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Correlation of Synovial Fluid Biomarkers With Cartilage Pathology and Associated Outcomes in Knee Arthroscopy
Correlation of Synovial Fluid Biomarkers With Cartilage Pathology and Associated Outcomes in Knee Arthroscopy Vanessa G. Cuéllar, M.D., Jason M. Cuéllar, M.D., Ph.D., Thorsten Kirsch, Ph.D., and Eric J. Strauss, M.D.
Purpose: To correlate the intraoperative concentrations of 20 synovial fluid biomarkers with preoperative symptoms, intraoperative findings, and postoperative outcomes in patients undergoing knee arthroscopy, with comparisons made to samples obtained from asymptomatic knees. Methods: Synovial fluid samples were obtained from 81 patients undergoing knee arthroscopy meeting the inclusion criteria, which included 70 samples from operative knees and 32 samples from contralateral knees. Preoperatively, baseline data obtained from clinical questionnaires including a visual analog scale (VAS) score, the Lysholm score, and the Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form were recorded. Synovial fluid was collected from both the operative knee and asymptomatic contralateral knee. Synovial fluid was stored with a protease inhibitor at 80 C until analysis. Intraoperative findings, procedures performed, and International Cartilage Repair Society (ICRS) cartilage status scores in all operative knees were documented. The concentrations of the following 20 biomarkers were measured using a multiplex magnetic bead immunoassay: matrix metalloproteinase (MMP) 3; MMP-13; tissue inhibitor of metalloproteinase (TIMP) 1; TIMP-2; TIMP-3; TIMP-4; fibroblast growth factor 2; eotaxin; interferon g; interleukin (IL) 10; platelet-derived growth factor BB; IL-1 receptor antagonist; IL1b; IL-6; monocyte chemotactic protein 1 (MCP-1); macrophage inflammatory protein 1a; macrophage inflammatory protein 1b; RANTES (regulated upon activation, normal T cell expressed and secreted); tumor necrosis factor a; and vascular endothelial growth factor. Clinical outcome scores were obtained in 83% of patients at a mean of 17 months’ follow-up postoperatively. Analysis of variance and Pearson correlation analysis were performed to determine statistical significance between preoperative data, intraoperative findings, postoperative outcomes, and synovial fluid biomarker concentrations compared with asymptomatic contralateral knees. Results: Analysis was performed on 70 operative and 32 contralateral samples. There were strong positive correlations between ICRS score and age, symptom duration, VAS score, and Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form. A strong positive correlation was found between MCP-1 and IL-6 concentrations, intraoperative ICRS score, and continued pain at the time of final follow-up. MCP-1 and IL-6 were the strongest predictors of severe cartilage lesions, whereas IL-1 receptor antagonist was inversely related. MMP-3 levels were consistently elevated in all operative samples and directly correlated to increased preoperative VAS scores. RANTES, vascular endothelial growth factor, and platelet-derived growth factor BB were the strongest predictors of postoperative improvement at final follow-up regardless of injury and cartilage status. Conclusions: Synovial fluid biomarkers have the capacity to reflect the intra-articular environment before surgery and potentially predict postoperative clinical outcomes. Recognition of key molecular players may yield future therapeutic targets, and large clinical trials exploring these discoveries are anticipated. Level of Evidence: Level III, therapeutic casecontrol study.
From the Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, New York, New York, U.S.A. The authors report the following potential conflict of interest or source of funding: V.G.C. receives support from Cytonics. J.M.C. receives support from Cytonics. E.J.S. receives support from DePuy Mitek and Jaypee Publishing. Presented at the American Academy of Orthopaedic Surgeons Annual Meetings, New Orleans, LA, March 11-15, 2014, and Las Vegas, NV, March 24-28, 2015, and the Arthroscopy Association of North America Annual Meeting, Hollywood, FL, May 1-3, 2014.
Received December 12, 2014; accepted August 25, 2015. Address correspondence to Eric J. Strauss, M.D., Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 333 E 38th St, Fourth Floor, New York, NY 10016, U.S.A. E-mail: [email protected] Ó 2015 by the Arthroscopy Association of North America 0749-8063/141046/$36.00 http://dx.doi.org/10.1016/j.arthro.2015.08.033
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espite advances in magnetic resonance imaging (MRI) techniques, sensitivity and specificity for intra-articular cartilage pathology remain imperfect. Furthermore, the ability to preoperatively predict which patients with or without cartilage lesions will improve after arthroscopic surgery is even more challenging. The ability to understand the molecular milieu associated with various cartilage pathologies through biomarker analysis may have an impact on clinical practice. The use of biomarkers as part of the diagnostic workup and treatment decision-making process is gaining considerable attention and has become widely acknowledged in modern medicine. Biomarkers may be identified in the blood, urine, or synovial fluid of patients and represent objective indicators of normal processes, pathology, or responses to therapeutic intervention. In the field of orthopaedics, biomarkers have been explored in the setting of a variety of pathologies, most frequently in cases of osteoarthritis, infection, or tumor.1-4 Most studies to date have investigated biomarkers in the blood or urine. However, it is becoming increasingly appreciated that intraarticular pathologies may be more accurately described by analysis of joint-specific synovial fluid in addition to serum analysis.5 Several protein biomarkers have been explored as diagnostic tools, prognostic indicators, and surrogate endpoints for therapeutic intervention in both in vitro and in vivo models of cartilage injury.6 Inflammatory cytokines, metalloproteinases, proteases, and degradation products of cartilage have all been profiled in the pathophysiology of cartilage degradation.7-10 It is well established that diagnosis of knee cartilage injury is underappreciated by MRI studies.11 In one study, chondral lesions were observed intraoperatively in 45% of patients previously unrecognized by MRI.12 In other studies, cartilage degeneration was detected using molecular analysis that was otherwise not apparent using traditional MRI.13-16 The impact of these unrecognized chondral defects discovered at the time of arthroscopy on surgical outcomes is not known. The goal of this study was to determine whether molecular biomarkers exist that could predict the severity of chondral defects found at the time of surgery, as well as whether these potential early markers have any prognostic significance on postoperative clinical outcomes. This study aimed to correlate the intraoperative concentrations of 20 synovial fluid biomarkers with preoperative symptoms, intraoperative findings, and postoperative outcomes in patients undergoing knee arthroscopy, with comparisons made with samples obtained from asymptomatic knees. Our hypothesis was that a combination of molecular markers such as proteases and inflammatory mediators
could predict more advanced (grade III and IV) chondral lesions found intraoperatively and predict continued pain postoperatively.
Methods Patient Enrollment and Study Questionnaires This was a single-center institutional review boardeapproved study in which 81 consecutive patients were prospectively enrolled over a 2-year period. All patients who were indicated for knee arthroscopy by 2 board-certified sports surgeons (E.J.S.) and met the inclusion criteria were invited to participate, and chart reviewdin addition to patient history and physical examination findingsdwas used to identify the presence of any exclusion criteria. The exclusion criteria included the following parameters: age younger than 18 years, systemic inflammatory disease (e.g., rheumatoid arthritis), autoimmune disease, intra-articular injection in the 3 months before surgery, prior knee surgery, knee surgery within the follow-up period, immunomodulatory drug use, chemotherapy within the past year, planned open procedure, or cartilage/ meniscal transplant in addition to arthroscopy. The 3month limit for intra-articular injections was selected based on our experience with standards imposed by prior clinical trials for injection treatments and our opinion that this time frame limits external influence from previous interventions. Preoperatively, enrolled patients completed clinical questionnaires including a visual analog scale (VAS), the Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS), and the Lysholm scale.17 All patients who reported no pain and no history of injury in the contralateral knee also completed clinical questionnaires with respect to their nonoperative knee and were invited to provide a sample from the contralateral knee at the time of surgery to serve as an internal control. In 11 patients who contributed synovial fluid samples from both the operative knee and the contralateral knee, the operative sample was excluded from analysis because of subsequent surgery within the follow-up period or failure to obtain an adequate amount of synovial fluid from the operative knee. All patients meeting the inclusion criteria for the contralateral kneedspecifically, no history of surgery or pain in the contralateral knee and a currently asymptomatic contralateral kneedwere asked to contribute contralateral samples. Among the 70 operative knees, 49 patients either did not meet the inclusion criteria for the contralateral knee or refused aspiration. Aspiration of each knee was performed only once in the operating room in all patients, and thus each knee provided 1 sample of synovial fluid. Patient samples were excluded from analysis if no synovial fluid was aspirated.
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Table 1. Description of Biomarkers Analyzed in Study Biomolecule MMP-3 MMP-13 TIMP-1, -2, -3, and -4 FGF-2 Eotaxin IFNg IL-10 PDGF-BB IL-1b IL-1Ra IL-6 MCP-1 MIP-1a and MIP-1b RANTES TNFa VEGF
Basic Functions Stromelysin that degrades collagen II, IV, IX, X, and XI, as well as proteoglycans, fibronectin, laminin, and elastin; activates other MMPs Collagenase that degrades collagen I, II, III, IV, IX, X, and XIV Inhibition of MMPs; chondroprotective role Promotes angiogenesis, wound healing, and granulation tissue formation Small chemokine that selectively recruits eosinophils Cytokine that is critical for innate and adaptive immunity against viral and bacterial infections; activator of macrophages and MHC-II expression Cytokine with anti-inflammatory effects through downregulation of Th1 cytokine expression, MHC-II antigens, and macrophage activators; enhances B-cell survival and blocks NF-kB activity Growth factor that plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis Cytokine produced by activated macrophages; important mediator of inflammatory response; activates cyclooxygenase 2 Cytokine that inhibits proinflammatory effects of IL-1b Cytokine secreted by T cells and macrophages acting in proinflammatory function, especially during infection or after trauma; can stimulate IL-10 and IL-1Ra production to act in negative feedback loop of inflammatory response Chemokine that recruits monocytes, memory T cells, and dendritic cells to site of inflammation by tissue injury or infection Chemokines produced by macrophages; activate neutrophils, eosinophils, and basophils in addition to other cytokines such as IL-1, IL-6, and TNFa Chemokine that recruits leukocytes to site of inflammation and activates natural killer cells Cytokine produced by macrophages, lymphocytes, natural killer cells, and neutrophils and involved in acute phase of inflammatory response; can induce cell apoptosis Growth factor that induces angiogenesis
FGF-2, fibroblast growth factor 2; IFNg, interferon g; IL, interleukin; IL-1Ra, interleukin 1 receptor antagonist; MCP-1, monocyte chemotactic protein 1; MHC-II, major histocompatibility complex II; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NF-kB, nuclear factor kB; PDGF-BB, platelet-derived growth factor BB; RANTES, regulated upon activation, normal T cell expressed and secreted; Th1, T helper cell type 1; TIMP, tissue inhibitor of metalloproteinase; TNFa, tumor necrosis factor a; VEGF, vascular endothelial growth factor.
Synovial Fluid Collection and Storage In the operating room, after sterile preparation but before arthroscopic portal creation, synovial fluid sampling was performed using the superolateral portal location, with an 18-gauge needle and a 10-mL syringe. The maximal amount of synovial fluid that egressed was collected; however, only a few microliters was necessary for molecular analysis. Synovial fluid samples were transferred to sterile tubes containing a protease inhibitor cocktail solution (Halt Protease Inhibitor Cocktail, EDTA free; Pierce Biotechnology, Rockford, IL). The samples were stored on ice during transport to the laboratory, where they were centrifuged at 1,350g for 10 minutes and supernatant aliquoted into sterile tubes, before storage at 80 C. Biomarker Analysis and Intraoperative Findings The samples were analyzed for the presence of 20 biomarkers using a multiplex bead assay (Milliplex; Millipore, Billerica, MA) including matrix metalloproteinase 3 (MMP-3); MMP-13; tissue inhibitor of metalloproteinase (TIMP) 1; TIMP-2; TIMP-3; TIMP-4; fibroblast growth factor 2; eotaxin; interferon g; interleukin (IL) 10; platelet-derived growth factor BB (PDGFBB); IL-1 receptor antagonist; IL-1b; IL-6; monocyte chemotactic protein 1 (MCP-1); macrophage inflammatory protein (MIP) 1a; MIP-1b; RANTES (regulated upon activation, normal T cell expressed and secreted);
tumor necrosis factor a; and vascular endothelial growth factor (VEGF). These specific biomarkers were chosen because they are known players in joint pain, inflammation, and cartilage degeneration (Table 1). Before the assay, the synovial fluid samples were treated with hyaluronidase18; they were then assayed according to the manufacturer’s protocol. Intraoperative arthroscopic findings were reported by the surgeon (E.J.S.) and recorded by a trained research assistant during the initial diagnostic arthroscopy, including soft-tissue injury and cartilage pathology using the International Cartilage Repair Society (ICRS) grading scale.19 For patients with multiple cartilage lesions, data on all lesions were recorded but the ultimate score assigned was based on the highest-grade lesion identified (Table 2). Postoperative Follow-up In addition to routine postoperative visits, patients were contacted for follow-up at 1 and 2 years after arthroscopy and completed clinical questionnaires (VAS, KOOS-PS, and Lysholm scale). These data were compared with preoperative data, intraoperative findings, and synovial fluid biomarker concentrations. Statistical Methods Clinical questionnaire data were analyzed for all operative and contralateral knee samples with respect
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Table 2. Profile of Intra-articular Injuries With Respect to Cartilage Grade at Time of Surgery Patients/Operative Samples Included in Analysis, n Injury Subgroup ACL tear only Meniscus tear only ACL plus meniscus tear Cartilage lesion only
ICRS Grade 1 7 6 9 0
ICRS Grade 2 1 6 5 2
ICRS Grade 3 2 18 5 3
ICRS Grade 4 0 5 1 0
ACL, anterior cruciate ligament; ICRS, International Cartilage Repair Society.
to biomarker concentrations using analysis of variance. Pearson correlation coefficients were used to compare patients’ age, duration of symptoms, and ICRS grade with respect to biomarker concentrations.
Results Study Population and Intraoperative Findings Clinical and operative data were collected from 81 patients enrolled in the study. Synovial fluid was collected from 70 operative knees and 32 contralateral knees. Clinical data were obtained for 67 patients at a minimum follow-up duration of 1 year (mean, 17 6 months), representing an 83% rate of follow-up. The 70 operative knees from which synovial fluid samples were collected included 10 isolated anterior cruciate ligament (ACL) injuries, 35 isolated meniscal injuries, 20 ACL and meniscal injuries, and 5 knees with isolated cartilage pathology. Among the operative knee samples, there were 22 ICRS grade 1 lesions, 14 ICRS grade 2 lesions, 28 ICRS grade 3 lesions, and 6 ICRS grade 4 lesions. There was no difference in age between samples collected from operative and contralateral knees except when contralateral samples were compared with samples from patients with ICRS grade 1 lesions; the mean age of patients contributing contralateral knee samples was 41 15 years compared with 28 8 years, 43 14 years, 46 14 years, and 53 6 years for patients with ICRS grade 1, grade 2, grade 3, and grade 4 lesions, respectively (P < .001). Preoperatively, the median VAS score, KOOS-PS, and Lysholm score were 4.0, 27, and 65, respectively, for operative knees and 0, 0, and 100, respectively, for contralateral knees. The median duration of symptoms in all patients was 3 months (range, 1 to 264 months). There was a significant and direct linear correlation between preoperative VAS score and ICRS score (Fig 1). Median VAS pain scores were subdivided by injury type, the most painful, on average, being combined meniscus and cartilage injuries, with a median VAS score of 5, followed by ACL and meniscus injuries with cartilage lesions (VAS score, 4), ACL injuries with cartilage lesions (VAS score, 3), cartilage lesions only
(VAS score, 3), and isolated meniscus or ACL injuries without cartilage lesions (VAS score, 3) (P < .001). Intra-articular Biomarkers The mean biomarker concentrations ( standard error of the mean) were significantly greater in the operative samples compared with samples from the contralateral knees (non-paired) for MMP-3 (481,341 23,433 pg/mL v 164,285 25,998 pg/mL; P < .001), TIMP-1 (69,564 2,440 pg/mL v 55,939 4,638 pg/mL; P < .01), IL-6 (298 62 pg/mL v 21 14 pg/mL; P < .01), and MIP-1b (15 8 pg/mL v 9 1.3 pg/mL; P < .01), whereas the inverse relation was observed for TIMP-2 (44,221 1,257 pg/mL v 52,547 4,415 pg/mL; P < .05). Compared with samples from the contralateral control knees, samples from ICRS grade 1 to 4 lesions had greater concentrations of MMP3 (contralateral, 164,285 19,972 pg/mL; ICRS grade 1, 514,667 49,059 pg/mL; ICRS grade 2, 441,727 68,680 pg/mL, ICRS grade 3, 451,706 68,353 pg/mL; and ICRS grade 4, 583,296 12,938 pg/mL) and IL-6 (contralateral, 21 10 pg/mL; ICRS grade 1, 312 184 pg/mL; ICRS grade 2, 266 137 pg/mL; ICRS grade 3, 254 135 pg/mL; and ICRS grade 4, 532 158 pg/mL) (P < .001). For both IL-6 and MMP-3 (data not shown), the greatest concentrations were measured within samples from knees with meniscus or ACL injuries without cartilage lesions. Conversely, RANTES concentrations were significantly elevated only in knees with isolated cartilage lesions (Fig 2). When subgrouping by VAS score was performed, increased concentrations of IL-6 and MMP-3 correlated with an increased preoperative VAS score (Figs 3 and 4). By use of correlation analysis, preoperative VAS scores
Fig 1. Preoperative median visual analog scale pain score with respect to International Cartilage Repair Society (ICRS) score in operative knees and contralateral (CL) knees. * Significant difference between each ICRS group and with the contralateral knee. y Significant difference between ICRS 2 patients and ICRS 1 and 4 patients. z Significant difference between ICRS 3 patients and ICRS 1 and 4 patients.
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Fig 2. (A) Interleukin 6 (IL-6) concentration (in picograms per milliliter) and (B) regulated upon activation, normal T cell expressed and secreted (RANTES) concentration (in picograms per milliliter) subdivided by injury category compared with contralateral samples. (ACL, anterior cruciate ligament.) * Significant difference compared to contralateral knees.
correlated with the concentrations of MMP-3 (r ¼ 0.4, P < .0001), TIMP-1 (r ¼ 0.21, P < .03), IL-10 (r ¼ 0.2, P < .03), and IL-6 (r ¼ 0.23, P < .01). In addition, the concentrations of several analytes correlated to preoperative clinical parameters (Table 3). The concentrations of several molecules were not significantly different in the operative knee synovial fluid samples compared with the contralateral knee samples (MCP-1, TIMP-4, VEGF, RANTES, PDGF-BB, MMP-13, eotaxin, interferon g, IL10, tumor necrosis factor a, MIP-1a, and IL-1b). Clinical Follow-up Data and Biomarkers Clinical data were obtained for 67 patients at a minimum follow-up duration of 1 year (mean, 17 6
months), representing an 83% rate of follow-up. For operative knees, the median follow-up VAS score, KOOS-PS, and Lysholm score were 1.0, 15, and 91, respectively, compared with 4.0, 27, and 65, respectively, preoperatively. Patients with isolated cartilage lesions reported higher VAS and KOOS-PS scores and lower Lysholm scores compared with patients without cartilage lesions (Fig 5). In general, patients with isolated cartilage lesions reported the least amount of improvement at follow-up, with no statistically significant difference between preoperative and follow-up VAS, Lysholm, or KOOS-PS scores (Fig 6). Patients without evidence of cartilage pathology intraoperatively had the best outcomes at the most recent
Fig 3. Interleukin 6 (IL-6) concentration (in picograms per milliliter) subdivided by visual analog scale (VAS) pain score preoperatively. * Significant difference compared to patients with a mean preoperative VAS score of 0-2.
Fig 4. Matrix metalloproteinase 3 (MMP-3) concentration (in picograms per milliliter) subdivided by visual analog scale (VAS) pain score preoperatively. * Significant difference compared to patients with a mean preoperative VAS score of 0-2.
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Table 3. Correlation Analysis to Determine Which Biomarkers Correlated With Preoperative Clinical Parameters Using Pearson Correlation Coefficient (r) Parameter (Preoperative) VAS VAS VAS VAS Lysholm score Lysholm score Lysholm score Lysholm score Lysholm score Lysholm score KOOS-PS KOOS-PS KOOS-PS KOOS-PS
Analyte MMP-3 TIMP-1 IL-10 IL-6 MMP-3 TIMP-2 FGF-2 IL-10 IL-6 MCP-1 MMP-3 TIMP-2 FGF-2 IL-6
Correlation Coefficient (Pearson r) 0.4 0.21 0.2 0.23 0.50 0.28 0.23 0.33 0.4 0.23 0.44 0.28 0.25 0.32
P Value .0001 .03 .03 .01 .0001 .0058 .02 .001 .0001 .0001 .0001 .019 .03 .007
FGF-2, fibroblast growth factor 2; IL, interleukin; KOOS-PS, Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form; MCP-1, monocyte chemotactic protein 1; MMP-3, matrix metalloproteinase 3; TIMP, tissue inhibitor of metalloproteinase; VAS, visual analog scale pain score.
follow-up, but there was not a significant difference between outcome and ICRS score when subdivided by ICRS scores 1 through 4. Improvement in VAS score correlated with preoperative PDGF-BB (r ¼ 0.29) and RANTES (r ¼ 0.28) levels, with the lowest levels predicting the best outcomes (P < .05). An increased concentration of TIMP-3 was a predictor of higher VAS scores at follow-up in patients who underwent ACL reconstruction but not in patients with meniscus injuries only (r ¼ 0.43, P < .05). Concentrations of MMP-3 (r ¼ 0.26, P < .017), TIMP1 (r ¼ 0.21, P < .05), IL-10 (r ¼ 0.34, P < .001), and MCP-1 (r ¼ 0.32, P < .003) weakly correlated with follow-up Lysholm scores. Follow-up KOOS-PS was also weakly correlated with MMP-3 (r ¼ 0.22, P < .04), IL-6 (r ¼ 0.22, P < .05), and MCP-1 (r ¼ 0.27, P < .01) concentrations. The ratio of particular biomarkers to one another was investigated for possible associations with clinical parameters, particularly the relation of MMPs to TIMPs. There was a moderate negative correlation between the MMP-3:TIMP-2 ratio and preoperative Lysholm score (the greater the ratio, the worse the Lysholm score; r ¼ 0.53, P < .0001) and between the MMP-3:TIMP-2 ratio and follow-up Lysholm score (r ¼ 0.21, P < .05). There were similar correlations between the MMP3:TIMP-3 ratio and preoperative VAS score (r ¼ 0.03, P < .005), Lysholm score (r ¼ 0.32, P < .005), and KOOS-PS score (r ¼ 0.29, P < .05). Contralateral Knees in Which Symptoms Developed Patients who initially had no pain in their contralateral knee and contributed a control sample from that
knee but in whom pain later developed in the contralateral knee, defined as a VAS score greater than 2, were more likely to have had elevated concentrations of MCP-1 (P < .05) and VEGF (P < .05) and decreased RANTES concentrations (P < .05) at baseline compared with patients whose knee remained asymptomatic at follow-up (Fig 7).
Discussion This study identified several synovial fluid biomarkers associated with intra-articular knee pathology, pain, and clinical outcomes in patients undergoing knee arthroscopy. MCP-1 and IL-6 were the strongest predictors of more severe cartilage lesions intraoperatively independent of associated pathologies and also predicted worse outcomes at follow-up. MMP-3 concentrations were consistently elevated in all operative samples and were directly correlated to increased preoperative VAS scores. Levels of PDGF-BB, RANTES, and VEGF were able to predict clinical outcomes better than preoperative VAS scores, and patients with elevated MCP-1, VEGF, and decreased RANTES concentrations in their asymptomatic knees were more likely to have subsequent pain develop at final followup. Our observation of an elevated IL-6 level as a predictor of severe cartilage injury supports prior observations.20-22 IL-6 has been characterized as having a “pleiotropic” role and being involved in both innate immunity and maintenance of chronic inflammation.23 IL-6 may also be consistently associated with painful joints, as it has been shown to sensitize C fibers innervating the knee joint24 through the prostaglandincyclooxygenase inflammatory pathway.25,26 Similarly, MCP-1 elevation in the setting of intraoperative pathology has been shown in prior studies.22 MCP-1 has a heterogeneous role in inflammation and response to injury and has been shown to recruit stem cells and activate transcription blockers for chondrogenesis.27 Comparably to IL-6 and other chemokines and cytokines, it also appears to be directly involved in sensitizing nociceptors and promotes long-term dorsal root ganglion excitability, thus contributing to chronic pain.28,29 In contrast, PDGF-BB concentrations were highest in contralateral knees and depressed across all operative knees, most strikingly in those with advanced cartilage degeneration. PDGF-BB is a growth factor known to antagonize catabolic factors and to inhibit IL1beinduced cartilage degeneration.30 Our study indicates a possible protective role of PDGF-BB, with its absence allowing IL-1beinduced cartilage degeneration to proceed unchecked. Moreover, these results suggest that across all patients, those with the highest levels of PDGF-BB had the lowest pain scores at follow-up. A mean increase in this biomarker predicted
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Fig 5. (A) Visual analog scale (VAS) pain score, (B) Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS) activity score, and (C) Lysholm activity score preoperatively (Pre-op) compared with score at final follow-up in patients with cartilage injury (International Cartilage Repair Society [ICRS] grade 2, 3, or 4) and without cartilage injury (ICRS grade 1).
improvement in VAS score, independent of intraoperative cartilage score or associated soft-tissue pathology. This observation of elevated levels in contralateral knees compared with severe cartilage injury in operative knees was similar to measured levels of RANTES, particularly in terms of predicting pain at follow-up. RANTES is similar to other cytokines in that it modulates the response of lymphocytes after injury; it has been identified in other synovial fluid studies as well.31,32 RANTES is known to promote IL-6 production and increase proteoglycan levels in cartilage33,34; therefore its relatively decreased levels in contralateral knees that go on to develop pain similar to those with more advanced cartilage injury may lend insight into the pathophysiology of chronic disease after injury. Not surprisingly, the converse was observed in measured VEGF levels. VEGF has been found in cartilage tissue after injury or osteoarthritis in many other studies, particularly late osteoarthritis, and appears to inhibit aggrecan and collagen II synthesis, both of which are important in cartilage regeneration.35-38 Thus the
higher levels of VEGF in those knees in which pain developed are not unexpected. We also observed an increased ratio of MMP-3 to TIMPs correlated with worse clinical parameters, which may suggest that TIMPs function in a protective role.39 The presence of MMPs and TIMPs in synovial fluid has been widely investigated and consistently shows a pivotal relation with cartilage degradation.8-10 Several studies have suggested that chondrocytes up-regulate MMP expression in the progression of cartilage degradation.40-43 It is currently accepted that variable mechanical loading may up-regulate MMP expression; however, it is not clear if this is the instigating event.44,45 MMPs increase during abnormal cartilage loading, such as after an ACL tear; however, they are also induced by proinflammatory cytokines, in particular IL-1 and tumor necrosis factor.7 The consistent association with cartilage matrix breakdown and inflammatory cytokines underscores the importance of MMPs in osteoarthritis.46 Those patients without cartilage lesions did the best at final follow-up, and the presence of any cartilage
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Fig 6. (A) Visual analog scale (VAS) pain scores at preoperative baseline (Pre-op) and at final follow-up subdivided by injury. (B) Preoperative (preop) and final follow-up Lysholm activity scores subdivided by injury. (C) Preoperative (Pre-op) and final followup Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS) activity scores subdivided by injury. (ACL, anterior cruciate ligament.) * Significant difference between pre-operative and post-operative scores.
lesions predicted a worse functional outcome overall. However, the difference between preoperative and postoperative clinical scores among all samples was not significant. This finding is important because it highlights the sensitivity of the biomarkers studied for detecting differences between these 2 groups. Preoperative clinical scores associated with cartilage injury severity were not able to predict progression, as was seen with certain biomarkers such as PDGFBB, RANTES, and VEGF. Patients who initially had no pain in their contralateral knee and contributed a control sample from that knee but in whom pain later developed, defined as a VAS score greater than 2, were more likely to have had elevated concentrations of MCP-1, VEGF, and decreased RANTES compared with those who remained asymptomatic at follow-up. The timing of fluid collection with respect to the date of injury or initial onset of pain is relevant to our results. Our patients reported a mean duration of symptoms of 14 months; however, a range of 1 to 264
months (median, 3 months) was present. Clearly, this reflects the fact that most of our patients undergoing surgery presented with meniscal tears, which are more heterogeneous in terms of clinical symptoms and history compared with our smaller cohort of patients with ACL tears, which tend to present earlier after injury. The differential profile of inflammatory markers with respect to time since injury is an area of active research. A few studies have focused on ACL tears in particular and have shown that there is an initial inflammatory response that differs from chronic cases.47-49 Our study was limited by the number of patients in our injury subgroups, which prevented adequate analysis of this factor independently. Our study aims to reflect a more general molecular milieu in “all-comers” that may help the clinician understand which patients are more likely to have continued pain despite surgical intervention. Future studies may distinguish differences based on the time of presentation alone, regardless of preoperative pain scores or cartilage status; however, it is possible that the molecular profile alone is informative,
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Acknowledgment The authors acknowledge Laith M. Jazrawi, M.D., Priya Mukhopadhyay, B.S., William Small, B.S., and Naveen Chadalavada, B.S.
References
Fig 7. Mean preoperative cytokine concentrations (in picograms per milliliter) of monocyte chemotactic protein 1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF) in contralateral knee samples in patients with no pain (visual analog scale [VAS] score of 0) and patients with pain (VAS score >2) at final follow-up. * Significant difference between patient groups.
regardless of length of symptoms, presence of meniscal tear, or number of cartilage lesions, for example. Limitations The specimens obtained from the contralateral knee provided only a clinical control because arthroscopic evaluation was not possible. Future studies will include MRI scans of the contralateral knees to identify any structural pathology present. Not all patients consented to undergo contralateral aspiration, and not all knees provided an adequate amount of synovial fluid for analysis, which may present a bias in our study samples. When largely grouped by cartilage pathology or pain, there are many differences; however, the subgroup analysis is limited by smaller numbers in each group (e.g., ACL tear only). There was variation in the time to presentation after injury and synovial fluid sampling, with some patients having symptoms for several months to years and other patients having a more acute presentation and treatment. Our findings are currently limited by the follow-up duration, and we anticipate that more differences may become apparent with longerterm observation and retrospective analysis.
Conclusions Synovial fluid biomarkers have the capacity to reflect the intra-articular environment before surgery and potentially predict postoperative clinical outcomes. Recognition of key molecular players may yield future therapeutic targets, and large clinical trials exploring these discoveries are anticipated.
1. Patra D, Sandell L. Recent advances in biomarkers in osteoarthritis. Curr Opin Rheumatol 2011;23:465-470. 2. Berbari E, Mabry T, Tsaras G, et al. Inflammatory blood laboratory levels as markers of prosthetic joint infection: A systematic review and meta-analysis. J Bone Joint Surg Am 2010;92:2102-2109. 3. Byrum S, Montgomery CO, Nicholas RW, Suva LJ. The promise of bone cancer proteomics. Ann N Y Acad Sci 2010;1192:222-229. 4. Lotz M, Martel-Pelletier J, Christiansen C, et al. Value of biomarkers in osteoarthritis: Current status and perspectives. Ann Rheum Dis 2013;72:1756-1763. 5. Ruiz-Romero C, Blanco FJ. Proteomics role in the search for improved diagnosis, prognosis and treatment of osteoarthritis. Osteoarthritis Cartilage 2010;18:500-509. 6. Harkey MS, Luc BA, Golightly YM, et al. Osteoarthritisrelated biomarkers following anterior cruciate ligament injury and reconstruction: A systematic review. Osteoarthritis Cartilage 2015;23:1-12. 7. Van de Loo FAJ, Joosten LAB, van Lent PLEM, Arntz OJ, van den Berg WB. Role of interleukin-1, tumor necrosis factor alpha, and interleukin-6 in cartilage proteoglycan metabolism and destruction: Effect of in situ blocking in murine antigen- and zymosan-induced arthritis. Arthritis Rheum 1995;38:164-172. 8. Freemont AJ, Hampson V, Tilman R, Goupille P, Taiwo Y, Hoyland JA. Gene expression of matrix metalloproteinase 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific. Ann Rheum Dis 1997;56:542-549. 9. Shlopov BV, Lie W-R, Mainardi CL, Cole AA, Chubinskaya S, Hasty KA. Osteoarthritic lesions: Involvement of three different collagenases. Arthritis Rheum 1997;40:2065-2074. 10. Woessner JF Jr, Gunja-Smith Z. Role of metalloproteinases in human osteoarthritis. J Rheumatol Suppl 1991;8:99-101. 11. Hjelle K, Solheim E, Strand T, Muri R, Brittberg M. Articular cartilage defects in 1,000 knee arthroscopies. Arthroscopy 2002;18:730-734. 12. Figueroa D, Calvo R, Vaisman A, Carrasco MA, Moraga C, Delgado I. Knee chondral lesions: Incidence and correlation between arthroscopic and magnetic resonance findings. Arthroscopy 2007;23:312-315. 13. Orita S, Koshi T, Mitsuka T, et al. Associations between proinflammatory cytokines in the synovial fluid and radiographic grading and pain-related scores in 47 consecutive patients with osteoarthritis of the knee. BMC Musculoskelet Disord 2011;12:144. 14. Catterall JB, Stabler TV, Flannery CR, Kraus VB. Changes in serum and synovial fluid biomarkers after acute injury (NCT00332254). Arthritis Res Ther 2010;12:R229.
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15. Vasara AI, Konttinen YT, Peterson L, Lindahl A, Kiviranta I. Persisting high levels of synovial fluid markers after cartilage repair: A pilot study. Clin Orthop Relat Res 2009;467:267-272. 16. Qi C, Changlin H. Levels of biomarkers correlate with magnetic resonance imaging progression of knee cartilage degeneration: A study on canine. Knee Surg Sports Traumatol Arthrosc 2007;15:869-878. 17. Collins NJ, Misra D, Felson DT, Crossley KM, Roos EM. Measures of knee function: International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form, Knee Injury and Osteoarthritis Outcome Score (KOOS), Knee Injury and Osteoarthritis Outcome Score Physical Function Short Form (KOOS-PS), Knee Outcome Survey Activities of Daily Living Scale (KOS-ADL), Lysholm Knee Scoring Scale, Oxford Knee Score (OKS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Activity Rating Scale (ARS), and Tegner Activity Score (TAS). Arthritis Care Res (Hoboken) 2011;63:S208-S228 (suppl 11). 18. Jayadev C, Rout R, Price A, et al. Hyaluronidase treatment of synovial fluid to improve assay precision for biomarker research using multiplex immunoassay platforms. J Immunol Methods 2012;386:22-30. 19. Brittbert M, Winalski CS. Evaluation of cartilage injuries and repair. J Bone Joint Surg Am 2003;85:58-69 (suppl 2). 20. Stannus OP, Jones G, Blizzard L, Cicuttini FM, Ding C. Associations between serum levels of inflammatory markers and change in knee pain over 5 years in older adults: A prospective cohort study. Ann Rheum Dis 2013;72:535-540. 21. Monibi F, Roller BL, Stoker A, Garner B, Bal S, Cook JL. Identification of synovial fluid biomarkers for knee osteoarthritis and correlation with radiographic assessment. J Knee Surg in press, available online 30 April, 2015. doi:10. 1055/s-0035-1549022. 22. Cuéllar JM, Scuderi GJ, Cuéllar VG, Golish SR, Yeomans DC. Diagnostic utility of cytokine biomarkers in the evaluation of acute knee pain. J Bone Joint Surg Am 2009;91:2313-2320. 23. Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol 2015;16:448-457. 24. Brenn D, Richter F, Schaible HG. Sensitization of unmyelinated sensory fibers of the joint nerve to mechanical stimuli by interleukin-6 in the rat: An inflammatory mechanism of joint pain. Arthritis Rheum 2007;56:351-359. 25. Anderson GD, Hauser SD, McGarity KL, Bremer ME, Isakson PC, Gregory SA. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J Clin Invest 1996;97:2672-2679. 26. Samad TA, Moore KA, Sapirstein A, et al. Interleukin1beta mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity. Nature 2001;410: 471-475. 27. Harris Q, Seto J, O’Brien K, et al. Monocyte chemotactic protein-1 inhibits chondrogenesis of synovial mesenchymal progenitor cells: An in vitro study. Stem Cells 2013;31:2253-2265. 28. Kao DJ, Li AH, Chen JC, et al. CC chemokine ligand 2 upregulates the current density and expression of TRPV1
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channels and Nav1.8 sodium channels in dorsal root ganglion neurons. J Neuroinflammation 2012;9:189. White FA, Sun J, Waters SM, et al. Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons chronic compression of the dorsal root ganglion. Proc Natl Acad Sci U S A 2005;102: 14092-14097. Montaseri A, Busch F, Mobasheri A, et al. IGF-1 and PDGFbb suppress IL-1b-induced cartilage degradation through down-regulation of NF-kB signaling: Involvement of Src/ PI-3K/AKT pathway. PLoS One 2011;6:e28663. Zlotnik A, Yoshie O. Chemokines: A new classification system and their role in immunity. Immunity 2000;12: 121-127. Nair A, Gan J, Bush-Joseph C, et al. Synovial chemokine expression and relationship with knee symptoms in patients with meniscal tears. Osteoarthritis Cartilage 2015;23: 1158-1164. Beekhuizen M, Gierman LM, van Spil WE, et al. An explorative study comparing levels of soluble mediators in control and osteoarthritic synovial fluid. Osteoarthritis Cartilage 2013;21:918-922. Tang CH, Hsu CJ, Fong YC. The CCL5/CCR5 axis promotes interleukin-6 production in human synovial fibroblasts. Arthritis Rheum 2010;62:3615-3624. Tsuchida AI, Beekhuizen M, ’t Hart MC, et al. Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes. Arthritis Res Ther 2014;16:441. Pfander D, Kortje D, Zimmermann R, et al. Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints. Ann Rheum Dis 2001;60:1070-1073. Chen XY, Hao YR, Wang Z, Zhou JL, Jia QX, Qiu B. The effect of vascular endothelial growth factor on aggrecan and type II collagen expression in rat articular chondrocytes. Rheumatol Int 2012;32:3359-3364. Ludin A, Sela JJ, Schroeder A, Samuni Y, Nitzan DW, Amir G. Injection of vascular endothelial growth factor into knee joints induces osteoarthritis in mice. Osteoarthritis Cartilage 2012;21:419-497. Tourville TW, Johnson RJ, Slauterbeck JR, et al. The relationship between synovial fluid ARGS, cytokines, MMPs & TIMPs following acute ACL injury. Osteoarthritis Cartilage 2013;21:S75 (suppl). Billinghurst RC, Dahlberg L, Ionescu M, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic cartilage. J Clin Invest 1997;99:1534-1545. Mehraban F, Kuo S-Y, Riera H, Chang C, Moskowitz RW. Prostromelysin and procollagenase genes are differentially up-regulated in chondrocytes from the knees of rabbits with experimental osteoarthritis. Arthritis Rheum 1994;37: 1189-1197. Shlopov BV, Gumanovskaya ML, Hasty KA. Autocrine regulation of collagenase 3 (matrix metalloproteinase 13) during osteoarthritis. Arthritis Rheum 2000;43: 195-205. Dahlberg L, Billinghurst RC, Manner P, et al. Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares
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collagenase 1 (matrix metalloproteinase 1). Arthritis Rheum 2000;43:673-682. Lin PM, Chen CT, Torzilli PA. Increased stromelysin-1 (MMP-3), proteoglycan degradation (3B3- and 7D4) and collagen damage in cyclically load-injured articular cartilage. Osteoarthritis Cartilage 2004;12:485-496. Lucchinetti E, Adams CS, Horton WE Jr, Torzilli PA. Cartilage viability after repetitive loading: A preliminary report. Osteoarthritis Cartilage 2002;10:71-81. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: Associations with degenerative changes. Arthritis Rheum 2001;44: 585-594. Struglics A, Larsson S, Kumahashi N, Frobell R, Lohmander LS. Changes in cytokines and Aagrecan
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ARGS Nnoepitope in synovial fluid and serum and in C-terminal crosslinking telopeptide of type II collagen and N-terminal crosslinking telopeptide of type I collagen in urine over five years after anterior cruciate ligament rupture: An exploratory analysis in the knee anterior cruciate ligament nonsurgical versus surgical treatment trial. Arthritis Rheumatol 2015;67:1816-1825. 48. Swärd P, Frobell R, Englund M, Roos H, Struglics A. Cartilage and bone markers and inflammatory cytokines are increased in synovial fluid in the acute phase of knee injury (hemarthrosis)dA cross-sectional analysis. Osteoarthritis Cartilage 2012;20:1302-1308. 49. Bigoni M, Sacerdote P, Turati M, et al. Acute and late changes in intraarticular cytokine levels following anterior cruciate ligament injury. J Orthop Res 2013;31: 315-321.
Purpose: To correlate the intraoperative concentrations of 20 synovial fluid biomarkers with preoperative symptoms, intraoperative findings, and postoperative outcomes in patients undergoing knee arthroscopy, with comparisons made to samples obtained from asymptomatic knees. Methods: Synovial fluid samples were obtained from 81 patients undergoing knee arthroscopy meeting the inclusion criteria, which included 70 samples from operative knees and 32 samples from contralateral knees. Preoperatively, baseline data obtained from clinical questionnaires including a visual analog scale (VAS) score, the Lysholm score, and the Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form were recorded. Synovial fluid was collected from both the operative knee and asymptomatic contralateral knee. Synovial fluid was stored with a protease inhibitor at 80 C until analysis. Intraoperative findings, procedures performed, and International Cartilage Repair Society (ICRS) cartilage status scores in all operative knees were documented. The concentrations of the following 20 biomarkers were measured using a multiplex magnetic bead immunoassay: matrix metalloproteinase (MMP) 3; MMP-13; tissue inhibitor of metalloproteinase (TIMP) 1; TIMP-2; TIMP-3; TIMP-4; fibroblast growth factor 2; eotaxin; interferon g; interleukin (IL) 10; platelet-derived growth factor BB; IL-1 receptor antagonist; IL1b; IL-6; monocyte chemotactic protein 1 (MCP-1); macrophage inflammatory protein 1a; macrophage inflammatory protein 1b; RANTES (regulated upon activation, normal T cell expressed and secreted); tumor necrosis factor a; and vascular endothelial growth factor. Clinical outcome scores were obtained in 83% of patients at a mean of 17 months’ follow-up postoperatively. Analysis of variance and Pearson correlation analysis were performed to determine statistical significance between preoperative data, intraoperative findings, postoperative outcomes, and synovial fluid biomarker concentrations compared with asymptomatic contralateral knees. Results: Analysis was performed on 70 operative and 32 contralateral samples. There were strong positive correlations between ICRS score and age, symptom duration, VAS score, and Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form. A strong positive correlation was found between MCP-1 and IL-6 concentrations, intraoperative ICRS score, and continued pain at the time of final follow-up. MCP-1 and IL-6 were the strongest predictors of severe cartilage lesions, whereas IL-1 receptor antagonist was inversely related. MMP-3 levels were consistently elevated in all operative samples and directly correlated to increased preoperative VAS scores. RANTES, vascular endothelial growth factor, and platelet-derived growth factor BB were the strongest predictors of postoperative improvement at final follow-up regardless of injury and cartilage status. Conclusions: Synovial fluid biomarkers have the capacity to reflect the intra-articular environment before surgery and potentially predict postoperative clinical outcomes. Recognition of key molecular players may yield future therapeutic targets, and large clinical trials exploring these discoveries are anticipated. Level of Evidence: Level III, therapeutic casecontrol study.
From the Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, New York, New York, U.S.A. The authors report the following potential conflict of interest or source of funding: V.G.C. receives support from Cytonics. J.M.C. receives support from Cytonics. E.J.S. receives support from DePuy Mitek and Jaypee Publishing. Presented at the American Academy of Orthopaedic Surgeons Annual Meetings, New Orleans, LA, March 11-15, 2014, and Las Vegas, NV, March 24-28, 2015, and the Arthroscopy Association of North America Annual Meeting, Hollywood, FL, May 1-3, 2014.
Received December 12, 2014; accepted August 25, 2015. Address correspondence to Eric J. Strauss, M.D., Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 333 E 38th St, Fourth Floor, New York, NY 10016, U.S.A. E-mail: [email protected] Ó 2015 by the Arthroscopy Association of North America 0749-8063/141046/$36.00 http://dx.doi.org/10.1016/j.arthro.2015.08.033
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espite advances in magnetic resonance imaging (MRI) techniques, sensitivity and specificity for intra-articular cartilage pathology remain imperfect. Furthermore, the ability to preoperatively predict which patients with or without cartilage lesions will improve after arthroscopic surgery is even more challenging. The ability to understand the molecular milieu associated with various cartilage pathologies through biomarker analysis may have an impact on clinical practice. The use of biomarkers as part of the diagnostic workup and treatment decision-making process is gaining considerable attention and has become widely acknowledged in modern medicine. Biomarkers may be identified in the blood, urine, or synovial fluid of patients and represent objective indicators of normal processes, pathology, or responses to therapeutic intervention. In the field of orthopaedics, biomarkers have been explored in the setting of a variety of pathologies, most frequently in cases of osteoarthritis, infection, or tumor.1-4 Most studies to date have investigated biomarkers in the blood or urine. However, it is becoming increasingly appreciated that intraarticular pathologies may be more accurately described by analysis of joint-specific synovial fluid in addition to serum analysis.5 Several protein biomarkers have been explored as diagnostic tools, prognostic indicators, and surrogate endpoints for therapeutic intervention in both in vitro and in vivo models of cartilage injury.6 Inflammatory cytokines, metalloproteinases, proteases, and degradation products of cartilage have all been profiled in the pathophysiology of cartilage degradation.7-10 It is well established that diagnosis of knee cartilage injury is underappreciated by MRI studies.11 In one study, chondral lesions were observed intraoperatively in 45% of patients previously unrecognized by MRI.12 In other studies, cartilage degeneration was detected using molecular analysis that was otherwise not apparent using traditional MRI.13-16 The impact of these unrecognized chondral defects discovered at the time of arthroscopy on surgical outcomes is not known. The goal of this study was to determine whether molecular biomarkers exist that could predict the severity of chondral defects found at the time of surgery, as well as whether these potential early markers have any prognostic significance on postoperative clinical outcomes. This study aimed to correlate the intraoperative concentrations of 20 synovial fluid biomarkers with preoperative symptoms, intraoperative findings, and postoperative outcomes in patients undergoing knee arthroscopy, with comparisons made with samples obtained from asymptomatic knees. Our hypothesis was that a combination of molecular markers such as proteases and inflammatory mediators
could predict more advanced (grade III and IV) chondral lesions found intraoperatively and predict continued pain postoperatively.
Methods Patient Enrollment and Study Questionnaires This was a single-center institutional review boardeapproved study in which 81 consecutive patients were prospectively enrolled over a 2-year period. All patients who were indicated for knee arthroscopy by 2 board-certified sports surgeons (E.J.S.) and met the inclusion criteria were invited to participate, and chart reviewdin addition to patient history and physical examination findingsdwas used to identify the presence of any exclusion criteria. The exclusion criteria included the following parameters: age younger than 18 years, systemic inflammatory disease (e.g., rheumatoid arthritis), autoimmune disease, intra-articular injection in the 3 months before surgery, prior knee surgery, knee surgery within the follow-up period, immunomodulatory drug use, chemotherapy within the past year, planned open procedure, or cartilage/ meniscal transplant in addition to arthroscopy. The 3month limit for intra-articular injections was selected based on our experience with standards imposed by prior clinical trials for injection treatments and our opinion that this time frame limits external influence from previous interventions. Preoperatively, enrolled patients completed clinical questionnaires including a visual analog scale (VAS), the Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS), and the Lysholm scale.17 All patients who reported no pain and no history of injury in the contralateral knee also completed clinical questionnaires with respect to their nonoperative knee and were invited to provide a sample from the contralateral knee at the time of surgery to serve as an internal control. In 11 patients who contributed synovial fluid samples from both the operative knee and the contralateral knee, the operative sample was excluded from analysis because of subsequent surgery within the follow-up period or failure to obtain an adequate amount of synovial fluid from the operative knee. All patients meeting the inclusion criteria for the contralateral kneedspecifically, no history of surgery or pain in the contralateral knee and a currently asymptomatic contralateral kneedwere asked to contribute contralateral samples. Among the 70 operative knees, 49 patients either did not meet the inclusion criteria for the contralateral knee or refused aspiration. Aspiration of each knee was performed only once in the operating room in all patients, and thus each knee provided 1 sample of synovial fluid. Patient samples were excluded from analysis if no synovial fluid was aspirated.
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Table 1. Description of Biomarkers Analyzed in Study Biomolecule MMP-3 MMP-13 TIMP-1, -2, -3, and -4 FGF-2 Eotaxin IFNg IL-10 PDGF-BB IL-1b IL-1Ra IL-6 MCP-1 MIP-1a and MIP-1b RANTES TNFa VEGF
Basic Functions Stromelysin that degrades collagen II, IV, IX, X, and XI, as well as proteoglycans, fibronectin, laminin, and elastin; activates other MMPs Collagenase that degrades collagen I, II, III, IV, IX, X, and XIV Inhibition of MMPs; chondroprotective role Promotes angiogenesis, wound healing, and granulation tissue formation Small chemokine that selectively recruits eosinophils Cytokine that is critical for innate and adaptive immunity against viral and bacterial infections; activator of macrophages and MHC-II expression Cytokine with anti-inflammatory effects through downregulation of Th1 cytokine expression, MHC-II antigens, and macrophage activators; enhances B-cell survival and blocks NF-kB activity Growth factor that plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis Cytokine produced by activated macrophages; important mediator of inflammatory response; activates cyclooxygenase 2 Cytokine that inhibits proinflammatory effects of IL-1b Cytokine secreted by T cells and macrophages acting in proinflammatory function, especially during infection or after trauma; can stimulate IL-10 and IL-1Ra production to act in negative feedback loop of inflammatory response Chemokine that recruits monocytes, memory T cells, and dendritic cells to site of inflammation by tissue injury or infection Chemokines produced by macrophages; activate neutrophils, eosinophils, and basophils in addition to other cytokines such as IL-1, IL-6, and TNFa Chemokine that recruits leukocytes to site of inflammation and activates natural killer cells Cytokine produced by macrophages, lymphocytes, natural killer cells, and neutrophils and involved in acute phase of inflammatory response; can induce cell apoptosis Growth factor that induces angiogenesis
FGF-2, fibroblast growth factor 2; IFNg, interferon g; IL, interleukin; IL-1Ra, interleukin 1 receptor antagonist; MCP-1, monocyte chemotactic protein 1; MHC-II, major histocompatibility complex II; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NF-kB, nuclear factor kB; PDGF-BB, platelet-derived growth factor BB; RANTES, regulated upon activation, normal T cell expressed and secreted; Th1, T helper cell type 1; TIMP, tissue inhibitor of metalloproteinase; TNFa, tumor necrosis factor a; VEGF, vascular endothelial growth factor.
Synovial Fluid Collection and Storage In the operating room, after sterile preparation but before arthroscopic portal creation, synovial fluid sampling was performed using the superolateral portal location, with an 18-gauge needle and a 10-mL syringe. The maximal amount of synovial fluid that egressed was collected; however, only a few microliters was necessary for molecular analysis. Synovial fluid samples were transferred to sterile tubes containing a protease inhibitor cocktail solution (Halt Protease Inhibitor Cocktail, EDTA free; Pierce Biotechnology, Rockford, IL). The samples were stored on ice during transport to the laboratory, where they were centrifuged at 1,350g for 10 minutes and supernatant aliquoted into sterile tubes, before storage at 80 C. Biomarker Analysis and Intraoperative Findings The samples were analyzed for the presence of 20 biomarkers using a multiplex bead assay (Milliplex; Millipore, Billerica, MA) including matrix metalloproteinase 3 (MMP-3); MMP-13; tissue inhibitor of metalloproteinase (TIMP) 1; TIMP-2; TIMP-3; TIMP-4; fibroblast growth factor 2; eotaxin; interferon g; interleukin (IL) 10; platelet-derived growth factor BB (PDGFBB); IL-1 receptor antagonist; IL-1b; IL-6; monocyte chemotactic protein 1 (MCP-1); macrophage inflammatory protein (MIP) 1a; MIP-1b; RANTES (regulated upon activation, normal T cell expressed and secreted);
tumor necrosis factor a; and vascular endothelial growth factor (VEGF). These specific biomarkers were chosen because they are known players in joint pain, inflammation, and cartilage degeneration (Table 1). Before the assay, the synovial fluid samples were treated with hyaluronidase18; they were then assayed according to the manufacturer’s protocol. Intraoperative arthroscopic findings were reported by the surgeon (E.J.S.) and recorded by a trained research assistant during the initial diagnostic arthroscopy, including soft-tissue injury and cartilage pathology using the International Cartilage Repair Society (ICRS) grading scale.19 For patients with multiple cartilage lesions, data on all lesions were recorded but the ultimate score assigned was based on the highest-grade lesion identified (Table 2). Postoperative Follow-up In addition to routine postoperative visits, patients were contacted for follow-up at 1 and 2 years after arthroscopy and completed clinical questionnaires (VAS, KOOS-PS, and Lysholm scale). These data were compared with preoperative data, intraoperative findings, and synovial fluid biomarker concentrations. Statistical Methods Clinical questionnaire data were analyzed for all operative and contralateral knee samples with respect
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Table 2. Profile of Intra-articular Injuries With Respect to Cartilage Grade at Time of Surgery Patients/Operative Samples Included in Analysis, n Injury Subgroup ACL tear only Meniscus tear only ACL plus meniscus tear Cartilage lesion only
ICRS Grade 1 7 6 9 0
ICRS Grade 2 1 6 5 2
ICRS Grade 3 2 18 5 3
ICRS Grade 4 0 5 1 0
ACL, anterior cruciate ligament; ICRS, International Cartilage Repair Society.
to biomarker concentrations using analysis of variance. Pearson correlation coefficients were used to compare patients’ age, duration of symptoms, and ICRS grade with respect to biomarker concentrations.
Results Study Population and Intraoperative Findings Clinical and operative data were collected from 81 patients enrolled in the study. Synovial fluid was collected from 70 operative knees and 32 contralateral knees. Clinical data were obtained for 67 patients at a minimum follow-up duration of 1 year (mean, 17 6 months), representing an 83% rate of follow-up. The 70 operative knees from which synovial fluid samples were collected included 10 isolated anterior cruciate ligament (ACL) injuries, 35 isolated meniscal injuries, 20 ACL and meniscal injuries, and 5 knees with isolated cartilage pathology. Among the operative knee samples, there were 22 ICRS grade 1 lesions, 14 ICRS grade 2 lesions, 28 ICRS grade 3 lesions, and 6 ICRS grade 4 lesions. There was no difference in age between samples collected from operative and contralateral knees except when contralateral samples were compared with samples from patients with ICRS grade 1 lesions; the mean age of patients contributing contralateral knee samples was 41 15 years compared with 28 8 years, 43 14 years, 46 14 years, and 53 6 years for patients with ICRS grade 1, grade 2, grade 3, and grade 4 lesions, respectively (P < .001). Preoperatively, the median VAS score, KOOS-PS, and Lysholm score were 4.0, 27, and 65, respectively, for operative knees and 0, 0, and 100, respectively, for contralateral knees. The median duration of symptoms in all patients was 3 months (range, 1 to 264 months). There was a significant and direct linear correlation between preoperative VAS score and ICRS score (Fig 1). Median VAS pain scores were subdivided by injury type, the most painful, on average, being combined meniscus and cartilage injuries, with a median VAS score of 5, followed by ACL and meniscus injuries with cartilage lesions (VAS score, 4), ACL injuries with cartilage lesions (VAS score, 3), cartilage lesions only
(VAS score, 3), and isolated meniscus or ACL injuries without cartilage lesions (VAS score, 3) (P < .001). Intra-articular Biomarkers The mean biomarker concentrations ( standard error of the mean) were significantly greater in the operative samples compared with samples from the contralateral knees (non-paired) for MMP-3 (481,341 23,433 pg/mL v 164,285 25,998 pg/mL; P < .001), TIMP-1 (69,564 2,440 pg/mL v 55,939 4,638 pg/mL; P < .01), IL-6 (298 62 pg/mL v 21 14 pg/mL; P < .01), and MIP-1b (15 8 pg/mL v 9 1.3 pg/mL; P < .01), whereas the inverse relation was observed for TIMP-2 (44,221 1,257 pg/mL v 52,547 4,415 pg/mL; P < .05). Compared with samples from the contralateral control knees, samples from ICRS grade 1 to 4 lesions had greater concentrations of MMP3 (contralateral, 164,285 19,972 pg/mL; ICRS grade 1, 514,667 49,059 pg/mL; ICRS grade 2, 441,727 68,680 pg/mL, ICRS grade 3, 451,706 68,353 pg/mL; and ICRS grade 4, 583,296 12,938 pg/mL) and IL-6 (contralateral, 21 10 pg/mL; ICRS grade 1, 312 184 pg/mL; ICRS grade 2, 266 137 pg/mL; ICRS grade 3, 254 135 pg/mL; and ICRS grade 4, 532 158 pg/mL) (P < .001). For both IL-6 and MMP-3 (data not shown), the greatest concentrations were measured within samples from knees with meniscus or ACL injuries without cartilage lesions. Conversely, RANTES concentrations were significantly elevated only in knees with isolated cartilage lesions (Fig 2). When subgrouping by VAS score was performed, increased concentrations of IL-6 and MMP-3 correlated with an increased preoperative VAS score (Figs 3 and 4). By use of correlation analysis, preoperative VAS scores
Fig 1. Preoperative median visual analog scale pain score with respect to International Cartilage Repair Society (ICRS) score in operative knees and contralateral (CL) knees. * Significant difference between each ICRS group and with the contralateral knee. y Significant difference between ICRS 2 patients and ICRS 1 and 4 patients. z Significant difference between ICRS 3 patients and ICRS 1 and 4 patients.
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Fig 2. (A) Interleukin 6 (IL-6) concentration (in picograms per milliliter) and (B) regulated upon activation, normal T cell expressed and secreted (RANTES) concentration (in picograms per milliliter) subdivided by injury category compared with contralateral samples. (ACL, anterior cruciate ligament.) * Significant difference compared to contralateral knees.
correlated with the concentrations of MMP-3 (r ¼ 0.4, P < .0001), TIMP-1 (r ¼ 0.21, P < .03), IL-10 (r ¼ 0.2, P < .03), and IL-6 (r ¼ 0.23, P < .01). In addition, the concentrations of several analytes correlated to preoperative clinical parameters (Table 3). The concentrations of several molecules were not significantly different in the operative knee synovial fluid samples compared with the contralateral knee samples (MCP-1, TIMP-4, VEGF, RANTES, PDGF-BB, MMP-13, eotaxin, interferon g, IL10, tumor necrosis factor a, MIP-1a, and IL-1b). Clinical Follow-up Data and Biomarkers Clinical data were obtained for 67 patients at a minimum follow-up duration of 1 year (mean, 17 6
months), representing an 83% rate of follow-up. For operative knees, the median follow-up VAS score, KOOS-PS, and Lysholm score were 1.0, 15, and 91, respectively, compared with 4.0, 27, and 65, respectively, preoperatively. Patients with isolated cartilage lesions reported higher VAS and KOOS-PS scores and lower Lysholm scores compared with patients without cartilage lesions (Fig 5). In general, patients with isolated cartilage lesions reported the least amount of improvement at follow-up, with no statistically significant difference between preoperative and follow-up VAS, Lysholm, or KOOS-PS scores (Fig 6). Patients without evidence of cartilage pathology intraoperatively had the best outcomes at the most recent
Fig 3. Interleukin 6 (IL-6) concentration (in picograms per milliliter) subdivided by visual analog scale (VAS) pain score preoperatively. * Significant difference compared to patients with a mean preoperative VAS score of 0-2.
Fig 4. Matrix metalloproteinase 3 (MMP-3) concentration (in picograms per milliliter) subdivided by visual analog scale (VAS) pain score preoperatively. * Significant difference compared to patients with a mean preoperative VAS score of 0-2.
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Table 3. Correlation Analysis to Determine Which Biomarkers Correlated With Preoperative Clinical Parameters Using Pearson Correlation Coefficient (r) Parameter (Preoperative) VAS VAS VAS VAS Lysholm score Lysholm score Lysholm score Lysholm score Lysholm score Lysholm score KOOS-PS KOOS-PS KOOS-PS KOOS-PS
Analyte MMP-3 TIMP-1 IL-10 IL-6 MMP-3 TIMP-2 FGF-2 IL-10 IL-6 MCP-1 MMP-3 TIMP-2 FGF-2 IL-6
Correlation Coefficient (Pearson r) 0.4 0.21 0.2 0.23 0.50 0.28 0.23 0.33 0.4 0.23 0.44 0.28 0.25 0.32
P Value .0001 .03 .03 .01 .0001 .0058 .02 .001 .0001 .0001 .0001 .019 .03 .007
FGF-2, fibroblast growth factor 2; IL, interleukin; KOOS-PS, Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form; MCP-1, monocyte chemotactic protein 1; MMP-3, matrix metalloproteinase 3; TIMP, tissue inhibitor of metalloproteinase; VAS, visual analog scale pain score.
follow-up, but there was not a significant difference between outcome and ICRS score when subdivided by ICRS scores 1 through 4. Improvement in VAS score correlated with preoperative PDGF-BB (r ¼ 0.29) and RANTES (r ¼ 0.28) levels, with the lowest levels predicting the best outcomes (P < .05). An increased concentration of TIMP-3 was a predictor of higher VAS scores at follow-up in patients who underwent ACL reconstruction but not in patients with meniscus injuries only (r ¼ 0.43, P < .05). Concentrations of MMP-3 (r ¼ 0.26, P < .017), TIMP1 (r ¼ 0.21, P < .05), IL-10 (r ¼ 0.34, P < .001), and MCP-1 (r ¼ 0.32, P < .003) weakly correlated with follow-up Lysholm scores. Follow-up KOOS-PS was also weakly correlated with MMP-3 (r ¼ 0.22, P < .04), IL-6 (r ¼ 0.22, P < .05), and MCP-1 (r ¼ 0.27, P < .01) concentrations. The ratio of particular biomarkers to one another was investigated for possible associations with clinical parameters, particularly the relation of MMPs to TIMPs. There was a moderate negative correlation between the MMP-3:TIMP-2 ratio and preoperative Lysholm score (the greater the ratio, the worse the Lysholm score; r ¼ 0.53, P < .0001) and between the MMP-3:TIMP-2 ratio and follow-up Lysholm score (r ¼ 0.21, P < .05). There were similar correlations between the MMP3:TIMP-3 ratio and preoperative VAS score (r ¼ 0.03, P < .005), Lysholm score (r ¼ 0.32, P < .005), and KOOS-PS score (r ¼ 0.29, P < .05). Contralateral Knees in Which Symptoms Developed Patients who initially had no pain in their contralateral knee and contributed a control sample from that
knee but in whom pain later developed in the contralateral knee, defined as a VAS score greater than 2, were more likely to have had elevated concentrations of MCP-1 (P < .05) and VEGF (P < .05) and decreased RANTES concentrations (P < .05) at baseline compared with patients whose knee remained asymptomatic at follow-up (Fig 7).
Discussion This study identified several synovial fluid biomarkers associated with intra-articular knee pathology, pain, and clinical outcomes in patients undergoing knee arthroscopy. MCP-1 and IL-6 were the strongest predictors of more severe cartilage lesions intraoperatively independent of associated pathologies and also predicted worse outcomes at follow-up. MMP-3 concentrations were consistently elevated in all operative samples and were directly correlated to increased preoperative VAS scores. Levels of PDGF-BB, RANTES, and VEGF were able to predict clinical outcomes better than preoperative VAS scores, and patients with elevated MCP-1, VEGF, and decreased RANTES concentrations in their asymptomatic knees were more likely to have subsequent pain develop at final followup. Our observation of an elevated IL-6 level as a predictor of severe cartilage injury supports prior observations.20-22 IL-6 has been characterized as having a “pleiotropic” role and being involved in both innate immunity and maintenance of chronic inflammation.23 IL-6 may also be consistently associated with painful joints, as it has been shown to sensitize C fibers innervating the knee joint24 through the prostaglandincyclooxygenase inflammatory pathway.25,26 Similarly, MCP-1 elevation in the setting of intraoperative pathology has been shown in prior studies.22 MCP-1 has a heterogeneous role in inflammation and response to injury and has been shown to recruit stem cells and activate transcription blockers for chondrogenesis.27 Comparably to IL-6 and other chemokines and cytokines, it also appears to be directly involved in sensitizing nociceptors and promotes long-term dorsal root ganglion excitability, thus contributing to chronic pain.28,29 In contrast, PDGF-BB concentrations were highest in contralateral knees and depressed across all operative knees, most strikingly in those with advanced cartilage degeneration. PDGF-BB is a growth factor known to antagonize catabolic factors and to inhibit IL1beinduced cartilage degeneration.30 Our study indicates a possible protective role of PDGF-BB, with its absence allowing IL-1beinduced cartilage degeneration to proceed unchecked. Moreover, these results suggest that across all patients, those with the highest levels of PDGF-BB had the lowest pain scores at follow-up. A mean increase in this biomarker predicted
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7
Fig 5. (A) Visual analog scale (VAS) pain score, (B) Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS) activity score, and (C) Lysholm activity score preoperatively (Pre-op) compared with score at final follow-up in patients with cartilage injury (International Cartilage Repair Society [ICRS] grade 2, 3, or 4) and without cartilage injury (ICRS grade 1).
improvement in VAS score, independent of intraoperative cartilage score or associated soft-tissue pathology. This observation of elevated levels in contralateral knees compared with severe cartilage injury in operative knees was similar to measured levels of RANTES, particularly in terms of predicting pain at follow-up. RANTES is similar to other cytokines in that it modulates the response of lymphocytes after injury; it has been identified in other synovial fluid studies as well.31,32 RANTES is known to promote IL-6 production and increase proteoglycan levels in cartilage33,34; therefore its relatively decreased levels in contralateral knees that go on to develop pain similar to those with more advanced cartilage injury may lend insight into the pathophysiology of chronic disease after injury. Not surprisingly, the converse was observed in measured VEGF levels. VEGF has been found in cartilage tissue after injury or osteoarthritis in many other studies, particularly late osteoarthritis, and appears to inhibit aggrecan and collagen II synthesis, both of which are important in cartilage regeneration.35-38 Thus the
higher levels of VEGF in those knees in which pain developed are not unexpected. We also observed an increased ratio of MMP-3 to TIMPs correlated with worse clinical parameters, which may suggest that TIMPs function in a protective role.39 The presence of MMPs and TIMPs in synovial fluid has been widely investigated and consistently shows a pivotal relation with cartilage degradation.8-10 Several studies have suggested that chondrocytes up-regulate MMP expression in the progression of cartilage degradation.40-43 It is currently accepted that variable mechanical loading may up-regulate MMP expression; however, it is not clear if this is the instigating event.44,45 MMPs increase during abnormal cartilage loading, such as after an ACL tear; however, they are also induced by proinflammatory cytokines, in particular IL-1 and tumor necrosis factor.7 The consistent association with cartilage matrix breakdown and inflammatory cytokines underscores the importance of MMPs in osteoarthritis.46 Those patients without cartilage lesions did the best at final follow-up, and the presence of any cartilage
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Fig 6. (A) Visual analog scale (VAS) pain scores at preoperative baseline (Pre-op) and at final follow-up subdivided by injury. (B) Preoperative (preop) and final follow-up Lysholm activity scores subdivided by injury. (C) Preoperative (Pre-op) and final followup Knee Injury and Osteoarthritis Outcome ScoreePhysical Function Short Form (KOOS-PS) activity scores subdivided by injury. (ACL, anterior cruciate ligament.) * Significant difference between pre-operative and post-operative scores.
lesions predicted a worse functional outcome overall. However, the difference between preoperative and postoperative clinical scores among all samples was not significant. This finding is important because it highlights the sensitivity of the biomarkers studied for detecting differences between these 2 groups. Preoperative clinical scores associated with cartilage injury severity were not able to predict progression, as was seen with certain biomarkers such as PDGFBB, RANTES, and VEGF. Patients who initially had no pain in their contralateral knee and contributed a control sample from that knee but in whom pain later developed, defined as a VAS score greater than 2, were more likely to have had elevated concentrations of MCP-1, VEGF, and decreased RANTES compared with those who remained asymptomatic at follow-up. The timing of fluid collection with respect to the date of injury or initial onset of pain is relevant to our results. Our patients reported a mean duration of symptoms of 14 months; however, a range of 1 to 264
months (median, 3 months) was present. Clearly, this reflects the fact that most of our patients undergoing surgery presented with meniscal tears, which are more heterogeneous in terms of clinical symptoms and history compared with our smaller cohort of patients with ACL tears, which tend to present earlier after injury. The differential profile of inflammatory markers with respect to time since injury is an area of active research. A few studies have focused on ACL tears in particular and have shown that there is an initial inflammatory response that differs from chronic cases.47-49 Our study was limited by the number of patients in our injury subgroups, which prevented adequate analysis of this factor independently. Our study aims to reflect a more general molecular milieu in “all-comers” that may help the clinician understand which patients are more likely to have continued pain despite surgical intervention. Future studies may distinguish differences based on the time of presentation alone, regardless of preoperative pain scores or cartilage status; however, it is possible that the molecular profile alone is informative,
9
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Acknowledgment The authors acknowledge Laith M. Jazrawi, M.D., Priya Mukhopadhyay, B.S., William Small, B.S., and Naveen Chadalavada, B.S.
References
Fig 7. Mean preoperative cytokine concentrations (in picograms per milliliter) of monocyte chemotactic protein 1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF) in contralateral knee samples in patients with no pain (visual analog scale [VAS] score of 0) and patients with pain (VAS score >2) at final follow-up. * Significant difference between patient groups.
regardless of length of symptoms, presence of meniscal tear, or number of cartilage lesions, for example. Limitations The specimens obtained from the contralateral knee provided only a clinical control because arthroscopic evaluation was not possible. Future studies will include MRI scans of the contralateral knees to identify any structural pathology present. Not all patients consented to undergo contralateral aspiration, and not all knees provided an adequate amount of synovial fluid for analysis, which may present a bias in our study samples. When largely grouped by cartilage pathology or pain, there are many differences; however, the subgroup analysis is limited by smaller numbers in each group (e.g., ACL tear only). There was variation in the time to presentation after injury and synovial fluid sampling, with some patients having symptoms for several months to years and other patients having a more acute presentation and treatment. Our findings are currently limited by the follow-up duration, and we anticipate that more differences may become apparent with longerterm observation and retrospective analysis.
Conclusions Synovial fluid biomarkers have the capacity to reflect the intra-articular environment before surgery and potentially predict postoperative clinical outcomes. Recognition of key molecular players may yield future therapeutic targets, and large clinical trials exploring these discoveries are anticipated.
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