Exploring the Early Molecular Pathogenesis of Osteoarthritis Using Differential Network Analysis of Human Synovial Fluid.

The molecular mechanisms that drive the onset and development of osteoarthritis (OA) remain largely unknown. In this exploratory study, we used a proteomic platform (SOMAscan assay) to measure the relative abundance of more than 6000 proteins in synovial fluid (SF) from knees of human donors with healthy or mildly degenerated tissues, and knees with late-stage OA from patients undergoing knee replacement surgery. Using a linear mixed effects model, we estimated the differential abundance of 6251 proteins between the three groups. We found 583 proteins upregulated in the late-stage OA, including MMP1, collagenase 3 and interleukin-6. Further, we selected 760 proteins (800 aptamers) based on absolute fold changes between the healthy and mild degeneration groups. To those, we applied Gaussian Graphical Models (GGMs) to analyze the conditional dependence of proteins and to identify key proteins and subnetworks involved in early OA pathogenesis. After regularization and stability selection, we identified 102 proteins involved in GGM networks. Notably, network complexity was lost in the protein graph for mild degeneration when compared to controls, suggesting a disruption in the regular protein interplay. Furthermore, among our main findings were several downregulated (in mild degeneration versus healthy) proteins with unique interactions in the healthy group, one of which, SLCO5A1, has not previously been associated with OA. Our results suggest that this protein is important for healthy joint function. Further, our data suggests that SF proteomics, combined with GGMs, can reveal novel insights into the molecular pathogenesis and identification of biomarker candidates for early-stage OA.

Identification and quantification of degradome components in human synovial fluid reveals an increased proteolytic activity in knee osteoarthritis patients vs controls.

Synovial fluid (SF) may contain cleavage products of proteolytic activities. Our aim was to characterize the degradome through analysis of proteolytic activity and differential abundance of these components in a peptidomic analysis of SF in knee osteoarthritis (OA) patients versus controls (n = 23). SF samples from end-stage knee osteoarthritis patients undergoing total knee replacement surgery and controls, that is, deceased donors without known knee disease were previously run using liquid chromatography mass spectrometry (LC-MS). This data was used to perform new database searches generating results for non-tryptic and semi-tryptic peptides for studies of degradomics in OA. We used linear mixed models to estimate differences in peptide-level expression between the two groups. Known proteolytic events (from the MEROPS peptidase database) were mapped to the dataset, allowing the identification of potential proteases and which substrates they cleave. We also developed a peptide-centric R tool, proteasy, which facilitates analyses that involve retrieval and mapping of proteolytic events. We identified 429 differentially abundant peptides. We found that the increased abundance of cleaved APOA1 peptides is likely a consequence of enzymatic degradation by metalloproteinases and chymase. We identified metalloproteinase, chymase, and cathepsins as the main proteolytic actors. The analysis indicated increased activity of these proteases irrespective of their abundance.

Vitamin E infused highly cross-linked cemented cups in total hip arthroplasty show good wear pattern and stabilize satisfactorily: a randomized, controlled RSA trial with 5-year follow-up.

Background and purpose - Vitamin E infused highly cross-linked polyethylene (VEPE) was introduced in order to enhance oxidative resistance in highly cross-linked polyethylene cups in total hip arthroplasty (THA). We have, with a follow-up of 5 years, evaluated wear characteristics of 2 identically designed cemented cups with the only difference being the material, VEPE or ultra-high molecular weight polyethylene (UHMWPE). Furthermore, we report cup migration and clinical outcome. Patients and methods - 48 patients with primary osteoarthritis were randomized to either UHMWPE or VEPE cups. Patients were followed with radiostereometric analysis (RSA) from the first postoperative day, at 3 months, 1, 2, and 5 years as well as with hip-specific outcome questionnaires. Results - At 3 months the mean proximal head penetration for UHMWPE was 0.07 mm (95% CI 0.03-0.11) and for VEPE 0.06 mm (-0.01 to 0.13). Thereafter, there was a continuous annual wear of 0.08 mm/year, up to 0.46 mm (0.36-0.57) at 5 years, for the UHMWPE cup. The VEPE cup showed low annual wear of 0.01 mm/year, up to 0.09 mm (0.02-0.16) at 5 years. In the first 3 months the UHMWPE cup migrated cranially 0.08 mm (0.03-0.13) whereas the VEPE cup migrated 0.17 mm (0.10-0.24), Thereafter, they showed similar migration patterns with stabilization between 2 and 5 years up to 0.21 mm (0.04-0.39) and 0.24 mm (0.13-0.36) respectively. The HOOS remained good up to 5 years, and no cup was revised. Interpretation - Compared with otherwise identical UHMWPE cups the cemented VEPE cup shows statistically significant reduction of wear up to 5 years and both cup types stabilize well with good clinical outcome.

Poor outcome after a surgically treated chondral injury on the medial femoral condyle: early evaluation with dGEMRIC and 17-year radiographic and clinical follow-up in 16 knees.

Background and purpose - The optimal treatment for traumatic cartilage injuries remains unknown. Contrast-enhanced MRI of cartilage (dGEMRIC) evaluates cartilage quality and a low dGEMRIC index may predict radiographic osteoarthritis (OA). The purpose of this study was (a) to explore the results 17 years after surgical treatment of an isolated cartilage knee injury and (b) to evaluate the predictive value of dGEMRIC. Patients and methods - 16 knees with an isolated traumatic cartilage injury of the medial femoral condyle had cartilage repair surgery either by microfracture or autologous cartilage implantation. dGEMRIC of the injured knee was performed 2 years after surgery and radiographic examinations were performed 17 years after the operation. Results - Radiographic OA was present in 12 of 16 knees. Irrespective of surgical method, the dGEMRIC index was lower in repair tissue compared with adjacent cartilage in the medial compartment, 237 ms vs. 312 ms (p < 0.001), which in turn had lower value than in the non-injured lateral cartilage, 312 ms vs. 354 ms (p < 0.008). The dGEMRIC index in the cartilage adjacent to the repair tissue correlated negatively with radiographic osteophyte score, r = -0.75 (p = 0.03). Interpretation - A traumatic cartilage injury is associated with a high prevalence of OA after 17 years. The low dGEMRIC index in the repair tissue 2 years postoperatively indicates fibrocartilage of low quality. The negative correlation between the dGEMRIC index in the adjacent cartilage and future OA suggests that the quality of the surrounding cartilage influences outcome after cartilage repair surgery.

Mineral Crystal Thickness in Calcified Cartilage and Subchondral Bone in Healthy and Osteoarthritic Human Knees.

Osteoarthritis (OA) is the most common joint disease, where articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. In particular, it is technically challenging to study calcified cartilage, where relevant but poorly understood pathological processes such as tidemark multiplication and advancement occur. Here, we used state-of-the-art microfocus small-angle X-ray scattering with a 5-µm spatial resolution to determine the size and organization of the mineral crystals at the nanostructural level in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from both medial and lateral compartments of medial compartment knee OA patients (n = 15) and cadaver knees (n = 10). Opposing the common notion, we found that calcified cartilage has thicker and more mutually aligned mineral crystals than adjoining bone. In addition, we, for the first time, identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32 nm thicker crystals compared to the rest of calcified cartilage. Finally, we found 0.2 nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process because the lateral compartment is typically less loaded in medial compartment knee OA. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee might be involved with the growth of mineral crystals, which is especially evident in the calcified cartilage. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Proteomic characterization of the normal human medial meniscus body using data-independent acquisition mass spectrometry.

Recent research suggests an important role of the meniscus in the development of knee osteoarthritis. We, therefore, aimed to analyze the proteome of the normal human meniscus body, and specifically to gain new knowledge on global protein expression in the different radial zones. Medial menisci were retrieved from the right knees of 10 human cadaveric donors, from which we cut a 2 mm radial slice from the mid-portion of the meniscal body. This slice was further divided into three zones: inner, middle, and peripheral. Proteins were extracted and prepared for mass spectrometric analysis using data-independent acquisition. We performed subsequent data searches using Spectronaut Pulsar and used fixed-effect linear regression models for statistical analysis. We identified 638 proteins and after statistical analysis, we observed the greatest number of differentially expressed proteins between the inner and peripheral zones (163 proteins) and the peripheral and middle zones (136 proteins), with myocilin being the protein with the largest fold-change in both comparisons. Chondroadherin was one of eight proteins that differed between the inner and middle zones. Functional enrichment analyses showed that the peripheral one-third of the medial meniscus body differed substantially from the two more centrally located zones, which were more similar to each other. This is probably related to the higher content of cells and vascularization in the peripheral zone, whereas the middle and inner zones of the meniscal body appear to be more similar to hyaline cartilage, with high levels of extracellular matrix proteins such as aggrecan and collagen type II.

Relating MR relaxation times of ex vivo meniscus to tissue degeneration through comparison with histopathology.

BACKGROUND: Quantitative magnetic resonance imaging (MRI), e.g. relaxation parameter mapping, may be sensitive to structural and compositional tissue changes, and could potentially be used to non-invasively detect and monitor early meniscus degeneration related to knee osteoarthritis. OBJECTIVE: To investigate MR relaxation times as potential biomarkers for meniscus degeneration through comparisons with histopathology. METHODS: We measured MR relaxation parameters in the posterior horn of 40 menisci (medial and lateral) at a wide range of degenerative stages. T1, T2 and T2* were mapped using standard and ultrashort echo time sequences at 9.4 T and compared to gold standard histology using Pauli's histopathological scoring system, including assessment of surface integrity, collagen organization, cellularity and Safranin-O staining. RESULTS: All three relaxation times increased with total Pauli score (mean difference per score (95% CI) for T2*: 0.62 (0.37, 0.86), T2: 0.83 (0.53, 1.1) and T1: 24.7 (16.5, 32.8) ms/score). Clear associations were seen with scores of surface integrity (mean difference per score for T2*: 3.0 (1.8, 4.2), T2: 4.0 (2.5, 5.5) and T1: 116 (75.6, 156) ms/score) and collagen organization (mean difference between highest and lowest score for T2*: 5.3 (1.6, 8.9), T2: 6.1 (1.7, 11) and T1: 204 (75.9, 332) ms). The results were less clear for the remaining histopathological measures. CONCLUSIONS: MR relaxation times T1, T2 and T2* of ex vivo human menisci are associated with histologically verified degenerative processes, in particular related to surface integrity and collagen organization. If confirmed in vivo, MR relaxation times may thus be potential biomarkers for meniscus degeneration.