Many epidemiological surveys have identified smoking as a risk factor for osteoporosis, but it is unclear whether smoking has a direct effect on bone metabolism and if such an effect could cause osteoporosis. Therefore, we examined whether smoking causes osteoporosis based on the impact of smoke exposure on the bones of rats. A rat model of passive cigarette smoking was prepared by breeding rats in a cigarette-smoking box for 4 or 8 weeks. Histological changes, micro-computed tomographic (CT) analysis, mechanical bone strength, and bone mineral density of the femur and lumbar vertebrae were examined in these rats and in control rats that were not exposed to smoke. Lower mechanical bone strength was observed in smoke-exposed rats, but these differences were not significant. Significantly lower bone mineral density was found in the femur (P<.01) and lumbar bones (P<.001) of 8-week smoke-exposed rats compared to controls. In a micro-CT scan of lumbar vertebrae, the bone volume, trabecular thickness, trabecular number, and trabecular separation differed significantly between smoke-exposed rats and controls. Histologically, the osteocytes in the smoke-exposed rats were small (approximately 25% of the size in controls), and decreased numbers of marrow cells and osteoblasts (P<.01), as well as a black carbon dust-like substance, were found in the bone of smoke-exposed rats. These results indicate that smoking significantly decreases bone mineral density, which causes osteoporosis, and the organizational changes in the bone suggest a direct effect of smoking on bone structure. Fewer marrow cells were present in the smoke-exposed rats, and a black carbon dust-like substance was observed.
Many epidemiological surveys have found that smoking is a risk factor for osteoporosis.1-6 The absolute risk of fracture was determined in a meta-analysis of data from a World Health Organization global cohort, and current smoking was selected as one of the risk factors.7 However, it is unclear whether smoking has a direct effect on bone metabolism and whether such an effect may cause osteoporosis. The mechanisms through which tobacco impairs bone health are not well understood, but may involve direct effects on osteoblasts8,9 and may be mediated by changes in calcium absorption,10,11 estrogen metabolism,12 and bone vascularization.13,14
Nicotine, the principal pharmacological agent in cigarettes, has been the focus of several studies evaluating the relationship between specific cigarette components and bone. However, the impact of nicotine on bone remains controversial, with some studies showing adverse skeletal effects14-17 and others finding no effects.18-21 Cigarette smoke exposure has been found to be more detrimental to bone and implant interfaces compared to nicotine treatment alone,22 which suggests that cigarette smoke constituents other than nicotine might be responsible for the negative impact of smoking on bone.
This study examined the impact of smoking on bone structure in a rat model of passive cigarette smoking to examine the mechanism of association of smoking with osteoporosis.
Materials and Methods
Twenty 8-week-old male Wistar King A rats were fed a standard solid diet under a light-dark cycle of 12 hours. Five rats each were bred in an environment with or without smoking for 4 or 8 weeks to prepare smoke-exposed and control animals. An acrylic resin chamber (60×30×30 cm) and automatic smoking equipment (smoking box) that could send cigarette smoke into the chamber were developed for preparation of the passive smoking rat model (Figure 1).23-25 Using this equipment, the time, amount, and interval of smoke to be sent into the chamber was defined, and 20 cigarettes could be set at a time (Engineering System Co, Nagano, Japan).
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Figure 1: The smoking box was an acrylic resin chamber fitted with automatic passive smoking equipment used to ignite cigarettes automatically at a designated time under computer control and send smoke into the box (A). Twenty cigarettes could be set at one time (B).
Untipped cigarettes (Short Peace; JT, Tokyo, Japan) were used. Cigarette smoke was sent for 5 minutes and then the box was ventilated with room air for 5 minutes. This procedure was repeated 20 times at 1-hour intervals such that the rats passively smoked 20 cigarettes per day. Continuous measurement of oxygen partial pressure (MT Technologies, Tokyo, Japan) was used to maintain an oxygen concentration of 20% (150 mm Hg) in the box.
The impact of passive smoking was evaluated by measuring blood nicotine concentrations. In a preliminary study, passive smoking of 20 cigarettes was found to give a blood nicotine concentration in rats exceeding that of heavy smokers (110 ng/mL),23-26 and therefore passive smoking of 20 cigarettes per day was used in the main study. All rats received humane care in accordance with the Guide for Animal Experimentation and Handling of Laboratory Animals of Nihon University School of Medicine.
The rats were sacrificed by intraperitoneal administration of sodium pentobarbital after 4 or 8 weeks, and the lumbar spine and both femurs were removed. After soft tissues were removed from the samples, the right femur and fifth lumbar vertebra were frozen at -20°C for storage. Subsequently, the mechanical bone strength of the femur was measured in a 3-point bending test to determine the maximum force required for fracture. The distance between the points and the bending speed were set at 13.00 mm and 10 mm/min, respectively. For the lumbar spine, a compression test was performed in which pressure was provided at a rate of 2 mm/min to measure the maximum force required for fracture (AG-2000E; Shimadzu Corp, Kyoto, Japan).
The left femur and the sixth lumbar vertebra were fixed with 70% ethanol to measure bone mineral density using dual energy x-ray absorptiometry (DEXA) (DCS-600; Aloka Co, Tokyo, Japan).
Micro-CT (ELE SCAN; Nittetsu Elex Co, Tokyo, Japan) was performed on the left femur and sixth lumbar vertebra after measurement of bone mineral density to examine changes in bone trabeculae. The bone volume, trabecular thickness, trabecular number, and trabecular separation were measured using TRY-BONE image analysis software (RATOC System Engineering, Tokyo, Japan).
A non-decalcified sample of the sixth lumbar (fixed with ethanol once daily for 7 days) was prepared. After embedding in methylmethacrylate resin and slicing of samples to 5 µm thickness in the midsagittal plane, hematoxylin-eosin and Villanueva-Goldner staining were performed. Changes in osteocytes, marrow cells, and osteoblasts in the growth plate cartilage and secondary cancellous bone obtained after removing the first layer of cancellous bone were examined by optical microscopy. ImageJ 1.37 (National Institutes of Health, Bethesda, Maryland) was used for image analysis.
All data are expressed as mean±SD. A Student t test was performed for comparison of average values between the 2 groups. SPSS 11.0 (SPSS, Inc, Chicago, Illinois) was used for statistical analysis with P<.05 considered to indicate a significant difference.
The blood nicotine concentrations in the 4- and 8-week smoke-exposed rats were 278.4±129.8 ng/mL (range, 115-421 ng/mL) and 329.4±90.3 ng/mL (range, 273-490 ng/mL), respectively. These concentrations in controls were 7.6±2.5 ng/mL (range, 5-10 ng/mL) and 6±1.7 ng/mL (range, 5-9 ng/mL), respectively. The blood nicotine concentrations in the smoke-exposed rats were significantly higher than those in controls. The mean blood nicotine concentration for 10 smokers who smoked 30 to 40 cigarettes daily (0.5 mg of nicotine per cigarette) was 110 ng/mL, indicating that the average blood nicotine concentration in the smoke-exposed rats was two- to threefold greater than the average for heavy smokers.
The femur mechanical bone strengths in the 4- and 8-week smoke-exposed rats were 256.4±19.0 N and 232.8±37.4 N, respectively, and those in the controls were 268.8±13.9 N and 252.1±25.7 N, respectively. The smoke-exposed animals had lower values at both time points, but no significant difference was noted between the groups at each time point (Figure 2A). The lumbar mechanical bone strengths in the 4- and 8-week smoke-exposed rats were 210.6±35.0 N and 251.2±74.3 N, respectively, and those in the controls were 264.8±30.1 N and 295.5±27.4 N, respectively. Similarly to the femur, lower values were observed in smoke-exposed animals, but again without a significant difference between the groups (Figure 2B).
Figure 2: To assess bone strength, a 3-point bending test was performed for the femur (A) and a compression test was performed for lumbar vertebrae (B). There was no significant difference in the results of the tests in A and B, but the 4- and 8-week smoke-exposed rats had low bone strengths for the femur and lumbar vertebrae.
The femur bone mineral densities in the 4- and 8-week smoke-exposed rats were 213.2±12.2 mg/cm2 and 191.4±5.4 mg/cm2, respectively, and those in the controls were 217.7±8.5 mg/cm2 and 212.8±7.2 mg/cm2, respectively. There was no significant difference between the bone mineral density at 4 weeks, but the bone mineral density in the smoke-exposed rats was significantly lower than that in controls at 8 weeks (P<.01) (Figure 3A). The lumbar spine bone mineral densities in the 4- and 8-week smoke-exposed rats were 156.6±4.4 mg/cm2 and 163.8±5.6 mg/cm2, respectively, and those in the controls were 167.3±15 mg/cm2 and 201.5±7.9 mg/cm2, respectively. Similarly to the femur, these data did not differ significantly at 4 weeks, but the bone mineral density in the lumbar spine was significantly lower in the smoke-exposed rats at 8 weeks (P<.001) (Figure 3B).
Figure 3: Clear decreases in femur (A) and lumbar (B) bone mineral densities were observed in 8-week smoke-exposed rats.
Reduced trabecular thickness, decreased trabecular number, and expanded trabecular separation were found in the lumbar spines of smoke-exposed rats. No significant differences were noted in the femur between smoke-exposed and control animals. A reduction of bone volume was found in the femurs and lumbar spines of smoke-exposed rats (Figure 4; Table).
Fewer osteoid were present in smoke-exposed rats, showing that the decrease in bone mass and fragility were not caused by a calcification disorder and suggesting histological characteristics of osteoporosis. The size of osteocytes in the smoke-exposed animals was approximately one-fourth that in the control group (Figure 5). Fewer marrow cells were present in the smoke-exposed rats, and a black carbon dust-like substance was observed (Figure 6), but this could not be identified due to difficulty with extraction of the substance. The number of osteoblasts in the secondary cancellous bone was significantly lower in smoke-exposed rats compared to controls (40.8±14.6 vs 261±11.8; P<.01) (Figure 6).
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Figure 5: Histological changes in osteocytes subjected to Villanueva-Goldner staining (×40). The size of osteocytes in smoke-exposed rats (B) was smaller than that in controls (A).
Our results show that the smoke-exposed rats developed osteoporosis based on a clear decrease in femur and lumbar bone mineral densities after 8 weeks of exposure. Small osteocytes, fewer osteoblasts and marrow cells, and a black carbon dust-like substance were found in the bones of smoke-exposed rats. These histological findings show that smoking has a direct impact on bone metabolism. The small osteocytes may have arisen from a lack of interconnected osteoblasts since the osteoblast count was low, and the absence of an intramedullary environment may have been due to necrosis of marrow cells. Since cancellous bone is highly sensitive to changes in blood supply and metabolism, smoking is likely to have a large impact on this bone if smoking affects bone metabolism.
The mechanisms through which cigarettes impair bone health have previously been suggested to involve direct effects on bone vascularization.13,14 A close relationship between passive smoking and Legg-Calvé-Perthes disease has also been suggested,27 since nicotine may further decrease the low blood flow to the femoral head. It has also been shown that the basivertebral blood flow decreases following intravenous administration of nicotine in a dog model.28 These data suggest that decreased blood flow to bone may have an impact on bone metabolism.
We did not examine bone blood flow in this study, but the small osteocytes and decreased counts of osteoblasts and marrow cells may reflect the decrease in blood flow. However, it is not certain that the decreased blood flow to bone is the cause of osteoporosis in the smoke-exposed rats, since nicotine may also have direct adverse effects on osteoblasts through dose-dependent suppression of cellular proliferation of osteoblast-like cells29 and inhibition of formation of osteoblasts.8,9 We also cannot explain the presence of the black carbon dust-like substance based on a decrease in blood flow to bone. This substance and phagocytotic processes may be important, and identification of the dust-like substance is required in a future study. However, the histologic results show a direct impact of smoking on bone, and to our knowledge this is the first in vivo study to show a foreign substance (black carbon dust) in marrow in a smoking model.
The blood nicotine concentrations of the smoke-exposed rats were significantly higher than those of the controls. The average blood nicotine concentration in smoke-exposed rats was two- to threefold greater than that in heavy smokers. Previous studies of nicotine administration in rats have shown no effect on biomechanical properties at low doses.18-21 Among the range of nicotine doses (4.5-9 mg/kg daily) used previously, only the highest dose (9 mg/kg daily) had limited harmful effects on vertebral bone mineral content.30 The blood nicotine concentration for this dose was approximately 230 ng/mL. Thus, the average blood nicotine concentration in smoke-exposed rats in the present study was 1.2- to 1.4-fold greater than the highest dose used in previous studies.
One difficulty in interpreting smoking studies is that cigarette smoke is composed of >4000 different substances31 that are present as gases or particles. The major gaseous components of cigarette smoke include carbon monoxide, carbon dioxide, sulfur dioxide, cresol, and amphenol, whereas the particles include nicotine, tar, and water. In the present study, the smoke-exposed rat model was evaluated based on the serum nicotine level. However, the rats were exposed to whole cigarette smoke in this study and not just to nicotine alone. Furthermore, the nicotine blood level fluctuates and there is considerable variability in the pattern of metabolism among individuals. The blood nicotine concentrations varied from 278.4±129.8 ng/mL to 329.4±90.3 ng/mL, which may reflect variability in the nicotine assay32 and differences in inhalation among individuals.
Passive smoking does not involve direct inhalation of cigarette smoke. We have also examined the effect of smoking on intervertebral disks using this method and found significant changes in gene expression during intervertebral disk degeneration induced by cigarette smoke inhalation.23-25 Therefore, we believe that our model is suitable for assessment of the effects of passive smoking.
Mechanical bone strength was unaffected in the smoke-exposed rats, but an effect on bone mineral density was observed. This suggests that the mechanical test may not adequately reflect changes in cancellous bone. The smoke-exposed rats showed a clear decrease in femur and lumbar bone mineral densities at 8 weeks, but no effect at 4 weeks, which may be due to differences in exposure time to blood nicotine. The blood nicotine concentration in the smoke-exposed rats was two- to threefold greater than that in heavy smokers, and this may have a harmful effect on bone.
The blood nicotine concentration did not differ significantly at 4 and 8 weeks, but there may be a harmful effect of longer smoke exposure for 8 weeks. Longer-term nicotine administration has previously been proposed to result in indirect cellular effects in bone as secondary responses to other systemic nicotine effects.20 We could have shown a threshold dose if we examined an intermediate time point. It will be of interest to perform this analysis in a future study.
Analysis of the fine structure of the bone using micro-CT showed a significant difference in trabecular number, trabecular separation, and trabecular thickness in the lumbar bone. In the femurs of smoke-exposed animals, there was a tendency for a decrease in trabecular number and thickness and a tendency for expansion of the trabecular separation, but there were no significant differences with controls. Bone volume reduction was found in both the femurs and lumbar spines of smoke-exposed rats. This shows that the overall trabecular bone in the smoke-exposed group is weaker, although micro-CT detected no defects in other parameters of bone structure.
Evaluation of the fine structure of cancellous bone by micro-CT is limited. In contrast to DEXA, an osteoid with low calcification is not distinguished from bone structure in micro-CT. This makes micro-CT unsuitable in osteoid-rich samples,33 but very little osteoid volume was apparent histologically in this study. Therefore, it is likely that the osteoid content had little effect on measurements made by micro-CT.
The model used in the present study reflects the effects of passive cigarette smoking, but not those of active smoking. Difficulties exist in drawing a direct comparison to smoking in humans, since humans inhale tobacco directly from cigarettes. Humans are active smokers, and we cannot comment on the effects of active smoking using our model. The results of this study may also have been influenced by the concentration of nicotine in the rats, which was two- to threefold greater than that in heavy smokers. The nicotine concentration of the control group was low but was not zero, since controls may have been exposed to some secondhand smoke in the animal facility. This model was evaluated based only on the serum nicotine level. We cannot evaluate other inhaled chemicals that could contribute. The difference in species also makes it difficult to extrapolate data from a rat model to humans. Within these limitations, our results suggest that smoking may have a direct impact on bone structure and may cause osteoporosis.
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Drs Ajiro, Tokuhashi, Matsuzaki, Nakajima, and Ogawa are from the Department of Orthopedic Surgery, Nihon University School of Medicine, Tokyo, Japan.
Drs Ajiro, Tokuhashi, Matsuzaki, Nakajima, and Ogawa have no relevant financial relationships to disclose.
Correspondence should be addressed to: Yasumitsu Ajiro, MD, 090-8941-6588, 30-1 Ooyaguchi Kamicho, Itabashi Ku, Tokyo, 173-861, Japan.