Characterization of synovial fluid cytokine profiles in chronic meniscal tear of the knee

Concentrations of pro‐ and anti‐inflammatory cytokines in synovial fluid samples collected from patients with chronic meniscal tears were investigated. An acute inflammatory response is generally reported 24–48 h after knee injury, but the largest body of data available in literature concerns anterior cruciate ligament injury and very little information is available about the balance of soluble factors in the synovial fluid of knees with chronic meniscal tears. Sixty‐nine patients (46 males and 23 females) with meniscal tear that occurred more than 3 months earlier were enrolled. According to cartilage integrity assessment by arthroscopic examination, patients were assigned to one of the following groups: (i) no chondral damage (n = 18); (ii) chondral damage graded from I to II (n = 15); and (iii) chondral damage graded from III to IV (n = 37). In all groups, levels of IL‐10 and inflammatory cytokines IL‐6, TNF‐α, and IL‐8 where greater compared with those reported in the intact population; by contrast, levels of IL‐1ra and IL‐1β were significantly lower. Interestingly, IL‐6 levels were higher in female than male patients. Cytokine levels did not correlate with degree of chondral damage. IL‐6 and IL‐1ra levels positively correlated with IL‐1β, and negatively correlated with TNF‐α. Interestingly, levels of IL‐1β and TNF‐α were inversely correlated. Our data demonstrate increased levels of pro‐inflammatory cytokines (IL‐6, IL‐8, and TNF‐α) in the chronic phase of meniscal trauma. This pro‐inflammatory state is maintained in the joint from the time of initial injury to several months later and could be a key factor in hampering cartilage regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:340–346, 2017.

     

Metabolic profiling of synovial fluid in a unilateral ovine model of anterior cruciate ligament reconstruction of the knee suggests biomarkers for early osteoarthritis

Joint injuries and subsequent osteoarthritis (OA) are the leading causes of chronic joint disease. In this work, we explore the possibility of applying magnetic resonance spectroscopy‐based metabolomics to detect host responses to an anterior cruciate ligament (ACL) reconstruction injury in synovial fluid in an ovine model. Using multivariate statistical analysis, we were able to distinguish post‐injury joint samples (ACL and sham surgery) from the uninjured control samples, and as well the ACL surgical samples from sham surgery. In all samples there were 65 metabolites quantified, of which six could be suggested as biomarkers for early post‐injury degenerative changes in the knee joints: isobutyrate, glucose, hydroxyproline, asparagine, serine, and uridine. Our results raise a cautionary note indicating that surgical interventions into the knee can result in metabolic alterations that need to be distinguished from those caused by the early onset of OA. Our findings illustrate the potential application of metabolomics as a diagnostic and prognostic tool for detection of injuries to the knee joint. The ability to detect a unique pattern of metabolic changes in the synovial fluid of sheep offers the possibility of extending the approach to precision medicine protocols in patient populations in the future. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:71–77, 2015.

     

PRG4 exchange between the articular cartilage surface and synovial fluid.

The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules, found both in synovial fluid (SF) and bound to the articular cartilage surface. Currently the mechanism by which PRG4 binds to the articular surface is not well understood. The objectives of this study were to determine (1) the effect of bathing fluid contents on PRG4 concentration at the articular surface ([PRG4](cart)), and (2) whether native PRG4 can be removed from the surface and subsequently repleted with PRG4 from synovial fluid. In one experiment, cylindrical cartilage disks were stored in solutions of various PRG4 concentrations, either in phosphate-buffered saline (PBS) or SF as the carrier fluid. In a separate experiment, cartilage disks were stored in solutions expected to remove native PRG4 from the articular surface and allow subsequent repletion with PRG4 from SF. [PRG4](cart) was independent of PRG4 concentration of the bathing fluid, and was similar for both carrier fluids. PRG4 was removed from cartilage by treatment with hyaluronidase, reduction/alkylation, and sodium dodecyl sulphate, and was repleted fully by subsequent bathing in SF. These results suggest that the articular surface is normally saturated with tightly bound PRG4, but this PRG4 can exchange with the PRG4 in SF under certain conditions. This finding suggests that all tissues surrounding the joint cavity that secrete PRG4 into the SF may help to maintain lubrication function at the articular surface.     

Oxidant and anti-oxidant systems of synovial fluid from patients with knee post-traumatic arthritis.

It has been suggested that patients with knee post-traumatic arthritis (PA), associated or not to haemarthrosis (HA), display altered oxidant and anti-oxidant systems in their synovial fluid. This study aimed to establish whether this is really the case. Synovial fluid samples were obtained by transdermal arthrocentesis from 69 patients with PA (36 of them had HA) and 22 control subjects. The activities of synovial fluid zinc-copper superoxide dismutase (ZnCuSOD) and manganese superoxide dismutase (MnSOD) isoenzymes, catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) enzymes, and malondialdehyde (MDA) concentration and synovial fluid viscosity were measured in the study groups. Patients with PA had significantly increased activities of all antioxidant enzymes, except CAT, and MDA concentration than did the controls. However, synovial fluid viscosity was found to be decreased in the study group, mainly in the HA subgroup. Results suggest that excessive free radicals production may exist in synovial fluid of PA patients and may contribute to knee joint destruction.     

关节液中脂联素水平与膝关节骨关节炎严重程度的相关性研究

背景：众所周知,肥胖是引起骨关节炎发生、发展的危险因素之一。肥胖人群容易发生关节炎的原因是超载作用和代谢因素。脂肪组织是一个活跃的器官,它的分泌脂肪细胞因子包括脂联素进入全身循环,参与了骨关节炎的发病。 目的：测量骨关节炎患者血清和关节滑液中脂联素水平,分析其与膝关节骨关节炎严重程度的相关性。 方法：64例膝关节骨关节炎患者（关节炎组）和19例因半月板或韧带损伤患者（对照组,行关节镜检查后排除软骨损伤）被纳入本研究。采用Kellgren-Lawrence（KL）标准对关节炎患者的膝关节前后位X线片进行评估分级。使用酶联免疫吸附试验检测脂联素在对照组和关节炎组患者血清和关节滑液中的表达水平。使用WOMAC量表（Western On-tario and McMaster Universities Arthritis Index）评估患者膝关节功能。 结果：骨关节炎组患者血清中的脂联素水平[（6177.8±991.0）ng/ml]高于对照组[（5793.4±673.3）ng/ml],但差异无统计学意义（P=0.12）。但在骨关节炎组患者,血清中的脂联素浓度[（6177.8±991.0）ng/ml]显著高于关节滑液中的脂联素[（824.3±304.7）ng/ml,P〈0.001）。关节炎患者关节液中脂联素浓度与评估疾病的严重程度呈负相关（r=-0.51,P〈0.001）,但血清脂联素浓度与疾病的严重程度关联不显著（r=-0.17,P=0.18）。血清脂联素水平、滑膜液脂联素水平与MOMAC疼痛评分均不显著相关（r=-0.01和r=-0.13,P＞0.05）。 结论：骨关节炎患者滑膜液中脂联素水平与膝关节炎严重程度呈负相关。检测关节滑液中的脂联素可能有助于早期发现关节炎。     

First in vivo detection and characterization of hyaluronan‐coated extracellular vesicles in human synovial fluid

Extracellular vesicles (EVs) function in intercellular signaling by transporting different membrane and cytosolic molecules, including hyaluronan (HA) and its synthesis machinery. As both EVs and HA are abundant in synovial fluid, we hypothesized that HA synthesized in synovial membrane would be carried on the surface of EVs. Synovial fluid (n = 15) and membrane samples (n = 5) were obtained from knee surgery patients. HA concentrations were analyzed in synovial fluid and HA and its synthesis machinery were examined with histochemical stainings in synovial membrane. To assess the size distribution of EVs in synovial fluid and to visualize HA on EVs, nanoparticle tracking analysis (NTA), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were utilized. The average HA concentration in synovial fluid was 2.0 ± 0.21 mg/ml without significant differences between the patients with trauma/diagnostic arthroscopy and primary or post‐traumatic osteoarthritis. Positive stainings of HA synthases (HAS1–3), HA and its receptor CD44 in synovial cells indicated active HA secretion in synovial membrane. According to NTA, EVs were abundant in synovial fluid and their main populations were ≤300 nm in diameter after differential centrifugation. There were no significant differences in the EV counts between the patients with primary or post‐traumatic osteoarthritis. TEM verified that HA‐positive particles detected by CLSM were lipid membrane vesicles surrounded by a HA coat. Our results provide the first in vivo evidence that human synovial fluid contains HA‐positive EVs, one source of which presumably is the long HAS‐positive protrusions of synovial fibroblasts. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1960–1968, 2016.     

Transcriptomic analysis of synovial extracellular RNA following knee trauma: A pilot study

Traumatic knee injuries often result in damage to articular cartilage and other joint structures. Such trauma is a strong risk factor for the future development and progression of osteoarthritis (OA). The molecular mechanisms and signaling pathways modulating response to knee joint trauma remain unclear. Moreover, investigations of biomarkers influencing responses have been targeted rather than broad, unbiased discovery studies. Herein, we characterize the complete complement of extracellular RNA (exRNA) in the synovial fluid of 14 subjects following knee injury. Fluid was collected during surgery from the injured knees, and from the contralateral knee in a subset, undergoing surgical repair of the ACL and/or meniscal repair/debridement. Arthroscopic grading of chondral damage in four knee compartments was performed using the Outerbridge classification. exRNA was extracted and subjected to massively parallel total RNA sequencing. Differential abundance of RNA was calculated between the subject cohorts of injured and non‐injured knee, average Outerbridge score ≥0.5 and less, and chronic and acute injury duration defined as ≤4 months till surgery or longer. Overall, expression of several thousand genes was identified in the synovial fluid. Furthermore, differential expression analysis suggests a role of exRNA fragments of matrix metalloproteinases and skeletal muscle fiber genes in the response to traumatic injury. Together, these data suggest that high‐throughput approaches can indicate exRNA molecular signatures following knee trauma. Future studies are required to more fully characterize the biological roles of these exRNA and the cadence of their respective release that may lead to translational treatment options for post‐traumatic OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1659–1665, 2018.

     

骨关节炎患者血清及关节液中白细胞介素-17表达及其意义

目的检测膝骨关节炎(osteoarthritis,OA)患者血清及关节液中自细胞介素-17(interleukin-17)的表达并评估其与OA严重程度的相关性。方法收集100例原发性膝OA患者血清及关节液标本,并以50例健康志愿者血清标本作为对照。OA严重程度及分级分别采用Lequesne指数和K-L分级系统。酶联免疫吸附法分别检测血清及关节液中IL-17浓度,并分析其与OA严重程度的相关性。结果 OA组患者血清中IL-17浓度[(5.292±1.470)pg/ml]明显高于对照组[(4.173±0.640)pg/ml],差异有统计学意义(P0.05)。K-L4级或3级OA患者关节液IL-17的浓度明显高于2级OA患者,差异有统计学意义(P0.05)。4级OA患者关节液IL-17的浓度[(6.161±1.120)pg/ml]明显高于3级OA患者[(4.474±0.816)pg/ml],差异有统计学意义(PO.05)。关节液IL-17浓度与Lequesne指数呈正相关(r=0.6232,P0.05);血清IL-17浓度与Lequesne指数之间无明显相关。结论 IL-17在OA患者血清及关节液中的表达明显升高,关节液中IL-17的水平与OA严重程度之间明显相关,提示IL-17的失衡表达在OA的发病机制中具有一定作用。     

Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis

We investigated whether mesenchymal stem cells (MSCs) in synovial fluid (SF) increased in the knee with degenerated cartilage and osteoarthritis. SF was obtained from the knee joints of 22 patients with anterior cruciate ligament (ACL) injury during ACL reconstruction, and cartilage degeneration was evaluated arthroscopically. SF was also obtained from the knee joints of 6 healthy volunteers, 20 patients with mild osteoarthritis, and 26 patients with severe osteoarthritis, in which the grading was evaluated radiographically. The cell component in the SF was cultured for analyses. Synovium (SYN) and bone marrow (BM) were also harvested during total knee arthroplasties. The MSC number in SF was correlated with the cartilage degeneration score evaluated by arthroscopy. The MSC number in the SF was hardly noticed in normal volunteers, but it increased in accordance with the grading of osteoarthritis. Though no significant differences were observed regarding surface epitopes, or differentiation potentials, the morphology and gene profiles in SF MSCs were more similar to those in SYN MSCs than in BM MSCs. We listed 20 genes which were expressed higher in both SYN MSCs and SF MSCs than in BM MSCs, and 3 genes were confirmed by quantitative RT-PCR. MSCs in SF increased along with degenerated cartilage and osteoarthritis.     

膝关节骨关节炎滑液和软骨骨桥蛋白水平与其病变程度的相关性

目的 探讨膝关节骨关节炎(osteoarthritis,OA)患者滑液和关节软骨骨桥蛋白(osteopontin,OPN)水平及其与病变严重程度的关系.方法 随机选取50例膝关节OA患者[男15例,女35例;年龄48～81岁,平均(61.8±7.4)岁]和10名健康对照者[男4例,女6例;年龄59～68岁,平均年龄(63.2±6.0)岁]作为研究对象.采用Mankin评分评价疾病严重程度,Kellgren-Lawrence标准进行放射学分级,酶联免疫吸附法测定关节滑液OPN水平,免疫组化方法测定关节软骨OPN光密度值.结果 OA患者与对照者相比,关节滑液OPN水平[(4519.60±1830.37)pg/ml:(1179.70±303.39)pg/ml)和关节软骨OPN光密度值[(0.60±-0.06):(0.43±0.07)]均明显升高.关节滑液OPN水平与关节软骨OPN表达呈正相关(r=0.411,P=0.003).关节滑液OPN水平与OA病变严重程度(KL分级)呈正相关(r=0.581,P＜0.001).关节软骨OPN表达与OA病变严重程度(Mankin评分)呈正相关(r=0.675,P＜0.001).结论 关节滑液和关节软骨OPN水平与病变严重程度相关.     

Results of total hip and total knee arthroplasties in patients with synovial chondromatosis

This retrospective analysis examines the outcome of total joint arthroplasty for severe arthritis in patients with synovial chondromatosis. All 11 patients treated with total hip arthroplasty (n = 7) or total knee arthroplasty (n = 4) returned for follow-up at a mean of 10.8 years after surgery. Pain and functional scores improved significantly in all patients. Knee range of motion improved in all patients. Synovial chondromatosis recurred in 1 knee (25%) and 1 hip (14%). Total joint arthroplasty is a valuable treatment option for these patients with predictable improvement in pain and function. Knee range of motion is likely to improve but may be less than expected for primary total knee arthroplasty. Patients remain at risk for recurrence.     

Metabolic analysis of knee synovial fluid as a potential diagnostic approach for osteoarthritis

Osteoarthritis (OA) is a leading cause of chronic joint pain in the older human population. Diagnosis of OA at an earlier stage may enable the development of new treatments to one day effectively modify the progression and prognosis of the disease. In this work, we explore whether an integrated metabolomics approach could be utilized for the diagnosis of OA. Synovial fluid (SF) samples were collected from symptomatic chronic knee OA patients and normal human cadaveric knee joints. The samples were analyzed using ¹H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography‐mass spectrometry (GC‐MS) followed by multivariate statistical analysis. Based on the metabolic profiles, we were able to distinguish OA patients from the controls and validate the statistical models. Moreover, we have integrated the ¹H NMR and GC‐MS results and we found that 11 metabolites were statistically important for the separation between OA and normal SF. Additionally, statistical analysis showed an excellent predictive ability of the constructed metabolomics model (area under the receiver operating characteristic curve = 1.0). Our findings indicate that metabolomics might serve as a promising approach for the diagnosis and prognosis of degenerative changes in the knee joint and should be further validated in clinical settings. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1631–1638, 2015.     

Characterization of synovial fluid from periprosthetic infection in revision total joint arthroplasty by single-molecule microscopy

Periprosthetic joint infection is among the most common and severe complications in total joint arthroplasty. Today, a combination of different methods is used for diagnosis because no single method with sufficient sensitivity and specificity is available. In this study, we explored the usability of single-molecule microscopy to characterize synovial fluid samples from periprosthetic joint infections. Patients (n = 27) that needed revision arthroplasty underwent the routine diagnostic procedures for periprosthetic joint infection of the University Hospital in Bonn. Additionally, the diffusion rate of two probes, dextran and hyaluronan, was measured in small volumes of periprosthetic synovial fluid samples using single-molecule microscopy. To evaluate the suitability of single-molecule microscopy to detect PJI the AUC for both markers was calculated. The diffusion rate of hyaluronan in periprosthetic synovial fluid from patients with septic loosening was faster than in samples from patients with aseptic loosening. Single-molecule microscopy showed excellent diagnostic performance, with an area under the receiver operating characteristic curve of 0.93, and allowed the detection of periprosthetic joint infection in patients that would be challenging to diagnose with current methods. For the first time, single-molecule microscopy was used to detect periprosthetic joint infection. Our results are encouraging to study the value of single-molecule microscopy in a larger patient cohort. The speed and accuracy of single-molecule microscopy can be used to further characterize synovial fluid, potentially allowing intraoperative diagnosis of periprosthetic joint infections in the future.     

Biomechanical properties of mixtures of blood and synovial fluid

Synovial fluid (SF) is a viscous ultrafiltrate of plasma that lubricates articulating joint motion. During acute trauma and certain cartilage repair procedures, blood is introduced into the joint and mixes with variable amounts of SF. The hypothesis of this study was that the dilution of blood with SF alters the rheological properties of the blood and the mechanical properties of the clot formed. The objectives were to determine the composition (solid fraction, protein content), coagulation (fibrin polymerization time, torsional strength), and mechanical (stiffness, permeability) properties of mixtures of blood with 10% or 50%SF. While the initial stages of coagulation of blood were not markedly affected by the presence of the SF, dilution with SF altered the coagulation torque profile over time, decreased the final clot structure mechanical stiffness (42–90% decrease), and increased the fluid permeability of the clots (41‐ to 468‐fold). Compared to diluting blood with PBS, SF had a smaller effect on the mechanical properties of the clot, possibly due to the presence of high molecular weight hyaluronan. These properties of blood/SF mixtures may facilitate an understanding of the repair environment in the joint and of mechanisms of cartilage repair. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:240–246, 2011     

关节镜+术后冲洗治疗骨性关节炎一氧化氮水平变化

目的比较关节镜+术后冲洗治疗膝关节骨性关节炎对关节液中一氧化氮(NO)水平变化的差异。方法将确诊为膝关节骨性关节炎的32例患者随机分成2组,每组16例。持续冲洗组在膝关节镜术后用3000ml生理盐水持续6h冲洗关节腔;非冲洗组在关节镜手术后不行关节腔冲洗。于术前、术后第24h、术后第1、2、4周分别抽取2ml的膝关节液,检测NO浓度。结果两组均能使关节液中的NO浓度水平下降。术后24h和第1、2周时,两组患者的NO检测结果差异有统计学意义,P值分别为0.0004、0.0024及0.0037,持续冲洗组明显优于非冲洗组。在术后4周时,持续冲洗组和非冲洗组的NO检测结果无差异(P=0.172)。结论膝关节镜下治疗膝关节骨性关节炎持续冲洗组和非冲洗组比较,在短期内关节液中NO的水平下降明显,可有效改善症状。     

Global and targeted metabolomics of synovial fluid discovers special osteoarthritis metabolites

To identify special metabolites in synovial fluid of osteoarthritis (OA) via a metabolomics approach. Synovial fluid of 35 participants (25 OA patients and 10 controls) was detected by GC‐TOF/MS and multivariate data analysis was applied to analyze correlation among the observations. Different metabolites were screened by VIP value (VIP > 1), student t‐test (p < 0.05), and fold change (fold >1.5), and verified with the standard metabolites in the synovial fluid of 24 OA patients and 11 controls by LC/MS. The classification performance of different metabolites was analyzed by receiver operating characteristic (ROC) analysis. The results showed that six different metabolites (glutamine, 1,5‐anhydroglucitol, gluconic lactone, tyramine, threonine, and 8‐aminocaprylic acid) were strongly associated with OA in global metabolomics. Verified results of the first three metabolites were the same as the identified results using targeted metabolomics. ROC curve analysis demonstrated that their concentrations in synovial fluid were strongly correlated to OA. In addition, the concentrations of gluconic lactone were significantly different between OA and RA. Metabolites with altered levels may be contributors to OA pathogenesis and can be used as potential diagnosis criteria for OA. Gluconic lactone may prove to be a novel criterion for differential diagnosis of OA from RA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1973–1981, 2017.

     

初次全膝关节置换术后并发症及翻修手术的原因分析

目的 探讨初次全膝关节置换术后30 d内相关并发症发生情况及翻修手术的相关因素.方法 收集2001年1月至2012年12月在北京协和医院骨科进行初次全膝关节置换术患者的临床资料,假体均为固定平台假体,采用骨水泥固定,排除翻修病例及血友病关节炎患者.共有1 920例患者(2 779例次全膝关节置换手术)纳入研究,男性323例,女性1 607例;年龄25～86岁,平均(66±9)岁.骨关节炎1 720例(89.58％),类风湿关节炎168例(8.75％),强直性脊柱炎12例(0.63％),继发骨关节炎20例(1.04％).随访患者术后30 d内发生的主要系统并发症、局部并发症及发生的翻修手术及相关因素.结果 随访截至2013年12月,共有1 854例患者(2 693个关节)获得随访,失访率为3.44％.术后平均随访67个月,死亡3例.41例(2.21％)患者出现系统并发症,其中最常见的为呼吸系统并发症(0.49％,9/1 854)及心血管并发症(0.38％,7/1 854).术后经超声证实的症状性深静脉血栓形成发生率为3.02％ (56/1 854),其中7例发生肺栓塞.术后发生局部并发症24例(1.29％),包括伤口愈合不良、伤口感染、神经损伤.59个关节接受翻修手术治疗,常见原因包括感染后松动(1.19％,32/2 693)和术后关节僵硬(0.37％,10/2693).结论 初次全膝关节置换术后30 d内最常见系统并发症为呼吸系统及心血管系统并发症.感染后松动是术后翻修最常见的原因.