Establishing disease biomarkers has been a long-sought after goal to improve Osteoarthritis (OA) diagnosis, prognosis, clinical and pharmaceutical interventions. Given the role of the synovium in contributing to OA, a meta-analysis was performed to determine significant synovial biomarkers in human OA tissue, compared to non-OA patients. Outcomes will direct future research on marker panels for OA disease modelling in vitro/in vivo, aiding clinical research into OA disease targets.

A PRISMA compliant search of databases was performed to identify potential biomarker studies analysing human, OA, synovial samples compared to non-OA/healthy participants. The Risk of Bias In Non-Randomised Studies of Interventions (ROBINS-I) tool assessed methodological quality, with outcome analysed by Grading of Recommendations Assessment, Development and Evaluation (GRADE). Meta-analyses were conducted for individual biomarkers using fixed or random effect models, as appropriate. Where three or more studies included a specific biomarker, Forest Plot comparisons were generated.

3230 studies were screened, resulting in 34 studies encompassing 25 potential biomarkers (1581 OA patients and 695 controls). Significant outcomes were identified for thirteen comparisons. Eleven favoured OA (IL-6, IL-10, IL-13, IP-10, IL-8, CCL4, CCL5, PIICP, TIMP1, Leptin and VEGF), two favoured non-OA controls (BMP-2 and HA). Notably, PIICP showed the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001, I² 99%), and TIMP1 resulted critically important (0.95 [0.65, 1.25], p <0.00001, I² 82%). Leptin and CCL4 showed lower effects (SMD 0.81 [0.33, 1.28], p =0.0009; 0.59 [0.32, 0.86], p <0.0001, respectively).

Thirteen significant synovial biomarkers showed links with OA bioprocesses including collagen turnover, inflammatory mediators and ECM components. Limitations arose due to bias risk from incomplete or missing data, publication bias of inconclusive results, and confounding factors from patient criteria. These findings suggest markers of potential clinical viability for OA diagnosis and prognosis that could be correlated with specific disease stages.

Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates.

The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture.

Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes.

RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment.

RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes.

Stratification is required to ensure that only those patients likely to benefit, receive Autologous Chondrocyte Implantation (ACI); ideally by assessing a biomarker in the blood. This study aimed to assess differences in the plasma proteome of individuals who respond well or poorly to ACI.

Isobaric tag for relative and absolute quantitation (ITRAQ) mass spectrometry and label-free proteomics analyses were performed in tandem as described previously by our group (Hulme et al., 2017; 2018; 2021) using plasma collected from ACI responders (n=10) compared with non-responders (n=10) at each stage of surgery (Stage I, cartilage harvest and Stage II, cell implantation).

iTRAQ using pooled plasma detected 16 proteins that were differentially abundant at baseline in ACI responders compared with non-responders (n=10) (≥±2.0 fold; p<0.05). Responders demonstrated a mean Lysholm (patient reported functional score from 0–100) improvement of 33±13 and non-responders a mean worsening of −13±13 points. The most pronounced plasma proteome shift was seen in response to Stage I surgery in ACI non-responders, with 48 proteins being differentially abundant between the two surgical procedures. We have previously noted this marked shift in response to initial surgery in the SF of ACI non-responders, several of these proteins were associated with the Acute Phase Response. One of these proteins, clusterin, could be confirmed in patients’ plasma using an independent immunoassay using individual samples. Label-free proteomic data from individual samples identified only cartilage acidic protein-1 (known to associate with osteoarthritis progression) to be significantly more abundant at Stage I in the plasma of non-responders.

This study indicates that proteins can be identified within the plasma that have potential use in ACI patient stratification. Further work is required to validate the findings of this discovery-phase work in larger ACI cohorts.

Anterior cruciate ligament (ACL) injuries have been increasing, especially amongst adolescents. These injuries can increase the risk for early-onset knee osteoarthritis (OA). The consequences of late-stage knee OA include structural joint change, functional limitations and persistent pain. Interleukin-6 (IL-6) is a pro-inflammatory biomarker reflecting knee joint healing, and increasing evidence suggests that IL-6 may play a critical role in the development of pathological pain. The purpose of this study was to determine the relationship between subjective knee joint pain and function, and synovial fluid concentrations of the pro-inflammatory cytokine IL-6, in adolescents undergoing anterior cruciate ligament reconstruction surgery.

Seven youth (12-17 yrs.) undergoing anterior cruciate ligament (ACL) reconstruction surgery participated in this study. They completed the Pedi International Knee Documentation Committee (Pedi-IKDC) questionnaire on knee joint pain and function. At the time of their ACL reconstruction surgery, synovial fluid samples were collected through aspiration to dryness with a syringe without saline flushing. IL-6 levels in synovial fluid (sf) were measured using enzyme linked immunosorbent assay. Spearman's rho correlation coefficient was used to determine the correlation between IL-6 levels and scores from the Pedi-IKDC questionnaire.

There was a statistically significant correlation between sfIL-6 levels and the Pedi-IKDC Symptoms score (-.929, p=0.003). The correlations between sfIL-6 and Pedi-IKDC activity score (.546, p = .234) and between sfIL-6 and total Pedi-IKDC score (-.536, p = .215) were not statistically significant.

This is the first study to evaluate IL-6 as a biomarker of knee joint healing in an adolescent population, reported a very strong correlation (-.929, p=0.003) between IL-6 in knee joint synovial fluid and a subjective questionnaire on knee joint pain. These findings provide preliminary scientific evidence regarding the relationship between knee joint pain, as determined by a validated questionnaire and the inflammatory and healing status of the patient's knee. This study provides a basis and justification for future longitudinal research on biomarkers of knee joint healing in patients throughout their recovery and rehabilitation process. Incorporating physiological and psychosocial variables to current return-to-activity (RTA) criteria has the potential to improve decision making for adolescents following ACL reconstruction to reduce premature RTA thereby reducing the risk of re-injury and risk of early-onset knee OA in adolescents.

Aim

Evaluate if Neutrophil Extracellular Traps related biomarkers (citrullinated histone H3 [H3Cit], cellfree DNA [cfDNA], and myeloperoxidase) are increased in synovial fluid of patients with PJI and investigate the diagnostic accuracy of NET formation biomarkers for PJI.

Multiple biochemical biomarkers have been previously investigated for the diagnosis, prognosis and response to treatment of articular cartilage damage, including osteoarthritis (OA). Synovial fluid (SF) biomarker measurement is a potential method to predict treatment response and effectiveness. However, the significance of different biomarkers and their correlation to clinical outcomes remains unclear. This systematic review evaluated current SF biomarkers used in investigation of cartilage degeneration or regeneration in the knee joint and correlated these biomarkers with clinical outcomes following cartilage repair or regeneration interventions.

PubMed, Institute of Science Index, Scopus, Cochrane Central Register of Controlled Trials, and Embase databases were searched. Studies evaluating SF biomarkers and clinical outcomes following cartilage repair intervention were included. Two researchers independently performed data extraction and QUADAS-2 analysis. Biomarker inclusion, change following intervention and correlation with clinical outcome was compared.

9 studies were included. Study heterogeneity precluded meta-analysis. There was significant variation in sampling and analysis. 33 biomarkers were evaluated in addition to microRNA and catabolic/anabolic ratios. Five studies reported on correlation of biomarkers with six biomarkers significantly correlated with clinical outcomes following intervention. However, correlation was only demonstrated in isolated studies.

This review demonstrates significant difficulties in drawing conclusions regarding the importance of SF biomarkers based on the available literature. Improved standardisation for collection and analysis of SF samples is required. Future publications should also focus on clinical outcome scores and seek to correlate biomarkers with progression to further understand the significance of identified markers in a clinical context.

After a hip fracture, infections are common, but signs of infection resemble those of systemic inflammatory response to trauma and surgery, and conventional infection markers lack specificity. Plasma-calprotectin, a novel marker of neutrophil activation, has shown potential as an infection marker in ER and ICU settings.

To investigate if plasma-calprotectin is superior compared to conventional infection biomarkers after hip fracture.

Prospective cohort study of hip fracture patients admitted to our department. Calprotectin, procalcitonin (PCT), C-reactive protein (CRP), and white blood cell (WBC) count were measured in blood plasma upon admission and on day 3 post-surgery. Patients with infection (pneumonia, UTI, sepsis, SSI, other soft tissue infections) pre- or post-surgery were compared to a control group without infection within 30 days.

Statistics: Wilcoxon rank-sum test, medians with interquartile range, and area under the curve (AUC) with 95% confidence intervals.

Pilot study comprises calprotectin obtained at least once for 60 patients at admission and 48 on day 3. Mean age 84 years (SD 8.4), 65% women.

9/60 patients (23%) were admitted with infections. They had higher levels of CRP (median 111 [73-149]) and PCT (0.35 [0.18–0.86]) compared to the control group (29 [16-64], p=0.037; 0.10 [0.07–0.17], p=0.007). Calprotectin (2.67 vs 2.51) and WBC (12.2 vs 9.3) did not differ significantly. AUC was highest for PCT (0.79 [CI 0.60–0.97]), followed by CRP (0.71 [0.46–0.96]), WBC (0.60 [0.35–0.84]), and calprotectin (0.58, [0.33–0.83]).

Day 3, 6/48 (13%) had infections, without significant differences between groups in any marker. The median levels were: calprotectin 3.5 vs 3.1, CRP 172 vs 104, WBC 12 vs 9, PCT 0.16 vs 0.17. Calprotectin had highest AUC 0.68 (0.41–0.93, n.s.). AUC for WBC was 0.67 (0.31–1.00), CRP 0.66 (0.38–0.94), PCT 0.56 (0.29–0.82).

Preliminary data show no significant associations with postoperative infection for any of the studied biomarkers. However, plasma-calprotectin might perform slightly better compared to conventional markers.

Abstract

Aim

We report on the performance of a simple algorithm using a combination of synovial fluid White blood cell count(WBC), C-reactive protein(CRP) and α-Defensin(AD) tests to aid in the diagnosis of prosthetic joint infections.

Background

About 85% of the patients with low back pain seeking medical care have nonspecific low back pain (NsLBP), implying that no definitive cause can be identified. Many pain conditions are linked with elevated serum levels of (pro-)inflammatory biomarkers.

Aim

Our goal is to assess diagnostic accuracy of synovial fluid testing in diagnosing prosthetic joint infection (PJI) as defined by the European Bone and Joint Infection Society (EBJIS). In addition to differential leukocyte count, simples and inexpensive biomarkers such as synovial fluid C-reactive protein (CRP), adenosine deaminase (ADA) and alpha-2-macrogloblulin(A2M) were also investigated and its possible role in increasing accuracy assessed.

Introduction

There are several potential biological mechanisms that may influence aseptic implant failure including excessive innate and adaptive immune responses to implant debris. We investigated the hypothesis that patients with painful total joint replacements will exhibit elevated levels of metal reactivity and inflammatory markers compared to patients with well-performing TJA. We evaluated this hypothesis by testing for metal hypersensitivity using in vitro LTT assay and analyzing serum levels of selected inflammatory markers.

Background

Structural bone allografts are an established treatment method for long-bone structural defects arising from such conditions as trauma, sarcoma, and osteolysis following total joint replacement. However, the quality of structural bone allografts is difficult to non-destructively assess prior to use. The functional lifetime of structural allografts depend on their ability to resist cyclic loading, which can lead to fracture even at stress levels well below the yield strength. Because allograft bone has limited capacity for remodeling, optimizing allograft selection for bone quality could decrease long-term fracture risk. Raman spectroscopy biomarkers can non-destructively assess the three primary components of bone (collagen, mineral, and water), and may predict the resistance of donor bone allografts to fracture from cyclic loads.

The purpose of this study was to prospectively assess the ability of Raman biomarkers to predict number of cycles to fracture (“cyclic fatigue life”) of human allograft cortical bone.

Aim

To evaluate a panel of peripheral blood and synovial fluid biomarkers for the identification of periprosthetic joint infection PJI.

There are numerous studies in the current literature that have demonstrated altered levels of various biomarkers in the serum of patients with implant loosening. Despite increasing interest in the biology of implant incorporation there are no studies investigating the changes in biological marker (of either osteoblastic or osteoclastic activity) levels during the integration of the bone-implant interface. Such a study would provide data about the biological profile of normal integration and would be helpful for future monitoring of implant prosthetic performance (either normal or abnormal).

We present data from a study performed on 100 osteoarthritic patients, who underwent cementless THA (Synergy, Reflexion Interfit, Smith & Nephew) and 100 non arthritic volunteers. Serial measurements of serum biochemical markers (bone formation and resorption), of cytokines and of other biological mediators and growth factors were evaluated at regular intervals over the course of six years. Curves of per cent changes from baseline and marker variability curves have been created for each marker which are indicative of the incorporation process.

Evaluating markers of osteoblastic activity, a first response, with average values below baseline, was observed at the level of the seventh day (perhaps as a response to local trauma). A second osteo-productive response was observed between the third week and 9 months (peak average values at the level of the 6^th month). At the 1st year time interval, average values reached baseline and remained at this level up to the 6th postoperative year. Evaluating markers of osteoclastic activity, a first response, with average values above baseline, was observed at the level of the seventh day (perhaps as a response to local trauma). A second osteoclastic response was observed between the third week and 3 months (perhaps a coupling response to enhanced osteoblastic activity). At 6 months, average values reached baseline and remained at this level up to the 6th postoperative year.

It seems that bone implant interface in cementless total hip arthroplasty remains active up to the 9^th postoperative month. Possible future deviation from such ‘individual normal’ curves will be indicative of the initiation of the osteolysis process and loss of fixation.

Osteoarthritis (OA)

is the most common arthritic condition. OA causes joint pain, loss of mobility and significantly affects the quality of life for the affected individual. The major burden to patients with arthritis is pain. However, often radiological joint destruction and the extent of pain do not correlate. This causes a dilemma for clinicians in advising timing for joint replacement surgery. In arthritis, concentrations of the neurotransmitter, glutamate is increased within the synovial fluid activating both peripheral pain mechanisms and pathological processes (1). Other pathological/pain related metabolites are also released into synovial fluid, which provides a real time snap shot of the joint pathology. We have tested the hypothesis that ‘The increased levels of pain and disease-related metabolites within human synovial fluids from arthritic joints can be detected and quantified ex vivo using high resolution 1H-NMR.’

Degenerative joint disease (DJD) involves the proteolysis of many extracellular matrix molecules (ECM) present in articular cartilage and other joint tissues such as tendon, meniscus and ligaments. Recent research has identified key enzymes involved in the catabolism of ECM. Two classes of enzyme the Matrix Metalloproteinases (MMP’s) MMP-2, MMP-3, MMP-13 and the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) of proteinases most notably, ADAMTS-1, -4 and −5, have been shown to be involved in the catabolism of ECM (such as type II collagen and cartilage aggrecan). The presence of several MMPs in the synovial fluid has been reported; however, little data has yet been gathered on the presence of ADAMTS-1, -4 or −5 (the aggrecanases) in synovial fluids. In this study we have used a recombinant artificial substrate and specific neoepitope antibodies that recognise either MMP- generated or aggrecanase -generated degradation products to measure the relative activity of these two enzyme families in the synovial fluid from human patients.

Methods: A recombinant substrate containing the interglobular domain of cartilage aggrecan , flanked by a complement regulator and the Fc region of IgG has been stably transfected into CHO cells. The recombinant protein has been purified from the medium using a Protein A column followed by gel chromatography using a Superose 12 column. Synovial fluid samples were depleted of serum immunoglobulin by pre-absorption with ProSepA. The recombinant substrate was then added to synovial fluid samples and incubated overnight as 37?C. The recombinant substrate was recovered from samples using ProsepA and then separated by SDS-PAGE (10% gels). Gels were transferred to nitrocellulose membranes and immunoblotted with antibodies recognising the undigested substrate and using neoeptiope antibodies specifically recognising MMP or aggrecanase –generated catabolites.

Results: Preliminary analysis by Western blot using the anti IGD neoepitopes BC-14 (detecting cleavage at the major MMP site) and BC-3 (detecting cleavage at the aggrecanase site) demonstrated that enzymes in human synovial fluid collected from patients diagnosed with rheumatoid arthritis cleaved the pro-drug at the MMP site with little or no evidence of aggrecanase catabolism. In contrast, synovial fluid collected from patients diagnosed with osteoarthritis indicted that there was cleavage at the aggrecanase site. In these preliminary studies we have also examined the enzyme activity in a set of clinical samples collected from patients that have undergone knee replacement surgery having been given either n-3 fatty acids or a placebo 10 weeks prior to surgery. Results indicate that aggrecanase generated fragments were found in synovial fluid from placebo patients, and reduced levels of enzyme activity were apparent in fluids tested from patients that had received n-3 fatty acids prior to surgery.

Discussion: This data suggests that the recombinant substrate will aid in the detection of MMP or aggrecanase activities in synovial fluid samples. The ratio of MMP to aggrecanase activity has potential as a biomarker for the severity of cartilage degeneration in degenerative joint diseases.

Aim

Our goal is to increase diagnostic accuracy of synovial fluid testing in differentiating prosthetic joint infection(PJI) by more exhaustively studying simple and inexpensive biomarkers. For that purpose, we sought to determine: 1) if synovial fluid C-reactive protein(CRP), alpha-2-macrogloblulin(A2M), procalcitonin and adenosine deaminase(ADA) concentrations are different between infected and aseptic cases; 2) performance and optimal cutoff values of each marker; 3) whether any such test may help improve diagnostic performance of traditional leukocyte count.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different soft and hard musculosk-eletal tissues. In the intervertebral disc (IvD) the major SLRPs involved in regulation of types I & II collagen fibril size are believed to be decorin, fibromodulin and lumican. Research into IvD degeneration and backpain is hampered by a lack of specific biomarkers to detect and monitor the disease process. We have discovered that two keratan sulphate (KS) substituted members of the SLRP family, Keratocan and Lumican (that are major KS-pro-teoglycans found in cornea) were unusually expressed in extracts from degenerative disc tissues.

Methods: Non-degenerate disc tissue (n=10) was obtained from 2 scoliosis patients and degenerate disc tissue from 11 patients undergoing surgery. The degenerate discs were graded using criteria described by Pfir-rman et al (Spine26: 1873; 2001). Tissue samples were extracted with 4M guanidine HCl and after dialysis subjected to SDS-PAGE and Western blot analyses using monoclonal antibodies that recognise epitopes on kera-tocan and lumican.

Results & Discussion: Keratocan was not found in the non-degenerate disc tissue but was present in all degenerate IvD tissues tested. Lumican showed and increased expression in extracts of degenative IvD tissues. Our working hypothesis is that the increased expression of these two SLRPs in degenerative disc tissue results from a reparative depostion of a type I collagen fibrillar ‘scar’. This unusual expression suggests their potential as biomarkers for detecting the onset of degenrative disc disease.

Introduction

Obesity is one of risk factors of anterior cruciate ligament tear in man or cranial cruciate ligament (CCL) tear in dog. Adipokines are biologically active mediators released from adipocytes, and correlate with changes in body mass index. In order to study the possibility that adipocytes play a role in the pathogenesis of CCL disease, we investigated alterations of the matrix degradation biomarker genes (matrix metalloproteinase-13 [MMP-13], aggrecan) in CCL cells after stimulating with adipokines.