Low trabecular bone score is associated with high C-reactive protein levels in systemic sclerosis


 Objectives

To evaluate trabecular bone score (TBS) in patients with systemic sclerosis (SSc) and to identify risk factors related to low TBS in SSc.
Methods

TBS and areal bone mineral density (aBMD) were assessed in patients with SSc (n = 57), rheumatoid arthritis (RA) (n = 47), and hand osteoarthritis (OA) (n = 37) using DXA. Osteoporosis risk factors, laboratory findings, SSc-specific organ involvement, and patterns of nailfold capillaroscopy (NFC) were also assessed. Multivariate linear regression analysis was performed to identify the risk factors associated with TBS in SSc patients.
Results

The median TBS (Q1, Q3) value was 1.378 (1.322, 1.425) in SSc patients, 1.336 (1.261, 1.396) for RA patients, and 1.430 (1.387, 1.438) for controls (p < 0.001). No significant differences were observed in the median lumbar spine TBS and aBMD at the lumbar spine, femoral neck, and total hip between the SSc and RA groups. The TBS was negatively correlated with the erythrocyte sedimentation rate (p = 0.042) and C-reactive protein (CRP) (p = 0.005) in the SSc group only and with cumulative glucocorticoid doses in the RA group only (p = 0.031). We found no association between TBS and SSc cutaneous subtype, internal organ involvement, autoantibody profile, NFC patterns, and use of immunosuppressive agents, such as cyclophosphamide. In the multivariate analyses, age, female sex, current, and average CRP were significantly associated with TBS.
Conclusions

TBS assessment revealed poor bone quality in patients with SSc, similar to those with RA. CRP levels were negatively correlated with TBS in patients with SSc, and higher CRP levels were independently associated with low TBS.

This study aimed to evaluate TBS in patients with SSc compared with those with RA and hand osteoarthritis (OA) and to identify risk factors related to low TBS in SSc.

Study population
A single-center cross-sectional study was conducted at the Soonchunhyang University Seoul Hospital (Seoul, South Korea) between July 2019 and December 2020. Patients with SSc, RA, and hand OA were enrolled as controls. The de nitive diagnosis of SSc, RA, and hand OA was made in accordance with the 2013 American College of Rheumatology/European League against Rheumatism (ACR/EULAR) classi cation criteria [13], 2010 ACR/EULAR classi cation criteria [14], and 1990 ACR classi cation criteria [15], respectively. Exclusion criteria included patients with in ammatory rheumatic diseases other than SSc and RA, premenopausal status, thyroid or parathyroid disorders, presence of chronic renal or liver disease, gastrectomy, bariatric surgery, malabsorption syndrome, and chronic obstructive pulmonary disease. The study was approved by the Institutional Review Board (IRB) for Human Research (2020-10-008) at Soonchunhyang University Seoul Hospital. Written consent was obtained from all participants.
TBS and aBMD assessment aBMD was measured at the lumbar spine (L1-L4) and left hip (femoral neck and total proximal femur) using DXA (Hologic Horizon W; Hologic Inc., Danbury, CT, USA). All measurements were taken by experienced operators using the same machine and standardised procedures for participant positioning. TBS was evaluated at the lumbar spine (L1-L4) on the same DEX acquisition used for aBMD assessment using TBS iNsight software (version 3.0; Med-Imaps, Merignac, France). Patients with SSc were divided into three TBS groups according to the risk of fracture in a recent meta-analysis: high risk, TBS < 1.23; intermediate risk, TBS 1.23-1.31; low risk, TBS above 1.31 [16].

Clinical and laboratory evaluation
For each patient, the following data were collected at the time of TBS assessment: demographics, disease duration, de ned as the time elapsed between the onset of the rst disease-related symptoms (except for Raynaud phenomenon in SSc) and enrollment, OP risk factors (smoking status, alcohol consumption, menopausal status, and family history of OP fracture). Laboratory tests were performed to determine the erythrocyte sedimentation rate (ESR) and the levels of C-reactive protein (CRP), 25 hydroxyvitamin D (25(OH)D), and bone turnover markers (bone alkaline phosphatase [ALP], serum alkaline phosphatase, procollagen type 1 intact N-terminal propeptide [P1NP], C-terminal telopeptide of type 1 collagen [CTX], and osteocalcin). CRP serum levels were also documented at every visit from the rst visit to our rheumatology clinic. Patients with SSc were classi ed into three groups according to CRP concentration: in ammatory, intermediate, and non-in ammatory SSc. Patients with SSc displaying elevated CRP levels (≥0.5 mg/dL ) at more than 80% of visits were de ned as in ammatory SSc, while patients with normal CRP levels on all visits were de ned as non-in ammatory SSc [6]. CRP elevations related to infections or medical interventions, as evidenced by a retrospective chart review, were excluded from the analysis. Laboratory ndings, including the presence of serum anti-nuclear antibody, anti-centromere, anti-topoisomerase, and anti-ribonucleoprotein, were also recorded.
Additionally, internal organ involvement in patients with SSc, including interstitial lung disease, gastrointestinal involvement [17], scleroderma renal crisis [18], and pulmonary arterial hypertension, were investigated [19]. The skin thickness score was obtained using the modi ed Rodnan total skin score [20]. Patterns of nailfold capillaroscopy (NFC) were obtained for patients with SSc as previously reported by Cutolo et al.: early, active, and late patterns [21].

Statistical analysis
Statistical analyses were performed using the SPSS software package (SPSS Inc., Chicago, IL, USA) for Windows v. 22.0. Differences among three groups were analysed using the Kruskal-Wallis test with Bonferroni post-hoc test. Data were compared using the chi-square test and Mann-Whitney U-test, as appropriate. The Spearman rank correlation test was used to assess the relationship between the TBS and continuous variables. Partial correlation analyses, adjusted for age, sex, and cumulative GC dose, were performed to assess the relationship between TBS and CRP levels. Generalised estimating equations (GEE) models were used to determine the independent variables associated with TBS. All items that were associated with TBS by univariate analysis with p-values less than 0.05 were included in the multivariate model. In the multivariate analysis, separate models for current CRP and average CRP were performed due to multicollinearity. The results were considered statistically signi cant at p<0.05.

Results
Baseline characteristics of the study population A total of 141 patients were included in the study: SSc (n = 57), RA (n = 47), and hand OA (n = 37). Table 1 summarises the baseline characteristics of the study population. The current GC doses were comparable between patients with SSc and those with RA. Cumulative doses of GC were signi cantly lower in patients with SSc than in those with RA (adjusted p<0.001). The use of BP was higher in the RA group than in the SSc and OA groups (p<0.001). The median (Q1, Q3) duration of BP use in the RA group was 1.9 (0.8, 4.15).  Table 2). No signi cant differences were observed in the median lumbar spine TBS and aBMD at the lumbar spine, femoral neck, and total hip between the SSc and RA groups.

Assessment of TBS and BMD
Correlation between clinical parameters, in ammatory markers, aBMD, and TBS In all groups, the TBS showed a signi cantly negative correlation with age and a positive correlation with the aBMD of the femoral neck and total hip ( Table 3). The TBS was negatively correlated with ESR (current and average) and current CRP in the SSc group only and with cumulative GC doses in the RA group only. The average CRP was negatively correlated with TBS in both the SSc and RA groups. However, partial correlation analysis for TBS and average CRP levels, adjusted for age, sex, and cumulative GC doses, showed a negative correlation in the SSc group (r=-0.316, p=0.020) and non-signi cant correlation in the RA group (r=-0.197, p=0.200). In both SSc and RA groups, no signi cant correlation between CRP (average and current) and aBMD (femoral neck, total hip, lumbar spine) was found.
Characteristics of patients with SSc strati ed according to TBS fracture risk groups Table 4 shows the clinical and laboratory characteristics of the TBS subgroups in patients with SSc according to the risk of fracture: high risk (n=3), intermediate risk (n=8), and low risk (n=46). Patients with intermediate to high risk of fracture were older and had higher cumulative GC doses for the last three months than those in the low-risk groups (p=0.014 and p=0.046, respectively). According to the CRP status, in ammatory and intermediate SSc were more prevalent in the intermediate-risk to high-risk TBS group than in the low-risk TBS group (27.3% and 63.6% vs. 2.2% and 28.3%, respectively) (p=0.003). About 70% of patients in the low-risk TBS group had non-in ammatory SSc.
The use of BP and supplemental vitamin D was more frequent in the intermediate-risk to high-risk group than in the low-risk group. There were no signi cant differences among the TBS subgroups of patients in terms of fracture risk with respect to smoking status, alcohol consumption, disease duration, autoantibody pro les, internal organ involvement, mRSS, and the use of immunosuppressive agents. When the three categories of NFC patterns were compared for the patients with SSc, no signi cant association was observed between NFC patterns and TBS, aBMD, and laboratory ndings (see Additional e 1).
Linear regression analysis for TBS among patients with SSc Table 5 shows the results of the univariate and multivariate analyses of TBS in patients with SSc. Univariate analysis revealed that TBS was associated with age, female sex, ESR, CRP, average ESR, average CRP, and alcohol consumption. In the multivariate analyses, age, female sex, CRP, and average CRP were signi cantly associated with TBS.

Discussion
In the present study, we investigated the bone microarchitecture based on TBS and risk factors affecting poor bone quality in patients with SSc. Decreased TBS and BMD values were observed in patients with SSc and RA compared to those with hand OA.
In ammatory markers, such as CRP and ESR, were negatively correlated with TBS in patients with SSc, but not in those with RA. Age, female sex, current, and average CRP were independent risk factors associated with low TBS.
Chronic in ammatory diseases are also associated with OP. Bone loss is related to increased osteoclast activity induced by proin ammatory cytokines, such as interleukin 6 (IL-6), IL-1, and tumour necrosis factor [22]. CRP is a general in ammatory marker produced by hepatocytes upon stimulation by IL-6. An inverse association between CRP levels and BMD has also been reported in healthy women [23]. In previous SSc studies, an association between elevated CRP levels and disease activity, mRSS, ILD progression, and high mortality has been demonstrated [6,24,25]. We found a positive correlation of CRP (current and average) with TBS, and higher CRP was independently associated with low bone quality in patients with SSc. Interestingly, CRP level was not associated with aBMD in patients with SSc. These results show the direct interaction between in ammation and bone microarchitecture in patients with SSc. Surveillance of OP, including bone quality, is needed to reduce OP complications, especially in patients with SSc with increased CRP levels.
Although the RA group had signi cantly older age and higher cumulative GC doses than the SSc group, the TBS did not differ between the two groups. Cumulative GC doses were marginally higher in patients with SSc with intermediate to high TBS risk scores than in those with low-risk scores. However, a correlation between cumulative GC dose and TBS was observed in the RA group, but not in the SSc group. Although GCs may alleviate bone resorption related to in ammation, GCs are known risk factors for bone loss [26]. Due to the higher surface area and rate of bone turnover, GCs affect the trabecular bone compartment more than the cortical bone compartment [27]. Koumakis et al. showed that daily GC doses were independently associated with low TBS in patients with SSc. Koumakis et al. reported that the mean cumulative GC doses and disease duration of patients with SSc were 8630 mg and 10.2 years, respectively [11]. In contrast, the median cumulative GC doses and disease duration in our study were 885 mg and 2.9 years, respectively. A much lower cumulative GC dose and shorter disease duration could have affected our observation that GC had little impact on bone quality in patients with SSc. In line with this, Koumakis et al. reported much lower absolute TBS values compared to our study. This could also be explained by the large difference in the cumulative GC doses. The proportion of BP users, vitamin D supplementation, and postmenopausal women were similar between the two studies [11].
We found no association of TBS with SSc cutaneous subtype and internal organ involvement pattern, which was consistent with a previously reported study [11]. According to the NFC pattern, we observed no signi cant differences in patients with SSc. Ruaro et al. reported that patients with SSc with late pattern had lower TBS than those with early and active patterns [12]. Ruaro et al. reported that 25(OH)D levels were signi cantly lower in patients with SSc with a late pattern. However, the three SSc groups according to the NFC pattern did not show a difference in 25(OH)D levels in our study. Ruaro et al. excluded patients with SSc on drug regimens that could in uence bone turnover, but we included patients with SSc undergoing OP treatment such as BP, calcium, and vitamin D. These distinct study designs could lead to differences in the results between the two studies.
CYC administration is related to premature ovarian failure, and an animal study has shown the suppressive effects of CYC on osteoblastogenesis [5,28], which leads to concerns about the development of OP in patients with SSc. One study showed that CYC use was associated with an increased risk of femoral neck fractures in patients with systemic lupus erythematosus (SLE) [29]. However, a cross-sectional study on a large SLE cohort failed to show an association between immunosuppression and BMD [30]. We could not nd an association between immunosuppressive agents and TBS in patients with SSc. Among the CYC users in our SSc population (n = 17), only 5 patients were premenopausal at the time of CYC administration, 8 patients were postmenopausal, and 4 patients were male. Postmenopausal status at CYC administration or reduction in the in ammatory response of CYC could affect the results of our study [31]. Further large studies are needed to reveal the relationship between CYC treatment and the development of OP in patients with SSc.
Our study had several limitations. First, this study was a cross-sectional observational design, and we could not determine a causal relationship between CRP and TBS in patients with SSc. Second, due to the relatively small number of patients with SSc with intermediate-to high-risk TBS scores, the regression analysis could be underpowered. Third, there was a lack of data on the history of non-vertebral fractures. Finally, we included patients with SSc who were taking OP medication. Patients with SSc with intermediate to high TBS risk used more BP and supplemental vitamin D, re ecting those already diagnosed with OP. Further studies on the relationship between TBS and CRP are needed after excluding the OP drug in uence in patients with SSc.

Conclusion
TBS assessment revealed poor bone quality in the lumbar spine in patients with SSc, similar to that in patients with RA. CRP levels were negatively correlated with TBS in patients with SSc, and higher CRP levels were independently associated with low TBS. These ndings suggest that in ammation plays a role in the decreased bone quality of patients with SSc.

Declarations Ethics approval and consent to participate
The study was approved by the Institutional Review Board (IRB) for Human Research (2020-10-008) at Soonchunhyang University Seoul Hospital.

Consent for publication
Written consent was obtained from all participants.

Availability of data and materials
The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by research funding from the Korean Society for Bone and Mineral Research and Soonchunhyang University.
Authors' contributions KAL, HJK, and HSK were involved in study conception and design. KAL, JSK, and WHC were involved in data acquisition. KAL and HSK performed data analysis and interpretation. All authors were involved in the drafting or critical revision of the article, and all authors approved the nal version for publication. HSK had full access to all of the data in the study and takes

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