Association of Serum Biochemical Biomarker Profiles of Joint Tissue Inflammation and Cartilage Metabolism With Posttraumatic Osteoarthritis-Related Symptoms at 12 Months After ACLR.

BACKGROUND: Anterior cruciate ligament injury and anterior cruciate ligament reconstruction (ACLR) are risk factors for symptomatic posttraumatic osteoarthritis (PTOA). After ACLR, individuals demonstrate altered joint tissue metabolism indicative of increased inflammation and cartilage breakdown. Serum biomarker changes have been associated with tibiofemoral cartilage composition indicative of worse knee joint health but not with PTOA-related symptoms. PURPOSE/HYPOTHESIS: The purpose of this study was to determine associations between changes in serum biomarker profiles from the preoperative sample collection to 6 months after ACLR and clinically relevant knee PTOA symptoms at 12 months after ACLR. It was hypothesized that increases in biomarkers of inflammation, cartilage metabolism, and cartilage degradation would be associated with clinically relevant PTOA symptoms after ACLR. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Individuals undergoing primary ACLR were included (N = 30). Serum samples collected preoperatively and 6 months after ACLR were processed to measure markers indicative of changes in inflammation (ie, monocyte chemoattract protein 1 [MCP-1]) and cartilage breakdown (ie, cartilage oligomeric matrix protein [COMP], matrix metalloproteinase 3, ratio of type II collagen breakdown to type II collagen synthesis). Knee injury and Osteoarthritis Outcome Score surveys were completed at 12 months after ACLR and used to identify participants with and without clinically relevant PTOA-related symptoms. K-means cluster analyses were used to determine serum biomarker profiles. One-way analyses of variance and logistic regressions were used to assess differences in Knee injury and Osteoarthritis Outcome Score subscale scores and clinically relevant PTOA-related symptoms between biomarker profiles. RESULTS: Two profiles were identified and characterized based on decreases (profile 1: 67% female; age, 21.4 ± 5.1 years; body mass index, 24.4 ± 2.4) and increases (profile 2: 33% female; age, 21.3 ± 3.2 years; body mass index, 23.4 ± 2.6) in sMCP-1 and sCOMP preoperatively to 6 months after ACLR. Participants with profile 2 did not demonstrate differences in knee pain, symptoms, activities of daily living, sports function, or quality of life at 12 months after ACLR compared to those with profile 1 (P = .56-.81; η2 = 0.002-0.012). No statistically significant associations were noted between biomarker profiles and clinically relevant PTOA-related symptoms (odds ratio, 1.30; 95% CI, 0.23-6.33). CONCLUSION: Serum biomarker changes in MCP-1 and sCOMP within the first 6 months after ACLR were not associated with clinically relevant PTOA-related symptoms.

Relationship between vitamin D status and clinical outcomes in dogs with a cranial cruciate ligament rupture.

Cranial cruciate ligament rupture (CCLR) is one of the most common orthopaedic disorders diagnosed in dogs yet the factors which influence postoperative clinical outcomes are poorly understood. Low vitamin D status has been linked to poorer clinical outcomes in human patients undergoing elective orthopaedic surgery. The aim of this study was to examine the relationship between pre-operative vitamin D status, as defined by serum 25 hydroxyvitamin D (25(OH)D) concentrations, and initial disease severity and clinical outcomes in dogs undergoing surgical treatment for a CCLR. Serum 25(OH)D concentrations were measured in 44 dogs with a CCLR on the day before surgery. C-reactive protein concentrations were measured at a median time of 1 day post-surgery and the patient's clinical and radiographic response to CCLR surgical treatment was assessed at a median timepoint of 60 days post-surgery. Serum 25(OH)D concentrations in dogs with a CCLR was not significantly different to a population of healthy dogs (median 74.1 nmol/L and 88.40 nmol/L, respectively). There was no significant correlation between pre-operative serum 25(OH)D concentrations and length of pre-diagnosis clinical signs, pre-operative lameness scores or day 1 post-operative CRP concentrations. Thirty nine of the 44 dogs were re-examined at a median 60 days post-surgery. There was no relationship between the day 60 lameness scores and pre-operative serum 25(OH)D concentrations. In summary, we discovered that the vitamin D status of dogs with a CCLR was not significantly lower than healthy dogs and pre-operative serum 25(OH)D concentrations were not correlated to either pre-surgical disease severity or post-operative clinical outcomes.

Control-Normalized Fisher Ratio Analysis of Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry Data for Enhanced Biomarker Discovery in a Metabolomic Study of Orthopedic Knee-Ligament Injury.

An innovative form of Fisher ratio (F-ratio) analysis (FRA) is developed for use with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) data and applied to the investigation of the changes in the metabolome in human plasma for patients with injury to their anterior cruciate ligament (ACL). Specifically, FRA provides a supervised discovery of metabolites that express a statistically significant variance in a two-sample class comparison: patients and healthy controls. The standard F-ratio utilizes the between-class variance relative to the pooled within-class variance. Because standard FRA is adversely impacted by metabolites expressed with a large within-class variance in the patient class, "control-normalized FRA" has been developed to provide complementary information, by normalizing the between-class variance to the variance of the control class only. Thirty plasma samples from patients who recently suffered from an ACL injury, along with matched controls, were subjected to GC × GC-TOFMS analysis. Following both standard and control-normalized FRA, the concentration ratio for the top 30 "hits" in each comparison was obtained and then t-tested for statistical significance. Twenty four out of 30 metabolites plus the therapeutic agent, naproxen (24/30), passed the t-test for the control-normalized FRA, which included 8/24 unique to control-normalized FRA and 16/24 in common with the standard FRA. Likewise, standard FRA provided 21/30 metabolites passing the t-test, with 5/21 undiscovered by control-normalized FRA. The complementary information obtained by both F-ratio analyses demonstrates the general utility of the new approach for a variety of applications.

Infrared spectroscopy of serum as a potential diagnostic screening approach for naturally occurring canine osteoarthritis associated with cranial cruciate ligament rupture.

OBJECTIVE: To evaluate infrared (IR) spectroscopy of serum as a screening tool to differentiate dogs affected by naturally occurring osteoarthritis (OA) associated with cranial cruciate ligament rupture (CrCLR) and controls. METHOD: 104 adult dogs with CrCLR (affected group) and 50 adult control dogs were recruited for this prospective observational study. Serum samples were collected preoperatively from CrCLR dogs and from a subset of these dogs at 4-, and 12-week post-surgical intervention to stabilize the affected stifles. Serum was collected once from control dogs. Dry films were made from serum samples, and IR absorbance spectra acquired. Data preprocessing, principal component analysis and multivariate analysis of covariance were performed to separate samples from the two groups, and to evaluate temporal differences. Weighted logistic regression with L1 regularization method was used to develop a predictive model. Model performance based on an independent test set was evaluated. RESULTS: Spectral data analysis revealed significant separation between the sera of CrCLR and control dogs (P < 0.0001), but not amongst different time points in the OA group. The sensitivity, specificity, AUC and accuracy of the test set were 84.62%, 96.15%, 93.20% and 92.31% respectively. CONCLUSIONS: These findings confirm the potential of IR-spectroscopy of serum with chemometrics methods to differentiate controls from dogs with OA associated with CrCLR. This is the first step in development of an economic, and comparatively simple IR-based screening serum test for OA. Utility of this tool as a clinical screening and diagnostic test requires further investigation and validation.

Upregulation of Systemic Inflammatory Pathways Following Anterior Cruciate Ligament Injury Relates to Both Cartilage and Muscular Changes: A Pilot Study.

In conjunction with cartilage breakdown, muscle maladaptation including atrophy and increased fibrosis have been observed in the quadriceps following anterior cruciate ligament (ACL) injury. Previously observed upregulated muscle-related proteins in the synovial fluid following ACL rupture allude to cellular communication between the joint and muscle. Therefore, the purpose of this study was to determine whether muscle-related analytes are differentially expressed in the serum. Sixteen patients with an acute ACL tear participated in this IRB-approved study. Serum was obtained at two different time points at a mean of 6 and 14 days post-injury, and serum was analyzed by a highly multiplexed assay of 1,300 proteins. Pathway analysis using DAVID was performed; genes included met three criteria: significant change between the two study time points using a paired t test, significant change between the two study time points using a Mann-Whitney non-parametric test, and significant Benjamini post hoc analysis. Twelve analytes significantly increased between time points. Proteins chitinase-3-like protein 1 (p = 0.01), insulin-like growth factor binding protein 1 (p = 0.01), insulin-like growth factor binding protein 5 (p = 0.02), renin (p = 0.004), and lymphotoxin alpha 1: beta 2 (p = 0.03) were significantly upregulated in serum following acute ACL injury. The current results confirm the inflammatory pattern previously seen in the synovial fluid thought to play a role in the progression of post-traumatic osteoarthritis after ACL injury, and this data also provides further insights into important communication between the joint and quadriceps group, whose function is important in long term health. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:387-392, 2020.

The Effects of Anterior Cruciate Ligament Reconstruction on Individual Quadriceps Muscle Thickness and Circulating Biomarkers.

Anterior cruciate ligament reconstruction (ACLR) frequently results in quadriceps atrophy. The present study investigated the effect of ACLR on the muscle thickness of the different constituent muscles of the quadriceps and circulating biomarkers related to muscle atrophy and hypertrophy. Fourteen subjects underwent anterior cruciate ligament reconstruction following injury. Quadriceps muscle thicknesses were measured using ultrasound, and circulating biomarkers in the blood were measured using enzyme-linked immunosorbent assays (ELISAs) at the preoperative visit (PRE) and at two postoperative visits (PO1, PO2) in the early stages post-surgery. Differences between time points were analyzed using one-way repeated measures analysis of variance (ANOVA) tests. The most important finding was that severe muscle atrophy occurred in the vastus intermedius (VI) after ACLR (PRE: 20.45 ± 6.82 mm, PO1: 16.05 ± 6.13 mm, PO2: 13.18 ± 4.7 mm, F = 59.0, p < 0.001). Furthermore, the myostatin level was slightly increased, and IGF-1 was significantly reduced throughout the entire period. Therefore, we suggest that inducing selective hypertrophy in the vastus intermedius during the process of rehabilitation would be important for athletes and individuals who engage in explosive sports. Moreover, inhibiting myostatin level increases and maintaining IGF-1 levels in the early phase of recovery after ACLR to prevent muscle atrophy may provide a pharmaceutical option for rehabilitation after anterior cruciate ligament injury.

Correlation of serum and synovial leptin concentrations with body condition scores in healthy and osteoarthritic dogs.

OBJECTIVE: To evaluate the relationship between serum and synovial fluid (SF) leptin concentrations and body condition score (BCS) in healthy and osteoarthritic dogs. STUDY DESIGN: Controlled, prospective, clinical study. ANIMALS: Nineteen healthy dogs and 29 dogs with osteoarthritis (OA) secondary to cranial cruciate ligament injury. METHODS: Synovial fluid was obtained from the femorotibial joint under sedation (healthy dogs) or during surgery (OA dogs). Serum and SF leptin and interleukin (IL)-1ß concentrations were measured via enzyme-linked immunosorbent assay. Dogs were classified as optimal weight (BCS 4-5/9) or overweight (BCS >5/9). Radiographs were scored for OA severity by a radiologist. Owners completed the Liverpool Osteoarthritis in Dogs (LOAD) questionnaire. RESULTS: Mean (± SD) SF leptin (4.09 ± 4 ng/mL) was lower than serum leptin (6.88 ± 5.52 ng/mL, P < .0001). Synovial fluid leptin was higher in overweight (5.28 ± 4.21) than in optimal body weight dogs (1.54 ± 1.72 ng/mL, P < .0001). Serum (P < .001) and SF leptin (P = .004) concentrations were associated with BCS. Concentration of SF leptin did not differ between healthy (2.4 ± 2.04 ng/mL) and OA (4.9 ± 4.3 ng/mL, P = .25) dogs. Synovial fluid leptin and LOAD scores were weakly associated (P = .03). No association was detected between SF leptin and radiographic score or IL-1ß (P = .73). CONCLUSION: Serum and SF leptin correlated with BCS in this population. Synovial fluid leptin was weakly associated with LOAD scores but not with radiographic severity of OA or IL-1ß. CLINICAL SIGNIFICANCE: Serum and SF leptin concentrations do not predict radiographic severity of canine OA but contribute to joint pain and dysfunction.

Metabolomic serum profiling after ACL injury in rats: A pilot study implicating inflammation and immune dysregulation in post-traumatic osteoarthritis.

ACL rupture is a major risk factor for post-traumatic osteoarthritis (PTOA) development. Little information exists on acute systemic metabolic indicators of disease development. Thirty-six female Lewis rats were randomized to Control or noninvasive anterior cruciate ligament rupture (ACLR) and to three post-injury time points: 72 h, 4 weeks, 10 weeks (n = 6). Serum was collected and analyzed by 1 H nuclear magnetic resonance (NMR) spectroscopy and combined direct injection and liquid chromatography (LC)-mass spectrometry (MS)/MS (DI-MS). Univariate and multivariate statistics were used to analyze metabolomic data, and predictive biomarker models were analyzed by receiver operating characteristic (ROC) analysis. Topological pathway analysis was used to identify perturbed pathways. Two hundred twenty-two metabolites were identified by 1 H NMR and DI-MS. Differences in the serum metabolome between ACLR and Control were dominated by medium- and long-chain acylcarnitine species. Further, decreases in several tryptophan metabolites were either found to be significantly different in univariate analysis or to play important contributory roles to multivariate model separation. In addition to acylcarnitines and tryptophan metabolites, glycine, carnosine, and D-mannose were found to differentiate ACLR from Control. Glycine, 9-hexadecenoylcarnitine, trans-2-Dodecenoylcarnitine, linoelaidyl carnitine, hydroxypropionylcarnitine, and D-Mannose were identified as biomarkers with high area under ROC curve values and high predictive accuracies. Our analysis provides new information regarding the potential contribution of inflammatory processes and immune dysregulation to the onset and progression of PTOA following ACL injury. As these processes have most commonly been associated with inflammatory arthropathies, larger-scale studies elucidating their involvement in PTOA development and progression are necessary. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1969-1979, 2018.

Molecular Biomarkers of Knee Pathology.

The identification of biomarkers has become increasingly important in our fundamental understanding of the molecular basis for disease and subsequently in the advancement of modern medicine. Biomarkers have been identified in a plethora of normal and pathologic conditions and are most often found in blood, tissue, or synovial fluid. Orthopaedic research has more recently focused on biomarkers of cartilage and joint diseases, with an emphasis on understanding the molecular underpinnings of their pathophysiology. This article focuses on the biomarkers identified to date in several select knee pathologies and how further research can contribute to new diagnostic tools and targeted therapeutics.

Early Cartilage Changes After Anterior Cruciate Ligament Injury: Evaluation With Imaging and Serum Biomarkers-A Pilot Study.

PURPOSE: To determine if magnetic resonance imaging markers of cartilage matrix and morphology and circulating serum biomarkers of inflammation and matrix degradation differ over time in patients with anterior cruciate ligament (ACL) injury and bone marrow edema lesions (BMELs) when compared with matched control subjects. METHODS: We performed a case-control study, in which 11 ACL-injured subjects scheduled to undergo reconstruction and 11 matched control subjects were scheduled for testing. Participants were selected for the ACL reconstruction (ACLR) group if they injured their ACL while participating in sports, were aged 14 to 30 years, had 1 or more BMELs, and were scheduled to undergo bone-patellar tendon-bone ACLR. Testing required patients to undergo magnetic resonance imaging for measurement of T2 relaxation times in standardized regions of interest over the medial and lateral tibial plateaus and femoral condyles and have blood drawn for measurement of cartilage oligomeric matrix protein (COMP) and C-reactive protein levels before ligament reconstruction and 1 year after surgery. RESULTS: ACL patients had prolonged T2 relaxation times, indicative of cartilage matrix degradation, in the superficial central lateral tibial plateau (P = .02) and deep medial tibial plateau when compared with control subjects (P = .0001). Prolonged T2 relaxation times were also noted over the lateral femoral condyle at baseline for ACL patients compared with control subjects (P = .001), but the differences resolved by 1 year (P = .98). Circulating serum COMP levels were greater in ACL patients (233.23 ± 88.26 ng/mL) compared with control subjects (169.05 ± 64.53 ng/mL, P = .05). CONCLUSIONS: T2 mapping showed prolonged relaxation times in the lateral compartment of the knee in ACLR patients with lateral BMELs. Furthermore, prolonged T2 relaxation times were apparent in the medial compartment of the knee in ACL-injured patients where bone marrow lesions were not present. Higher serum COMP levels were present in ACL-injured subjects when compared with control subjects. LEVEL OF EVIDENCE: Level II, prospective case-control study.