Trabecular bone score in active or former smokers with and without COPD

Background Smoking is a recognized risk factor for osteoporosis. Trabecular bone score (TBS) is a novel texture parameter to evaluate bone microarchitecture. TBS and their main determinants are unknown in active and former smokers. Objective To assess TBS in a population of active or former smokers with and without Chronic Obstructive Pulmonary Disease (COPD) and to determine its predictive factors. Methods Active and former smokers from a pulmonary clinic were invited to participate. Clinical features were recorded and bone turnover markers (BTMs) measured. Lung function, low dose chest Computed Tomography scans (LDCT), dual energy absorptiometry (DXA) scans were performed and TBS measured. Logistic regression analysis explored the relationship between measured parameters and TBS. Results One hundred and forty five patients were included in the analysis, 97 (67.8%) with COPD. TBS was lower in COPD patients (median 1.323; IQR: 0.13 vs 1.48; IQR: 0.16, p = 0.003). Regression analysis showed that a higher body mass index (BMI), younger age, less number of exacerbations and a higher forced expiratory volume-one second (FEV1%) was associated with better TBS (β = 0.005, 95% CI:0.000–0.011, p = 0.032; β = -0.003, 95% CI:-0.007(-)-0.000, p = 0.008; β = -0.019, 95% CI:-0.034(-)-0.004, p = 0.015; β = 0.001, 95% CI:0.000–0.002, p = 0.012 respectively). The same factors with similar results were found in COPD patients. Conclusions A significant proportion of active and former smokers with and without COPD have an affected TBS. BMI, age, number of exacerbations and the degree of airway obstruction predicts TBS values in smokers with and without COPD. This important information should be considered when evaluating smokers at risk of osteoporosis.


Introduction
Osteoporosis affects an estimated 200 million people, being a major cause of morbidity and mortality worldwide and resulting in approximately 9 million new fractures annually [1]. The agreed international definition of osteoporosis [2] highlights the notion that both low bone mineral density (BMD) and other bone abnormalities such as microarchitectural impairment contribute to skeletal fragility. However, the diagnosis of osteoporosis is based solely on the measurement of BMD by dual energy absorptiometry (DXA) scan [3]. Interestingly, an impaired bone microarchitecture, independent of BMD, is also associated with a greater risk of bone fracture [4]. Therefore, it is recognized that the evaluation of bone microarchitecture might enhance the accuracy of risk fracture assessment.
Lumbar spine trabecular bone score (TBS) was developed as an innovative grey-level texture parameter that can be applied to DXA acquisitions. TBS is calculated from experimental variograms of the 2D projection DXA images, quantifying variation in grey-level texture as a function of distance from one pixel to the others. It is not exactly a measurement of bone microarchitecture, but approximately represents the 3D bone characteristics: trabecular separation, trabecular number, and connectivity density [4,5]. Therefore, higher TBS corresponds to more homogeneously textured bone, representing strong and fracture resistant microarchitecture and vice versa. This novel score does not require further patient tests and is easily calculated in seconds by commercially available software applied prospectively or retrospectively to standard DXA images [6].
Smoking has long been identified as a risk factor for osteoporosis, with studies showing that older smokers have decreased bone mineral density measured by DXA scan and increased fracture risk compared to nonsmokers, particularly at the hip [7]. Moreover, the increase in fracture risk in this population is out of proportion to the alterations seen on bone density; this indicates, perhaps, a deficiency in bone quality [8].
To better understand the potential clinical utility of TBS in smokers, the main objective of this study is to describe TBS and its predictive factors in a population of active and former smokers with and without COPD.

Participants
Study subjects were active and former smokers evaluated at the pulmonary clinic of Clínica Universidad de Navarra between August 2014 and March 2016. Invited patients were men and postmenopausal women, 50 years of age or older, with a smoking history of �10 pack-years. Previous diagnosis of osteoporosis, use of preventive osteoporosis treatment and �6 month between the DXA scan and the blood draw were the exclusion criteria. All subjects signed a consent form previously approved by the Institution's ethics committee (Comite de Ética Clínica Universidad de Navarra, n˚151/2014).
The initial visit included a complete medical history, physical examination, blood samples collection, pulmonary function tests (PFTs), 6 minute walking distance (6MWD), BMD measurement and a low-dose chest computed tomography (LDCT). A questionnaire was administered by a single investigator including age, race, body mass index (BMI), menopause information, personal and family fracture history, tobacco and alcohol intake history, medication use in the past and present (including oral and inhaled corticosteroids) and number of respiratory exacerbations. Respiratory exacerbations were defined as an acute worsening of respiratory symptoms (cough, sputum production, dyspnea, wheezing) which result in the use of additional therapy [9,10], as defined by global initiative for chronic obstructive lung disease (GOLD) [11]. the young normal reference mean BMD. Diagnosis of osteoporosis was based on the World Health Organization (WHO) criteria [15].

Trabecular bone score (TBS)
Lumbar spine TBS was obtained using the DXA images of each patient. TBS was calculated using TBS iNsight software (Version 2.2.0, Med-Imaps, Bordeaux, France) by one of the authors (MR) who was blinded to the clinical data of the participants. Since there is a lack of reference values for TBS in a Mediterranean population like the present one, we used the three categories recommended by the software provider (iNsight): 1) normal microarchitecture: TBS�1.300; 2) partially deteriorated microarchitecture: TBS<1.300 and >1.200 and 3) degraded microarchitecture: TBS�1.200.

Low dose chest CT (LDCT)
LDCT examinations were obtained with the patient in supine position, in cranio-caudal direction and at end-inspiration. Patients were studied with a 64 slice multidetector CT scanner (Somatom Sensation 64, Somatom Definition, Siemens Healthcare, Erlangen, Germany), also at a low-dose setting (120 kV tube voltage, 40 mAs tube current, 64x0.6 mm slice collimation, 0.5 s gantry rotation time, 1.4 pitch, 1 mm slice thickness, 1 mm reconstruction interval).

Assessment of emphysema on LDCT
Emphysema was visually assessed in the chest CT by one reader (JG) for presence, type and severity, using a validated criteria established by the Fleischner Society [16]. Scoring procedures used a five-level semi quantitative scale based on criteria used in the National Emphysema Treatment Trial [17].

Biochemistry
All the samples were drawn in all patients early in the morning of the visit day and in fasting status. Samples were collected into Vacutainer tubes, and aliquots were stored frozen at -80˚C until analysis. Measurements of BTM were performed at the Laboratory of Biochemistry of the Clínica Universidad de Navarra. Details of the measurements have been previously described [18].

Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences version 20.0 (SPSS Inc). Normal distribution was assessed by the Shapiro-Wilks test. Quantitative data are represented as mean ± SD or median (interquartile range), depending on the data distribution; relative frequencies were used for qualitative data. Differences between study groups were evaluated by the Student's t test, Mann-Whitney U test, and χ 2 statistics accordingly. Simple bivariate Pearson correlation coefficients were examined between TBS and other continuous variables (age, BMI, lumbar spine BMD), showing a weak inverse correlation with age (-0.25), a moderate positive correlation with BMI (0.33) and a strong positive correlation with lumbar spine BMD (0.56). Uni and multivariable linear regression analysis with TBS as a dependent variable were performed to study the potential independent association of the studied parameters. The multivariable linear regression analysis included those variables that were statistically significant in the univariable analysis and those clinically soundable. Two multivariable analyses were performed, one in the entire population and another focused on COPD patients. All analysis was adjusted for age, sex, body mass index (BMI) and lumbar spine BMD, based on the correlation analysis.

Results
The study population characteristics are shown in Table 1. This study sample included patients in their 6 th decade with a slight predominance of males (56%) and somewhat overweight (mean 27.2 Kg/m 2 ). Two thirds of the patients met COPD criteria with a mild degree of airway limitation.
Emphysema was visually detected in 56.5% of the population, with centrilobular and paraseptal (47.5%) being the most frequent type, followed by centrilobular alone (44%) and paraseptal alone (8.5%). The majority of the population had low bone mass according to WHO criteria (51%) and 23.4% had osteoporosis. In 13 patients, the BTM levels could not be measured.
Median TBS in our population was 1.334. The distribution of the different categories of TBS levels by the results of DXA scan is shown in Table 2. Importantly, 55 out of 145 (38%) smokers had abnormal TBS values (<1.300) and we also highlight that 8.3% of the patients with normal densitometry values have TBS values in the range of partially deteriorated and degraded microarchitecture. Interestingly, we found that 56 patients (62%) with normal TBS values were diagnosed with osteopenia and osteoporosis using the DXA scan. Table 3 shows the comparison of smokers with and without COPD. COPD patients were significantly older and had a lower BMI. Moreover, there was a higher proportion of active smokers with a higher smoking history. As expected, emphysema was more frequent and more severe in COPD patients. A main finding was that COPD patients had lower TBS (1.3± 0.13 vs 1.4 ±0.16, p = 0.003) compared with smokers without COPD. Comparing BTMs, we only found statistically significant differences in P1NP values (35.2±19.6 vs 41.5±30.6 respectively, p = 0.0045). Table 4 shows the independent association of each studied parameter with TBS. Age, FEV 1 , higher BMI and number of exacerbations were associated with higher TBS values. Table 5 shows this association but only in those with COPD. The same predictors were found in both groups.

Discussion
The most important and novel information of the present study is that it reports for the first time TBS values in a high risk population of active and former smokers with and without COPD. We found lower TBS in COPD patients and, interestingly, that at least 40% of the studied individuals have abnormal (<1.300) TBS values, which the literature reported to be associated with an increased risk of major osteoporotic fracture, independent of BMD values [19,20,21,22,23]. We also found that the best predictors of TBS values were lung function, number of exacerbations, age and BMI. Moreover, TBS values were, as expected, lower in women than in men, but only in smokers without COPD, after adjusting for other relevant factors.
It is well known that one of the most common extra pulmonary manifestations of COPD is osteoporosis [24,25,26]. The present study showed that patients with COPD attending a pulmonary clinic have lower TBS compared with those without. This is in line with a study performed on 61 COPD males, where BMD and TBS were independently associated with 2 or 3 vertebral fractures in these patients [27]. Similarly, in a study that explores the clinical factors associated with trabecular bone score in 29.047 postmenopausal women from Manitoba, COPD was statistically significant in the multiple linear regression analysis [28]. This is also in agreement with previous findings indicating that COPD patients have reduced bone stiffness and failure load estimated by finite elements analysis based on in-vivo high resolution images of the distal radius and tibia [14]. Furthermore, a recent study in postmenopausal women with COPD demonstrated that the microarchitectural deterioration could be evidenced by low trabecular number, connectivity, and thickness, additionally with an increase in trabecular spacing [29]. Therefore, the evaluation reporting reduced TBS values in these COPD patients  could imply that they have decreased bone strength [30], placing them at an increased fracture risk [31,32]. The present work also found that lung function, age and BMI were the most important factors associated with TBS values in this population. Lung function represented by FEV 1 % was one of the most important factors independently associated with TBS values in the entire population and in COPD patients. Previous studies have also demonstrated an association between pulmonary function and bone density or bone architecture in COPD patients [33]. Moreover, Kulak et al. showed that patients with a more severe degree of airway limitation (GOLD spirometric grades 3 and 4) presented a significantly lower bone formation rate when compared with patients with mild to moderate disease (0.028±0.009 versus 0.016±0.011 μm 3 / μm 2 /day, p = .04) (29). Misof et al., also found that heterogeneity of cancellous bone mineralization was significantly lower in the most severe COPD patients (GOLD III and IV) [34]. As also reported in the general population, lower BMI is associated with a lower TBS [35]. A low BMI is usually present in approximately 15-20% of COPD patients [36] and it also plays an important role in osteoporosis and risk fracture [37,38]. COPD patients are predisposed to low body weight due to multiple reasons including malnutrition, depression and hyper-catabolism related to increased energy cost (breathing, tissue hypoxia, use of beta-agonist, and  chronic systemic inflammation) [39,40,41]; therefore, it is not a surprise that the present study results indicate that it is also associated with low TBS. The independent association between exacerbations and TBS values is another finding from the present study. Exacerbations play an important role in COPD patients because they have negative impacts in several aspects, such as pulmonary function [42,43], health status [10], survival [44,45], the BODE index [46] and also socioeconomic cost [47]. This impact also affects and causes deterioration in risk facts related to osteoporosis. In this regard, exacerbations are associated with physical inactivity [46], systemic inflammation [48] and the use of corticosteroids. A longitudinal study of 42 COPD patients demonstrated that patients with a history of exacerbations have a higher decrease in bone mineral density assessed on chest computed tomography compared with those without a history of exacerbations [49]. Interestingly, TBS is abnormal (<1.300) in a significant proportion of the smokers (40%) which did not exactly match with the ones that had abnormal BMD values (Table 1), suggesting the complementary role of this technique in evaluating risk fracture in this high risk population. We found two scenarios that confirmed the complementary information provided by BMD and TBS: 47 (63.5%) patients diagnosed with osteopenia by DXA had normal TBS and 9 (26.5%) osteoporotic patients by DXA scan had normal TBS. These results suggest that these techniques provide different information about bone status, indicating that they are complementary and maybe useful to identify individuals at risk of bone fracture. Further studies should explore the potential complementary role of these techniques to better identify those smokers at high risk for bone fractures.
The present study has limitations. Firstly, this is a highly selected population of smokers from a tertiary academic Pulmonary Clinic; therefore, results should be confirmed in other populations. Secondly, this is a relatively small population lacking a never-smoker control group to complete the entire spectrum of population types, this could have helped us determine the normal values representative of our local population. Thirdly, fortunately these patients are currently under follow up to determine if those with abnormal TBS values and normal bone mineral density will develop bone fractures, enabling us to provide that relevant information.

Conclusions
A significant proportion of active and former smokers with and without COPD have an affected TBS, an easy way to measure skeletal microarchitecture and therefore, a potential predictor of risk fracture. Lung function, age, BMI and number of exacerbations were their main predictors. TBS seems to provide complementary information in the evaluation of risk fracture in this high-risk population. Further studies should validate these important findings.