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Inflammatory cytokines and matrix metalloproteinases in the synovial fluid after intra-articular elbow fracture
J Shoulder Elbow Surg (2019) -, 1–7
www.elsevier.com/locate/ymse
Inflammatory cytokines and matrix metalloproteinases in the synovial fluid after intra-articular elbow fracture Elizabeth P. Wahl, MD*, Alexander J. Lampley, MD, Angel Chen, BA, Samuel B. Adams, MD, Dana L. Nettles, PhD, Marc J. Richard, MD Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, USA Background and hypothesis: Post-traumatic elbow contracture remains a common and challenging complication with often unsatisfactory outcomes. Although the etiology is unknown, elevated or abnormal post-fracture synovial fluid cytokine levels may result in the migration of fibroblasts to the capsule and contribute to capsular pathology. Thus, the purpose of this study was to characterize the cytokine composition in the synovial fluid fracture hematoma of patients with intra-articular elbow fractures. Methods: The elbow synovial fluid fracture hematoma of 11 patients with intra-articular elbow fractures was analyzed for CTXII (C-terminal telopeptides of type II collagen [a cartilage breakdown product]) as well as 15 cytokines and matrix metalloproteinases (MMPs) including interferon g, interleukin (IL) 1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor a, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. The uninjured, contralateral elbow served as a matched control. Mean concentrations of each factor were compared between the fluid from fractured elbows and the fluid from control elbows. Results: The levels of 14 of 15 measured cytokines and MMPsdinterferon g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor a, MMP-1, MMP-3, MMP-9, and MMP-10dwere significantly higher in the fractured elbows. In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power. The mean concentration of CTXII was not significantly different between groups. Conclusions: These results demonstrate a proinflammatory environment after fracture that may be the catalyst to the development of post-traumatic elbow joint contracture. The cytokines with elevated levels were similar, although not identical, to the cytokines with elevated levels in studies of other weightbearing joints, indicating the elbow responds uniquely to trauma. Level of evidence: Basic Science Study; Molecular Biology Ó 2019 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved. Keywords: Elbow contracture; elbow fracture; elbow stiffness; intra-articular; synovial fluid; intra-articular elbow fracture; elbow synovial fluid
This study was approved by the Duke University Health System Institutional Review Board for Clinical Investigations (Pro00072559).
*Reprint requests: Elizabeth P. Wahl, MD, Department of Orthopaedic Surgery, Duke University Medical Center, 4709 Creekstone Dr, Ste 200, Durham, NC 27703, USA. E-mail address: [email protected] (E.P. Wahl).
1058-2746/$ - see front matter Ó 2019 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved. https://doi.org/10.1016/j.jse.2019.09.024
2 Post-traumatic elbow joint contracture, a wellrecognized and debilitating sequela after intra-articular elbow fractures, is characterized by heterotopic bone formation, failure of fracture healing, soft tissue contracture (fibrosis), capsular thickening, and/or arthrosis30,31 and often results in significant disability.12,30,33-35,40,42,45 Despite considerable efforts to understand and prevent elbow joint contracture, the pathogenesis remains unclear, with surgical release being required in up to 15% of patients to restore motion.35 Furthermore, approximately 20% of patients will require a subsequent release with reported rates of complications (eg, ulnar nerve dysfunction) as high as 9%,30,40 and elbow range of motion is often not restored to normal, with patient satisfaction reported as fair at best.33 This has resulted in considerable efforts to elucidate the processes involved in this pathology. Hildebrand et al23 have studied post-traumatic knee joint capsules of rabbits and contracted elbow capsules of humans and found significantly elevated numbers of myofibroblasts and mast cells in the capsules of the contracted joints compared with healthy donor joints. Cohen et al4 found increases in collagen disorganization and the fibroblast presence in the diseased capsules compared with the healthy capsules, along with elevated staining for matrix metalloproteinase (MMP) 1, MMP-2, and MMP-3. A subset of stiff elbows will also display heterotopic ossification (HO) owing to progenitor cell differentiation into osteoblasts in the soft tissues of the elbow.30,31,36 Although these features of contracture may take several weeks to months to manifest, it is hypothesized that the processes that lead to this pathology may begin immediately after the traumatic event.15,44 In an effort to understand post-traumatic pathologies in other synovial joints, several studies have analyzed the synovial fluid (SF) content after trauma in the knee and ankle and demonstrated a highly proinflammatory and catabolic environment1,14; however, differential profiles and long-term outcomes between these joints suggest a joint-specific response to insult.13 Although a similar unfavorable environment would be expected in the elbow after joint trauma, the pathology of joint contracture in the elbow is of greater concern than that in the knee or ankle, suggesting a unique response by elbow joint tissues. Thus, the purpose of this study was to compare the acute-phase SF cytokine profile after elbow fracture with SF from healthy elbows (contralateral elbows of the same patients) to identify factors that could contribute to an increased risk of development of various features of post-traumatic elbow capsular contracture and serve as potential early targets for intervention.
E.P. Wahl et al. with a mean age of 56 years (range, 27-80 years), comprising 7 women and 4 men. An intra-articular elbow fracture was defined as any fracture of the humerus, ulna, or radius in which the fracture line extended into the articular surface of the elbow joint. The inclusion criteria consisted of an operatively treated intra-articular elbow fracture, an uninjured contralateral elbow that was pain free, and no history of trauma in either elbow. Of the patients, 2 had isolated humeral fractures; 1 had an isolated olecranon fracture; 1 had an isolated radial head fracture; and 7 had combined humeral, ulnar, and/or radial fractures. All fractures were displaced at least 2 mm and were isolated injuries. The uninjured elbow served as a matched control. Patients were excluded if they were younger than 18 years; were pregnant; or had a history of cancer, diabetes, hemophilia, or systemic inflammatory disease. Participation followed informed consent in accordance with an approved institutional review board protocol at our institution.
Elbow joint SF aspiration and lavage SF was obtained from the injured elbow at the time of initial presentation to the emergency department if the patient was seen by a consulting orthopedist, as described previously.3 SF was also obtained from the bilateral elbows at the time of surgery in the same manner. As the aspirate from the injured side contained considerable fracture hematoma, it is more appropriately characterized as SF fracture hematoma. After bilateral upper-extremity surgical scrubbing and draping, arthrocentesis was performed on the elbow joints in sequential fashion using an 18-gauge needle and a 10-mL syringe. The needle was inserted through the standard posterolateral approach as previously described.3 If no fluid was obtained or an inadequate amount (<2 mL) was obtained from the control side on initial arthrocentesis, the 10-mL syringe was removed from the needle and a sterile saline solution flush syringe was attached to the needle. One to two milliliters of sterile saline solution was injected into the elbow joint, the sterile saline solution flush syringe was removed, and the 10-mL syringe was reattached to the needle for elbow re-aspiration. There were no incidences in which the injured elbow required lavage. The fluid samples were transferred to a 15-mL conical centrifuge tube (Falcon; Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at room temperature to remove cells and particulate matter (3500 rpm, 15 minutes27); the resultant supernatant was then aspirated, aliquoted, labeled, and stored at –80 C in cryopreservation tubes (Nalgene; Thermo Fisher Scientific).
Serum collection If the control elbow joint required lavage, then a whole blood sample was collected concurrently to measure serum urea concentrations to correct for dilution through lavage, as described later. The blood was allowed to sit for 20 minutes in glass tubes and then centrifuged at room temperature, and the serum fraction was then processed and stored in the same manner as the SF sample.
Materials and methods Correction for dilution through lavage Patient enrollment, population, and study design This was a prospective case-control study of patients with unilateral intra-articular elbow fractures. We enrolled 11 patients in the study,
Lavage of the control elbow without knowing the starting amount of SF in the joint would prohibit calculation of the cytokine concentration. However, the proportion of urea in SF relative to
Synovial fluid analysis after elbow fracture serum, in both healthy and diseased states, has been shown to be a constant, fixed ratio.27,28 Urea has been described as a passive transport marker for arthritis biomarker studies when direct aspiration of joint fluid is difficult.1,2,27 To correct for the dilution effects of lavage, we measured the urea concentrations in both the serum and the SF using a colorimetric assay (QuantiChrom Urea Assay Kit; BioAssay Systems, Hayward, CA, USA). The dilution factor of the SF due to lavage was calculated by dividing the serum urea concentration by the SF urea concentration for each patient requiring lavage. Cytokine measurements were corrected for dilution factors prior to statistical analysis. This technique has been reported for evaluating the SF cytokine concentration, MMP composition, and cellular metabolites of other joints after intraarticular fracture.1,2
Cytokine analysis Fluid samples were analyzed for 15 cytokines and MMPs including interferon (IFN) g, interleukin (IL) 1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor (TNF) a, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. Concentrations of cytokines were determined from the fluid using a human proinflammatory panel (IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL10, IL-12p70, IL-13, TNF-a). Levels of MMP-1, MMP-3, and MMP-9 were quantified using an MMP 3-plex immunoassay, and levels of MMP-2 and MMP-10 were quantified using a human MMP 2-plex sandwich immunoassay. All assays described earlier were performed according to the manufacturer’s instructions (Meso Scale Discovery, Rockville, MD, USA).
Type II collagen catabolism Levels of degradation products of C-terminal telopeptides of type II collagen (CTXII), reflective of cartilage degradation, were quantified from fluid samples using a competitive immunoassay (Cartilaps; Immunmodiagnostic Systems, Scottsdale, AZ, USA). For this assay, all samples were pretreated with Streptomyces hyaluronidase (H-3884; Sigma-Aldrich, St Louis, MO, USA) for 1 hour at 37 C at 5 U/mL to reduce viscosity. The remainder of the assay was performed according to the manufacturer’s instructions.
Statistical methods Measured values for any sample that fell below the lower limit of detection (LLOD) were replaced by one-half the LLOD for statistical analysis. It should be noted that this only occurred for samples from the control sample population. Distributions of all cytokines and MMPs, as well as CTXII, from both the control and fracture groups were evaluated for normality using the ShapiroWilk W test. A paired t test was used to evaluate differences between the control and fracture groups for IL-1b, as this variable was found to be normally distributed in both the control and fracture populations. Differences between the control and fracture groups for all other variables were determined using the nonparametric Wilcoxon rank sum test. Differences were considered significant at P < .05. Statistical analysis was performed using JMP Pro software (version 14; SAS Institute, Cary, NC, USA). A post hoc power analysis was also performed for all measured variables.
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Results Eleven patients were enrolled in this study; in 1 patient, aspiration of the injured elbow was performed on the day of injury, whereas in the other 10, aspiration of the injured elbow was performed at the time of surgery. Five patients required lavage of the uninjured elbow at the time of surgery. The median time from fracture to surgery was 2.5 days (range, 0-17 days). After correction for dilution, mean concentrations of cytokines and MMPs including IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-a, MMP-1, MMP3, MMP-9, and MMP-10 were significantly higher in the fractured elbows compared with the healthy, contralateral elbows (Fig. 1). In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power (Table I). It is important to note that cytokine levels of many of the samples from the healthy group fell below the LLOD. In addition to cytokines, samples were analyzed for evidence of type II collagen breakdown through measurement of CTXII concentrations. The concentrations of CTXII between the healthy and fracture groups were not significantly different (P ¼ .97).
Discussion Our study provides an important step in understanding the environment surrounding the elbow after fracture. Similarly to the knee and ankle, the SF fracture hematoma is acutely proinflammatory and catabolic with elevated levels of soluble factors capable of initiating several signaling cascades involved in the processes that lead to elbow contracture.9,11,26,38,47 Although it is unknown why this pathology develops in only a portion of elbows, it is reasonable to hypothesize that in some individuals, the processes necessary for normal wound healing fail to subside, allowing abnormal tissue remodeling. Because the etiology in the elbow is still under investigation, it is helpful to examine the literature concerning contractures affecting other organ systems (eg, skin, lung, kidney, or liver),39,41,43 as some of these systems have processes in common with the elbow. One of the main causes of elbow contracture is the development of fibrosis surrounding the capsule. Studies in other organ systems have suggested that the myofibroblast is a key cell involved in this response to injury owing to its expression of the contractile protein a smooth muscle actin.25 During normal wound healing by secondary intent, the myofibroblast reduces the size of the wound using the cells’ contractile properties. Then, after the wound is healed, the myofibroblasts disappear through apoptosis.37 However, dysregulation of this healing response has been implicated as the cause of tissue fibrosis. In fact, studies have shown that the differentiation of the fibroblast into the
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Figure 1 Median (horizontal line), interquartile range (box), and minimum and maximum values (whiskers) for measured cytokine and matrix metalloproteinase (MMP) concentrations in synovial fluid from uninjured elbows (white bars) and fractured elbows (gray bars). *Statistically significant difference. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; CTX-II, C-terminal telopeptides of type II collagen; CTL, control.
myofibroblast is associated with other fibrotic conditions such as skin fibrosis6 and Dupuytren contracture.29 Likewise, in both human elbow joint contracture capsules and rabbit knee contracture models, Hildebrand et al17-24 have demonstrated that the myofibroblast is an ‘‘effector cell’’ in
Table I
the development of post-traumatic joint contracture through myofibroblast hyperplasia and excessive collagen production. Although the exact mechanism of this problematic myofibroblast hyperplasia is unknown, studies have
Cytokine and MMP levels in synovial fluid from healthy and fractured elbows
Cytokine or MMP
Healthy elbows
IFN-g, pg/mL IL-10, pg/mL IL-12p70, pg/mL IL-13, pg/mL IL-1b, pg/mL IL-2, pg/mL IL-4, pg/mL IL-6, ng/mL IL-8, ng/mL TNF-a, pg/mL MMP-1, mg/mL MMP-2, mg/mL MMP-3, mg/mL MMP-9, mg/mL MMP-10, ng/mL CTXII, ng/mL
0.33 0.15 0.07 2.39 0.17 0.42 0.10 0.004 0.012 0.80 0.002 0.09 0.10 0.03 1.74 0.51
0.39 0.29 0.08 4.92 0.15 0.7 0.23 0.007 0.009 0.82 0.002 0.05 0.07 0.04 1.82 0.24
Fractured elbows 8.37 2.89 5.62 11.65 4.47 1.36 45.6 1.76 1.38 2.58 0.24 0.09 1.3 0.28 3.48 0.60
5.32 1.84 2.91 5.79 3.81 0.93 93.70 0.37 0.80 0.60 0.43 0.04 0.91 0.25 2.74 0.44
P value
Power, %
<.0001 <.0001 <.0001 .0008 <.0001 .004 .0001 <.0001 <.0001 .0006 .0001 .86 .0002 .0043 .038 .97
99.9 99.8 100 98.1 96.2 76.4 36.3 100 100 100 45 N/S 98.9 90.1 42 N/S
IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; CTXII, C-terminal telopeptides of type II collagen; N/S, not significant. Data are presented as mean standard deviation with P values for the Wilcoxon rank sum test or Student t test (significance declared at P < .05) and percentages for post hoc power analysis.
Synovial fluid analysis after elbow fracture suggested that an excess of growth factors and cytokines that promote myofibroblast differentiation and inhibit myofibroblast apoptosis may cause the development of post-traumatic capsular contracture.10,48 These findings led to the hypothesis that the activation of mast cells, which results in synthesis and secretion of growth factors and cytokines known to have mitogenic effects on myofibroblasts, is important in the development of post-traumatic capsular contracture.16 This hypothesis was substantiated by Monument et al,32 who demonstrated that ketotifen, a mast cell stabilizer, was effective in significantly reducing not only the severity of joint capsule contracture but also mast cell and myofibroblast hyperplasia in a rabbit model of post-traumatic joint contracture. Although the literature supports the theory of mast cell–driven post-traumatic capsular contracture, it is still unclear what is activating the mast cell after injury. We hypothesize that a proinflammatory response after intraarticular fracture demonstrated in our study may contribute to molecular events that activate the mast cell. We found the concentrations of 10 cytokines and 4 MMPs to be significantly higher in the fractured elbows compared with the healthy, contralateral elbows. Although many are involved in a normal wound- and bone-healing process, they may also be involved in directly activating fibroblasts or indirectly activating them by initiating the mast cell–driven post-traumatic capsular contracture. For example, both transforming growth factor b and TNF-a are potent stimulators of fibroblasts, and IL-13 has been implicated in the fibrotic response, as it is a potent inducer of MMPs.20,46 MMP-2 and MMP-9 can further upregulate transforming growth factor b.47 In addition, IL-6 and TNFa enhance IFN-g production by mast cells, and IL-10 can directly enhance the mast cell number in the presence of IL-3 and stem cell factor.26 In contrast, IL-4 can inhibit stem cell factor, which is required for mast cell activation. Taken together, these findings suggest that soluble factors whose levels are elevated during normal wound healing may initiate joint pathology if homeostasis is delayed. Indeed, Desai et al5 compared the outcomes of patients undergoing operative fixation of closed elbow injury with an ulnohumeral dislocation, radial head fracture, and coronoid fracture (terrible-triad elbow injury) and found that the patients treated with steroids had significantly improved elbow range of motion at 24 weeks’ follow-up compared with those who did not receive steroids. The steroid, a potent anti-inflammatory, likely inhibited inflammatory signaling pathways, which may have resulted in inhibiting mast cell activation and the formation of arthrofibrosis. In addition to fibrosis, HO is a concern in the elbow and can result from stimulation of progenitor cells to become bone-forming cells in the presence of inflammation.38 HO contributes to loss of range of motion in up to 40% of elbow fractures.7,8 Although the mechanisms involved in HO are still under investigation, there is significant
5 evidence to suggest that prolonged inflammation, particularly IL-1b and TNF-a, may stimulate an HO cascade through a bone morphogenetic protein 2, prostaglandin E2, and cyclooxygenase 2 pathway.9,38 Both IL-1b and TNF-a levels were significantly elevated in fractured elbows in our study, and numerous studies have shown that prophylactic administration of nonsteroidal anti-inflammatory medications lowers the risk of HO,38 suggesting that this early inflammation may initiate HO immediately after trauma and/or surgery. It is interestingly to note that, compared with the SF after intra-articular fracture of the ankle and knee, our study demonstrated a similar proinflammatory profile. For example, Adams et al1 compared the SF between acutely fractured ankle joints and normal ankle joints in 21 patients and found that after intra-articular fracture, the SF of the injured ankle joint exhibited a largely proinflammatory and extracellular matrix–degrading environment, specifically IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, and MMP10. Similarly, Haller et al14 performed SF analysis in 45 patients after tibial plateau fractures and found a largely proinflammatory response with elevated concentrations of IL-1b, IL-6, IL-8, IL-10, IL-1 receptor antagonist, and monocyte chemoattractant protein 1 compared with the uninjured knee SF. However, differences in the potential long-term pathologies among these joints suggest that jointspecific mechanisms are involved. Indeed, hypoxia, mechanical factors, pH, and micronutrients all play a role in the etiology of joint contractures and post-traumatic arthritis, suggesting that a combination of factors is likely involved in specific pathologies. This study has several limitations. Not all elbows underwent aspiration at the initial time of injury; thus, no conclusions about baseline or peak concentrations can be made. However, evidence suggests that mechanisms involved in joint contracture may be initiated immediately after trauma,1 suggesting that the composition of SF within the first 3 weeks after trauma, as reported here, may be important for elucidating these mechanisms. A second limitation of this study is the relatively small sample size; however, post hoc power analysis demonstrated greater than 90% power for 11 of 16 variables, further suggesting an abnormal acutely fractured joint environment. In addition, the fractured elbow had significant hemarthrosis. The serum of each patient was not evaluated for the same biomarkers as those evaluated in the fracture hematoma, which is a limitation of this study. Although investigating the serum biomarkers was not the aim of this study, it would be a first step in determining if the concentrations of biomarkers in the serum reflect the concentrations observed within the affected elbow. Finally, the focus of this study was to simply compare SF composition between healthy and fractured joints; therefore, this study did not investigate specific mechanisms that may contribute to joint contractures in the approximately 20% of patients in whom this pathology develops.
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Conclusion 8.
This study provides critical and new data toward the goal of determining the causative markers of posttraumatic capsular contracture of the elbow, as neither the normal elbow nor acutely injured elbow SF environment has been previously described. These data are instructive for developing future studies capable of determining differences between patients in whom posttraumatic capsular contracture develops and those in whom it does not, toward the goals of identifying at-risk patients and identifying therapeutic targets to prevent contracture. This study represents a necessary and innovative step in characterizing the acute healthy and post-traumatic intra-articular SF environment in the elbow.
10.
Disclaimer
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The study was completed with an educational grant in the amount of $10,000 from the Piedmont Orthopedic Foundation (Durham, NC, USA). The outside source of funds was involved in data collection and data analysis but was not involved in preparation or editing of the manuscript. The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.
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Synovial fluid analysis after elbow fracture 26. Kirshenbaum A. Regulation of mast cell number and function. Hematol Oncol Clin North Am 2000;14:497-516.v. 27. Kraus VB, Huebner JL, Fink C, King JB, Brown S, Vail TP, et al. Urea as a passive transport marker for arthritis biomarker studies. Arthritis Rheum 2002;46:420-7. https://doi.org/10.1002/art.10124 28. Kraus VB, Stabler TV, Kong SY, Varju G, McDaniel G. Measurement of synovial fluid volume using urea. Osteoarthritis Cartilage 2007;15: 1217-20. https://doi.org/10.1016/j.joca.2007.03.017 29. Krause C, Kloen P, Ten Dijke P. Elevated transforming growth factor beta and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren’s disease fibroblasts. Fibrogenesis Tissue Repair 2011;4:14. https://doi.org/10.1186/1755-1536-4-14 30. Lindenhovius AL, Jupiter JB. The posttraumatic stiff elbow: a review of the literature. J Hand Surg Am 2007;32:1605-23. https://doi.org/10. 1016/j.jhsa.2007.09.015 31. Mellema JJ, Lindenhovius AL, Jupiter JB. The posttraumatic stiff elbow: an update. Curr Rev Musculoskelet Med 2016;9:190-8. https:// doi.org/10.1007/s12178-016-9336-9 32. Monument MJ, Hart DA, Befus AD, Salo PT, Zhang M, Hildebrand KA. The mast cell stabilizer ketotifen fumarate lessens contracture severity and myofibroblast hyperplasia: a study of a rabbit model of posttraumatic joint contractures. J Bone Joint Surg Am 2010; 92:1468-77. https://doi.org/10.2106/jbjs.i.00684 33. Morrey BF. The posttraumatic stiff elbow. Clin Orthop Relat Res 2005;431:26-35. https://doi.org/10.1097/01.blo.0000152366.58660.ea 34. Morrey BF. Post-traumatic contracture of the elbow. operative treatment, including distraction arthroplasty. J Bone Joint Surg Am 1990;72:601-18. 35. Myden C, Hildebrand K. Elbow joint contracture after traumatic injury. J Shoulder Elbow Surg 2011;20:39-44. https://doi.org/10.1016/ j.jse.2010.07.013 36. Nandi S, Maschke S, Evans PJ, Lawton JN. The stiff elbow. Hand (N Y) 2009;4:368-79. https://doi.org/10.1007/s11552-009-9181-z 37. Nedelec B, Ghahary A, Scott PG, Tredget EE. Control of wound contraction. basic and clinical features. Hand Clin 2000;16:289-302.
7 38. Ranganathan K, Loder S, Agarwal S, Wong VW, Forsberg J, Davis TA, et al. Heterotopic ossification: basic-science principles and clinical correlates. J Bone Joint Surg Am 2015;97:1101-11. https://doi. org/10.2106/JBJS.N.01056 39. Reisdorf P, Lawrence DA, Sivan V, Klising E, Martin MT. Alteration of transforming growth factor-beta1 response involves downregulation of Smad3 signaling in myofibroblasts from skin fibrosis. Am J Pathol 2001;159:263-72. 40. Ring D, Adey L, Zurakowski D, Jupiter JB. Elbow capsulectomy for posttraumatic elbow stiffness. J Hand Surg Am 2006;31:1264-71. https://doi.org/10.1016/j.jhsa.2006.06.009 41. Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima A, Saika S, et al. Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev 2006;17:19-27. https://doi.org/10.1016/j.cytogfr. 2005.09.008 42. Shahriaree H, Sajadi K, Silver CM, Sheikholeslamzadeh S. Excisional arthroplasty of the elbow. J Bone Joint Surg Am 1979;61:922-7. 43. Vaughan MB, Howard EW, Tomasek JJ. Transforming growth factorbeta1 promotes the morphological and functional differentiation of the myofibroblast. Exp Cell Res 2000;257:180-9. 44. Viola RW, Hastings H II. Treatment of ectopic ossification about the elbow. Clin Orthop Relat Res 2000;(370)::65-86. 45. Wilson PD. Capsulectomy for the relief of flexion contractures of the elbow following fracture. 1944. Clin Orthop Relat Res 2000;(370): 3-8. 46. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008;214:199-210. https://doi.org/10.1002/path.2277 47. Zent J, Guo LW. Signaling mechanisms of myofibroblastic activation: outside-in and inside-out. Cell Physiol Biochem 2018;49:848-68. https://doi.org/10.1159/000493217 48. Zhang HY, Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am J Respir Cell Mol Biol 1999;21: 658-65.
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Inflammatory cytokines and matrix metalloproteinases in the synovial fluid after intra-articular elbow fracture Elizabeth P. Wahl, MD*, Alexander J. Lampley, MD, Angel Chen, BA, Samuel B. Adams, MD, Dana L. Nettles, PhD, Marc J. Richard, MD Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, USA Background and hypothesis: Post-traumatic elbow contracture remains a common and challenging complication with often unsatisfactory outcomes. Although the etiology is unknown, elevated or abnormal post-fracture synovial fluid cytokine levels may result in the migration of fibroblasts to the capsule and contribute to capsular pathology. Thus, the purpose of this study was to characterize the cytokine composition in the synovial fluid fracture hematoma of patients with intra-articular elbow fractures. Methods: The elbow synovial fluid fracture hematoma of 11 patients with intra-articular elbow fractures was analyzed for CTXII (C-terminal telopeptides of type II collagen [a cartilage breakdown product]) as well as 15 cytokines and matrix metalloproteinases (MMPs) including interferon g, interleukin (IL) 1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor a, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. The uninjured, contralateral elbow served as a matched control. Mean concentrations of each factor were compared between the fluid from fractured elbows and the fluid from control elbows. Results: The levels of 14 of 15 measured cytokines and MMPsdinterferon g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor a, MMP-1, MMP-3, MMP-9, and MMP-10dwere significantly higher in the fractured elbows. In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power. The mean concentration of CTXII was not significantly different between groups. Conclusions: These results demonstrate a proinflammatory environment after fracture that may be the catalyst to the development of post-traumatic elbow joint contracture. The cytokines with elevated levels were similar, although not identical, to the cytokines with elevated levels in studies of other weightbearing joints, indicating the elbow responds uniquely to trauma. Level of evidence: Basic Science Study; Molecular Biology Ó 2019 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved. Keywords: Elbow contracture; elbow fracture; elbow stiffness; intra-articular; synovial fluid; intra-articular elbow fracture; elbow synovial fluid
This study was approved by the Duke University Health System Institutional Review Board for Clinical Investigations (Pro00072559).
*Reprint requests: Elizabeth P. Wahl, MD, Department of Orthopaedic Surgery, Duke University Medical Center, 4709 Creekstone Dr, Ste 200, Durham, NC 27703, USA. E-mail address: [email protected] (E.P. Wahl).
1058-2746/$ - see front matter Ó 2019 Journal of Shoulder and Elbow Surgery Board of Trustees. All rights reserved. https://doi.org/10.1016/j.jse.2019.09.024
2 Post-traumatic elbow joint contracture, a wellrecognized and debilitating sequela after intra-articular elbow fractures, is characterized by heterotopic bone formation, failure of fracture healing, soft tissue contracture (fibrosis), capsular thickening, and/or arthrosis30,31 and often results in significant disability.12,30,33-35,40,42,45 Despite considerable efforts to understand and prevent elbow joint contracture, the pathogenesis remains unclear, with surgical release being required in up to 15% of patients to restore motion.35 Furthermore, approximately 20% of patients will require a subsequent release with reported rates of complications (eg, ulnar nerve dysfunction) as high as 9%,30,40 and elbow range of motion is often not restored to normal, with patient satisfaction reported as fair at best.33 This has resulted in considerable efforts to elucidate the processes involved in this pathology. Hildebrand et al23 have studied post-traumatic knee joint capsules of rabbits and contracted elbow capsules of humans and found significantly elevated numbers of myofibroblasts and mast cells in the capsules of the contracted joints compared with healthy donor joints. Cohen et al4 found increases in collagen disorganization and the fibroblast presence in the diseased capsules compared with the healthy capsules, along with elevated staining for matrix metalloproteinase (MMP) 1, MMP-2, and MMP-3. A subset of stiff elbows will also display heterotopic ossification (HO) owing to progenitor cell differentiation into osteoblasts in the soft tissues of the elbow.30,31,36 Although these features of contracture may take several weeks to months to manifest, it is hypothesized that the processes that lead to this pathology may begin immediately after the traumatic event.15,44 In an effort to understand post-traumatic pathologies in other synovial joints, several studies have analyzed the synovial fluid (SF) content after trauma in the knee and ankle and demonstrated a highly proinflammatory and catabolic environment1,14; however, differential profiles and long-term outcomes between these joints suggest a joint-specific response to insult.13 Although a similar unfavorable environment would be expected in the elbow after joint trauma, the pathology of joint contracture in the elbow is of greater concern than that in the knee or ankle, suggesting a unique response by elbow joint tissues. Thus, the purpose of this study was to compare the acute-phase SF cytokine profile after elbow fracture with SF from healthy elbows (contralateral elbows of the same patients) to identify factors that could contribute to an increased risk of development of various features of post-traumatic elbow capsular contracture and serve as potential early targets for intervention.
E.P. Wahl et al. with a mean age of 56 years (range, 27-80 years), comprising 7 women and 4 men. An intra-articular elbow fracture was defined as any fracture of the humerus, ulna, or radius in which the fracture line extended into the articular surface of the elbow joint. The inclusion criteria consisted of an operatively treated intra-articular elbow fracture, an uninjured contralateral elbow that was pain free, and no history of trauma in either elbow. Of the patients, 2 had isolated humeral fractures; 1 had an isolated olecranon fracture; 1 had an isolated radial head fracture; and 7 had combined humeral, ulnar, and/or radial fractures. All fractures were displaced at least 2 mm and were isolated injuries. The uninjured elbow served as a matched control. Patients were excluded if they were younger than 18 years; were pregnant; or had a history of cancer, diabetes, hemophilia, or systemic inflammatory disease. Participation followed informed consent in accordance with an approved institutional review board protocol at our institution.
Elbow joint SF aspiration and lavage SF was obtained from the injured elbow at the time of initial presentation to the emergency department if the patient was seen by a consulting orthopedist, as described previously.3 SF was also obtained from the bilateral elbows at the time of surgery in the same manner. As the aspirate from the injured side contained considerable fracture hematoma, it is more appropriately characterized as SF fracture hematoma. After bilateral upper-extremity surgical scrubbing and draping, arthrocentesis was performed on the elbow joints in sequential fashion using an 18-gauge needle and a 10-mL syringe. The needle was inserted through the standard posterolateral approach as previously described.3 If no fluid was obtained or an inadequate amount (<2 mL) was obtained from the control side on initial arthrocentesis, the 10-mL syringe was removed from the needle and a sterile saline solution flush syringe was attached to the needle. One to two milliliters of sterile saline solution was injected into the elbow joint, the sterile saline solution flush syringe was removed, and the 10-mL syringe was reattached to the needle for elbow re-aspiration. There were no incidences in which the injured elbow required lavage. The fluid samples were transferred to a 15-mL conical centrifuge tube (Falcon; Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at room temperature to remove cells and particulate matter (3500 rpm, 15 minutes27); the resultant supernatant was then aspirated, aliquoted, labeled, and stored at –80 C in cryopreservation tubes (Nalgene; Thermo Fisher Scientific).
Serum collection If the control elbow joint required lavage, then a whole blood sample was collected concurrently to measure serum urea concentrations to correct for dilution through lavage, as described later. The blood was allowed to sit for 20 minutes in glass tubes and then centrifuged at room temperature, and the serum fraction was then processed and stored in the same manner as the SF sample.
Materials and methods Correction for dilution through lavage Patient enrollment, population, and study design This was a prospective case-control study of patients with unilateral intra-articular elbow fractures. We enrolled 11 patients in the study,
Lavage of the control elbow without knowing the starting amount of SF in the joint would prohibit calculation of the cytokine concentration. However, the proportion of urea in SF relative to
Synovial fluid analysis after elbow fracture serum, in both healthy and diseased states, has been shown to be a constant, fixed ratio.27,28 Urea has been described as a passive transport marker for arthritis biomarker studies when direct aspiration of joint fluid is difficult.1,2,27 To correct for the dilution effects of lavage, we measured the urea concentrations in both the serum and the SF using a colorimetric assay (QuantiChrom Urea Assay Kit; BioAssay Systems, Hayward, CA, USA). The dilution factor of the SF due to lavage was calculated by dividing the serum urea concentration by the SF urea concentration for each patient requiring lavage. Cytokine measurements were corrected for dilution factors prior to statistical analysis. This technique has been reported for evaluating the SF cytokine concentration, MMP composition, and cellular metabolites of other joints after intraarticular fracture.1,2
Cytokine analysis Fluid samples were analyzed for 15 cytokines and MMPs including interferon (IFN) g, interleukin (IL) 1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor (TNF) a, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10. Concentrations of cytokines were determined from the fluid using a human proinflammatory panel (IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL10, IL-12p70, IL-13, TNF-a). Levels of MMP-1, MMP-3, and MMP-9 were quantified using an MMP 3-plex immunoassay, and levels of MMP-2 and MMP-10 were quantified using a human MMP 2-plex sandwich immunoassay. All assays described earlier were performed according to the manufacturer’s instructions (Meso Scale Discovery, Rockville, MD, USA).
Type II collagen catabolism Levels of degradation products of C-terminal telopeptides of type II collagen (CTXII), reflective of cartilage degradation, were quantified from fluid samples using a competitive immunoassay (Cartilaps; Immunmodiagnostic Systems, Scottsdale, AZ, USA). For this assay, all samples were pretreated with Streptomyces hyaluronidase (H-3884; Sigma-Aldrich, St Louis, MO, USA) for 1 hour at 37 C at 5 U/mL to reduce viscosity. The remainder of the assay was performed according to the manufacturer’s instructions.
Statistical methods Measured values for any sample that fell below the lower limit of detection (LLOD) were replaced by one-half the LLOD for statistical analysis. It should be noted that this only occurred for samples from the control sample population. Distributions of all cytokines and MMPs, as well as CTXII, from both the control and fracture groups were evaluated for normality using the ShapiroWilk W test. A paired t test was used to evaluate differences between the control and fracture groups for IL-1b, as this variable was found to be normally distributed in both the control and fracture populations. Differences between the control and fracture groups for all other variables were determined using the nonparametric Wilcoxon rank sum test. Differences were considered significant at P < .05. Statistical analysis was performed using JMP Pro software (version 14; SAS Institute, Cary, NC, USA). A post hoc power analysis was also performed for all measured variables.
3
Results Eleven patients were enrolled in this study; in 1 patient, aspiration of the injured elbow was performed on the day of injury, whereas in the other 10, aspiration of the injured elbow was performed at the time of surgery. Five patients required lavage of the uninjured elbow at the time of surgery. The median time from fracture to surgery was 2.5 days (range, 0-17 days). After correction for dilution, mean concentrations of cytokines and MMPs including IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-a, MMP-1, MMP3, MMP-9, and MMP-10 were significantly higher in the fractured elbows compared with the healthy, contralateral elbows (Fig. 1). In addition, post hoc power analysis revealed that 10 of 14 significant differences were detected with greater than 90% power (Table I). It is important to note that cytokine levels of many of the samples from the healthy group fell below the LLOD. In addition to cytokines, samples were analyzed for evidence of type II collagen breakdown through measurement of CTXII concentrations. The concentrations of CTXII between the healthy and fracture groups were not significantly different (P ¼ .97).
Discussion Our study provides an important step in understanding the environment surrounding the elbow after fracture. Similarly to the knee and ankle, the SF fracture hematoma is acutely proinflammatory and catabolic with elevated levels of soluble factors capable of initiating several signaling cascades involved in the processes that lead to elbow contracture.9,11,26,38,47 Although it is unknown why this pathology develops in only a portion of elbows, it is reasonable to hypothesize that in some individuals, the processes necessary for normal wound healing fail to subside, allowing abnormal tissue remodeling. Because the etiology in the elbow is still under investigation, it is helpful to examine the literature concerning contractures affecting other organ systems (eg, skin, lung, kidney, or liver),39,41,43 as some of these systems have processes in common with the elbow. One of the main causes of elbow contracture is the development of fibrosis surrounding the capsule. Studies in other organ systems have suggested that the myofibroblast is a key cell involved in this response to injury owing to its expression of the contractile protein a smooth muscle actin.25 During normal wound healing by secondary intent, the myofibroblast reduces the size of the wound using the cells’ contractile properties. Then, after the wound is healed, the myofibroblasts disappear through apoptosis.37 However, dysregulation of this healing response has been implicated as the cause of tissue fibrosis. In fact, studies have shown that the differentiation of the fibroblast into the
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Figure 1 Median (horizontal line), interquartile range (box), and minimum and maximum values (whiskers) for measured cytokine and matrix metalloproteinase (MMP) concentrations in synovial fluid from uninjured elbows (white bars) and fractured elbows (gray bars). *Statistically significant difference. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; CTX-II, C-terminal telopeptides of type II collagen; CTL, control.
myofibroblast is associated with other fibrotic conditions such as skin fibrosis6 and Dupuytren contracture.29 Likewise, in both human elbow joint contracture capsules and rabbit knee contracture models, Hildebrand et al17-24 have demonstrated that the myofibroblast is an ‘‘effector cell’’ in
Table I
the development of post-traumatic joint contracture through myofibroblast hyperplasia and excessive collagen production. Although the exact mechanism of this problematic myofibroblast hyperplasia is unknown, studies have
Cytokine and MMP levels in synovial fluid from healthy and fractured elbows
Cytokine or MMP
Healthy elbows
IFN-g, pg/mL IL-10, pg/mL IL-12p70, pg/mL IL-13, pg/mL IL-1b, pg/mL IL-2, pg/mL IL-4, pg/mL IL-6, ng/mL IL-8, ng/mL TNF-a, pg/mL MMP-1, mg/mL MMP-2, mg/mL MMP-3, mg/mL MMP-9, mg/mL MMP-10, ng/mL CTXII, ng/mL
0.33 0.15 0.07 2.39 0.17 0.42 0.10 0.004 0.012 0.80 0.002 0.09 0.10 0.03 1.74 0.51
0.39 0.29 0.08 4.92 0.15 0.7 0.23 0.007 0.009 0.82 0.002 0.05 0.07 0.04 1.82 0.24
Fractured elbows 8.37 2.89 5.62 11.65 4.47 1.36 45.6 1.76 1.38 2.58 0.24 0.09 1.3 0.28 3.48 0.60
5.32 1.84 2.91 5.79 3.81 0.93 93.70 0.37 0.80 0.60 0.43 0.04 0.91 0.25 2.74 0.44
P value
Power, %
<.0001 <.0001 <.0001 .0008 <.0001 .004 .0001 <.0001 <.0001 .0006 .0001 .86 .0002 .0043 .038 .97
99.9 99.8 100 98.1 96.2 76.4 36.3 100 100 100 45 N/S 98.9 90.1 42 N/S
IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; CTXII, C-terminal telopeptides of type II collagen; N/S, not significant. Data are presented as mean standard deviation with P values for the Wilcoxon rank sum test or Student t test (significance declared at P < .05) and percentages for post hoc power analysis.
Synovial fluid analysis after elbow fracture suggested that an excess of growth factors and cytokines that promote myofibroblast differentiation and inhibit myofibroblast apoptosis may cause the development of post-traumatic capsular contracture.10,48 These findings led to the hypothesis that the activation of mast cells, which results in synthesis and secretion of growth factors and cytokines known to have mitogenic effects on myofibroblasts, is important in the development of post-traumatic capsular contracture.16 This hypothesis was substantiated by Monument et al,32 who demonstrated that ketotifen, a mast cell stabilizer, was effective in significantly reducing not only the severity of joint capsule contracture but also mast cell and myofibroblast hyperplasia in a rabbit model of post-traumatic joint contracture. Although the literature supports the theory of mast cell–driven post-traumatic capsular contracture, it is still unclear what is activating the mast cell after injury. We hypothesize that a proinflammatory response after intraarticular fracture demonstrated in our study may contribute to molecular events that activate the mast cell. We found the concentrations of 10 cytokines and 4 MMPs to be significantly higher in the fractured elbows compared with the healthy, contralateral elbows. Although many are involved in a normal wound- and bone-healing process, they may also be involved in directly activating fibroblasts or indirectly activating them by initiating the mast cell–driven post-traumatic capsular contracture. For example, both transforming growth factor b and TNF-a are potent stimulators of fibroblasts, and IL-13 has been implicated in the fibrotic response, as it is a potent inducer of MMPs.20,46 MMP-2 and MMP-9 can further upregulate transforming growth factor b.47 In addition, IL-6 and TNFa enhance IFN-g production by mast cells, and IL-10 can directly enhance the mast cell number in the presence of IL-3 and stem cell factor.26 In contrast, IL-4 can inhibit stem cell factor, which is required for mast cell activation. Taken together, these findings suggest that soluble factors whose levels are elevated during normal wound healing may initiate joint pathology if homeostasis is delayed. Indeed, Desai et al5 compared the outcomes of patients undergoing operative fixation of closed elbow injury with an ulnohumeral dislocation, radial head fracture, and coronoid fracture (terrible-triad elbow injury) and found that the patients treated with steroids had significantly improved elbow range of motion at 24 weeks’ follow-up compared with those who did not receive steroids. The steroid, a potent anti-inflammatory, likely inhibited inflammatory signaling pathways, which may have resulted in inhibiting mast cell activation and the formation of arthrofibrosis. In addition to fibrosis, HO is a concern in the elbow and can result from stimulation of progenitor cells to become bone-forming cells in the presence of inflammation.38 HO contributes to loss of range of motion in up to 40% of elbow fractures.7,8 Although the mechanisms involved in HO are still under investigation, there is significant
5 evidence to suggest that prolonged inflammation, particularly IL-1b and TNF-a, may stimulate an HO cascade through a bone morphogenetic protein 2, prostaglandin E2, and cyclooxygenase 2 pathway.9,38 Both IL-1b and TNF-a levels were significantly elevated in fractured elbows in our study, and numerous studies have shown that prophylactic administration of nonsteroidal anti-inflammatory medications lowers the risk of HO,38 suggesting that this early inflammation may initiate HO immediately after trauma and/or surgery. It is interestingly to note that, compared with the SF after intra-articular fracture of the ankle and knee, our study demonstrated a similar proinflammatory profile. For example, Adams et al1 compared the SF between acutely fractured ankle joints and normal ankle joints in 21 patients and found that after intra-articular fracture, the SF of the injured ankle joint exhibited a largely proinflammatory and extracellular matrix–degrading environment, specifically IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, and MMP10. Similarly, Haller et al14 performed SF analysis in 45 patients after tibial plateau fractures and found a largely proinflammatory response with elevated concentrations of IL-1b, IL-6, IL-8, IL-10, IL-1 receptor antagonist, and monocyte chemoattractant protein 1 compared with the uninjured knee SF. However, differences in the potential long-term pathologies among these joints suggest that jointspecific mechanisms are involved. Indeed, hypoxia, mechanical factors, pH, and micronutrients all play a role in the etiology of joint contractures and post-traumatic arthritis, suggesting that a combination of factors is likely involved in specific pathologies. This study has several limitations. Not all elbows underwent aspiration at the initial time of injury; thus, no conclusions about baseline or peak concentrations can be made. However, evidence suggests that mechanisms involved in joint contracture may be initiated immediately after trauma,1 suggesting that the composition of SF within the first 3 weeks after trauma, as reported here, may be important for elucidating these mechanisms. A second limitation of this study is the relatively small sample size; however, post hoc power analysis demonstrated greater than 90% power for 11 of 16 variables, further suggesting an abnormal acutely fractured joint environment. In addition, the fractured elbow had significant hemarthrosis. The serum of each patient was not evaluated for the same biomarkers as those evaluated in the fracture hematoma, which is a limitation of this study. Although investigating the serum biomarkers was not the aim of this study, it would be a first step in determining if the concentrations of biomarkers in the serum reflect the concentrations observed within the affected elbow. Finally, the focus of this study was to simply compare SF composition between healthy and fractured joints; therefore, this study did not investigate specific mechanisms that may contribute to joint contractures in the approximately 20% of patients in whom this pathology develops.
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Conclusion 8.
This study provides critical and new data toward the goal of determining the causative markers of posttraumatic capsular contracture of the elbow, as neither the normal elbow nor acutely injured elbow SF environment has been previously described. These data are instructive for developing future studies capable of determining differences between patients in whom posttraumatic capsular contracture develops and those in whom it does not, toward the goals of identifying at-risk patients and identifying therapeutic targets to prevent contracture. This study represents a necessary and innovative step in characterizing the acute healthy and post-traumatic intra-articular SF environment in the elbow.
10.
Disclaimer
14.
The study was completed with an educational grant in the amount of $10,000 from the Piedmont Orthopedic Foundation (Durham, NC, USA). The outside source of funds was involved in data collection and data analysis but was not involved in preparation or editing of the manuscript. The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.
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