferential directions, whereas sagittally prepared slices allowed diffusivity measurement in the radial and axial directions. For a given motion segment, diffusivities from the total of 6–9 FRAP tests (2–3 replicates per slice; 3 slices per motion segment) were averaged for each disc region.
Three-way analyses of variance (ANOVA) were performed in R (R Core Team, 2023) to identify the effects of smoke exposure, timepoint, and region independently on solute diffusivity, diffusivity anisotropy, and porosity (Table S1). Additionally, one-way ANOVA tests were conducted to clarify the effects of treatments (control, smoke exposure, cessation control, smoke cessation) on solute diffusivity, diffusivity anisotropy, tissue porosity, and AF bundle coherency. Post hoc comparisons were made to further understand the relationships between smoke treatments and tissue regions on the measured outcomes. A Holm-Bonferroni p-value adjustment was implemented to control multiplicity error. All data are represented as mean ± SD. Findings were considered significant for p-values < 0.05. Levels of significance are denoted by asterisks, where * is for p-values < 0.05, and ** is for p-values < 0.005.
RESULTS
Solute diffusivity
The diffusivities of fluorescein in each disc region differed between treatment groups (AFradial: p < 0.001, NP: p < 0.001, CEP: p < 0.001) (Figure 3A,B, Table S2). AF radial diffusivity was significantly greater in the control and cessation control groups relative to the smoke and smoke cessation groups (Control: 324.34 ± 30.06 μm2/s, Smoke Exposure: 281.13 ± 23.34 μm2/s, p = 0.0059; Cessation Control: 290.56 ± 8.72 μm2/s, Smoke Cessation: 238.70 ± 15.52 μm2/s, p = 0.0015; Figure 3A). Axial and circumferential diffusivities for the AF were not significantly different between the groups. AF diffusivity anisotropy also varied with treatment (AF: p = 0.010). It was significantly less in the cessation control group relative to the smoke cessation group (Cessation Control: 0.261 ± 0.043, Smoke Cessation: 0.364 ± 0.036, p = 0.0153; Figure 3C). The NP average diffusivity was significantly greater in the cessation control group compared to the smoke cessation group (Cessation Control: 504.01 ± 33.42 μm2/s, Smoke Cessation: 362.40 ± 41.75 μm2/s, p = 0.0013; Figure 3B). The CEP average diffusivity was greater in the control and cessation control groups relative to the smoke and smoke cessation groups (Control: 382.30 ± 45.84 μm2/s, Smoke Exposure: 326.18 ± 34.30 μm2/s, p = 0.0187; Cessation Control: 301.84 ± 21.31 μm2/s, Smoke Cessation: 245.64 ± 29.23 μm2/s, p = 0.0187; Figure 3A).
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The diffusivities varied significantly between the time points in each region (AFradial: p = 0.008, NP: p = 0.036, CEP: p < 0.001). The AF radial diffusivity was significantly greater in the control and smoke exposure groups relative to the cessation control and smoke cessation groups (Control: 324.34 ± 30.06 μm2/s, Cessation Control: 290.56 ± 8.72 μm2/s, p = 0.0114; Smoke Exposure: 281.13 ± 23.34 μm2/s, Smoke Cessation: 238.70 ± 15.52 μm2/s, p = 0.0059; Figure 3A). The NP average diffusivity was significantly greater in the smoke exposure group relative to the smoke cessation group (Smoke Exposure: 477.57 ± 59.56 μm2/s, Smoke Cessation: 362.40 ± 41.75 μm2/s, p = 0.0066; Figure 3B). The CEP average diffusivity was significantly greater in the control and smoke exposure groups relative to the cessation control and smoke cessation groups (Control: 382.30 ± 45.84 μm2/s, Cessation Control: 301.84 ± 21.31 μm2/s, p = 0.0021; Smoke Exposure: 326.18 ± 34.30 μm2/s, Smoke Cessation: 245.64 ± 29.23 μm2/s, p = 0.0021; Figure 3B). Diffusion testing also revealed the AF to be more anisotropic than the NP and CEP regions (Table S1).
Structural composition
CEP porosity trended greater in the control and cessation control groups relative to the smoke and smoke cessation groups, respectively, but was not significantly different (Control: 0.583 ± 0.123 Smoke Exposure: 0.506 ± 0.147; Cessation Control: 0.473 ± 0.114 Smoke Cessation: 0.380 ± 0.067; Figure 3D). The control group, however, was significantly greater than the smoke cessation group (Control: 0.583 ± 0.123 Smoke Cessation: 0.380 ± 0.067 p = 0.0333; Figure 3D). No significant differences in porosity were seen in the AF or NP tissue between groups.
MPE imaging revealed a spatially unique remodeling pattern as a result of smoke exposure and smoke cessation. AF tissue showed a narrowing of collagen fiber bundles in the radial direction after smoke exposure and smoke cessation, especially in the interior AF regions (Figure 4). A quantitative assessment showed that the average AF collagen bundle diameter decreased from the outer AF to the interior AF regions in all groups. Bundle diameters 2, 3 and 4, outer AF bundles, were lower in the smoke and smoke cessation groups when compared to the control and cessation control groups (Figure S3b). The 2D solute diffusivity anisotropy and MPE coherency (structural anisotropy) measured in the transverse and sagittal planes showed similar trends between the treatment groups (Figure S3c). NP tissue revealed increased ECM remodeling and decreased cellularity following smoke exposure (Figure 4). The CEP tissue showed small calcification spots through the central region of the disc in the smoke exposure group and increased calcification in the smoke cessation group (Figure 4). MPE imaging demonstrated a temporally unique remodeling pattern as well. AF fiber bundles were more closely packed, and the NP region showed some increased remodeling after a period of cessation (Figure 4). The CEP region underwent some calcification, specifically in the peripheral region of the tissue in the cessation control group. It experienced more extensive calcification in the smoke cessation group relative to the smoke exposure group (Figure 4).
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DISCUSSION
The diffusion of fluorescein molecules was quantified in the radial, axial, and circumferential directions of the AF, NP, and CEP regions of the IVD using FRAP (Figure 3A, Table S2). This revealed that smoke exposure resulted in alterations to diffusivity spatially within the IVD. This study found that diffusivities in the AF region of the disc decreased only in the radial direction under smoke exposure conditions. MPE imaging (Figure 4), analysis of AF collagen bundle diameter (Figure S3b), and previous histological observations of fiber disorganization19,23,27,31 suggested that the collagen bundles in the AF region became denser, hindering the transport of nutrients in the radial direction without altering the diffusion in the axial or circumferential directions. As the NP and CEP regions were relatively isotropic (Figure 3C), their average diffusivity was reported. Following smoke exposure, diffusivity in the CEP decreased, while no change was observed in the NP region. MPE imaging revealed aberrant calcification and mineralization in the CEP (Figure 4) which, along with previous histological studies noting ossification of the endplate,29 suggest degenerative structural remodeling which is made evident by the decreased diffusion in the CEP. The regional remodeling pattern, particularly the decreases seen in the diffusivities of the AF (radial direction only) and the CEP due to smoke exposure, indicates that the NP region is somewhat insulated from the effects of two-month smoke exposure. Furthermore, the decreasing trend in porosity and aberrant calcification suggest pronounced structural changes in the CEP region, making it particularly susceptible to smoke exposure.
Diffusivity was also quantified after a period of smoke cessation in each region to examine if cessation reversed the effects of smoke exposure. This study found that, after 5 months of smoke cessation, the radial diffusivity of the AF region and the average diffusivities of the NP, and CEP regions all decreased relative to the cessation controls. MPE imaging (Figure 4), analysis of AF collagen fiber bundle diameter (Figure S3c), porosity (Figure 3D), and histologic bundle disorganization observed by Nemoto et al. suggest that the collagen fiber bundles continue to undergo structural remodeling even after smoke exposure has ceased.19 Additionally, MPE imaging observed fibrotic remodeling and decreased cellularity in the NP and further calcification of the CEP. The smoke cessation results demonstrate diffusivity continues to decrease and that cessation does not reverse the effects of smoke exposure. Porosity underlined apparent remodeling, revealing a decreasing trend in the AF and CEP regions though it was not statistically significant (Figure 3D). Further mechanistic study on the irreversibility of IVD degenerative remodeling under smoke cessation conditions will be critical in future research, given that the number of active smokers in the United States has decreased (from approximately 20% to 10% of the population over the course of 20 years), creating a larger population of smoke cessation patients.1,55
Disc aging effects were examined by comparing the control group (i.e., 8 months old) to the cessation control (i.e., 13 months old). A decrease in the diffusivity of the AF radial component and the CEP average diffusivity in the cessation control group suggests that these regions could experience greater structural alterations with aging compared to the NP. Structurally, the NP seems to be insulated from remodeling, where instead aforementioned patterns of collagen fiber bundle narrowing in the AF and calcification in the CEP are more notable (Figure 4). Interestingly, the diffusivity results were similar in both the aging and smoke-exposed groups insinuating that smoke exposure could have a similar effect to that of aging. MPE imaging, however, revealed different patterns of AF fiber bundle remodeling and CEP calcification (smoke exposure: calcification in the central region of the CEP, aging: calcification in the peripheral region of the CEP, Figure 4), indicating that the mechanisms behind the remodeling may differ between these groups.
Additionally, porosity was measured to provide quantitative structural information on the IVD regions to further support the observed changes in diffusivity. Previously, porosity was shown to be a direct correlate of small solute (molecular weight < 5000 Da) diffusivity.34,52,56,57 Porosity was expected to decrease since water content, which has been correlated with diffusion,40 is known to decrease with disc degeneration,58,59 however, the currently reported porosity was lower than indicated in prior IVD literature.34,49 Where the relationship between porosity and diffusivity may be more accurately described as exponential,50,52 for tissues with a lower porosity, structural features may mediate diffusivity outcomes more than porosity. Although porosity did not differ between tissue regions, it was observed to be lower in the smoke exposure group in the CEP, though not significantly so (Figure 3D). Therefore, the decreases in radial AF and CEP diffusivity in the smoking groups could be driven primarily by structural remodeling of these regions (Figure 4) rather than the porosity.
To our knowledge, this study is the first to quantify the effects of smoke exposure and smoke cessation on the IVD diffusion properties at each disc region in a rat model. The disc, being avascular, relies mainly on diffusion from the vertebral body through the CEP and the soft tissue surrounding the outer AF for small molecule solute transport.60 Interestingly, the diffusivity of the AF radial direction and CEP is significantly lower than the diffusivities of the NP and AF axial and circumferential directions. Any alteration in solute transport, particularly in the radial direction of the AF tissue and the CEP, could significantly disrupt the homeostasis of the extracellular environment, a disruption that would be more pronounced in large animal models and humans. Holms and Nachemson further emphasize that smoking, which induces vasoconstriction, may reduce blood flow and subsequently hinder solute transport between the disc tissue and surrounding vascular network.61 This vasoconstriction could lead to a more harsh extracellular environment, subsequently causing potential cellular protein and mitochondrial damage.24–26 This could have a long-lasting impact during smoke cessation, potentially explaining why we see alterations in the IVD at a structural level.
Previous in vitro models use arbitrary concentrations of nicotine or CSE over short periods to simulate smoking; however, these approaches primarily focus on the direct effects of these chemicals on cell populations without accounting for the systemic impact of smoking. Previous nicotine pump models focus on controlled nicotine levels in the blood over extended periods without accounting for the additional toxic chemicals in cigarette smoke that may affect IVD pathophysiology. Second-hand smoke exposure models addressed this by exposing animals to second-hand cigarette smoke in a manner closer to physiological conditions, but without including the first-hand smoke that would be directly inhaled through the cigarette butt. The smoke exposure apparatus used in this study can expose the animals to both second-hand and first-hand cigarette smoke (Figure S1). This was achieved by pumping smoke from the burning cigarette (second-hand smoke) and the smoke through the cigarette butt (first-hand smoke) into the housing chamber of the animal (Video S1). Smoke exposure was monitored by measuring the total particulate matter on exhaust filters, while dosage was controlled by monitoring the level of serum cotinine (i.e., nicotine metabolite), simulating heavy smoking starting in adulthood (Figure S2).44,45,47 By mimicking a more physiological smoking scenario, this model is suited for examining the spatial–temporal remodeling pattern of the IVD in the context of cigarette smoke exposure. By varying dosage, study duration, or specimen age, the model could be adapted to simulate various smoking scenarios for future mechanistic studies.
This study has several limitations. First, it utilizes a small animal model (i.e., SD rat), which can't fully represent human smokers' IVD pathophysiology since rat IVD structures are much smaller. Large animal models, such as porcine or bovine models, possess IVD structures and transport distances comparable to those in humans but are significantly more costly to maintain and require longer experimental durations. Rats also have a much shorter lifespan and metabolize nicotine faster than humans. Two months of a rat's lifespan equates to 5 human years,45 meaning the rats in this study experienced the equivalent of 5 years of smoke exposure followed by 12.5 years of smoke cessation. Despite simulating a human smoker who has quit, the rats' faster nicotine metabolism limits the translational relevance of this model. As a second limitation, two different motion segments were used to obtain the three component diffusivities through 2D FRAP measurements. While the motion segments were adjacent to one another, variability based on the motion segment level may have affected the results, although no statistical effect of motion segment level was found. Thirdly, while this study measured fluorescein diffusion, which mimics small molecule transport (i.e., glucose and lactate), there are many larger biomolecules, such as proteins and growth factors, which mediate IVD homeostasis through cell signaling. Solute diffusion has been shown to be inversely related to solute size through experimental studies on different biological tissues.33,62,63 However, the role that biochemical composition and structural organization play on diffusion remain to be elucidated,62,63 making large solute diffusivity an area of particular interest for future smoke exposure studies. Fourthly, porosity was measured using IVD tissues from two or three caudal motion segments to ensure sufficient tissue. Therefore, no correlation was analyzed between the porosity and solute diffusivity. Fifthly, this study performed tests on motion segments from different regions of the spine. Lumbar motion segments, except for one disc, have been harvested in a histologic and imaging study (to be published). Thoracic and caudal discs adjacent to the lumbar portion, with similar IVD structure and size, were used for FRAP and porosity measurements, respectively. Finally, it must be noted that the rats used in this study were originally part of a femur fracture healing study,64 potentially interfering with the baseline body physiology of the rats in this study. However, any effect of the fracture healing study would be seen in all animals and would be minimal on IVD degeneration progression.
In conclusion, the SD rat model demonstrates that two-month cigarette smoke exposure significantly deteriorates IVD structure, and alters its composition and solute diffusion properties, particularly in the CEP interface, while smoke cessation fails to reverse these effects. Additionally, it represents an advancement over previous in vitro and in vivo models to simulate physiological smoking scenarios.
AUTHOR CONTRIBUTIONS
Funding Acquisition: Yongren Wu, Hai Yao; Study Conceptualization: Yongren Wu, Hai Yao, Tong Ye; Methodology: Joshua Kelley, Hongming Fan, Glenn Hepfer, Avery Madden; Experimentation: Joshua Kelley, Avery Madden; Data Analysis: Josh Kelley, Nathan Buchweitz, Avery Madden; Writing and Review: Joshua Kelley, Nathan Buchweitz, Yongren Wu, Michael Kern, Danyelle M. Townsend, Hai Yao.
ACKNOWLEDGMENTS
This work was supported by the NIH/NIGMS COBRE: South Carolina Translational Research Improving Musculoskeletal Health (SC TRIMH; P20GM121342), NIH/DCR (R01DE021134), and the Cervical Spine Research Society Seed Starter Grant. We would like to thank Dr. Vincent D. Pellegrini, Jr. and Dr. Russell A. Reeves for their support on the development of the custom smoke exposure room and smoke exposure protocol. Additionally, we would like to thank Ivy Mignone for her assistance with specimen preparation.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
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Abstract
Background
Cigarette smoking is a recognized risk factor for orthopedic disorders, particularly intervertebral disc (IVD) degenerative disease. However, the IVD pathophysiology, especially the spatial–temporal remodeling progression in the context of cigarette smoking, remains unclear. This study aimed to address this knowledge gap through a quantitative assessment of IVD structural composition and diffusion properties using a Sprague–Dawley rat model.
Methods
Twenty‐four rats were divided into control and smoke exposure cohorts, each with two sub‐groups of six rats. One smoke exposure sub‐group was sacrificed after 2 months of daily cigarette smoke exposure in a custom smoking apparatus, while the other was sacrificed after an additional 5 months of smoke cessation. The control groups were age‐matched to the smoke exposure groups. A fluorescent recovery after photobleaching (FRAP) technique was used to determine solute diffusivities and multi‐photon excitation (MPE) imaging was performed to characterize structural changes in the annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP).
Results
A decrease in diffusivity was observed in the CEP and the AF (radial direction only) after 2 months of smoke exposure. MPE imaging showed aberrant CEP calcification and reduced AF radial collagen fiber bundle diameter, suggesting that the IVD exhibits regionally dependent structural remodeling due to smoke exposure. Furthermore, the smoke cessation group showed deteriorating alterations of structure and diffusivities in all three‐disc regions, including the NP, indicating that five‐month smoke cessation alone didn't reverse the progression of IVD degenerative remodeling during aging.
Conclusion
This study advances the understanding of IVD pathophysiology in the context of cigarette smoke exposure and cessation, laying the groundwork for potential earlier diagnosis and optimized interventions.
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Details
1 Department of Bioengineering, Clemson University, Charleston, South Carolina, USA
2 Department of Regenerative Medicine & Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA
3 Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, USA
4 Department of Bioengineering, Clemson University, Charleston, South Carolina, USA, Department of Regenerative Medicine & Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA
5 Department of Bioengineering, Clemson University, Charleston, South Carolina, USA, Department of Oral Health Sciences, Medical University of South Carolina, Charleston, South Carolina, USA
6 Department of Bioengineering, Clemson University, Charleston, South Carolina, USA, Department of Orthopaedics and Physical Medicine, Medical University of South Carolina, Charleston, South Carolina, USA