Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages

INTRODUCTION: Certain amino acids within proteins have been reported to change from the L form to the D form over time. This process is known as racemization and is most likely to occur in long-lived low-turnover tissues such as normal cartilage. We hypothesized that diseased tissue, as found in an osteoarthritic (OA) joint, would have increased turnover reflected by a decrease in the racemized amino acid content. METHODS: Using high-performance liquid chromatography methods, we quantified the L and D forms of amino acids reported to racemize in vivo on a biological timescale: alanine, aspartate (Asp), asparagine (Asn), glutamate, glutamine, isoleucine, leucine (Leu), and serine (Ser). Furthermore, using a metabolically inactive control material (tooth dentin) and a control material with normal metabolism (normal articular cartilage), we developed an age adjustment in order to make inferences about the state of protein turnover in cartilage and meniscus. RESULTS: In the metabolically inactive control material (n = 25, ages 13 to 80 years) and the normal metabolizing control material (n = 19, ages 17 to 83 years), only Asp + Asn (Asx), Ser, and Leu showed a significant change (increase) in racemization with age (P < 0.01). The age-adjusted proportions of racemized to total amino acid (D/D+L expressed as a percentage of the control material) for Asx, Ser, and Leu when compared with the normal articular cartilage control were 97%, 74%, and 73% in OA meniscal cartilage and 97%, 70%, and 78% in OA articular cartilage. We also observed lower amino acid content in OA articular and meniscal cartilages compared with normal articular cartilage as well as a loss of total amino acids with age in the OA meniscal but not the OA articular cartilage. CONCLUSIONS: These data demonstrate comparable anabolic responses for non-lesioned OA articular cartilage and OA meniscal cartilage but an excess of catabolism over anabolism for the meniscal cartilage

Post-translational aging of proteins in osteoarthritic cartilage and synovial fluid as measured by isomerized aspartate

INTRODUCTION: Aging proteins undergo non-enzymatic post-translational modification, including isomerization and racemization. We hypothesized that cartilage with many long-lived components could accumulate non-enzymatically modified amino acids in the form of isomerized aspartate and that its liberation due to osteoarthritis (OA)-related cartilage degradation could reflect OA severity. METHODS: Articular cartilage and synovial fluid were obtained from 14 randomly selected total knee arthroplasty cases (56 to 79 years old) and non-arthritis cartilage from 8 trauma cases (51 to 83 years old). Paired lesional cartilage and non-lesioned OA cartilage were graded histologically using a modified Mankin system. Paired cartilage and synovial fluids were assayed for isomerized aspartate, phosphate-buffered saline/EDTA (ethylenediaminetetraacetic acid) extractable glycosaminoglycans, and total protein. Macroscopically normal non-lesioned OA cartilage was separated into superficial and deep regions when cartilage thickness was at least 3 mm (n = 6). RESULTS: Normalized to cartilage wet weight, normal cartilage and deep non-lesioned OA cartilage contained significantly (P < 0.05) more isomerized aspartate than superficial non-lesioned OA cartilage and lesioned cartilage. Synovial fluid isomerized aspartate correlated positively (R2 = 0.53, P = 0.02) and glycosaminoglycans correlated negatively (R2 = 0.42, P = 0.04) with histological OA lesion severity. Neither synovial fluid isomerized aspartate nor glycosaminoglycans nor total protein correlated with histological scores of non-lesioned areas. CONCLUSIONS: We show for the first time that human cartilage and synovial fluid contain measurable quantities of an isomerized amino acid and that synovial fluid concentrations of isomerized aspartate reflected severity of histological OA. Further assessment is warranted to identify the cartilage proteins containing this modification and to assess the functional consequences and biomarker applications of this analyte in OA

A cohort study of 4,190 patients treated with low-intensity pulsed ultrasound (LIPUS): findings in the elderly versus all patients

BACKGROUND: Patient age is one of many potential risk factors for fracture nonunion. Our hypothesis is that older patients (≥ 60) with fracture risk factors treated with low-intensity pulsed ultrasound (LIPUS) have similar heal rate (HR) to the population as a whole. We evaluate the impact of age in conjunction with other risk factors on HR in LIPUS-treated patients with fresh fracture (≤ 90 days old). METHODS: The Exogen Bone Healing System is a LIPUS device approved in 1994 to accelerate healing of fresh fracture. After approval, the FDA required a Post-Market Registry to assess performance. Patient data collected from October 1994 until October 1998 were individually reviewed and validated by a registered nurse. Four distinct data elements were required to report a patient: date fracture occurred; date treatment began; date treatment ended; and a dichotomous outcome of healed v. failed, by clinical and radiological criteria. Data were used to calculate two derived variables; days to treatment (DTT) and days on treatment (DOT). Every validated fresh fracture patient with DTT, DOT, and outcome is reported. RESULTS: The validated registry had 5,765 patients with fresh fracture; 73% (N = 4,190) are reported, while 13% of patients were lost to follow-up, 11% withdrew or were non-compliant, and 3% died or are missing outcome. Among treatment-compliant patients, HR was 96.2%. Logistic estimates of the odds ratio for healing are equivalent for patients age 30 to 79 years and all age cohorts had a HR > 94%. Open fracture, current smoking, diabetes, vascular insufficiency, osteoporosis, cancer, rheumatoid arthritis, and prescription NSAIDs all reduced HR, but older patients (≥ 60) had similar HRs to the population as a whole. DTT was significantly shorter for patients who healed (p < 0.0001). CONCLUSIONS: Comorbid conditions in conjunction with aging can reduce fracture HR. Patients with fracture who used LIPUS had a 96% HR, whereas the expected HR averages 93%. Time to treatment was significantly shorter among patients who healed (p < 0.0001), suggesting that it is beneficial to begin LIPUS treatment early. Older patients (≥ 60) with fracture risk factors treated with LIPUS exhibit similar heal rates to the population as a whole

Risk factors for nonunion following surgically managed, traumatic, diaphyseal fractures:a systematic review and meta-analysis

BACKGROUND: There are several studies on nonunion, but there are no systematic overviews of the current evidence of risk factors for nonunion. The aim of this study was to systematically review risk factors for nonunion following surgically managed, traumatic, diaphyseal fractures. METHODS: Medline, Embase, Scopus, and Cochrane were searched using a search string developed with aid from a scientific librarian. The studies were screened independently by two authors using Covidence. We solely included studies with at least ten nonunions. Eligible study data were extracted, and the studies were critically appraised. We performed random-effects meta-analyses for those risk factors included in five or more studies. PROSPERO registration number: CRD42021235213. RESULTS: Of 11,738 records screened, 30 were eligible, and these included 38,465 patients. Twenty-five studies were eligible for meta-analyses. Nonunion was associated with smoking (odds ratio (OR): 1.7, 95% CI: 1.2–2.4), open fractures (OR: 2.6, 95% CI: 1.8–3.9), diabetes (OR: 1.6, 95% CI: 1.3–2.0), infection (OR: 7.0, 95% CI: 3.2–15.0), obesity (OR: 1.5, 95% CI: 1.1–1.9), increasing Gustilo classification (OR: 2.2, 95% CI: 1.4–3.7), and AO classification (OR: 2.4, 95% CI: 1.5–3.7). The studies were generally assessed to be of poor quality, mainly because of the possible risk of bias due to confounding, unclear outcome measurements, and missing data. CONCLUSION: Establishing compelling evidence is challenging because the current studies are observational and at risk of bias. We conclude that several risk factors are associated with nonunion following surgically managed, traumatic, diaphyseal fractures and should be included as confounders in future studies

Epidemiology of Fracture Nonunion in 18 Human Bones

Importance Failure of bone fracture healing occurs in 5% to 10% of all patients. Nonunion risk is associated with the severity of injury and with the surgical treatment technique, yet progression to nonunion is not fully explained by these risk factors. Objective To test a hypothesis that fracture characteristics and patient-related risk factors assessable by the clinician at patient presentation can indicate the probability of fracture nonunion. Design, Setting, and Participants An inception cohort study in a large payer database of patients with fracture in the United States was conducted using patient-level health claims for medical and drug expenses compiled for approximately 90.1 million patients in calendar year 2011.The final database collated demographic descriptors, treatment procedures as per Current Procedural Terminology codes; comorbidities as per International Classification of Diseases, Ninth Revision codes; and drug prescriptions as per National Drug Code Directory codes. Logistic regression was used to calculate odds ratios (ORs) for variables associated with nonunion. Data analysis was performed from January 1, 2011, to December 31, 2012, Exposures Continuous enrollment in the database was required for 12 months after fracture to allow sufficient time to capture a nonunion diagnosis. Results The final analysis of 309 330 fractures in 18 bones included 178 952 women (57.9%); mean (SD) age was 44.48 (13.68) years. The nonunion rate was 4.9%. Elevated nonunion risk was associated with severe fracture (eg, open fracture, multiple fractures), high body mass index, smoking, and alcoholism. Women experienced more fractures, but men were more prone to nonunion. The nonunion rate also varied with fracture location: scaphoid, tibia plus fibula, and femur were most likely to be nonunion. The ORs for nonunion fractures were significantly increased for risk factors, including number of fractures (OR, 2.65; 95% CI, 2.34-2.99), use of nonsteroidal anti-inflammatory drugs plus opioids (OR, 1.84; 95% CI, 1.73-1.95), operative treatment (OR, 1.78; 95% CI, 1.69-1.86), open fracture (OR, 1.66; 95% CI, 1.55-1.77), anticoagulant use (OR, 1.58; 95% CI, 1.51-1.66), osteoarthritis with rheumatoid arthritis (OR, 1.58; 95% CI, 1.38-1.82), anticonvulsant use with benzodiazepines (OR, 1.49; 95% CI, 1.36-1.62), opioid use (OR, 1.43; 95% CI, 1.34-1.52), diabetes (OR, 1.40; 95% CI, 1.21-1.61), high-energy injury (OR, 1.38; 95% CI, 1.27-1.49), anticonvulsant use (OR, 1.37; 95% CI, 1.31-1.43), osteoporosis (OR, 1.24; 95% CI, 1.14-1.34), male gender (OR, 1.21; 95% CI, 1.16-1.25), insulin use (OR, 1.21; 95% CI, 1.10-1.31), smoking (OR, 1.20; 95% CI, 1.14-1.26), benzodiazepine use (OR, 1.20; 95% CI, 1.10-1.31), obesity (OR, 1.19; 95% CI, 1.12-1.25), antibiotic use (OR, 1.17; 95% CI, 1.13-1.21), osteoporosis medication use (OR, 1.17; 95% CI, 1.08-1.26), vitamin D deficiency (OR, 1.14; 95% CI, 1.05-1.22), diuretic use (OR, 1.13; 95% CI, 1.07-1.18), and renal insufficiency (OR, 1.11; 95% CI, 1.04-1.17) (multivariate P < .001 for all). Conclusions and Relevance The probability of fracture nonunion can be based on patient-specific risk factors at presentation. Risk of nonunion is a function of fracture severity, fracture location, disease comorbidity, and medication use

Total Hip Arthroplasty or Hemiarthroplasty for Hip Fracture.

BACKGROUND: Globally, hip fractures are among the top 10 causes of disability in adults. For displaced femoral neck fractures, there remains uncertainty regarding the effect of a total hip arthroplasty as compared with hemiarthroplasty. METHODS: We randomly assigned 1495 patients who were 50 years of age or older and had a displaced femoral neck fracture to undergo either total hip arthroplasty or hemiarthroplasty. All enrolled patients had been able to ambulate without the assistance of another person before the fracture occurred. The trial was conducted in 80 centers in 10 countries. The primary end point was a secondary hip procedure within 24 months of follow-up. Secondary end points included death, serious adverse events, hip-related complications, health-related quality of life, function, and overall health end points. RESULTS: The primary end point occurred in 57 of 718 patients (7.9%) who were randomly assigned to total hip arthroplasty and 60 of 723 patients (8.3%) who were randomly assigned to hemiarthroplasty (hazard ratio, 0.95; 95% confidence interval [CI], 0.64 to 1.40; P = 0.79). Hip instability or dislocation occurred in 34 patients (4.7%) assigned to total hip arthroplasty and 17 patients (2.4%) assigned to hemiarthroplasty (hazard ratio, 2.00; 99% CI, 0.97 to 4.09). Function, as measured with the total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) total score, pain score, stiffness score, and function score, modestly favored total hip arthroplasty over hemiarthroplasty. Mortality was similar in the two treatment groups (14.3% among the patients assigned to total hip arthroplasty and 13.1% among those assigned to hemiarthroplasty, P = 0.48). Serious adverse events occurred in 300 patients (41.8%) assigned to total hip arthroplasty and in 265 patients (36.7%) assigned to hemiarthroplasty. CONCLUSIONS: Among independently ambulating patients with displaced femoral neck fractures, the incidence of secondary procedures did not differ significantly between patients who were randomly assigned to undergo total hip arthroplasty and those who were assigned to undergo hemiarthroplasty, and total hip arthroplasty provided a clinically unimportant improvement over hemiarthroplasty in function and quality of life over 24 months. (Funded by the Canadian Institutes of Health Research and others; ClinicalTrials.gov number, NCT00556842.)

Protein Modification by Deamidation Indicates Variations in Joint Extracellular Matrix Turnover

As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage

Interobserver reliability of classification and characterization of proximal humeral fractures: a comparison of two and three-dimensional CT

Interobserver reliability for the classification of proximal humeral fractures is limited. The aim of this study was to test the null hypothesis that interobserver reliability of the AO classification of proximal humeral fractures, the preferred treatment, and fracture characteristics is the same for two-dimensional (2-D) and three-dimensional (3-D) computed tomography (CT). Members of the Science of Variation Group--fully trained practicing orthopaedic and trauma surgeons from around the world--were randomized to evaluate radiographs and either 2-D CT or 3-D CT images of fifteen proximal humeral fractures via a web-based survey and respond to the following four questions: (1) Is the greater tuberosity displaced? (2) Is the humeral head split? (3) Is the arterial supply compromised? (4) Is the glenohumeral joint dislocated? They also classified the fracture according to the AO system and indicated their preferred treatment of the fracture (operative or nonoperative). Agreement among observers was assessed with use of the multirater kappa (&kappa;) measure. Interobserver reliability of the AO classification, fracture characteristics, and preferred treatment generally ranged from &quot;slight&quot; to &quot;fair.&quot; A few small but statistically significant differences were found. Observers randomized to the 2-D CT group had slightly but significantly better agreement on displacement of the greater tuberosity (&kappa; = 0.35 compared with 0.30, p &lt; 0.001) and on the AO classification (&kappa; = 0.18 compared with 0.17, p = 0.018). A subgroup analysis of the AO classification results revealed that shoulder and elbow surgeons, orthopaedic trauma surgeons, and surgeons in the United States had slightly greater reliability on 2-D CT, whereas surgeons in practice for ten years or less and surgeons from other subspecialties had slightly greater reliability on 3-D CT. Proximal humeral fracture classifications may be helpful conceptually, but they have poor interobserver reliability even when 3-D rather than 2-D CT is utilized. This may contribute to the similarly poor interobserver reliability that was observed for selection of the treatment for proximal humeral fractures. The lack of a reliable classification confounds efforts to compare the outcomes of treatment methods among different clinical trials and reports