Rheumatoid arthritis is associated with higher 90-day systemic complications compared to osteoarthritis after total shoulder arthroplasty: a cohort study

Article information

Clin Shoulder Elb. 2024;27(3):353-360
Publication date (electronic) : 2024 August 9
doi : https://doi.org/10.5397/cise.2024.00374
1Division of Shoulder and Elbow Surgery, Rothman Orthopaedic Institute, Philadelphia, PA, USA
2Department of Orthopaedic Surgery, Southern California Permanente Medical Group, Panorama City, CA, USA
3Palm Beach Orthopaedic Institute, Palm Beach Gardens, FL, USA
Corresponding Author: Peter Boufadel Division of Shoulder and Elbow Surgery, Rothman Orthopaedic Institute, 925 Chestnut St, Philadelphia, PA 19107, USA Tel: +1-2676664716, E-mail: paboufadel@gmail.com
Received 2024 May 7; Revised 2024 June 24; Accepted 2024 July 7.

Abstract

Background

Total shoulder arthroplasty (TSA) in patients with rheumatoid arthritis (RA) can present unique challenges. The aim of this study was to compare both systemic and joint-related postoperative complications in patients undergoing primary TSA with RA versus those with primary osteoarthritis (OA).

Methods

Using the TriNetX database, Current Procedural Terminology and International Classification of Diseases, 10th edition codes were used to identify patients who underwent primary TSA. Patients were categorized into two cohorts: RA and OA. After 1:1 propensity score matching, postoperative systemic complications within 90 days following primary TSA and joint-related complications within 5 years following anatomic TSA (aTSA) and reverse shoulder arthroplasty (RSA) were compared.

Results

After propensity score matching, the RA and OA cohorts each consisted of 8,523 patients. Within 90 days postoperation, RA patients had a significantly higher risk of total complications, deep surgical site infection, wound dehiscence, pneumonia, myocardial infarction, acute renal failure, urinary tract infection, mortality, and readmission compared to the OA cohort. RA patients had a significantly greater risk of periprosthetic joint infection and prosthetic dislocation within 5 years following aTSA and RSA, and a greater risk of scapular fractures following RSA. Among RA patients, RSA had a significantly higher risk of prosthetic dislocation, scapular fractures, and revision compared to aTSA.

Conclusions

Following TSA, RA patients should be considered at higher risk of systemic and joint-related complications compared to patients with primary OA. Knowledge of the risk profile of RA patients undergoing TSA is essential for appropriate patient counseling and education.

Level of evidence

III.

INTRODUCTION

Rheumatoid arthritis (RA) is a systemic autoimmune condition characterized by widespread joint inflammation, synovitis, and other systemic symptoms [1,2]. While medical management of RA has improved recently with the development of disease-modifying anti-rheumatic drugs (DMARDs), uncontrolled RA remains relevant to shoulder surgeons due to its potential to lead to erosive arthritis of the glenohumeral joint [3]. In addition, the use of corticosteroids in the treatment of RA may lead to avascular necrosis of the humeral head, necessitating treatment [3]. The treatment options for RA patients experiencing RA-related glenohumeral arthritis are largely limited to either anatomic total shoulder arthroplasty (aTSA) or reverse shoulder arthroplasty (RSA). However, performing TSA in patients with RA entails a greater complication profile, such as a heightened risk for infection from immunosuppressive medications [4,5], compromised rotator cuff integrity [1,6], and impaired bone quality from the systemic inflammation and corticosteroid use, making these patients more susceptible to acromial stress fractures [7,8], intraoperative fractures [7], and the sequelae of poor implant fixation [1].

Despite these challenges, a multicenter study by Lévigne et al. [9] demonstrated that RSA is a durable and effective treatment option for RA-related destruction of the glenohumeral joint, with a survivorship of 96% at 7-year follow-up. Conversely, in a systematic review of 279 shoulders, Haleem et al. [1] showed an expectedly lower revision-free survivorship of aTSA in RA patients, 87% at less than 7 years of follow-up. While numerous studies have reported on the outcomes of shoulder arthroplasty in patients with RA, there is a paucity of large-cohort studies that assess the risks of RA in comparison to primary osteoarthritis (OA). Understanding the comprehensive risk profile of RA patients is important for informed decision-making when considering surgical intervention. As such, the aim of this study was to compare both systemic and joint-related postoperative complications in patients undergoing primary TSA with RA versus those with primary OA.

METHODS

Study Design and Data Source

The TriNetX research database was retrospectively queried on January 20, 2024, for patients who underwent primary TSA from 2008 to 2024. TriNetX is a global health collaborative research platform that includes deidentified electronic health record data from 81 healthcare organizations and more than 170 million patients within the research network [10]. Data available include patient demographics, medications, lab values, diagnoses, and procedures. This study was exempt from institutional review board approval due to the use of solely deidentified patient information. TriNetX is a HIPAA-compliant national database that does not collect patient-identifying information. We did not interact directly with any of the patients included in this study, and thus informed consent could not be obtained.

Cohort Selection and Outcomes

Patients who underwent primary TSA, including both aTSA and RSA, were identified using Current Procedural Terminology code 23472 and International Classification of Diseases, 10th edition (ICD-10) codes Z96.611 and Z96.612. The initial pool of patients was divided into two cohorts using ICD-10 codes: RA and OA. The RA cohort consisted of patients who underwent primary TSA with a prior diagnosis of RA (M05 and M06). The OA cohort consisted of patients who underwent primary TSA with a diagnosis of primary OA of the shoulder (M19.012, M19.011), excluding RA (M05 and M06). The primary outcomes assessed were major systemic complications within 90 days following primary TSA, including total complications, wound dehiscence, superficial surgical site infection (SSI), deep SSI, deep vein thrombosis, pulmonary embolism, myocardial infarction (MI), pneumonia, acute renal failure, urinary tract infection, readmission, stroke, death, and blood transfusions within 72 hours postoperatively.

A secondary analysis was performed to assess postoperative joint complications between RA and OA patients within 5 years following aTSA and RSA, respectively. Patients who underwent RSA were identified using ICD-10 codes specific to reverse prosthetic total arthroplasty: 0RRK00Z and 0RRJ00Z and SNOMED codes 733591007 and 733592000. RSA patients were divided into two cohorts, RA-RSA and OA-RSA, and outcomes assessed were scapular fracture, periprosthetic fracture, prosthetic dislocation, periprosthetic joint infection (PJI), and revision shoulder arthroplasty. Patients who underwent aTSA were identified using ICD-10 codes Z96.61 and Z96.612 and excluding ICD-10 codes K00Z and 0RRJ00Z and SNOMED codes 733591007 and 733592000. aTSA patients were divided into two cohorts, RA-aTSA and OA-aTSA, and outcomes assessed were periprosthetic fracture, prosthetic dislocation, PJI, and revision shoulder arthroplasty. Additionally, postoperative joint complications of aTSA and RSA in RA patients were compared.

Patients were excluded during the analysis if a studied outcome was present prior to surgery. Propensity (1:1) matching was employed to account for age, sex, race, essential (primary) hypertension, diabetes mellitus, obesity, heart failure, chronic obstructive pulmonary disease (COPD), cerebrovascular disease, chronic kidney disease (CKD), liver disease, and tobacco use for all analyses assessing outcomes between RA and OA patients.

Statistical Analysis

The TriNetX platform incorporates analytical software that enables cohort selection, propensity score matching, and advanced data exploration and analysis. All statistical analyses were conducted through the TriNetX platform. Univariate analysis was performed using two-tailed Student t-tests for continuous variables and chi-square tests for categorical variables. Continuous variables were expressed as mean and standard deviation, and categorical variables were expressed as percentage. Statistical significance was determined at P<0.05.

RESULTS

Demographics

Prior to propensity score matching, the RA cohort consisted of 8,588 patients, and the OA cohort consisted of 54,976 patients. Significant differences between the two cohorts were noted for age, sex, race, essential hypertension, diabetes mellitus, overweight and obesity, heart failure, COPD, cerebrovascular disease, CKD, liver disease, and tobacco use (P<0.05).

Following 1:1 propensity score matching, our analysis included 8,523 patients in each cohort. After matching, there were no significant differences in patient demographics or comorbid characteristics (P>0.05), except for American Indian or Alaska Native race (P=0.032). In the RA cohort, the mean age was 68.9±10.3 years, and 70.9% were female. In the OA cohort, the mean age was 69.2±9.9 years, and 71.5% were female. In both cohorts, most patients, 70.9% and 71.5%, respectively, self-identified as white (Table 1). Of the 8,523 RA patients, 5,308 (62.3%) were rheumatoid factor- and/or anti-CCP antibody-positive. In the secondary analysis, postoperative joint complications were compared between RA and OA patients who underwent aTSA and RSA, respectively. Following 1 to 1 propensity score matching, the analysis included 5,720 patients in each of the RA-aTSA and OA-aTSA cohorts and 2,332 patients in each of the RA-RSA and OA-RSA cohorts.

Patient demographics and comorbid characteristics of non-matched and matched RA-TSA and OA-TSA patients

Postoperative Systemic Complications

Within 90 days following TSA, patients with RA had a significantly increased risk of total complications (P<0.001; odds ratio [OR], 1.50; 95% confidence interval [CI], 1.21–1.85), wound dehiscence (P=0.001; OR, 2.92; 95% CI, 1.47–5.79), deep SSI (P=0.024; OR, 2.30; 95% CI, 1.10–4.84), MI (P=0.005; OR, 1.82; 95% CI, 1.19–2.17), pneumonia (P=0.004; OR, 1.60; 95% CI, 1.16–2.20), acute renal failure (P<0.001; OR, 1.63; 95% CI, 1.24–2.15), urinary tract infection (UTI; P=0.003; OR, 1.61; 95% CI, 1.17–2.23), readmission (P=0.012; OR, 1.37; 95% CI, 1.07–1.76), and mortality (P<0.001; OR, 2.00; 95% CI, 1.45–2.75) compared with OA patients. Patients in the RA cohort also had a significantly increased risk of a blood transfusion within 72 hours following TSA (P=0.048; OR, 1.52; 95% CI, 1.00–2.30) compared to patients in the OA cohort (Table 2).

Ninety-day postoperative systemic complications for matched RA and OA patients

Postoperative Joint Complications

Within 5 years following aTSA, patients with RA had a significantly higher risk of prosthetic dislocation (P=0.001; OR, 1.58; 95% CI, 1.22–2.05) and PJI (P=0.004; OR, 1.69; 95% CI, 1.18–2.44) compared with OA patients (Table 3). Within 5 years following RSA, RA patients had a significantly higher risk of scapular fracture (P<0.001; OR, 2.15; 95% CI, 1.54–2.99), prosthetic dislocation (P<0.001; OR, 1.78; 95% CI, 1.32–2.45), and PJI (P=0.003; OR, 1.89; 95% CI, 1.23–2.92) compared with OA patients (Table 4). There were no significant differences in the risks of periprosthetic fracture and revision TSA between RA and OA patients following aTSA or RSA.

Five-year postoperative joint complications for matched RA-aTSA and OA-aTSA

Five-year postoperative joint complications for matched RA-RSA and OA-RSA

When comparing RSA and aTSA outcomes in RA patients, those who underwent RSA had a significantly higher risk of prosthetic dislocation (P<0.001; OR, 2.15; 95% CI, 1.55–2.97), PJI (P<0.001; OR, 2.56; 95% CI, 1.59–4.12), scapular fracture (P<0.001; OR, 2.52; 95% CI, 1.73–3.66), and revision (P<0.001; OR, 2.24; 95% CI, 1.60–3.12) compared to those who underwent aTSA (Table 5).

Five-year postoperative joint complications for matched RA-RSA and RA-aTSA

DISCUSSION

Although several studies have reported on the outcomes of shoulder arthroplasty in patients with RA, there is a paucity of large-cohort studies evaluating the risks relative to patients with primary OA. In this study, we compared both systemic and joint-related complications following TSA in 1 to 1 matched cohorts of patients with RA and OA using a large, up-to-date national database. Within 90 days following TSA, RA patients had significantly higher risk of total complications, wound dehiscence, deep SSI, MI, pneumonia, acute renal failure, UTI, readmission, and mortality. Additionally, RA patients exhibited an increased likelihood of a blood transfusion within 72 hours following surgery. Furthermore, within 5 years following aTSA or RSA, RA patients showed a significantly higher risk of prosthetic dislocation and PJI, and a significantly higher risk of scapular fractures was observed in patients treated with RSA.

Our study found a greater incidence of systemic complications in RA patients compared to OA patients in the first 90 days following TSA. As a systemic autoimmune disease, RA causes damage not only to joints, but also to other tissues and organs [11]. The incidence of cardiovascular disease in RA patients ranges from 30% to 60%, and such disease is the most common cause of death in this population [11,12]. Our study found RA patients to have a two-fold greater incidence of MI and mortality within 90 days after surgery compared to OA patients. This contrasts reported outcomes in the total hip/total knee arthroplasty (THA/TKA) literature, in which no significant difference in MI and mortality rates was found between RA and OA patients [13,14]. In addition, a significantly higher incidence of pneumonia was found in the RA cohort. Respiratory complications can occur in 30%–40% of RA patients and is the second leading cause of death [11,12].

Significantly greater postoperative rates of wound complications including dehiscence, deep SSIs, and readmissions were found in RA patients, which coincides with outcomes reported in the THA/TKA literature [13-17]. The greater rate of aseptic wound complications exhibited in the RA cohort may be attributed to the abundant presence of tumor necrosis factor-alpha, which can inhibit granulation tissue formation and disrupt wound healing [18]. Patients with RA were more likely to receive a blood transfusion within 72 hours postoperatively. Anemia is a common comorbidity in patients with RA, prevalent in 33%–60% of patients, and may explain the greater incidence of blood transfusions in RA patients [19]. Though some cases may result from iron deficiency caused by gastrointestinal bleeding secondary to non-steroidal anti-inflammatory drugs use, the anemia observed in RA patients is more likely a consequence of chronic inflammation and increased production of proinflammatory cytokines [19].

Furthermore, RA patients had a significantly higher risk of PJI and prosthetic dislocation within 5 years following surgery for aTSA or RSA. While superficial SSIs were similar in the two cohorts, RA patients had significantly higher rates of deep SSIs and PJIs postoperatively. Nezwek et al. [20] conducted a retrospective review of 902 RSA patients to explore risk factors for infection within 3 months postoperatively. In their study, patients with RA had a 3.5 times greater risk of developing a PJI compared to non-RA patients [20]. Patients with RA may be susceptible to infections due to immunosuppressive drugs, such as steroids and DMARDs. Therefore, physicians should maintain a heightened index of suspicion of postoperative infection in RA patients who present with persistent unexplained shoulder pain postoperatively [20]. The chronic inflammatory effects of RA may compromise the soft tissue around the shoulder as well, which may predispose patients to higher rates of prosthetic dislocation. Within 5 years following RSA, RA patients demonstrated a more than two-fold greater incidence of scapular fractures. This outcome aligns with previous studies that have demonstrated RA to be a strong predictor of postoperative acromial and scapular spine stress fractures [8,21-25]. RA can cause significant bone loss and reduced bone strength, resulting in a higher risk of fracture [26]. However, no significant difference in the incidence of periprosthetic fractures was found. With increased postoperative joint complications in RA patients, the revision rate was slightly increased compared to OA patients following RSA, but this did not reach statistical significance (4.06% vs. 3.12%, P=0.093).

Reverse and anatomic TSA are both indicated in the setting of end-stage glenohumeral joint arthritis secondary to RA. While RSA has become increasingly favored over aTSA in the treatment of RA patients due to concerns over the integrity of the rotator cuff and managing glenoid bone loss, in our study, the RA-aTSA cohort (5,720 patients) was larger than the RA-RSA cohort size (2,332 patients). Nevertheless, both surgical options are effective in restoring function and reducing pain [1,25,27]. Garcia et al. [27] performed a retrospective study comparing clinical outcomes and complications of aTSA and RSA in 86 patients with inflammatory arthritis, with a mean follow-up of 51.6 months. The authors showed that both aTSA and RSA significantly improved patient-reported outcome measures and range of motion; although, aTSA exhibited significantly greater improvement and final postoperative outcome scores than RSA [27]. While the authors demonstrated no significant difference in complication rates between aTSA and RSA in RA patients [27], our study found that patients who underwent RSA had significantly higher risk of prosthetic dislocation, PJI, and scapular fracture. Additionally, we found that patients who underwent RSA had a higher revision rate compared to those who underwent aTSA. However, this contrasts with findings of a systematic review of 279 shoulders by Lévigne et al. [9] showing RSA to be a durable and effective option with a revision-free survivorship of 96% at a 7-year follow-up, which was greater than the 87% revision-free survivorship reported for aTSA [1].

Our study had several limitations. First, the data were sourced from electronic medical record databases of healthcare organizations, originally intended for clinical practice rather than research. Additionally, the dataset was limited to electronic medical records and lacked chart review data. Hence, certain essential health information may have been underreported, and inaccurate diagnoses and coding of clinical events may have occurred. The use of ICD codes may not consistently classify all relevant diagnoses, procedures, or adverse events, which may have affected the cohorts created and the outcome variables studied. In particular, we were unable to control for rotator cuff health in patients undergoing TSA for primary OA. Despite these limitations, this study is the first to assess and compare postoperative complications after TSA in patients with RA to those in patients with OA.

CONCLUSIONS

Following TSA, RA patients should be considered at a higher risk compared to patients with primary OA. RA is associated with significantly higher systemic complication rates within 90 days, including wound dehiscence, deep SSI, MI, pneumonia, acute renal failure, urinary tract infection, blood transfusion, readmission, and mortality. Patients with RA also have an elevated 5-year risk of PJIs and prosthetic dislocations following aTSA and RSA, as well as scapula fractures following RSA in comparison to patients with OA. Knowledge of the comprehensive risk profile of RA patients undergoing TSA is essential for appropriate patient counseling and education. Further studies are necessary to enhance our understanding of the increased risks associated with RA and to explore postoperative management following shoulder arthroplasty in this higher-risk patient population.

Notes

Author contributions

Conceptualization: PB, MYF. Data curation: PB, JL, RL, MD. Formal analysis: PB, JL. Investigation: PB, JL, RL, MYF, AZK, BWH, JAA. Methodology: PB, RL, MYF, MD, AZK, BWH, JAA. Resources: JL. Software: JL. Supervision: AZK, BWH, JAA. Validation: PB, MYF. Writing – original draft: PB, RL. Writing – review & editing: PB, MYF, MD, AZK, BWH, JAA.

Conflict of interest

AZK would like to disclose to receive support for education from Arthrex, Medical Device Business Services, and Elite Orthopedics and hospitality payments from Stryker and Exactech. JAA would like to disclose royalties from: DJO Global, Zimmer-Biomet, Smith and Nephew, Stryker, Globus Medical Inc. Research support from: Lima Corporation - Italy, Orthofix, Arthrex, OREF. Royalties, financial or material support from: Wolters Kluwer. Board member/committee appointments for: American Shoulder and Elbow Society, Pacira.

Funding

None.

Data availability

Contact the corresponding author for data availability.

Acknowledgements

None.

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Article information Continued

Table 1.

Patient demographics and comorbid characteristics of non-matched and matched RA-TSA and OA-TSA patients

Characteristic Non-matched patient
Matched patient
RA OA P-value RA OA P-value
Total 8,588 54,976 8,523 8,523
Patient demographics
 Age (mean±SD, yr) 68.9±10.3 68.5±10.0 <0.001 68.9±10.3 69.2±9.9 0.058
  Female sex (%) 70.9 49.5 <0.001 70.9 71.5 0.379
 Race (%)
  White 78.0 79.9 <0.001 78.0 79.1 0.090
  Black or African American 8.8 6.8 <0.001 8.8 8.4 0.543
  Hispanic or Latino 4.3 3.0 <0.001 4.3 3.8 0.094
  American Indian or Alaska Native 0.4 0.3 0.327 0.4 0.2 0.032*
  Other race 1.4 1.6 0.346 1.4 1.1 0.056
  Unknown race 10.3 10.5 0.734 10.3 9.6 0.119
Comorbidity (%)
  Essential hypertension 68.5 59.1 <0.001 68.5 69.4 0.196
  Diabetes mellitus 26.0 21.5 <0.001 26.0 26.1 0.889
  Overweight and obesity 34.0 28.4 <0.001 33.9 33.7 0.734
  Heart failure 15.0 8.4 <0.001 15.0 14.6 0.463
  Chronic obstructive pulmonary disease 17.4 9.7 <0.001 17.4 17.1 0.656
  Cerebrovascular disease 16.4 11.0 <0.001 16.4 16.0 0.467
  Chronic kidney disease 17.8 10.5 <0.001 17.8 17.8 0.984
  Liver disease 13.0 9.1 <0.001 13.0 12.8 0.648
  Tobacco use 4.1 3.8 0.176 4.1 3.8 0.307

RA: rheumatoid arthritis, TSA: total shoulder arthroplasty, OA: primary osteoarthritis, SD: standard deviation.

*

Significant P-value <0.05.

Table 2.

Ninety-day postoperative systemic complications for matched RA and OA patients

Outcome Incidence (%)
OR 95% CI P-value
RA OA
Total complications 3.61 2.45 1.50 1.21–1.85 0.000*
Wound dehiscence 0.38 0.13 2.92 1.47–5.79 0.001*
Superficial SSI 0.33 0.23 1.40 0.79–2.45 0.248
Deep SSI 0.27 0.12 2.30 1.10–4.84 0.024*
Deep vein thrombosis 0.63 0.41 1.54 0.99–2.40 0.052
Pulmonary embolism 0.50 0.56 0.90 0.59–1.38 0.632
Myocardial infarction 0.74 0.41 1.82 1.19–2.79 0.005
Pneumonia 1.30 0.82 1.60 1.16–2.20 0.004*
Acute renal failure 1.81 1.11 1.63 1.24–2.15 0.000*
Urinary tract infection 1.46 0.91 1.61 1.17–2.23 0.003*
Readmission 1.76 1.29 1.37 1.07–1.76 0.012
Stroke 0.38 0.31 1.21 0.71–2.06 0.485
Mortality 1.32 0.67 2.00 1.45–2.75 0.000*
Blood transfusion <72 hours 0.66 0.43 1.52 1.00–2.30 0.048*

RA: rheumatoid arthritis, OA: primary osteoarthritis, OR: odds ratio, CI: confidence interval, SSI: surgical site infection.

*

Significant P-value <0.05.

Table 3.

Five-year postoperative joint complications for matched RA-aTSA and OA-aTSA

Outcome Incidence (%)
OR 95% CI P-value
RA-aTSA OA-aTSA
Periprosthetic fracture 1.09 0.86 1.27 0.87–1.85 0.212
Prosthetic dislocation 2.65 1.69 1.58 1.22–2.05 0.001*
Periprosthetic joint infection 1.39 0.83 1.69 1.18–2.44 0.004*
Revision TSA 2.16 2.25 0.96 0.75–1.23 0.749

TSA: total shoulder arthroplasty, RA-aTSA: anatomic TSA patients with rheumatoid arthritis, OA-aTSA: anatomic TSA patients with primary osteoarthritis, OR: odds ratio, CI: confidence interval.

*

Significant P-value <0.05.

Table 4.

Five-year postoperative joint complications for matched RA-RSA and OA-RSA

Outcome Incidence (%)
OR 95% CI P-value
RA-RSA OA-RSA
Periprosthetic fracture 1.86 2.20 0.84 0.56–1.27 0.409
Prosthetic dislocation 5.13 2.92 1.78 1.32–2.45 0.000*
Periprosthetic joint infection 2.61 1.39 1.89 1.23–2.92 0.003*
Revision TSA 4.06 3.12 1.32 0.96–1.81 0.093
Scapular fracture 4.92 2.35 2.15 1.54–2.99 0.000*

RSA: reverse shoulder arthroplasty, RA-RSA: RSA patients with rheumatoid arthritis, OA-RSA: RSA patients with primary osteoarthritis, OR: odds ratio, CI: confidence interval, TSA: total shoulder arthroplasty.

*

Significant P-value <0.05.

Table 5.

Five-year postoperative joint complications for matched RA-RSA and RA-aTSA

Outcome Incidence (%)
OR 95% CI P-value
RA-RSA RA-aTSA
Periprosthetic fracture 1.95 1.27 1.55 0.97–2.46 0.063
Prosthetic dislocation 5.14 2.46 2.15 1.55–2.97 <0.001*
Periprosthetic joint infection 2.61 1.04 2.56 1.59–4.12 <0.001*
Revision 4.18 1.70 2.52 1.73–3.66 <0.001*
Scapular fracture 4.88 2.24 2.24 1.60–3.12 <0.001*

RA-RSA: reverse shoulder arthroplasty patients with rheumatoid arthritis, RA-aTSA: anatomic total shoulder arthroplasty patients with rheumatoid arthritis, OR: odds ratio, CI: confidence interval.

*

Significant P-value <0.05.